Application of hsp70 as regulator of enzymatic activity

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biotechnology. The claimed invention relates to the field of modulation of enzymatic activity by application of interaction between a molecular chaperone Hsp70 and lysosomal phospholipid bis(monoacylglycero)phosphate (BMP). The invention can be used in medicine.

EFFECT: interaction of Hsp70-BMP modulates activity of lysosomal compartment enzymes, interacting with BMP, therefore the claimed invention represents means of reversing pathology of lysosomal storage diseases.

21 cl, 16 dwg, 2 tbl, 3 ex

 

This application is nepredvidatelne application patent application DK PA 2008 00885 filed June 26, 2008, which is incorporated into this description in full by reference. All patent and non-patent references listed in the preliminary application or in the present application is also included in the present description in full by reference.

The scope of the invention

The present invention relates to the field of modulation of enzymatic activity by using the interaction between the molecular chaperone Hsp70 and the lysosomal phospholipid bis(monoacylglycero)phosphate (BMP, also known as LBPA). The interaction of the Hsp70-BMP modulates the activity of lysosomal enzymes compartment interacting with BMP, and thus, the present invention provides a means for reversing the pathology of lysosomal storage diseases.

The level of technology

Molecular chaperones are found in all compartments of the cell, where conformational rearrangements of the protein, while protein synthesis is the main source nevernude peptides in the cell, the problem of cells, which occurs due to high temperature or other incentives that can make proteins structurally labile and, therefore, subject to netvertising and aggregation, faces JV is civicism cell response, involving the production of protective proteins. This response is a phenomenon, which is observed in all types of cells from prokaryotes to eukaryotes and is known as the response to heat shock or stress. Induced this response proteins known as heat shock proteins (HSP), among which there are several collections.

The main example of a family of chaperones are proteins Hsp70. Recently, this family was linked with other aspects of cellular homeostasis, in addition to functioning as a chaperone, most notably because of its antiapoptotic properties, their functions in the immune system and the explicit dependence of malignant cells from stimulation of Hsp70. In addition, Hsp70 may play a role in maintaining the integrity of lysosomes. However, the molecular mechanisms remain unclear.

Lysosomal storage disorders are a rare group of diseases characterized by the accumulation of substances in the lysosomal compartment and leading to its destabilization that has a devastating effect on the affected individuals. Substances accumulate in the lysosomal compartment due to deficiency of enzymes involved in their catabolism.

To date, there is not treatment available of most lysosomal storage diseases. The main cause of this disease is the inability specifications the ical lysosomal enzymes effectively metabolismrelated specific lysosomal substances, such as lipids. Thus, for a subset of these diseases, including Gaucher disease and Fabry disease, apply substitution therapy enzymes (ERT) by providing to the patient a recombinant enzyme. However, ERT is an expensive form of therapy, which may limit its use in some regions, and is effective only against a specific type of disease for which the obtained recombinant enzyme. The aim of the present invention is to provide new means for the treatment of lysosomal storage diseases.

The invention

In the present invention the molecular basis for the involvement of Hsp70 in the stability of lysosomal membranes describe by explaining the molecular basis of the Association between Hsp70 and cellular membranes, in particular, cytoplasmic and lysosomal membranes.

From literature it is known that Hsp70 may play a role in maintaining the integrity of lysosomes. However, the molecular mechanism remains unclear. In addition, the question is whether this property is specific to most-induced stress Hsp70 (HspA1A/A1Β identified as Hsp70 throughout this study) or whether other members of the Hsp70 family the same characteristics also were not considered.

Data unanswered questions prompted one of the main C the lei of the present invention, representing the molecular basis of lysosomal-protective action of Hsp70. In this regard, developed a method of obtaining a recombinant Hsp70 and its mutants, as a way subcellular fractionation based on ultracentrifugation in a gradient of iodixanol. Analysis for direct estimation of the integrity of lysosomal membranes based on induced photooxidation permeability of lysosomes, which makes it possible microscopic approach to evaluating the effects of Hsp70 and other components in real time in relation to their ability to make sensitive or to protect the lysosomal membrane. In various in vitro systems have investigated the interaction of recombinant Hsp70 and mutants with different lipids, including measurement of light scattering liposomes at an angle of 90°, the shift of tryptophan fluorescence and surface plasmon resonance (BIAcore). Modeling electrostatic surface Hsp70 in silico helped in creating a conceptual model of the interaction of Hsp70-BMP. To test in vivo compliance observed in systems in vitro interaction of the lipid interaction of BMP-Hsp70 were marked for both components. To further demonstrate the potential application of this mechanism was described by way of cell death induced by introduction of cisplatin, and in this system, cell death methyl the lysosomal Hsp70 in tumor lines and untransformed cells.

For the consideration of the molecular basis of the contribution of Hsp70 in the stability of lysosomal membranes, the authors sought to establish a system that addressed the influence of cytosolic Hsp70, i.e. targeting Hsp70 directly on complementary mechanism. Photos electron microscopy Nylandsted et al. it is shown that Hsp70 may be present inside the lysosomes, and thus decided to develop a method of obtaining a recombinant human Hsp70 (rHsp70) in the hope to use the mechanism of endocytosis as the shipping method rHsp70 directly in complementary mechanism. The authors of the present invention to overcome, thus the need to add lysosomal sorting signals Hsp70, potentially compromising the function and avoiding complications that can occur due to overexpression. In addition, endocytotic approach makes it possible titration amounts of rHsp70 and in the long run, opens the possibility of studying the mechanism of absorption of extracellular Hsp70.

Having developed a method of obtaining Hsp70, to assess endocytosis was labelled with fluorophore Alexa Fluor 488 (Hsp70-AF488). Using confocal imaging showed that rHsp70 in reality, therefore, is aimed at complementary mechanism. Then, to evaluate the impact on the stability of lysosomal membranes, the authors have developed a method for quantitative determination of permeability of lysosomal m is mpany at the level of individual lysosomes and the application of this method for evaluating the effect subjected to endocytosis rHsp70. These methods form the basis of examples 1 and 2, in which the authors show that Hsp70 enhances the survival of cells through the stabilization of lysosomes via a pH-dependent high-affinity binding endolysosomal anionic phospholipid bis(monoacylglycero)phosphate (BMP). Positively charged ATPase domain of Hsp70 is responsible for the binding, but the substrate-binding domain is also necessary for effective stabilization of lysosomes. Interestingly, this interaction and protective action, which it has, dependent on tryptophan 90, localized in positively charged wedge ATPase domain. It is important that you can achieve the cytoprotective actions delivery rHsp70 by endocytosis and specifically cancel it by extracellular injection of BMP antibodies or inhibitors of Hsp70.

In addition, the authors also sought to combine the protection mechanism of lysosomal membranes Hsp70 with symptoms of cancer and programmed cell death. Thus, the authors characterized the program of cell death initiated by the introduction of conventional chemotherapeutic agents cisplatin, and found that it is not dependent on caspase, but it is characterized by lysosomal release of proteases. Transgenic, as well as subjected to endocytosis, Hsp70 can enhance survival cleto is exposed to cisplatin through the stabilization of lysosomal membranes. Interestingly, the authors showed that labeled lysosomal Hsp70 itself or its lysosomal partner interaction bis(monoacylglycero)phosphate (BMP) make transformed, but not normal, lines of prostate cells are sensitive to this drug called cisplatin, which is the experimental confirmation of application interaction BMP-Hsp70 as a pharmacological target for the treatment of malignant tumors.

Interestingly, despite the fact that Hsp70-2 has 86% homology in amino acid sequence with Hsp70, he is not able to protect the lysosomal membrane directly. However, the depletion of Hsp70-2 also affects the permeability of the lysosomal membrane and subsequent programmed cell death. This action does not depend on direct interaction of Hsp70-2 and lysosomal compartment, but rather is controlled by suppressing the growth factor of the epithelium of the crystalline lens (LEDGF) in response to the depletion of Hsp70-2.

The methods and results of this study are discussed in detail in the Examples section below.

Explaining herein the molecular basis of the cytoprotective action of Hsp70 by interacting with lysosomal BMP, contributing to the stabilization of lysosomal, the data obtained provide the basis for therapeutic effects on lysosomal storage disorders.

To date, demonstrated that, unexpectedly, the introduction of recombinant Hsp70 in cells effectively reverses the pathology of lysosomal storage diseases, as shown in this document for disease Niemann-pick disease and Farber. In addition, the introduction into the cells of the inducer of Hsp70, benzyl alcohol, effectively reverses the pathology of lysosomal storage diseases, as shown in this document for disease Niemann-pick.

Thus, the present invention relates to a method for the treatment of lysosomal storage diseases by increasing directly or indirectly intracellular concentration and/or activity of Hsp70 in need of individuals through the introduction of Hsp70 and its functional fragment or variant or through the introduction of the inductor or conductor Hsp70.

In one aspect the present invention relates to the bioactive agent capable of increasing the intracellular concentration and/or activity of Hsp70 for use as a medicament or for use in the treatment of lysosomal storage diseases.

In one of the embodiments specified bioactive agent is a Hsp70, or a functional fragment or variant.

In another embodiment, the specified bioactive agent is an inducer or conductor the m Hsp70.

Another aspect of the present invention relates to a method for treating lysosomal storage diseases, including the introduction of bioactive means of the present invention to the needy in the individual.

In one embodiment, the implementation of the said treatment is prophylactic, healing or soothing.

In one of the embodiments specified lysosomal disease accumulation selected from the group consisting of disease, Niemann-pick, Farber disease, Krabbe disease, Fabry disease, Gaucher disease, sialidosis, metachromatic leukodystrophy and deficit sabotinov.

In another embodiment, the specified lysosomal disease accumulation is characterized as having a residual enzymatic activity of the defective enzyme involved in the pathology.

The present invention also relates to a method for treating lysosomal storage disorders, including the introduction of bioactive means of the present invention in combination with at least one other treatment method.

An additional aspect of the present invention relates to a method of modulating the enzymatic activity of the enzyme, where the specified enzyme interacts with BMP (bis(monoacylglycero)phosphate), comprising the steps

i) the introduction of bioactive means of the present invention,

ii) assumptions wsimages the via between BMP and Hsp70, and

iii) modulation of the enzymatic activity of an enzyme that interacts with BMP.

In another aspect, the present invention relates to Hsp70, or a functional fragment or variant for use as a medicine.

In one aspect the present invention relates to a method of modulating the enzymatic activity of the enzyme, where the specified enzyme interacts with the BMP, including the introduction of Hsp70, or a functional fragment or variant in the form which allows the interaction between BMP and Hsp70 or its specified functional fragment or variant, and thus, modulation of the enzymatic activity of an enzyme that interacts with BMP.

Preferably Hsp70 or its specified functional fragment or variant forms a covalent or non-covalent complex with BMP.

Preferably BMP interacts with saposiem.

Preferably specified saponin selected from the group consisting of saposin A, saposin B, saposin C and saposin D.

Preferably the specified enzyme selected from the group consisting of sphingomyelinase, sour sphingomyelinase, sialidase, alpha-galactosidase, beta-galactosidase, beta-galactosylceramidase, glucosylceramidase and sour ceramidase.

Preferably, the specified modulation of enzyme activity is the stimulation of the enzyme is effective activity of the specified enzyme.

In another aspect, the present invention relates to Hsp70, or a functional fragment or variant for use as a drug. Preferably specified Hsp70, or a functional fragment or variant can be used in the treatment, alleviation or prevention of lysosomal storage diseases such as Niemann-pick disease, Gaucher disease, Farber disease, Krabbe disease, Fabry disease and sialidase.

In another aspect, the invention relates to a method of increasing the capture compounds, comprising the step of introducing the specified connection together with Hsp70, or a functional fragment or variant. In one of the embodiments specified Hsp70, or a functional fragment or variant covalently associated with the specified connection. In another embodiment, the specified Hsp70, or a functional fragment or variant ecovalence associated with the specified connection.

A variant embodiment of the invention relates to a method of stimulation of the enzymatic activity of the enzyme associated with the lysosomal disease accumulation, such as Niemann-pick disease, Gaucher disease, Farber disease, Krabbe disease, Fabry disease and sialidase. Preferably specified lysosomal disease accumulation is Niemann-pick disease.

Because lysosomal storage disorders arise is due to lack enzymatic activity, the aim of the invention is to increase the enzymatic activity to mitigate or cure the breach.

It is shown that Hsp70 interacts with BMP. Because BMP acts as a cofactor for various other proteins, the interaction between Hsp70 and BMP can modulate the function of these various other proteins. For example, BMP acts as a cofactor for aSM. Thus, the interaction between Hsp70 and BMP can increase the activity aSM. As Niemann-pick disease associated with a reduction in activity aSM, Hsp70 can mitigate or cure the Niemann-pick disease by increasing the activity aSM. Similarly, BMP acts as a cofactor for saposin A, saposin B, saposin C and saposin D. Data proteins saposin involved in other lysosomal storage disorders, and thus, Hsp70 can mitigate or cure other lysosomal storage disorders by enhancing activity saposin or enzyme associated with the specified saposiem.

In the embodiment of the invention used in conjunction with Hsp70 substitution enzyme therapy in the treatment of lysosomal storage disorders. Thus, the required amount of enzyme can be significantly reduced by the enzyme-activating action of Hsp70.

In another embodiment, Hsp70 is used to facilitate capture of enzymes in substitution treatment is AI enzyme thus, increasing the amount of enzyme that are captured in the relevant cells.

Definitions and abbreviations

aSMa/ASM: sour sphingomyelinase

ADD70: trap Hsp70 derived AIF

AIF: Apoptosis-inducing factor

AO: Acridine orange

Apaf-1: Apoptotic protease-activating factor 1

Bag-1: Bcl-2-associated Aminogen-1

Bcl-2: B-cell lymphoma/leukemia 2

Bid: BH3-agonist interacting domain death

BMP: bis(monoacylglycero)phosphate

CARD: Domain activation of caspase

The caspase: Containerspecific protease

CHIP: C-end of the Hsp70-binding protein

CytC: Cytochrome C

DD: death Domain

DED: effector Domain death

dsrnas: double-Stranded RNA

eHsp70: Extracellular Hsp70

ER: Endoplasmic network

ERT: replacement therapy enzyme

FADD: Fas-associated protein containing a death domain

HIP: Hsp70-interacting protein

HRP: horseradish Peroxidase

HS: Heat shock/stress

HSE: Element heat shock

HSF: Factor heat shock

Hsp: heat shock Protein

HspBP1: Protein 1, binding heat shock proteins

IAP: Protein-inhibitor of apoptosis

iMEF: Immortalized embryonic fibroblasts mouse

JNK: c-jun NH2-terminal kinase

LAMP-1/-2: Lysosomal membrane protein-1/-2

LBPA: Osobistewymiana acid

LDGF: growth Factor epithelium of the crystalline lens

LMP: the permeability of the lysosomal membrane

MIC-1: Macrophage inhibitory cytokine 1

MOMP: Permeability of the outer mitochondrial membrane

MPR: the Receptor for mannose-6-phosphate

MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazole bromide

NPD: Niemann-pick Disease

NPDA: Niemann-pick Disease, type A

NPDB: Niemann-pick Disease, type b

NPDC: Niemann-pick Disease, type C

NPDD: Niemann-pick Disease, type D

PCD:Programmed cell death

PKC: protein kinase C

POPC: Palmitoyloleoylphosphatidylcholine

POPS: Palmitoyloleoylphosphatidylcholine

RNAi: RNA interference

ROS: reactive oxygen species

SD: Standard deviation

siRNA: Small interfering RNA

Smac/Diablo: the Second mitochondrial activator of caspase

tBid: Truncated Bid

TNF: tumor necrosis Factor

TNFR: TNF receptor

TRADD: TNFR-associated protein containing a death domain

TRAF: TNFR-associated factor

Lysosomal storage disorders (LSD): the term "lysosomal violation of accumulation and lysosomal disease accumulation" are used as synonyms.

Functional Hsp70 fragment: the term "functional fragment of Hsp70" interpret to mean any fragment of Hsp70 with the desired function. In relation to the modulation of the enzymatic activity of a functional fragment is a fragment capable of fashion is activated enzymatic activity. Increased capture substances functional Hsp70 fragment is a fragment that can increase the capture of the specified substances. It should be understood that the exact quantitative effect of the functional fragment can vary from full-sized molecules. In some cases, a functional fragment may actually be more effective than a full-sized molecule. In addition, the use of fragments instead of full-sized molecules can be advantageous from the point of view of smaller fragments.

A functional variant of Hsp70: the term "functional variant of Hsp70" interpret the meaning of any variant of Hsp70 with the desired function. In relation to the modulation of the enzymatic activity of the functional variant is a variant that is able to modulate the enzymatic activity. To increase the capture substance is a functional variant of Hsp70 is a fragment capable of increasing the capture of the specified substances. It is clear that the exact quantitative effect of the functional variant may differ from a full-sized molecules. In some cases, a functional variant, in fact, may be more effective than a full-sized molecule.

"Bioactive agent" (i.e. biologically active substance/tool) is any with what edstam, drug, compound, composition of materials, or a mixture, which provides some pharmacologic, often beneficial, effect that can be demonstrated in vivo or in vitro. In this document this term also includes any physiologically or pharmacologically active substance that has a local or a systemic effect on the individual. Additional examples of supplements include as non-limiting examples of means, including or consisting of an oligosaccharide, products containing or consisting of a polysaccharide, tools, optionally containing or consisting of a glycosylated peptide, tools, optionally containing or consisting of a glycosylated polypeptide, products containing or consisting of nucleic acids, products containing or consisting of the oligonucleotide, products containing or consisting of polynucleotide, products containing or consisting of a lipid, products containing or consisting of fatty acids, products containing or consisting of complex fatty acid ester, and means containing or consisting of a secondary metabolite. It can be used prophylactically, therapeutically in connection with the treatment of the individual, such as a human or any other animal. In this document the bioactive agent is a substance, capable of increasing the intracellular concentration and/or activity of Hsp70.

The terms "drug" or "drug" in this document include biologically, physiologically, or pharmacologically active substances which can locally or systemically acting on the body of man or animal.

The terms "treatment" and "therapy" in this document equivalent is used for curative therapy, prophylactic or preventive therapy and softening or palliative therapy. The term includes the approach of obtaining beneficial or desired physiological results that can be installed clinically. For the purposes of this invention, beneficial or desired clinical results include, as non-limiting examples of reducing symptoms, reducing the extent of disease, stabilized (i.e. not worsening) state, stopping or slowing down the progress or deterioration of the condition/symptoms, mitigate or alleviate the condition or symptoms, and remission (partial or complete), detectable or not detectable. In this document the term "relief" and its variants mean that the extent and/or undesirable manifestations of physiological condition or symptom decline and/or progress slowed or extended in time compared to marinkovski the composition of the crust is Adamu invention.

"Treatment effect" or "therapeutic effect" is shown, if there is a change in the state affected by the treatment, which is judged by the criterion that comprises the definition of the term "treatment". This "shift" state, subjected to treatment exists, if exists, at least 5% improvement, preferably 10% improvement, more preferably at least 25%, even more preferably at least 50%, such as at least 75%, and most preferably at least 100% improvement. The change may be based on the improvements of gravity is affected by the treatment status of the individual, or the difference frequency of the improving conditions in the population of individuals with treatment and without treatment bioactive agent or bioactive agent in combination with the pharmaceutical composition of the present invention.

"Pharmacologically effective amount", "pharmaceutically effective amount" or "physiologically effective amount "bioactive money" is so many bioactive tools present in the pharmaceutical composition as described herein that is required to maintain the desired level of active funds in the bloodstream or at the site of action of the individual (for example, in the lungs, digestive system, colorectal system, etc is Stateline gland, and so on) to ensure with the aim of obtaining the expected physiological response when the introduction of such compositions. The exact number depends on many factors, such as active funds activity of the composition applied by means of delivery of the physical characteristics of the composition, the intended use of the patient (i.e. the number of doses that are given in the day), respect for the patient's treatment regimen, etc. and it can easily identify the person skilled in the art depending on the information provided in this document. "Effective amount" bioactive means you can enter in an injection or through repeated administration of the number constituting in total, an effective amount, preferably within 24-hour period. It can be determined by standard clinical methods for determining the appropriate quantities and timing of administration. It should be understood that "an effective amount" may be the result of empirical and/or individualized (for each case) determination by the attending medical professional and/or individual.

The terms "increase" and "improvement" of the positive effect and their variants in this document relate to therapeutic effect of bioactive funds compared to placebo or to increase therapeutic effect of medical treatment on the modern level of technology achieved over usually when the pharmaceutical composition is administered without bioactive means in this invention. "Increasing therapeutic effect is manifested when there is an acceleration and/or increase in intensity and/or duration of therapeutic effects achieved by the introduction of bioactive funds (funds). It may also include the increased duration of therapeutic effects. This can also manifest itself when to achieve the same effects as in the joint introduction of the bioactive agent (means)that is provided by the present invention, compared with the introduction of a larger amount of the pharmaceutical composition without bioactive tools require a smaller amount of the pharmaceutical composition. The strengthening effect is preferably, but not necessarily, leads to the treatment of acute symptoms, for which the pharmaceutical composition separately ineffective or less therapeutically effective. The gain achieved when there is at least a 5% strengthening of therapeutic effects, such as at least 10% strengthening of therapeutic effects, when the bioactive agent of the present invention is administered together with a pharmaceutical composition in comparison with the introduction of the pharmaceutical compositions separately. Preferably an increase of at least 25%, more preferably at least 50%, even more preferably, IU the greater extent, 75%, most preferably at least 100%.

"Co-administration" bioactive funds (funds) or supplements and medicines at the modern level of technology in this document refers to the introduction of one or more bioactive means of the present invention or introduction of one or more bioactive means of the present invention and pharmaceutical compositions according to the modern level of technology in a certain period of time. Preferred is a period of time less than 72 hours, such as 48 hours, for example less than 24 hours, such as less than 12 hours, for example less than 6 hours, such as less than 3 hours. However, these terms also indicate that the bioactive agent and therapeutic composition can be entered together.

The term "individual" refers to a vertebrate, in particular to the representatives of species of mammals, preferably primates, including humans. In a preferred embodiment, the individual in this document is a person, man or woman of any age.

"In need of this individual" refers to an individual who may benefit from the present invention. In one of the embodiments indicated needing this individual is a sick individual, where specified, the TB is evania is a lysosomal storage disease of accumulation.

The term "natural nucleotide" or "nucleotide" refers to any of the four deoxyribonucleotides, dA, dG, dT, and dC (DNA elements) and four ribonucleotides, A, G, U and C items (RNA), as they exist in nature. Every natural nucleotide contains or essentially consists of the balance of sugar (ribose or deoxyribose)indicated phosphate residue and residue natural/standard bases. Natural nucleotides bind with complementary nucleotides on well-known rules of pairing bases (Watson and Crick), where adenine (A) can be paired with thymine (T) or uracil (U); and where the guanine (G) can be paired with cytosine (C), where the corresponding pairs of nucleotides are part of the complementary antiparallel chains of nucleotides. The base pairing leads to specific hybridization between pre-defined and complementary nucleotides. The base pairing is the basis, in accordance with which the enzymes capable of catalyzing the synthesis of the oligonucleotide, complementary matrix to the oligonucleotide. In this synthesis of the building blocks (usually triphosphates of RIBO - or deoxyribo-derivatives of A, T, U, C or G) are directed by the matrix oligonucleotide for the formation of a complementary oligonucleotide with the correct complementary sequence. The recognition sequence of the oligonucleotides on it is omplementary sequence is mediated by the corresponding and interacting bases which form base pairs. In the nature of the specific interactions that lead to mating grounds, governed by the size of the grounds and structure of donor and acceptor hydrogen bonds in the grounds. Large purine base (A or G) is coupled with a small pyrimidine base (T, U or C). Additionally, the recognition of base pairs between pairs is influenced by hydrogen bonds formed between bases. In the geometry of the base pairs of the Watson-Crick six-membered ring (pyrimidine in natural oligonucleotides) is mapped to a ring system consisting of a fused six-membered ring and the five-membered ring of the purine in natural oligonucleotides) with hydrogen bond in the middle, connecting the two atoms of ring, and hydrogen bonds on either side of the ring connecting the functional group attached to each ring, with donor groups, coupled with acceptor groups.

Herein "nucleic acid" or "nucleic acid molecule" refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments obtained by polymerase chain reaction (PCR), and fragments obtained by ligation, cleavage, action endonuclease or actions exon is sleazy. The nucleic acid molecules can be monomers, which are natural nucleotides (such as DNA and RNA), or analogs of natural nucleotides (e.g., alpha-enantiomeric forms of natural nucleotides), or a combination. Modified nucleotides may be differences in sugar residues and/or residues pyrimidine or purine bases. Modification of sugar, including, for example, replacement of one or several hydroxyl groups, Halogens, alkyl groups, amines, and azide groups, or sugar can be functionalitywith as ethers or esters. In addition, a residue can be replaced steric and electron-similar structures, such as asakhara and carbocyclic sugar analogues. Examples of modifications of residues grounds include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other commonly known heterocyclic substitutes. The monomers of nucleic acids can be associated phosphodiesterase bonds or analogs of such links. Analogues fosfolipidnyh links include phosphothioate, phosphodithioate, phosphosaline, phosphodiester, phosphonylation, postanalytic, phosphoamide, etc. the Term "nucleic acid molecule" also includes, for example, so-called "peptide nucleic acid", which contain natural is whether the modified nucleic acid bases, attached to a polyamide backbone. You can apply single-stranded or double-stranded nucleic acids.

The term "complementary nucleic acid molecule" refers to a nucleic acid molecule having a complementary nucleotide sequence and reverse orientation as compared with the original nucleotide sequence. For example, the sequence 5' ATGCACGGG 3' complementary to the 5' CCCGTGCAT 3'.

"The selected nucleic acid molecule" is a nucleic acid molecule that is not integrated in the genomic DNA of the organism. For example, the DNA molecule, which encodes a growth factor and which was separated from the genomic DNA of the cell, is selected DNA molecule. Another example of a selected DNA molecule is chemically synthesized nucleic acid molecule that is not integrated into the genome of the organism. A nucleic acid molecule that was isolated from individual species, less than a complete DNA molecule of a chromosome of this species.

"Design of a molecule of nucleic acid" is a nucleic acid molecule, a single - or double-stranded molecule of nucleic acid-modified human intervention in such a way as to contain segments of nucleic acids, combined and mapped into non-existent in nature order.

Linear DNA" means Callaway the DNA molecule, having a free 5'- and 3'-ends. Linear DNA can be obtained from closed circular DNA molecules such as plasmids, by enzymatic degradation or physical destruction.

"Complementary DNA (cDNA)is single-stranded DNA molecule obtained from a matrix of mRNA using the enzyme reverse transcriptase. Typically, to initiate reverse transcription using primer, complementary parts of the mRNA. Professionals in this field also use the term "cDNA" to refer to double-stranded DNA molecule consisting of single-stranded DNA molecules, and she complementary DNA chain. The term "cDNA" also applies to clone the cDNA molecules synthesized by the RNA matrix.

"Heterologous DNA" refers to a DNA molecule or combination of DNA molecules that do not exist in nature in a certain cell host. Heterologous to a single host cell, the DNA molecule may contain DNA from species of the host cell (i.e. endogenous DNA), provided that the host DNA combined with DNA is not the owner (i.e. exogenous DNA). For example, a DNA molecule containing the DNA segment does not host encoding a polypeptide that is functionally associated with a segment of the host DNA containing a transcriptional promoter, considered as a heterologous DNA molecule. On the contrary, the heterologous DNA molecule may contain e is Doheny gene functionally linked to an exogenous promoter. As another illustration, a DNA molecule containing a gene from wild-type cells, considered as a heterologous DNA if the DNA molecule is embedded in a mutant cell with a lack of the gene of the wild type.

"Polypeptide" is a polymer of amino acid residues, preferably of the United solely by peptide bonds, obtained from natural or synthetic means. The polypeptide obtained by the expression of the DNA molecule are not the host, is "heterologous" peptide or polypeptide. In this document, the term "polypeptide" includes proteins, peptides and polypeptides, where these proteins, peptides or polypeptides can excision modify or not to modify. Post-translational modification may include phosphorylation, methylation and glycosylation.

The term "expression" refers to the biosynthesis of a gene or gene product.

"Hybridizing" means the annealing of chains of nucleic acids from different sources, which is the formation of base pairs between complementary regions of two DNA chains that source is not coupled. The term "hybridization under strict conditions" was defined by Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), 1101-1104. Preferably hybridization in simple terms means that after about the myuki for 1 hour with a single SSC and 0.1% SDS at 50°C, preferably at 55°C, more preferably at 62°C and most preferably at 68°C, in particular 1 hour in 0.2 times SSC and 0.1% SDS at 50°C, preferably at 55°C, more preferably at 62°C and most preferably at 68°C, see a positive hybridization signal.

The plot is full of homology" is defined as the coincidence of paired nucleotides in length sequences of the interacting nucleotides; in natural RNA pairing A with U and G with C.

"Promoter" is a nucleotide sequence that directs transcription of a structural gene. Typically, the promoter is localized in the 5'non-coding region of a gene, proximal to the site of the start of transcription of the structural gene. The sequence elements within promoters that function in the initiation of transcription, often described as the consensus nucleotide sequence. If the promoter is an inducible promoter, the level of transcription increases in response to an inducing agent. In contrast, the level of transcription is not regulated by an inducing agent, if the promoter is a constitutive promoter. Also known repressed promoters.

"Regulatory element" is a nucleotide sequence that modulates the activity of the promoter. For example, a regulatory element may content the th sequence of nucleotides, which is associated with cellular factors that trigger transcription exclusively or preferentially in specific cells, tissues, or organelles. These types of regulatory elements typically associated with genes that are expressed "cell-specific", "Dane-specific" or "organelle-specific way."

"Enhancer" is a type of regulatory element, which can increase the efficiency of transcription regardless of the distance or orientation of the enhancer relative to the site of the start of transcription.

"Cloning vector" is a nucleic acid molecule such as a plasmid, cosmid or bacteriophage, which has the ability to autonomously replicate in the cell host. Typically, cloning vectors contain one or a small number of recognition sites of restriction endonucleases, which makes possible the insertion of nucleic acid molecules in a certain way without losing important biological function of the vector, as well as the sequence of nucleotides that encodes a marker gene that is suitable for use in the identification and selection of transformed cells with the cloning vector. Typically, a marker gene include genes that confer resistance to tetracycline and ampicillin.

"Expressing vector" is a molecule nucleotide sequence that is the new acid, encodes a gene which is expressed in the cell host. As a rule, expressing the vector contains a transcriptional promoter, gene and terminator of transcription. Typically, gene expression is under the control of the promoter, and this gene is referred to as "functionally associated" with the promoter. Similarly, a regulatory element and a nuclear promoter are functionally related if a regulatory element modulates the activity of nuclear promoter. More than a simple vector, called "transcription vectors"that can only be transcribed, but not be broadcast: they can be replicated in the target cell, but not expressed, in contrast to expressing vectors. Transcription of the vector used for the amplification of their inserts.

"Recombinant host is a cell that contains a heterologous nucleic acid molecule, such as a cloning vector or expressing vector.

Transfection describes the introduction of foreign material in eukaryotic cells. The term "transfection" for non-viral methods most often used in relation to mammalian cells, while the term "transformation" is preferred to describe the transfer of non-viral DNA in bacteria and eukaryotic cells animal origin, such as fungi, algae and plants. For tra is speccie can be applied chemical, and physical methods.

"Polypeptide" is a polymer of amino acid residues, preferably connected by peptide bonds, obtained from natural or synthetic means. Produced by expression of DNA molecules do not host polypeptide is "heterologous" peptide or polypeptide. In this document, the term "polypeptide" includes proteins, peptides and polypeptides, where these proteins, peptides and polypeptides can excision modify or not to modify. For example, post-translational modification may include phosphorylation, methylation and glycosylation.

"Amino acid residue" may be natural or unnatural amino acid residue linked by peptide bonds or bonds that differ from the peptide bonds. Amino acid residues may be D-configuration or L - configuration. Amino acid residue contains aminobenzene part (NH2and carboxykinase part (COOH), separated by the Central part containing a carbon atom or a chain of carbon atoms, of which at least one atom contains at least one side chain or functional group. NH2refers to the amino group present on aminocore amino acid or peptide, and COOH refers to the carboxyl group present on the carboxyl end of aminox the slots or peptide. The generic term amino acid includes both natural and unnatural amino acids. Natural amino acids according to the standard nomenclature, as described in J. Biol. Chem., 243:3552-59 (1969) and adopted at 37 C.F.R., section 1.822(b)(2), belong to the group of amino acids listed below in table 1 of this document. Unnatural amino acids are those that do not are shown in table 1. Examples of unnatural amino acids are those described in, for example, in 37 C.F.R. section 1.822(b)(4), all of which are incorporated herein by reference. Unnatural amino acid residues also include as non-limiting examples of modified amino acid residues, L-amino acid residues and stereoisomers D-amino acid residues.

Table 1
Natural amino acids and their corresponding codes
DesignationAmino acid
1-letter3-letter
YTyrtyrosine
GGlyglycine
F Phephenylalanine
MMetmethionine
AAlaalanine
SSerserine
IIleisoleucine
LLeuleucine
TThrthreonine
VValvaline
PProProline
KLyslysine
HHishistidine
QGlnglutamine
EGluglutamic acid
WTrp tryptophan
RArgarginine
DAspaspartic acid
NAsnasparagine
CCyscysteine

"Equivalent amino acid residue" refers to amino acid residue capable of substituting another amino acid residue in the polypeptide that no significant differences in the structure and/or function of the polypeptide. Thus, the equivalent amino acids have similar properties, such as the size of the side chain, the polarity of the side chain (polar or nonpolar), hydrophobicity (hydrophobic or hydrophilic), pH (acidic, neutral or alkaline) and the organization of the carbon side chain (aromatic/aliphatic). In this regard, "equivalent amino acid residues can be seen as "conservative substitutions of amino acids".

Classification of equivalent amino acids is in one of the embodiments in the following classes: 1) HRK, 2) DENQ, 3) C, 4) STPAG, 5) MILV and 6) FYW

In this document the term "equivalent replacement amino acids, one amino acid can be the t to replace another one of the embodiments in groups of amino acids, following in this document:

i) Amino acids having polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr, Tyr, and Cys);

ii) Amino acids having non-polar side chains (Gly, Ala, Val, Leu, Ile, Phe, Trp, Pro, and Met);

(iii) Amino acids having aliphatic side chains (Gly, Ala, Val, Leu, Ile);

iv) Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro);

v) Amino acids having aromatic side chains (Phe, Tyr, Trp);

vi) Amino acids having acidic side chains (Asp, Glu);

(vii) Amino acids with alkaline side chains (Lys, Arg, His);

(viii) Amino acids having amide side chains (Asn, Gln);

(ix) Amino acids having hydroxyl side chains (Ser, Thr);

x) Amino acids having sulfur-containing side chains (Cys, Met);

xi) Neutral, laboratornye amino acids (Pro, Ala, Gly, Ser, Thr);

xii) a Hydrophilic, acidic amino acids (Gln, Asn, Glu, Asp), and

xiii) Hydrophobic amino acids (Leu, Ile, Val).

The present invention also relates to variants of Hsp70 or its fragments, where the replacement model through computer analysis, which used the homology sequences to predict the effect of substitutions on the function of a protein (for example, Pauline C. Ng and Steven Henikoff, Genome Research, Vol. 11, Issue 5, 863-874, May 2001).

Due to the inaccuracy of the standard analytical methods of molecular weight and length of the polymers considered as priblizitelen the e value. When such a value is expressed as "approximately" X or "approximately" X, the accuracy of the explicit values of X considered as +/-20%, such as +/-10%, for example +/-5%.

Description of the drawings

Figure 1

Effect of Hsp70 binding aSMa with BMP and levels of ceramide. (A) Binding 0.2 μm aSM with BMP-containing liposomes at pH 4.5 as a function predskazannogo Hsp70 (experimental analogue of example 1, see materials and methods in this document). Hsp70 was allowed to dissociate for 10 minutes, thereby achieving lower asymptotes for the dissociation before adding aSMa. (B) Confocal microscopy and quantitative analysis of the levels of ceramide in iMEF wild-type (WT) and transgenic for Hsp70 (Hsp70-TG) iMEF. Immunological analysis was performed with a monoclonal antibody mouse anti ceramide (clone 15b4). Conducted quantitative analysis, based on the received laser scanning micrographs of the 6 pre-defined regions, after which quantitative analysis was carried out using the software LSM Duo.

Figure 2

Action rHsp70 on the activity of the acidic sphingomyelinase in iMEF-WT (immortalized embryonic fibroblasts mouse wild type). Cells were injected rHsp70 at a concentration of 3 nm, 30 nm and 300 nm and measured the activity aSM (A500 is the value produced ceramide, which increases tightly the TB). Control cells were treated with BSA (bovine serum albumin).

Figure 3

The activity of acid SM in different fibroblasts. NPDA: Niemann-pick Disease type A.

Figure 4

The scheme of the hydrolysis of the main sphingolipid. Ecohydrological the breakdown of sphingolipids with short hydrophilic head groups requires nonenzymatic cofactors, proteins, activators of sphingolipids (SAP or saposin). Hereditary deficiencies of the corresponding enzyme, as well as the corresponding protein-activator called lysosomal lipid accumulation and lead to the development of various sphingolipidoses. From Ferlintz et al., Chem. Phys. Lipids, (102) 35-43, 1999.

Figure 5

Lysosomal Hsp70 stabilizes lysosomal membranes. (a) Presents confocal pictures of cells U-2-OS, which were incubated with 300 nm rHsp70-AF488 (green) for 24 hours, fixed and stained for the presence of lysosomal integral membrane protein-1 (LIMP-1; red). For colocalization with markers of other organelles see Fig. 9. (b) Prior to quantitative analysis of rHsp70-AF488 in the membranes (MEM.) and supernatant (sup.), obtained through repeated cycles of freezing-thawing and centrifugation light membrane fraction (LMF), cells U-2-OS cells were then incubated with 300 nm rHsp70-AF488 within 24 hours. The immunoblot lysosome-associated membrane protein 2 (LAMP-2) and cathepsin is Β (Cat B) demonstrates the applicability of the method of fractionation. (c) photographs of cells U-2-OS subjected to photo-oxidation (acridine orange and blue light). The loss of integrity of lysosomes were visualized by the loss of the red and the increase of green coloring. (d and e) Cells U-2-OS cells were then incubated with the indicated recombinant proteins (300 nm) for 24 hours and analyzed the integrity of lysosomes after photooxidation. As stated, before adding recombinant proteins (e) cells were treated with the indicated siRNA for 48 hours. Values represent values ± SD for three (d) five (e) independent experiments. Presents immunoblot these proteins from cells U-2-OS were left untreated or were treated with control or Hsp70 siRNA they are shown on the right. Bars: 20 μm (a and c).

Figure 6

the pH-dependent interaction between Hsp70 and BMP stabilizes lysosomal membranes, (a) Relative changes in light scattering at an angle of 90° liposomes after adding rHsp70 (0.12 nm aliquot) to the liposomes containing the lipids (χ=0,2) at a pH of 7.4 (left) and pH of 6.0 (right). (b) Cells U-2-OS were left untreated (-) or incubated with 50 µg/ml of anti-BMP or control IgG for 7 hours before adding the filling (-) or 300 nm rHsp70 within 24 hours and analyzed the integrity of lysosomes after photooxidation. (c) Cells U-2-OS were left untreated or incubated with 0 μg/ml anti-BMP or control IgG for 7 hours before adding the filling (-) or 25 μm cisplatin for 24 hours and analyzed the morphology of apoptotic cells after staining hagstom 33342. (d) Interaction rHsp70 and its mutants with POPC liposomes/BMP (χΒΜΡ=0,2) at a pH of 6.0 was evaluated by changes in the relative intensities of peaks of fluorescence. Protein concentration was 0.36 μm (rHsp70), 0.5 µm (ΔΑΤΡ) and 0.35 μm (ΔPBD) (left) or 0.43 μm (right), and was added to the liposomes in 10 mm aliquot. (e) Analysis of interactions BIAcore between rHsp70 wild-type (WT) and deletion (ΔΑΤΡ and ΔPBD) and the point heteroplasmy mutation (W90F and W580F) with immobilized LUV at pH 4.5 (average diameter: 100 nm; the total lipid concentration: 0.1 mm; composition: sphingomyelin (χ=0,1), phosphatidylcholine (χ=0.5), and cholesterol (χ=0.2) and BMP (χ=0,2)). Contributed liposomes to achieve equilibrium (100) and made the indicated concentration (left) or 300 nm (right) of recombinant protein in a buffer of sodium acetate (50 mm, pH 4.5) to 200 C at a flow rate of 20 ál/min with a subsequent stage of dissociation within 10 minutes ΔRU defined as the difference between the response signal after reaching equilibrium liposomes and balance the protein-liposome. (f and g) Cells U-2-OS were left untreated (control) or incubated with the indicated recombinant proteins Hsp70 (300 nm) for 24 hours and analyzed the integrity of lysosomes after photooxidation (f), or were treated filler (white columns) or 25 μm cisplatin (black bars) for 24 hours and analyzed apoptosis-like morphology (g). (h) a Ribbon model and the molecular model the surface of the ATPase domain of Hsp70. ATP (representing the van der Waals surface) can be visualized bound in the ATP-binding pocket. Green and purple spheres represent the van der Valavu surface coordinated ions of calcium and sodium, respectively. Marked positively charged part of the domain at the bottom and at the position of the W90. Values represent values ± SD for at least five independent experiments (b, c, f and g).

Figure 7

Hsp70 stimulates activity of ASM, which, in turn, stabilizes the complementary mechanism. (a) Biacore Analysis of binding of 200 nm rASM with BMP-containing liposomes at pH 4.5 as a function predskazannogo rHsp70. The experiments were conducted as described in the comments to Fig. 6e, with rASM added within 180 sec after 10 min of stage rHsp70-dissociation followed another 10 - minute stages of dissociation. (b) the ASM Activity in MEF lysates of wild-type (WT) and transgenic for Hsp70 (Hsp70) MEF (left panel) and WT MEF incubated with 300 nm rHsp70 within 24 or 48 hours, as indicated. (c and d) Viability (MTT reduction; c) and the activity of cytosolic cathepsin (zFRa; (d) in WT and Hsp70 iMEF treated with the indicated concentrations of desipramine for 3 hours. (e) in Vivo separately-cell visualization of the loss of integrity of lysosomes (photo-oxidation in WT and Hsp70 MEF, as well as Hsp70 MEF, incubated for 3 hours with 12.5 and 25 μm desipramine (left and right panel, respectively). Loss to what asnau (left panel) and an increase in green fluorescence (right panel) continuously measured to obtain the total kinetic curves of the loss of integrity of lysosomes. 25-60 cells investigated pr. the experiment of the predefined regions), p<0.001 for Hsp70 vs. WT and Hsp70 + desipramine vs. Hsp70. All values represent values ± SD for at least 3 independent experiments.

Figure 8

rHsp70 stimulates activity of ASM, complementary mechanism stabilizes and reduces the amount of lysosomes in NPDA fibroblasts. (a) Intravital separately-cell visualization of the stability of lysosomes from primary fibroblasts from a patient with NPDA analyzed as in Fig. 3e, p<0,001. (b) the Activity of ASM NPDA fibroblasts left untreated or treated with 300 nm rHsp70 within 48 hours (left panel) or 150 nm rASM alone or in combination with 300 nm rHsp70 within 24 hours (right panel). Calculated values of p obtained from the performance of the enzyme (DA500/mg protein/min). The photograph on the right shows the absorption rASM by endocytosis (green) and its localization in the lysosomal compartment, as visualized through staining Lysotracker Red. (c) the Stability of lysosomes NPDA fibroblasts that were left untreated or treated for 24 hours with 300 nm rHsp70, 150 nm aSMa or a combination of rHsp70 and aSMa, analyzed as in Fig. 3e. p<0.001 for all treatments compared to untreated cells. (d) Quantitative analysis of lysosomal region confocal cross-sectional areas of the cells in NPDA fibroblasts, the OS is awashima raw or processed within 24 hours of 300 nm BSA, 300 nm rHsp70, 150 nm rASM (150 nm) or a combination of rHsp70 and rASM. The photograph on the right shows the effect of rHsp70 (green) on the amount of lysosomal compartment (red) in NPDA fibroblasts. White arrows mark subjected to endocytosis rHsp70 and reduced lysosomal compartment. Values represent values ± SD for 3 independent experiments. The scale=20 µm. UT=untreated.

Figure 9

Colocalization subjected to endocytosis rHsp70-AF488 with lysosomes. Presents confocal pictures of cells U-2-OS, incubated with 300 nm rHsp70-AF488 (green) for 24 hours, fixed and stained for the following markers organelles (red): lysosomal membrane protein-1 (LAMP-1; complementary mechanism), LAMP-2 (complementary mechanism), LBPA/BMP (6C4; endolysosomal compartment), cyt c (mitochondria), SERCA (ER) and golgin-97 (Golgi apparatus). Bars: 20 μm (LAMP-1, LAMP-2 and BMP) or 10 μm (Cyt c, SERCA and Golgin-97).

Figure 10

Interaction rASM (recombinant aSMa) and BMP in the presence of rHsp70. (a) Interaction with rASM immobilized anionic liposomes (average diameter of 100 nm, the total lipid concentration of 0.1 mm, and composition; 10 mol.% sphingomyelin, 50 mol.% phosphatidylcholine, 20 mol.% cholesterol and 20 mol.% BMP) at pH 4.5. The response signals were measured after binding of liposomes, which is defined as zero. (b) Effect predskazannogo rHsp70 on the subsequent binding rASM. Specified to the number of rHsp70 incubated with immobilized anionic liposomes is identical with (a). After 10 min of stage dissociation rHsp70 was added 200 nm rASM for 180 sec with a subsequent 10 min dissociation.

Figure 11

Effect of low molecular weight compounds inducer of Hsp70, benzyl alcohol, in the fibroblasts of a patient with Niemann-pick disease type A (NPDA). (A) Induction of Hsp70 in NPDA Götz benzyl alcohol, depending on the dosage (protein expression). (B) Increasing the stability of lysosomes NPDA Götz after treatment of cells NPDA Götz 40 mm benzyl alcohol. (C) Reducing pathology in cells NPDA Götz after treatment 40 mm benzyl alcohol, as measured by cross-sectional area of lysosomes (method hereinafter described in detail in example 2).

Figure 12

The effect of depletion aSMa on the stability of lysosomes. Small interfering RNA (siRNA) to acidic sphingomyelinase (si938, si1257, si1340) and control siRNA (mm) was transfusional cells U-2O-S through oligofectamine (Invitrogen) according to the manufacturer's instructions. The concentration of miRNAs: 50 nm. After 72 hours, the reduction was confirmed by RT-PCR (not included) and cells were analyzed to determine the stability of lysosomes by intravital visualization of individual cells mediating the photooxidation acridine orange. The increase in green fluorescence continuously measured to obtain the total kinetic curves of the loss of integrity of lysosomes. How should that shape chart cells, the process is designed miRNAs to aSMa, demonstrate a significant increase in the stability of lysosomes. Optionally, the method explained in example 2.

Figure 13

Treatment of all cell lines NPDA/B rHsp70 significantly reverses lysosomal pathology, i.e. reduces the cross-sectional area of lysosomes. Quantitative analysis of lysosomal confocal region of the squares of the cross-section of fibroblast cells with Niemann-pick disease type A and Β (NPDA/NPDB) and normal fibroblasts (BJ)that were left untreated or treated for 24 hours with 300 nm BSA or dextran as a control, or processed within 24 hours of 300 nm rHsp70, 150 nm rhaSMa or a combination thereof. Cells NPDA processed within 24 hours of 300 nm rHsp70-W90F (W90F), noninteracting with BMP mutant Hsp70 had an effect comparable with that of control cells. For methods, see example 2.

Figure 14

Increased activity aSM in transgenic for Hsp70 fibroblasts and fibroblasts NPDA processed rHsp70. Mass spectrometric analysis of lipid spectrum (sphingomyelin and ceramide, as indicated) in immortalized embryonic mouse fibroblasts (iMEF) wild-type (WT) or transgenic for Hsp70 (TG) (A and B), and fibroblasts of patients with Niemann-pick disease type A (NPDA 83/24)that were left untreated or treated rHsp70 (C). Lower levels of sphingomyelin and higher levels of ceramide indicate HC is the increase in the activity of acid sphingomyelinase.

Figure 15

Reversal of pathology in fibroblasts of patients with Farber disease. Quantitative analysis of lysosomal region confocal cross-sectional areas of the cells of patients with Farber disease. The fibroblasts of a patient with the disease Farber (Farber 89/73 and Farber 89/78) were left untreated or were treated for 24 hours with 300 nm BSA or 300 nm rHsp70, as indicated. As follows from the figures, the treatment of the fibroblasts of a patient with the disease Farber rHsp70 essentially reverses the abnormal lysosomes, i.e. reduces the cross-sectional area of lysosomes. For a description of the methods see example 2.

Figure 16

Hsp70 increases the capture of other molecules via endocytosis. Panel A: an immortalized embryonic mouse fibroblasts (iMEF) wild-type (WT) or transgenic for Hsp70 (TG) were incubated with 20 μg/ml labeled Alexa Fluor-488 BSA (BSA*) within 24 hours. Capture via endocytosis was determined by fluorescence microscopy (not given) (see example 2). Then the cells were collected and analyzed capture BSA*. As follows from the figures, Hsp70 transgenic iMEF have substantially more grip BSA*than iMEF wild type. Panel B: cell osteosarcoma U-2O-S were incubated with 20 μg/ml BSA* within 24 hours with 3000 nm rHsp70 or without, as specified. Uptake through endocytosis was determined by fluorescence microscopy (not given (see example 2). The cells are then collected and analyzed the uptake of BSA*. As follows from the figure, the cells U-2O-S, which together added BSA* rHsp70 and have significantly greater absorption of BSA*than cells incubated only with BSA*.

Detailed description of the invention

As demonstrated by the authors of the present invention, the main part of its cytoprotective action of Hsp70 has through direct interaction with endolysosomal membranes; interaction controls specific phospholipid called (bis(monoacylglycero)phosphate). BMP is only present in late endosomes and lysosomes. The authors demonstrate that the interaction of the Hsp70-BMP depends on N-terminal ATPase domain of Hsp70, particularly tryptophan 90, and, in addition, the interaction depends on the pH. The interaction between Hsp70 and BMP is essential for membrane-stabilizing action of Hsp70, as it provides a platform to modulate the stability of a specific subgroup of lysosomal enzymes and prevents destabilization of lysosomal membranes and subsequent release of lysosomal enzymes. These data form the basis of a new and promising method for the treatment of lysosomal storage diseases, as described in this document.

Complementary mechanism

Since the discovery of lysosomes Duve in 1955 in view of these organelles was dominated by the dogma about h is about they are exclusively the final stage of endocytosis in animal cells - a compartment accommodating a wide range of hydrolases, which in the case of release into the cytosol cause necrosis and inflammation of tissue. This idea of lysosomes, how about installing waste disposal in the best case and how about non-specific means of suicide in the worst case, has changed dramatically in the light of recent discoveries, providing proof considerably large specific functions of lysosomes and their contents.

Lysosomal hydrolases

As the main compartment intracellular degradation, and subsequent recycling of the cell, the complementary mechanism to receive my hetero - and automagically cargo in the lumen of this organelle reaches its destination. Degradation has a number of acid hydrolases (phosphatases, nucleases, glycosidase, proteases, peptidases, sulfatase, lipase etc)able to degrade all the major macromolecules of the cell. Among the most studied lysosomal proteases there exists a family Katasonov proteases. Cathepsins can be divided into three subgroups based on the amino acid in their active center, i.e. cysteine (B, C, H, F, K, L, O, S, V, W and X/Z), aspartate (D and E) and serine (G) cathepsins. Cathepsins function optimally at acidic pH in lysosomes (pH 4-5), although they can function at neutral pH outside of lysosomes, albeit with reduced stability and/or change specificn the property.

Until recently believed that the function of cathepsins limited endolysosomal the turnover of proteins and degradation of extracellular matrix during secretion. However, in the last few years many cathepsins attributed to more specific functions, including a role in the remodeling of bone tissue, antigen presentation, the homeostasis of the epidermis, the processing of prohormones, the protection of cytotoxic lymphocytes from self-destruction after degranulation, the protection of the Central nervous system of the mouse, angiogenesis, invasion of malignant cells, and programmed cell death (PCD).

In addition to the breakdown of proteins, complementary mechanism and the later endosome also responsible for the metabolism of lipids in cells, such as glycosphingolipids, through groups endolysosomal enzymes and coenzymes, whose own function depends on the lipid composition endolysosomal membranes. The importance of metabolism functional endolysosomal lipids can be easily estimated, taking into account that clinical disease is identified in the case of dysfunction at any stage of the metabolism of sphingolipids, leading to such diseases as disease Tay-Sachs disease Sandhoff, Farber disease, Fabry disease, Gaucher disease, Krabbe disease, and Niemann-pick disease.

Transport in complementary mechanism to and from

The endocytotic transport membranes plays the significant role in the cell of a mammal due to the delivery of components of membranes, various dissolved molecules and related receptor ligands in a number of intracellular compartments. While various ways of endocytosis until recently were simple, where the main pathways converge on the lysosomes, which are degradation and possible recycling back to the plasma membrane, recent evidence shows that these pathways are more complex than it seemed at first glance.

The path of endocytosis

Endocytosis is the most understandable in terms of receptor-mediated endocytosis of molecules through the formation of coated clatrina recesses, although identified a number of ways endocytosis, mediated not by Katrina (for example, macropinocytosis, phagocytosis, engagement through the formation of kahweol and uncovered clatrina recesses). Terminology system endocytosis is not fully standardized and ubiquitous term "early endosome" actually describes two separate endosomal compartment - sorting endosome and endocytotic recirculating compartment (ERC). In normal receptor-mediated route of endocytosis receptors such as the transferrin receptor, the receptor for low density lipoprotein and the receptor for mannose-6-phosphate (MPR), concentrated in covered clatrina recesses on the surface cytoplas eticheskoi membrane through interactions between conservative motifs in their cytoplasmic tails and elements latinboy shell. After separation latinboy oblucki new formed endosome merges with other endosomes and existing sorting endosomes to be sorting by endosomes. As the name implies, its primary task is to sort the emerging components on the appropriate locations. Three well-known destination include cytoplasmic membrane, late of endosome and ERC. When ripe sorts of endosome she is experiencing a decrease in pH, which promotes the release associated with receptor ligands into the lumen of endosome. However, until full ripening sorts of endosome in late endosome processed molecules to be sorted. I believe that this process occurs through the removal of small tubes, a process that contributes to the sorting of membrane proteins from dissolved molecules, because the ratio of surface to volume of the tubes is higher than that of vesicular sorting of endosome. Split tubes can pass membrane proteins directly back into the cytoplasmic membrane (direct return path) or ERC. Basically ERC is accumulation of tubular organelles, the localization of which varies in different types of cells. While the ERC is able to sort molecules in several different destinations, the majority of the mod is ekul, passing ERC, returns to the cytoplasmic membrane.

When sorting endosome ripening, the pH in the lumen continuously decreasing, mainly through the action of the proton ATPase using type (V-ATPase), at the same time there is a shift in the content of membrane proteins and lipids. Membrane transport from the sorting of endosome in late endosome and then lysosome is cause for some debate. The discussion was related to whether it's better to explain this transport by vesicular transport or by ripening sorts of endosome. Both models represent the average between sorting and late endosomes. While the model is maturation States that the vesicles, which reaches the late endosome, is what is left after the removal of components from the former sorts of endosome, the model pre-existing compartment argues that transport molecules in the later endosome occurs through endocytotic transport vesicles (ECV), a specific transport vesicles between preexisting sorting and late endosomal the compartment. And sorting, and late endosomal compartment is considered as structurally more complex and have more specialized functions than the transport vesicles. Recent imaging studies of living cells is regulated mechanistic aspects of both models, however, as vesicles arise from the active network of early endosomes, may be subject to debate how they lose small GTPase RAB5 and acquire RAB7, a marker of late endosomes. Although the organization way of endocytosis functionally well defined, the terminology can be confusing. Functionally, the path determined by endocytosis receptors of the household (e.g., transferrin receptor) and other lipids and proteins, passed through early endosome/sorting endosome, where there is a separation of the ligand to the receptor, but not through late endosome where it can happen proteolysis. However, apart from these functional criteria, the picture becomes more complicated when it comes to terminology, not least when the condition for the formation of intraluminal vesicles, beginning in early endosomes and becoming more and more visible during the maturation of late endosomes, leading to the emergence of the term "multivesicular Taurus" (MVB). This term is interchangeably used for ECV and late endosomes, as well as for all endocytotic vesicles containing multivesicular areas or elements, including hybrid organelles, which are formed when complementary mechanism fuse with late endosomes (which contain multivesicular patterns). However, the later endosome contain b is the larger of intraluminal vesicles and, thus, often the compartment described by the term "multivesicular calf."

Ultimately, a significant amount of confusion in this area arose from the separation of late endosomes and lysosomes or its lack. Both compartment equally acidic, and most, if not all, of the proteins present in the lysosomes, also found in late endosomes. Based on the model of the later maturing endosome are precursors of lysosomes, but tend to speed development, as might be expected from theory, and it is very difficult to achieve strict classification. Recently, however, presented evidence that complementary mechanism and the later endosome are separate compartments, which are then subjected to "kiss" (temporary merger), as well as a full merger, after which complementary mechanism can be converted from hybrid granell.

Biosynthetic path

In addition to endocytosis, late of endosome also get the cargo through the path MPR from the network to the TRANS-Golgi (TGN) (biosynthetic path). The cation-dependent MPR and the cation-independent receptor MPR/insulin-like growth factor-II (IGF-II) divide the function of delivering newly synthesized acid hydrolases from the TGN in a complementary mechanism. Detection of acid hydrolases MPR requires additional carbohydrates in the endoplasmic reticulum and subsequent modificat and and phosphorylation of carbohydrate residues to residues mannose-6-phosphate in the CIS-Golgi. MPR-associated hydrolases first delivered in endosome, where they dissociate from the receptor due to decrease in pH in the cavity, thus allowing the receptors to return back to the TGN. Protein mainly responsible for sorting MPR-covered clatrina indentations on TGN is adaptery protein-1 (AP-1), while also playing the part localized in the Golgi apparatus containing γ-loop binding factor ADP-ribosylate proteins (GGA). Currently, it is known that AP-1 and GGA work together or actually aimed at two MPR to different subcellular localizations. AP-1 is part of a family adapting proteins, consisting of four members, all of which are heterotetrameric proteins, widely used in the secretory and endocytotic pathways. In addition to the above the role of AP-1-coated clatrina recesses formed in the TGN, AP-1 and AP-2 are used in covered clatrina pits during endocytosis at the plasma membrane, whereas AP-3 and AP-4 function in the transport of lysosomal membrane proteins (LAMP).

The path of autophagy

The autophagy is the third well characterized by the penetration of macromolecules into lysosome. The autophagy is an evolutionary conservative way, involved in the turnover of long-lived proteins and organelles. Generally, it acts on the low b is the basal level, although it can be induced, for example, under conditions of dietary restriction. Under these conditions, macroautophagy is the main way responsible for the delivery of a substance in a complementary mechanism. Macroautophagy is characterized by a flat membrane tanks around cytoplasmic organelles and/or part of cytosol, thus forming a closed membrane bound vacuole, autophagosome. Ultimately, autophagosome fused with lysosomes, forming autophagosome/autolysosome where the degradation and recycling of absorbed macromolecules. The origin of the membrane autophagosome still not clear. Endoplasmic network, Golgi apparatus, studied membrane compartment, called pagefor, as well as de novo synthesis, considered as sources of membrane autophagosome. Recent progress by the genetics of the yeast and the subsequent opening of the homologues in mammals rapidly improves the understanding of the process of autophagy, and it is hoped that in the near future they will shed light on the source of the membrane autophagosome.

There are also other ways of getting autopaging cargo in complementary mechanism. More chaotic process called macroautophagy, is characterized by absorption of cytosole the lysosomes through invagination of the lysosomal membrane. In addition to macromolecules that are present in oglashennya the cytosol, this process may also involve the capture of organelles, such as peroxisomes. Ultimately chaperone-mediated transport proteins cytosol into the lumen of the complementary mechanism is a more focused and selective form of autophagy. This path depends on the presence of constitutively expressed member of the family of heat shock proteins 70, Hsc70, on both sides of the lysosomal membranes. Moreover, the process depends on the recognition of LAMP-2a conservative KDEL motif on the target protein.

Restructuring of lysosomes and lysosomal secretion

After the fusion of lysosomes with late endosomes or autophagosome complementary mechanism rebuilt formed from hybrid organelles through the sequestration of membrane proteins and condensation of the contents of the lumen. Of membrane proteins that need to be removed or recycled from hybrid organelles, the most notable are the MPR, because they are by definition not in the lysosomes. However, complementary mechanism cannot be considered as the final stage of the path of endocytosis, as they are also capable of forming secretory complementary mechanism by merging with secretory granules, Ca2+-dependent process, which was first discovered in the secreting cells of hemopoiesis. However, there is also evidence of Ca2+-regulated membrane-proximal lysosomal compartment, CTE is committed to exocytosis in " non-secretory cells. The process of exocytosis is dependent on protein Rab27a, member of the Rab protein family, which includes more than 60 members. Rab are small GTPase that play a key regulatory role in most phases of membrane transport, including the formation of vesicles, mobility, connection, and merge. At least 13 Rab proteins are used in the path of endocytosis to determine the fate of various subjected to endocytosis of molecules and vesicles.

Programmed cell death

Regulation of the total number of cells, as well as the number of cells of various tissues, together with the need for the mechanism of elimination of unwanted cells are a fundamental requirement in multicellular organisms. Thus, programmed cell death gives a multicellular organism is a potential - free yourself from unwanted cells without loss of the cell and, thus, to avoid inflammation associated with necrosis, the principal alternative to programmed cell death.

Apoptosis

The word apoptosis is used in Greek to describe the "falling" of the petals or leaves from the trees, and was first put into circulation Currie and co-authors in 1972 to describe the General type of programmed cell death, which the authors observed for a number of tissues and cell types. The authors noted that the observed is mi phenomena have considerable morphological similarity, which differs from the morphological features that characterize the cells exposed to pathological, necrotic death, and assumed that these General morphological traits may be due to the identical fundamental process.

When cells die by apoptosis, they undergo a series of transformative events. Among these events are essential for typical apoptotic phenotype is the activation of the caspase - family of cysteine endopeptidase, which decompose substrates at specific aspartate residues, and thus got its name. Activation of caspases leads to the proteolytic processing of other caspases, as well as many other changes in the total activity of the proteins in the cells, ultimately leading to characteristic morphological features associated with activation of caspases and thus, by definition, with apoptosis. Classical features of apoptosis include compression cells and vacuolization cytoplasmic membrane, condensation of chromatin in regular geometric shape, fragmentation of DNA at ~200 BP fragments, the so-called nucleosomal ladder, Department of cell from neighboring cells and disintegration of the cells in a small closed vesicles called apoptotic cells. In a multicellular environment data of apoptotic cells in the end is th result undergo phagocytosis by macrophages or neighboring cells, thus, completing the destruction of unwanted cells.

Programmed cell death

Programmed cell death (PCD) is not synonymous with apoptosis, although some might think so, based on the literature, in which this term is used indiscriminately. Gradually accept the term PCD, although still used the term apoptosis to describe the program of cell death is regulated by the activation of caspases, in particular caspase-3. However, the ability of some cells to survive after activation of proapoptotic caspases, as well as PCD with a complete lack of activation of caspases and the activation of caspases, leading to neuropetides PCD, show significant plasticity of the program (s) cell death and, thus, the PCD can more accurately be defined as cell death-dependent signals and activities within the dying cells. Suggest that PCD can be divided into apoptosis, apoptosis-like, necrose-like PCD morphology of the nuclei of dying cells, each definition is due to the different morphological characteristics, which is the main form of chromatin condensation or her absence, although it is more preferable to distinguish PCD, based on the signal transduction pathways underlying each set of conditions leading to PCD. However, this method of separation of different types of PD are not yet used since it is necessary to classify the road leading to different types of cell death.

Necrosis

Necrosis is the principal alternative to the PCD and cannot be prevented in any other way, except by removing incentives that cause necrosis. This type of cell death, usually observed in pathological damage to the organism.

Molecular mechanism of programmed cell death

Apoptosis

As indicated in the previous section, apoptosis is determined by the activation of members of the family of cysteine endopeptidase known as caspase, and morphology associated with their activation. Caspase present in cells as inactive Imogene that can be quickly activated by proteolytic processing. Processing occurs as a hierarchical cascade, in which the apoptotic stimulus activates initiating caspase (e.g. caspase-8 and -9), which, in turn, activates the next level in the hierarchy, the effector caspase (e.g. caspase-3, -6 and -7). The latter is considered as executors of apoptosis, because they break down a number of substrates, the processing of which ultimately leads to the phenotype associated with apoptosis. The program of apoptosis can be activated by various stimuli, which roughly can be divided into extracellular and intracellular stimuli, recent systems which have mitochondrion as an important participant. Extracellular stimuli and the subsequent response, inducing apoptosis, also known as the external transmission signal, it includes a series of events, beginning with the activation of one of the various death receptors such as Fas/Apo-1/CD95, TNFR or TRAIL. After binding of their respective ligands of these receptors activates containing death domain (DD) adaptorname molecules, such as TRADD (TNFR1-associated protein containing a death domain) and FADD (Fas-associated protein containing a death domain), through interactions with DD, which is present in the receptor. Then the data adaptorname molecules present in the receptor complex of caspase 8, wherein the caspase is activated, possibly induced by spatial proximity autocatalytical processing. Then in some cells (the so-called cells type I) caspase-8 directly cleaves and activates procaspase-3, while in cells of type II substrate for caspase-8 is a cytoplasmic protein Bid. Cleavage of Bid generates fragment (truncated Bid (tBid), which induces oligomerization, translocation and insertion of two proapoptotic members of Bcl-2, Bax and Bak, on the outer mitochondrial membrane. This mediates insertion of escaping electrons to cytochrome c (CytC) from the intermembrane space of mitochondria together with mn is a number of other proteins, the most significant of which are the factor induction of apoptosis (AIF), Smac/DIABLO, which counteracts the effects of proteins, proteins known as inhibitors of apoptosis (IAP), and endonuclease G, Ankasa. It should be noted that although this is an important moment in theories activation of caspases through the mitochondria, in terms of how the insertion of Bax and Bak facilitates the release of cytochrome c, there is no conclusive evidence. After release from mitochondria CytC accumulates in the cytoplasm, where it binds with the protein Apaf-1 (apoptotic protease-activating factor-1), which leads to conformational changes that promote oligomerization Apaf-1. Then the oligomer is associated with procaspase-9 through homotypic interactions between domains, activation of caspase (CARD), which leads to the formation of a complex called apoptosome. The formation of this complex leads to a significant increase in enzymatic activity procaspase-9 activity, which leads to proteolytic activation of caspase-3.

Also apoptosis can run the intracellular factors that increase the permeability of the outer mitochondrial membrane (MOMP), a process known as internal transmission signal. These factors include secondary messengers associated with cellular stress, such as Ca2+NO and arachidonic acid, as well as bilirubin, bile salts and incentives, which can cause denaturation of proteins and damage to nuclear and mitochondrial DNA, such as ionizing radiation, heat stress, reactive oxygen species (ROS) and chemotherapeutic agents. In case of damage to nuclear DNA in control of various protein kinases, which depend on the form of DNA damage, but also detrimental to its increase. The activity of these kinases induces the accumulation of p53, which then can act as a transcription factor, causing increased transcription of proapoptotic genes such as Bax, Noxa and PUMA, all of which can induce MOMP. At the level of mitochondria p53 induces the expression of mitochondrial enzymes, which locally generate ROS, and mitochondrial matrix protein (p53AIP1), overexpression of which triggers the fall of the potential of mitochondria and apoptosis.

Induction of MOMP by p53 or through the actions described above, the internal incentives is the point of intersection of the internal and external transmission signal, where the internal transmission signal should described above for the external transmission signal with the release of cytochrome C, formation of apoptosome and activation of caspase-3, the components of the final stages of the death of undesirable cells.

Alternatives to apoptosis

For the last decade challenged the exclusive role of caspases as artists PCD, and an increasing amount of evidence suggests greater things in life, and especially death, the cells than can be attributed only to the caspase.

Recently developed caspase-specific pharmacological inhibitors, as well as inactivation of the transmission path of the signal through caspase factors, such as energy depletion, nitrative/oxidative stress and members of the protein family of inhibitors of apoptosis (IAP), not always stop the progression towards death, they initiate or even reinforce the subgroup of the fundamental caspase-independent programs death. These programs include initiated by death receptor transmission signal and the transmission signal initiated anticancer drugs, lack of growth factors, staurosporine, Bax-related proteins and depletion of Hsp70. The morphological characteristics of these caspase-independent programs death often resemble observed in classical apoptosis, and growing experimental evidence of the role of other proteases, such as cathepsins, calpain and serine proteases, as essential cofactors of upstream and downstream caspases. This argument is reinforced by data that many nakazanie protease is able to cleave at least some of classicisticcastle caspases, that may explain some of the similarities observed between the caspase-dependent and-independent programs death.

Although you can dispute the importance of such programs death, because they are masked by the efficiency of caspase accumulate evidence of evolutionary conservative role of lysosomal Katasonov proteases in cell death is initiated in response to various stimuli, such as the death receptor family of receptor tumor necrosis factor, hypoxia, oxidative stress, osmotic stress, high temperature and anticancer drugs.

The involvement of lysosomes in the programmed cell death

While the role of lysosomes and their hydrolases in the cleansing phase of PCD, i.e. the uptake of apoptotic cells and Taurus neighboring cells or phagocytes, well-studied, understanding the importance of lysosomes and lysosomal hydrolases in the intermediate events PCD took time. One of the reasons for this delay may be that methylketone inhibitors peptides, commonly used to evaluate the role of caspases in PCD (for example, zVAD-fmk, Ac-DEVD-fmk, Boc-D-fmk and so on), also inhibit other cysteine proteases, including some of cysteine cathepsins. Even nine years after the discovery of their cross-reaction of the protective effects of these inhibitors at concentrations capable Engibarov the th nakazanie protease, often interpreted as evidence of caspase-mediated pathways of death and continue to underestimate the role of other cysteine proteases in PCD. Opening lysosomal PCD can optionally be delayed due to the fact that the ultrastructure of lysosomes seems to be intact in apoptotic cells, analyzed by electron microscopy. Thus, the destruction of lysosomes until recently considered as a change in the principle of "all or nothing" in the later stages of uncontrolled necrotic cell death and autolysis of the tissue. However, new ways to more accurately assess the integrity of the lysosomal membrane, allowed to reveal that complementary mechanism with normal ultrastructure can skip some of their enzymes, and this partial permeability of lysosomal membranes (LMP) not only had a place in many models of death, but may in fact run apoptosis and apoptosis-like PCD.

The permeability of lysosomal membranes (LMP) and its consequences

Studies with a variety of compounds that directly target the integrity of lysosomal membranes, such as H2O2, methyl ester L-leucyl-L-leucine, osmotic stress, sphingosine, lysosomotropic antibiotics norfloxacin and ciprofloxacin and photooxidative damage of lysosomes (f is talis), prove that the moderate permeability of lysosomes can lead to PCD. The quantitative relationship between the degree of destruction of lysosomes and type of cell death suggests an explanation for the wide differences in the morphological outcome of the LMP. In this model, low intensity stress launches a limited release of the contents of the lysosome into the cytoplasm and subsequent apoptosis or apoptosis-like cell death, while high intensity stress leads to generalized destruction of lysosomes and rapid necrosis of the cells. Thus, a low concentration of sphingosine acid metabolite ceramide synthesized by ceramidase with detergent-like properties at low pH induces partial LMP and caspase-mediated apoptosis, whereas high concentrations cause massive LMP and caspase-independent necrotic cell death. In this model run partial LMP death can inhibit pharmacological inhibitors of cysteine and aspartate of cathepsins, and increased activity of cytosolic cathepsins is preceded by activation of caspases and changes the potential of the mitochondrial membranes, suggesting a direct role of cytosolic cathepsins in the process of death. It is important that the role of LMP and cathepsins in cell death is not limited experimental the material models using direct destroyers of lysosomes. LMP also participates in the execution of cell death in response to a wide range of classical apoptotic stimuli, such as activation of death receptors of the family of receptors of the tumor necrosis factor (TNF), interleukin-1, activation of p53, lack of growth factors, stabilizing microtubules tools, etoposide, activation of the Sigma-2 receptor, synthetic retinoid CD437, activation of the receptor B-cells, staurosporin, osmotic stress, and low molecular weight compounds identified in the screening for new anticancer drugs that induce p53-independent apoptosis.

LMP as trigger the mitochondrial pathway of apoptosis

Cytotoxic effects LMP often depend, at least in part, from activation of the mitochondrial pathway of death. An elegant study by microinjection demonstrates that the localization in the cytosol separate the lysosomal hydrolase, cathepsin D, is sufficient to start the permeability of the outer mitochondrial membrane and apoptosis during cellular doses corresponding to half of the total cellular activity of cathepsin D. However, cathepsin D insufficient to start PCD in all models of cell death involving LMP. Other well-studied mediators run LMP PCD include cysteine cathepsins B and L, and the active form is ikorodu. However, it must be emphasized that accordingly does not exclude the role of other lysosomal hydrolases, lysosomal secondary messengers and LMP-mediated acidification cytosole. One of the links between cathepsins and permeability of the mitochondrial membrane may be Bid, a proapoptotic exclusively BH3 protein from the Bcl-2, which can processional and activate some of cysteine cathepsins, but not cathepsin D in cytosolic pH. However, assume that cathepsin D activates and cleaves Bid in the acidic environment endolysosomal compartment after internalization of the receptor-1 TNF (TNF-R1). In this model, the endocytosis of activated ligand TNF-R1 leads to a mediated sour sphingomyelinase the synthesis of ceramide, which is then associated with inactive cathepsin D and activates it through autocatalytical processing. Cathepsin D can also activate Bax Bid-dependent manner that demonstrated treated with staurosporine T-cells. In addition, treated with ciprofloxacin fibroblasts LMP runs the permeability of the mitochondrial membrane through a Bid-dependent activation of Bax and Bak. In this model system, the activation of Bax is not dependent on cathepsin D, but instead depends on reactive oxygen species. It should be noted that induced by ciprofloxacin p is oncemore mitochondrial membrane is not completely inhibited in cells with the lack of Bax and Bak. An alternative mechanism connecting the LMP and the permeability of the mitochondrial membrane may include direct effects of reactive oxygen species and/or lipid mediators, such as arachidonic acid, which can be synthesized in cathepsin B-dependent manner.

Studies using immortalized embryonic mouse fibroblasts (MEF) from mice with deficiency of individual cathepsins clearly revealed that in the execution of cell death, depending on the triggering LMP incentives involved various cathepsins. Immortalized MEF from mice with deficiency of cathepsins B and L, but not from mice with deficiency of cathepsin D, vysokopitatelny to TNF, while the opposite picture is observed when processing cells by staurosporine. Extensive research induced TNF pathways of cell death, among other things, revealed that the role of individual cathepsins in PCD depends on the type of the studied cells. As described above, induced TNF death of immortalized MEF depends on of cysteine cathepsins, but not cathepsin D. in Addition, depletion of cathepsin D effectively protects cells cervical cancer HeLa from induced TNF and cisplatin cytotoxicity. This difference is not due to the General differences between human cells and mouse, because cathepsin B separately and together with other the cysteine cathepsins is also luceville effective induced TNF destruction of cells in the cell lines of cervical cancer (ME-180) and breast cancer (MCF-7). The explanation of this diversity is still not clear, but it can play the role of different levels of expression of individual cathepsins and their inhibitors in various cell lines. Thus, the different ability of different incentives death to regulate the levels of expression of individual cathepsins and their inhibitors may explain the differences in response to various stimuli. For example, it is known that adriamycin and etoposide enhance the expression of cathepsin D by the activation of p53. Alternatively, other transmission path of the signal induced by various stimuli may act in conjunction with specific cathepsins.

Mitochondrion-independent path loss induced LMP

It is important that the lethal effects of LMP and cytosolic cathepsins are not limited to activation of the internal path of apoptosis. In the cells of small cell lung cancer treated with stabilizing microtubules drugs (paclitaxel, epothilone Β and discodermolide), LMP is in the process of cell death, and cysteine cathepsins mediate micronucleate and the death of cells of caspase-independent manner. In TNF treated cell lines carcinoma human LMP occurs as a result of the permeability of the outer mitochondrial membrane. However, inhibition of activity or expression of cysteine cathepsins gives things is the only protection against induced TNF cell death without significant inhibition of the activation of effector caspases. In addition, cathepsin B is responsible for apoptosis-like changes, such as chromatin condensation, the output of phosphatidylserine and vacuolization cytoplasmic membrane in the absence of active caspases in TNF treated cells fibrosarcoma mouse WEHI-S. in Addition, depletion of heat shock protein 70 (Hsp70) in different cells of human malignant tumors, as well as corobimilla activation of T cells starts LMP and mediated by cathepsins apoptosis-like PCD without activating the inner path of apoptosis. In accordance with these data cathepsin B can induce nuclear apoptosis in isolated nuclei. Thus, cathepsins, presumably, able to act as initiation, and as effector protease of programmed cell death depending on the stimulus and context of the cell. Because of their ability to mediate PCD in cancer cells where mitochondrial pathway of death is blocked, e.g. due to overexpression of Bcl-2, increased the hope that treatments that induce LMP, can prove its effectiveness in the treatment of malignant tumors that are resistant to inducers of classical apoptosis. In addition, this idea is supported by data showing that immortality and transformation can make the cells sensitive to lysosomal cell death.

Transmission the signal LMP

As described above, LMP, which occurs after the release of the contents of a complementary mechanism, particularly cathepsins into the cytosol, is considered a key step in the activation of lysosomal path of destruction. However, the transmission of the signal, leading to LMP, is only beginning to be elucidated. One of the most studied mechanisms is the transmitted signal from receptor 1 tumor necrosis factor, although the explanation of this transmission signal to the LMP is very difficult because of very different response in different target cells.

Thus, TNF can induce caspase-dependent and-independent LMP depending on the context of the cell. In addition, related to TNF ligands FasL, TRAIL and TWEAK also associated with the caspase-independent PCD, and apoptotic and necrotic morphology. Pharmacological and genetic studies suggest that the caspase-mediated path that leads from TNF to LMP depends on caspase-8 and -9, while the activation of caspase-9 is very different in human cells and mouse. The relationship between caspase and LMP is still unknown, although induced and TNF mediated by caspase-8 cleavage of Bid suggests the involvement of these genes, these data are not confirmed by induced TNF LMP in iMEF deficit Bid. In addition, assume that a Bid is a target for cathepsins in lysosomal path of destruction involving Bid sooner rather than later, LMP.

Also the TNF stimulates the breakdown of sphingomyelin to phosphorylcholine and ceramide through activation of neutral sphingomyelinase (SM) on the plasma membrane and sour SM (aSM) in the lysosomal compartment. Both events involved in induced TNF path cell death, but until now only neutral SMa associated with LMP through factor associated with neutral SMa (FAN). Research on iMEF deficit FAN, as well as human fibroblasts expressing a dominant negative form of FAN, showed that FAN not only mediates induced TNF production of ceramide, but is also involved in the processing of caspase-8 and cell death. Because induced TNF LMP in mouse hepatocytes is dependent on caspase-8, reducing its processing may explain the reduced LMP in TNF treated hepatocytes expressing dominant negative FAN. However, we cannot exclude the role of ceramide and its metabolites. Their role in induced TNF signaling the death is confirmed by the decrease induced TNF and Fas-induced hepatotoxicity in mice with deficiency of aSMa, which is activated after caspase-8. In particular, synthesized from ceramide in the reaction catalyzed by lysosomal enzyme acid ceramidase, sphingosine is an attractive candidate because it, in contrast to ceramide, can act as a detergent, directly destabilizing the lysosomal membrane. In addition to increased synthesis of the precursor of sphingosine, ceramide, by the activation of SMase, TNF also regulates the levels of sphingosine through mediated by cathepsin B poniz is ment regulation sphingosine-1, the enzyme that converts proapoptotic sphingosine in anti-apoptotic sphingosine-1-phosphate. This activity of cathepsin B can lead to the accumulation of sphingosine in lysosomes and thus can, at least partially, explain the involvement of cathepsin B in effective LMP in TNF treated hepatocytes.

In addition, TNF can run LMP and cell death in the presence of caspase inhibitors. This path is not dependent on caspase-8, but requires interaction with the receptor containing death domain protein-1 (RIP-1) and involves the synthesis of active forms of oxygen. Together with vnutrirezonatornym iron oxidative stress can generate oxygen radicals through the Fenton reaction and, thus, may cause the oxidation of lipids lysosomal membranes, leading to destabilization of the membrane and release the contents of lysosomes. Molecular link between RIP-1, oxidative stress and LMP is still not clear.

Induction of cell death in several classical inducers of apoptosis (i.e. p53, etoposide and staurosporine) also involves LMP with subsequent cathepsin-dependent permeability of the mitochondrial membrane. However, the transmission path of the signal from these incentives to LMP remain outstanding.

Mechanisms to protect cells from LMP

Taking into account the potential fatal outcome LMP, not surprisingly, choclette has developed a number of ways to counteract this, inhibiting himself LMP or protecting cells from acid hydrolases that fall into the cytosol due to LMP.

It is reported that, in addition to many other functions, phosphatidylinositol-3-kinase (PI3K) protects complementary mechanism of destabilization. Inhibition of PI3K in cells of the vascular endothelial person induces the release of cathepsin B into the cytosol, indicating a rather direct role of PI3K in preserving the integrity of lysosomal membranes. In addition, inhibitors of PI3K make the cells sensitive to induced TNF and interleukin-1 lysosomal pathways of death. The modified functions of lysosomes and increased levels of expression of cathepsins in cancer cells may represent a threat in the form of lower stability of lysosomes. Thus, PI3K, which is usually activated in malignant human cells might also contribute to the stability of lysosomes in tumor cells and, thus, to increase the resistivity data cells to death. While the role of PI3K in the stability of lysosomes of tumor cells only discusses recent data indicate a role of Hsp70 in protection of lysosomes from destroying the membrane incentives. This research is mainly conducted on tumor cells, which are also often demonstrate the localization of Hsp70 on the plasma membrane, as well as in endolysosomal the compartment.

In alluniversity of lysosomal proteases into the cytosol after LMP cytosolic inhibitors of proteases are protected from the harmful effects. While endogenous inhibitors of cathepsin D is unknown, cysteine cathepsins can effectively inhibit at least three cytosolic protease inhibitors, i.e. cystatine A and B and inhibitor of serine proteases 2A (Spi2A), recently discovered that they also have a potential inhibitory activity against some of cysteine cathepsins (B, H, K, L, and V) and cathepsin G. the Importance of these inhibitors in the prevention of PCD in physiological and pathological conditions show through mice with deficiency of cystatin B, which show increased apoptosis-grains of the cerebellum. In addition, after treatment of TNF expression of Spi2A is induced via NF-κB and inhibits induced TNF activity of cytosolic cathepsin B and cell death MEF. Interestingly, recently reported that in C. Elegans cytosolic inhibitor of serine proteases (serpin)-6 may protect against induction, as well as lethal effects from damage to the lysosomes caused hypoosmotic stress, and a variety of other lysosomal stress, demonstrating that protection from LMP is an evolutionary conservative mechanism.

Lysosomal storage disorders

Lysosomal storage disorders (LSD) are a group of approximately 40 rare inherited metabolic disorders that result is of arusei functions of lysosomes. LSD is caused by dysfunction of the lysosomes, usually due to a deficiency of certain enzyme necessary for the metabolism of lipids, glycoproteins or mucopolysaccharides. Although each disease is the result of mutations in different genes, leading to deficiency of enzyme activity, they all have common biochemical characteristics of all lysosomal storage diseases begin with abnormal accumulation of substances within a complementary mechanism.

Separately LSD occur with a frequency of less than 1:100000, but as a group they occur with a frequency of approximately 1:5000-1:10000. Most of these diseases are autosomal recessive, but a few are inherited X-coalesced heritable genes. as Fabry disease.

Mainly lysosomal storage disorders are classified by the nature of the primary material, and they can roughly be divided as follows:

- disease accumulation of lipids (lipids), mostly sphingolipids (including Gaucher disease and Niemann-pick);

º gangliosidoses (including disease Tay-Sachs);

º leukodystrophy;

- mucopolysacharides (including hunter's syndrome and disease Hurler);

- disease accumulation of glycoproteins (glycoproteins);

- mucolipidosis.

Depending on the severity of the disease patients die at a young and unpredictable age, many within a few months or years from m the ment of birth, while others live up to early adulthood, eventually dying of various pathologies their individual disease. Symptoms of LSD vary depending on individual diseases and can vary from mild to severe. These may include developmental delay, impaired movement, convulsions, dementia, deafness and/or blindness. Some individuals with LSD enlarged liver (hepatomegaly) and enlarged spleen (splenomegaly), there are problems with the cardiovascular and respiratory systems and abnormal bone growth.

Most patients are initially screened through a set of enzymes, which is the most effective way to final diagnosis. In some families, which are known for causing the disease mutation (mutation), and some genetic isolates can analyze mutations. Since there are several possible mutations, sometimes for confirmation of the diagnosis requires sequencing of the gene encoding the specific damaged enzyme. Prenatal diagnosis can be useful when a known genetic risk factor.

In one of the embodiments the present invention relates to a method for the treatment of lysosomal storage diseases.

The lysosomal hydrolysis of sphingolipids

p> In the lysosomal catabolism of sphingolipids (or glycosphingolipids) involved many enzymes (see figure 4). Each of these enzymes, or, more specifically, hydrolases, is responsible for the degradation of specific sphingolipid.

Lysosomal hydrolases of sphingolipids interact with proteins activators of sphingolipids (SAP or saponine) to stimulate the activity of these hydrolases. It is believed that SAP facilitate the interaction of the enzyme/substrate between a water-soluble enzymes and membrane-bound substrates.

In addition, the lipid composition of late endosomal and lysosomal compartments is characterized by the presence of negatively charged phospholipids, such as BMP and PI (phosphatidylinositol), which also stimulate the activity of some hydrolases. BMP-dependent lysosomal hydrolases include sialidase, α-galactosidase A, glucosylceramidase, β-galactosylceramidase, arylsulfatase A, acidic ceramidase and sphingomyelinase.

Cfactory saposin

Saposin are small lysosomal proteins, which serve as activators of various destructive lysosomal lipids enzymes. They may act by allocating the lipid substrate from the membrane environment, thus making it more accessible to Deplete dissolved EN zymes is impressive. All saposin mammals are synthesized as a single molecule precursor (prosaposin), which contains four domains saposin-B which is active saposin after proteolytic cleavage, and two domain saposin-A, which are removed in the activation reaction. Domains saposin-B also exist in other proteins, many of which operate in the lysis of the membrane.

Prosaposin (PSAP) is a protein that in humans is encoded by gene PSAP. This gene encodes a highly conserved glycoprotein, which is a precursor of 4 products splitting: sabotinov A, B, C, and D. Saposin is short protein activator of sphingolipids or SAP. Each domain protein precursor contains approximately 80 amino acid residues with a number of identical spaced cysteine residues and glycosylation sites. Saposin A-D are initially localized in the lysosomal compartment, where they facilitate the catabolism of glycosphingolipids with short oligosaccharide groups. The precursor protein exists as a secretory protein, and as an integral membrane protein and has neurotrophic activity. Saposin A-D are required for hydrolysis of some specific sphingolipids by lysosomal hydrolases.

Saposin are important coactivators sialidase (SAP-B), α-galactosidase A (SAP-B), Glu is sinceridade (SAP-C), β-galactosylceramidase (SAP-C), Ukrainian (SAP-B) and acidic ceramidase (SAP-D). Sour sphingomyelinase (aSMa) does not depend critically on any of the known proteins, activators, however, the presence of sabotinov increases the activity of this enzyme. Also described fifth saposin GM2-activator protein.

BMP

Bis(monoacylglycero)phosphate (BMP), also known as osobistewymiana acid, is the main part of the lipid composition of late endosomal and lysosomal compartments. He is a negatively charged phospholipid, more specifically, glycerophospholipids.

BMP was first isolated from rabbit lung, but it is now known that he is a minor component of the tissues of all animals. Its stereochemical configuration is different from that of the other glycerophospholipids animals that fosfodiesterzy balance associated with the positions sn-1 and sn-1' of glycerol, more than the position sn-3. It remains unclear whether the position sn-3 and 3' or sn-2 and sn-2' in the residues of glycerol esterified with fatty acids. Whatever the position of fatty acids on the glycerol backbone, their composition can be distinguished by frequent 18:1 (n-9) and 18:2(n-6), 20:4 and 22:6(n-3), although this is highly dependent on specific tissues, cell types or organelles. This distinctive composition suggests perfectly with ecificatio functions some of which need to be identified.

As a rule, BMP is preferably a minor component of animal tissue. However, he strongly promoted in the lysosomes of the liver and other tissues, where it can reach 15% or more from membrane phospholipids, and now he is recognized as a marker of these organelles. This is the later endosome and complementary mechanism containing a unique lipid BMP. Undoubtedly, there are involved the internal membranes of late endosomes, containing up to 70% of phospholipids as a BMP file.

If the reported presence of BMP in some alcaliphilic strains of Bacillus species can be confirmed, this would be the only known exception to the rule stating that the lipid has come strictly from mammals and is not present in prokaryotes, yeast and higher plants.

There is good evidence that BMP is synthesized from phosphatidylglycerol primarily in endosomal system. That is probably the main route for the first stage of phospholipase A2 removes a fatty acid from position sn-2 of phosphatidylglycerol. In the second phase lysophosphatidylserine azetiliruetsa position sn-2' head group of the residue of glycerol to produce sn-3:sn-1' mesobisphosphines acid by reaction transsilvania with lysophosphatidylserine in quality and acyl donor, and AC is a high acceptor. The third stage is sufficiently still necessary to describe, but it should include the removal of fatty acids from position sn-1 of the primary glycerin link and move phosphoryl ester from position sn-3 sn-1. The final position sn-2 primary glycerin link etherification, possibly through reaction transsilvania with other phospholipid as a donor (thus differing in fatty acid composition). Other biosynthesis pathway.

Actively exploring the function of BMP in the lysosomes. It may play a structural role in the formation of a complex system intraluminal membranes, contributing to the tendency to form a bilayer. He is a cone-shaped molecule and enhances the fusion of membranes at pH in the endosomes. In addition, its unique stereochemistry means that it is resistant to the action of phospholipases, so he interferes with or prevents camaraderie lysosomal membranes. Components of fatty acids can change quickly with transsilvanien, but glycerophosphate frame is stable. In addition, it's possible that this lipid may bind to specific proteins in membrane domains that are functionally similar to the rafts. This suggests that the characteristic network of rich BMP membranes contained within multivesicular the late endosome, regulates transport the tons of cholesterol, acting as a point of accumulation and redistribution. For example, when lysosomal membranes were incubated with antibodies to BMP, cholesterol tended to accumulate. The process is under the control of Alix/AIP1, which is a protein that specifically interacts with BMP and involved in the sorting multivesicular of endosome.

It is known that BMP significantly stimulates enzymes involved in the degradation glucosylceramide, such as proteins are activators of sphingolipids, such as saponine. In this case it just might work, providing a suitable environment for interaction glycosphingolipids and their activators. In addition, it plays a dynamic role in the supply of arachidonic acid for the synthesis of eicosanoids in alveolar macrophages.

For BMP-dependent enzymes degree of hydrolysis increases significantly when BMP is present in the membrane, for aSMa even without the presence of the protein activator, such as saponin. In figure 4 the dotted circle indicated enzymes or disease in which the enzyme is defective, which indicates the dependence on the BMP.

BMP is involved in the pathology of lysosomal storage diseases such as Niemann-pick disease type C (accumulation of cholesterol) and induced some drugs lipidosis. In the circumstances of its composition which has a tendency to change to a favorable molecular States, which contain less polyunsaturated components. He is an antigen found in autoimmune serum of patients with rare and poorly understood disease known as antiphospholipid syndrome, so it is a factor in the pathological basis of the disease.

In one of the embodiments this invention relates to a method for the treatment of lysosomal storage diseases by applying the interaction between Hsp70 and BMP.

Disease accumulation of lipids

Disease accumulation of lipids (or lipids) are a subgroup of lysosomal storage diseases in which harmful amounts of lipids accumulate in the intracellular space due to reduced expression or function of enzymes necessary for the metabolism of lipids. Over time, this excessive accumulation of lipids may cause permanent damage to cells and tissues, especially in brain, peripheral nervous system, liver, spleen and bone marrow.

Lipids are a broad group of natural molecules, including fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, e and K), monoglycerides, diglycerides, phospholipids, and others. The main biological functions of lipids include energy storage, they are structural components of cell membranes and important is passed signaling molecules.

Lipids can be roughly divided into hydrophobic or amphiphilic low-molecular compounds; amphiphilic nature of some lipids allows them to form structures such as vesicles, liposomes, or membranes in an aqueous environment. Biological lipids are formed in whole or in part from two distinct types of biological subunits: ketoacyl and isoprene groups. Using this approach, lipids can be divided into eight categories: fatty atilov, glycerolipid, glycerophospholipids, sphingolipids, charalabidis and are polyketides (formed by condensation ketoacyl subunits) and sterols and prenolone lipids (formed by condensation of isoprene subunits).

Although the term lipid is sometimes used as a synonym for fat, the fats are a subgroup of lipids called triglycerides. Lipids also include molecules such as fatty acids and their derivatives (including tri-, di -, and monoglycerides and phospholipids), as well as other Sterol-containing metabolites such as cholesterol.

Describe some lysosomal storage disorders characterized by the accumulation of lipids (i.e. disease accumulation of lipids); they are briefly described later in this document.

In one of the embodiments the present invention relates to a method of treatment of diseases of the accumulation of lipids.

The disease is of Inanna Peak

The Niemann-pick disease (NPD) is called the defect of the enzyme acid sphingomyelinase (aSMa) systematic name sphingomonadaceae. The bulk of the membrane sphingomyelin hydrolyzes lysosomal enzyme aSMa to get ceramide (and phosphocholine). Sphingomyelin contains ceremeny membrane anchor, which is associated with short phosphorylcholine balance.

Sphingomyelinase does not depend critically on any known proteins, activators, making the expected intramolecular activator domain aSMa and the presence of negatively charged lipids in liposome essential for the circulation of sphingomyelin. Thus, aSMa does not require the presence of sabotinov as cofactors; however, the presence of sabotinov in the future invariably stimulates the activity of this enzyme (Ferlinz et al., 1999). BMP stimulates activity aSMa.

When sphingomyelin cannot be metabolized properly, it accumulates within the cell, ultimately leading to cell death and malfunction of the major organ systems. Symptoms may include lack of muscle coordination, brain degeneration, learning problems, loss of muscle tone, increased tactile sensitivity, spasticity, difficulty in feeding and swallowing, slow of speech, and increased liver ilesanmi. In addition, there may be corneal opacity and characteristic cherry-red spot, which is formed around the center of the retina.

The Niemann-pick disease (NPD) has 4 related types; types A, B, C, and D. All types of NPD inherited in an autosomal recessive manner and affect both men and women. In types A and B deficient enzyme activity aSMa causes the accumulation of toxic quantities of sphingomyelin. The disease occurs when both copies of the gene aSMa the individual (both alleles) have a mutation.

The Niemann-pick disease type A (NPDA) is the most common type, occurs in children. It is characterized by jaundice, liver enlargement and profound brain damage. There is no effective treatment for individuals with type a, and patients with type As die in infancy, usually up to 18 months.

The Niemann-pick disease type Β (NPDB) includes enlarged liver and spleen, which usually occurs in young children, as well as common respiratory disorders. The increase in organs and respiratory disorders may cause cardiovascular stress and can lead to heart disease in old age. As a rule, patients with NPDB have either a small neurological problems, or do not have them. For several patients with type B was performed bone marrow transplantation and reported to shift the data results. Future development of replacement therapy enzyme and gene therapy can also help those who have the disease type B. Children with type B can live a relatively long time, but may require oxygen support due to pulmonary disorders.

NPDA and NPDB caused by one and the same deficiency of the enzyme, and increasing evidence that the two forms represent the opposite end of the range. Individuals with NPDA, generally have small products aSMa or not have it (less than 1% of normal), while those with NPDB, have approximately 10% of the normal level aSMa.

There are approximately 1,200 cases of NPA and NPB around the world with a predominance of Β-type or intermediate forms.

NPDA and NPDB is diagnosed by measuring the level of activity aSMa in leukocytes from a blood sample. Because this test identifies individuals with types A and B, it is not very reliable for identifying individuals who are carriers (those who possess only one functional copy of the gene ASM). In addition, the test shows reduced activity aSMa, but he can't always predict whether an individual type A, or type B, or an intermediate variant of the disease; this requires clinical trials of the individual.

In some populations of specific mutations are the cause of a high percentage of the cases of deficit aSMa. For NPDA R496L mutation, fsP330 and L302P are the cause of more than 95% of causing disease genetic changes in the population of Jews are Ashkenazi. To identify the media used direct testing of individuals in a given population data 3 mutations. In other populations, before conducting DNA analysis of media among family members, it is first necessary to identify the mutation in the affected individual.

For NPDB mutations aSMa H421Y and K576N responsible for 85% of the population NPDB in Saudi Arabia; mutation L137P, fsP189 and L549P responsible for 75% of the Turkish population NPDB; mutation S379P, R441X and R474W responsible for 55% of the Portuguese population NPDB; mutation A196P is responsible for 42% of English/Scottish population NPDB, and mutation F480L and DeltaR608 was identified as causing disease in patients with NPDB.

The Niemann-pick disease type C (NPDC) is significantly different from type A or B. Patients with NPDC is not able to properly metabolize cholesterol and other lipids in the cell and are characterized by a defect that disrupts the transport of cholesterol between brain cells. Thus, excessive amounts of cholesterol and other lipids accumulate in the liver, spleen and brain. NPDC secondary causes reduced activity aSMa that allows us to consider all three types as forms of the same disease.

There is a significant time shall ICIE symptoms when type C first appeared in the progression of the disease. Symptoms can occur as early as within a few months of life, and as late as adulthood. Violation of eye movements in the vertical plane (the inability to move eyes up and down), enlargement of the liver or jaundice in young children are strong signs for suspected NPDC. As a rule, in the early stages of the disease appear only one or two symptoms. In most cases, neurological symptoms begin to appear between the ages of 4 and 10 years. In General, the later start of neurological symptoms, the more slowly progressing disease.

Worldwide diagnosed approximately 500 cases of Niemann-pick type C. However, it is believed that the number of people affected by NPDC, above, but the difficulties in the diagnosis does not accurately assess the frequency of occurrence. Originally NPDC was diagnosed as a learning disability, mental retardation, clumsiness and delayed development of motor skills.

Currently, Niemann-pick disease type D consider a variant of type C. As a rule, type D occurs in humans, occurring from Nova Scotia. Individuals with types C and D are often prescribed low-cholesterol diet, but its clinical benefit is unconvincing. The life expectancy of individuals with the AMI C and D varies, however, the disease is always fatal. The vast majority of children die before the age of 20.

NPDC is a rare and highly variable condition and may not be recognized by some medical professionals. For those professionals who suspect this diagnosis in a patient, it can be determined by a biopsy of the skin, cultivation of fibroblasts and study of their ability to transport and accumulate cholesterol. The transport of cholesterol into the cells examined by measuring the conversion of cholesterol from one form to another (esterification). Cholesterol accumulation assessed by staining cells with reagent (Filipina), which glows under UV light.

In 1997 identified a gene NPDC1. Mutations or cause of the disease changes in this gene are responsible for approximately 95% of all cases NPDC. Since in this gene and the second gene NPDC, NPDC2 identified more than 250 different genetic mutations related to NPDC. In addition, approximately 95% of cases it is possible to identify the genetic changes that cause disease if the diagnosis NPDC for the first time confirmed the above testing. However, due to the fact that there are so many unique yet unidentified mutations, a sub-optimal use of genetic testing is as the primary diagnostic tool for NPDC.

The Niemann-pick disease affects all segments of the population with reported cases in North America, South America, Europe, Africa, Asia and Australia. However, a higher incidence is observed in some populations:

• the population of Jewish Ashkenazi (NPDA and NPDB);

• the population of French Canadians in Nova Scotia (type D,is considered to be a variant of the NPDC);

• the Maghreb region (Tunisia, Morocco and Algeria in North Africa (NPDB);

• Spanish-American population of southern new Mexico and Colorado (NPDC).

In one of the embodiments the present invention relates to a method of treatment of a disease Niemann-pick through the modulation of the enzymatic activity of acid sphingomyelinase (aSMa).

Disease Farber

The Farber disease is caused by a defect of the enzyme acid ceramidase. Sour ceramidase is responsible for the conversion of ceramide to sphingosine (and fatty acid); thus, the defect leads to the accumulation of ceramide. BMP stimulates its activity, and it depends on sabotinov.

Sour ceramidase also known as N-acylsphingosine, it encodes the ASAH1 gene. It is a heterodimeric protein containing deglycosylated alpha-subunit and a glycosylated beta subunit, which in the Mature enzyme cleaved excision.

The Farber disease is also known as lipogranulomas Headlights the EPA, the deficit ceramidase, fibrillary dissacharides and lipogranulomas. This is an extremely rare autosomal recessive disease characterized by abnormalities of the joints, liver, throat, tissues and the Central nervous system. Also can affect the liver, heart and kidneys. Typically, the symptoms found in the first few weeks of life, these include violation of motor and mental abilities, and difficulty swallowing. Other symptoms may include arthritis, increase lymph nodes and joints, hoarseness, subcutaneous nodules (and sometimes in the lungs and other parts of the body), chronic contraction of the muscles and tendons around the joints and vomiting. Affected individuals may need in the introduction of the endotracheal tube. In severe cases, an enlarged liver and spleen.

At the present time there is no specific treatment for the disease Farber. Corticosteroids can help reduce the pain. The nodes in some cases can be treated with bone marrow transplantation or surgically reduce or remove. Most children with the classic form of the disease Farber die at the age of 2 years, usually from lung disease. Individuals with a moderate form of the disease can live to adolescence.

In one of the embodiments the present invention relates to a method of treatment of a disease Farber pic what edstam modulation of the enzymatic activity of acid ceramidase.

Krabbe disease

Krabbe disease is caused by a defect of the enzyme β-galactosylceramidase. β-galactosylceramidase is responsible for the transformation of galactosylceramide in ceramide; thus, the defect leads to the accumulation of galactosylceramide. BMP stimulates its activity, it depends on sabotinov.

Krabbe disease is also known as globoid cell leukodystrophy or galactosylceramide the lipidosis. This is a rare often fatal degenerative autosomal recessive disease that affects the myelin sheath of the nervous system. It occurs in approximately 1 case per 100,000 newborns. A higher incidence of approximately 1 in 6000, was found in Arab communities in Israel.

Krabbe disease is caused by mutations in the GALC gene, which cause a deficiency of the enzyme galactosylceramidase. Lipid accumulation effect on the growth of the protective myelin sheath of nerves (the coating that insulates many nerves and causes severe degeneration of motor skills.

Children with Krabbe disease are normal at birth. Symptoms appear between 3 and 6 months and include irritability, fever, stiffness of the limbs, convulsions, difficulty feeding, vomiting, and slow mental and motor development. In the early stages of the disease, doctors often mistake them for symptoms of de the ski cerebral palsy. Other symptoms include muscle weakness, spasticity, deafness, optic atrophy and blindness, paralysis, and difficulty swallowing. Can also be long-lasting weight loss. There is also Krabbe disease with early in youth and adulthood, which have similar symptoms, but at a slower progression. In General, in children, the disease is fatal to 2 years. Patients with late-onset Krabbe disease have a tendency to slower progression of the disease and live much longer.

Although there is no treatment of Krabbe disease, it is shown that bone marrow transplantation improves the early stages of the disease. In General, treatment is symptomatic and supportive. Physiotherapy can help to maintain or increase muscle tone and circulation. A recent study reported that transplantation of umbilical cord blood successful in stopping the disease as long as it is given before the appearance of obvious symptoms.

In one of the embodiments the present invention relates to a method for the treatment of Krabbe disease through modulation of the enzymatic activity of β-galactosylceramidase.

Fabry disease

Fabry disease is caused by a defect of the enzyme α-galactosidase A. α-galactosidase a is responsible for prevremeni globotriaosylceramide at lactosylceramide; thus, the defect leads to the accumulation of globotriaosylceramide (also abbreviated as Gb3, GL-3 or cerametrics). BMP stimulates its activity, and it depends on sabotinov.

Fabry disease is also known as the disease of Anderson-Fabry, diffuse angiokeratoma torso syndrome Ruiter-Pompano-Weyers, cerametrics and disease Build-Klionsky. This X-linked recessive (inherited) disease that affects gameshot male and heterozygotes and homozygotes of the female sex; men have the most severe clinical symptoms, while in women they range from the visible absence of symptoms to severe as in men. Assume that this diversity is caused by the nature of the inactivation of X-chromosome during embryonic development of women.

Symptoms include anhidrosis (lack of sweating), weakness, angiokeratoma (benign damage to the capillaries of the skin), burning pain in the extremities and visual impairment. Angiokeratoma are small painful papules that appear at any region of the body, but are predominant on the thighs, buttocks, lower abdomen and groin. External visual impairment may represent a cornea verticillata (also known as keratopathy vortex). Keratopathy may be present in asymptomatic carriers, and its well is but to differentiate from other causes of vortex keratopathy (i.e. accumulation of drugs in the cornea). Other visual indicators that can be seen include aneurysm of the conjunctiva, the rear seceptible cataract, swelling of the optic nerve, swelling of the yellow spots, optic atrophy and dilation of vessels of the retina. Renal complications are a common and serious effect of the disease; renal failure may worsen throughout life. Proteinuria is often the first sign of kidney involvement. Also have heart complications; related to heart effects can deteriorate with age and can lead to increased risk of heart disease. Cerebrovascular effects lead to an increased risk of stroke. Other symptoms include tinnitus, dizziness, nausea, and diarrhea.

Typically, symptoms first appear in early childhood and are difficult to recognize; the rarity of Fabry disease for many doctors sometimes leads to incorrect diagnosis or neglect. Typically, the manifestation of the disease increases in the number and severity with age of the individual.

Until recently, the treatment of Fabry disease were aimed at symptomatic effects. However, recently it was treated by replacement therapy enzyme (ERT) using agalsidase alpha (replagal) and agalsidase beta (fabrazyme). the cost of these medicinal products is an issue (approximately $250,000 in US per year per patient) and remains a barrier for many patients in some countries. Substitution therapy with enzyme (usually injected every two weeks) can be performed in a patient at home by the patient. Replacement therapy enzyme does not bring healing and for maximum benefit you must enter a regular basis.

In one of the embodiments the present invention relates to a method for the treatment of Fabry disease by modulation of the enzymatic activity of α-galactosidase A.

Gaucher disease

Gaucher disease is caused by a defect of the enzyme glucosylceramidase (also known as glucocerebrosidase or acid β-glucosidase); protein weight of 55.6 kDa, length of 497 amino acids. Glucosylceramidase is responsible for the transformation of glucosylceramide (or glucocerebroside) in ceramide; thus, the defect leads to the accumulation of glucosylceramide. BMP stimulates its activity, which depends on sabotinov.

Gaucher disease is the most common of the lysosomal storage diseases. The fatty material may accumulate in the spleen, liver, kidneys, lungs, brain and bone marrow.

Symptoms may include enlarged spleen and liver, liver dysfunction, disorders of the skeleton and bone injuries that can cause pain, severe neurological complications, enlargement of lymph nodes and (occasionally) adjacent joints, the increase in the abdomen, brownish the skin tone, anemia, low platelets in blood and yellow fatty deposits in the sclera. Affected most seriously individuals may be more susceptible to infection.

The disease is autosomal recessive and, therefore, affects both men and women. Various mutations of glucosylceramidase determine the residual activity of the enzyme and, in many respects, the phenotype. Studies suggest that heterozygotes for specific mutations of glucosylceramidase have an increased risk of Parkinson's disease and malignancies (non-Hodgkin's lymphoma, melanoma and pancreatic cancer).

Glucosylceramide is a component of the membrane of erythrocytes and leukocytes. Macrophages, which remove these cells are unable to remove waste products that accumulate in the fibrils and turn into Gaucher cells, which appear at the light microscopy, reminding the jammed paper.

Gaucher disease has three common clinical subtype. Each type is associated with specific mutations. In total there are approximately 80 mutations.

• Type I (or nonaromatizable type) is the most common form of the disease, occurring in approximately 1 case per 50,000 live births. It is most often found among the Jews, Ashkenazi, 100 times more often than in the General population. The sympto is s can occur in childhood or in adulthood and include enlargement of the liver and severe enlargement of the spleen (collectively hepatosplenomegaly); spleen may break down and cause additional complications. The weakness of the skeleton and bone disease can be severe. Enlargement of the spleen and substitution of bone marrow, causing anemia, thrombocytopenia and leukopenia. The brain is not affected, but there may be abnormalities in the lungs and less frequently in the kidneys. Typically, patients in this group are easily formed bruising (due to low platelet levels), and they have a weakness due to low level of red blood cells. Depending on the onset and severity of disease in patients with type 1 can survive to maturity. Many patients have moderate form of the disease, or may not show symptoms.

• Type II (or acute infantile neuropathic Gaucher disease), usually begins within 6 months from birth and has a prevalence of approximately 1 per 100,000 live births. Symptoms include enlargement of the liver and spleen, extensive and progressive brain damage, disorders of eye movements, spasticity, convulsions, rigidity of the limbs and low ability to sucking and swallowing. Affected children usually die at the age of 2 years.

• Type III (chronic neuropathic form) can begin at any age and occurs in approximately 1 case per 100,000 Jivaro is established. It is characterized by slowly progressive, but more moderate, compared with the acute form or type 2 neurological symptoms. The main symptoms include an enlarged spleen and/or liver disease, seizures, poor coordination, skeletal disorders, disorders of eye movement, blood disorders, including anemia, and disorders of the respiratory system. Patients often live up to early adolescence and adulthood.

The National Gaucher Foundation States that approximately 1 in 100 people in the General population of the United States is a carrier of Gaucher disease type 1, resulting in a prevalence of 1 in 40000; among Jews, Ashkenazi ratio of the carriers is substantially higher, approximately 1 to 15. Gaucher disease type 2 does not show the particular dominance in any ethnic group. Gaucher disease type 3 is particularly prevalent in the population of the Northern region of Sweden Norrbotten, where the frequency of occurrence of the disease 1 to 50000.

For patients with type 1 and especially type 3 replacement enzyme therapy with intravenous recombinant glucosylceramidase can reduce the size of the liver and spleen, to reduce skeletal anomalies and to reverse other manifestations. The rarity of the disease means that it is difficult to conduct research on finding the dose, therefore, there is debate about the optimal dose and frequency of administration. Because of iskai occurrence it is a rare drug in many countries. Current treatment of disease by terazima (Cerezyme®) (imiglucerase for injection) is worth up to $of 550,000 annually for one patient, and treatment should continue for the whole life. Another drug approved for this disease in 2003, is miglustat.

Successful bone marrow transplantation cures nevrologicheskie manifestations of the disease, because it makes the population of monocytes with active glucosylceramidase. However, this method carries a significant risk and it is rarely performed on patients with Gaucher disease. Surgery to remove the spleen (splenectomy) may be required in rare cases, if the patient has anemia or increased when the body prevents the convenience of the patient. Blood transfusions may benefit some patients with anemia. Other patients may require surgery to replace joints to improve mobility and quality of life. Other treatment options include antibiotics for infections, antiepileptic drugs from convulsions, bisphosphonates for damaged bones and liver transplants.

Therapy of the lower substrate may show efficacy in stopping disease type 2, because it can penetrate the blood-brain barrier into the brain. Currently there is no effective treatment of severe povrezhdeniya, which may occur in patients with Gaucher disease type 2 and 3.

In one of the embodiments the present invention relates to a method for the treatment of Gaucher disease by modulation of the enzymatic activity of glucosylceramidase.

Sialidase

Sialidase, or mucolipidosis type I (ML I), is called the defect of the enzyme sialidase (or alpha-neuraminidase). Sialidase is responsible for the transformation of GM3 in lactosylceramide; thus, the defect leads to the accumulation of GM3. BMP stimulates its activity, and it depends on sabotinov.

Sialidase inherited autosomal recessive manner. Symptoms are present at birth or develop during the first year of life. Many of the affected children at birth noted extensive swelling throughout the body. These children are often born with coarse facial features, such as flat nose, swollen eyelids, enlarged gums and increased the size of the tongue (macroglossia). Many children are also born with a deformity of the skeleton, such as dislocation of the hip. Children often show sudden involuntary muscle contractions (called myoclonus) and have red spots in the eyes (cherry-red macula). They are often unable to coordinate voluntary movements (called ataxia). Also present tremor, visual disturbances and seizures. Tests reveal abnormal enlargement of the liver (Hep is tomehane) and spleen (splenomegaly) and extreme abdominal swelling. In General, children have a lack of muscle tone (hypotonia) and mental retardation, which is the source or progressively heavy. Many patients suffer from developmental delays and recurrent respiratory infections. Most children with ML I die before reaching 1 year of life.

Sialidase can be distinguished by age of onset of symptoms and types of the presented symptoms. The effects of the disease can vary from mild to severe.

Sialidase is a rare disease without racial predisposition. Available very small population data, but the study in the Netherlands showed a rate of approximately 1 case per 2175000 live births. However, this ratio may not apply to all populations, some of which have a higher occurrence; in addition, loss of clinical recognition is an important factor when newborn screening is not a matter of choice.

Treatment options sialidase remain limited and directly aimed at supporting the treatment and relief of symptoms.

In one of the embodiments the present invention relates to a method of treating sialidase through the modulation of the activity of sialidase.

Metachromatic leukodystrophy

Metachromatic leukodystrophy (MLD) or deficit arylsulfatase is called A defect of the enzyme arylsulfatase A (or cerebrospinalis). Arylsulfatase A is responsible for the transformation of sulfatide (or cerebroside-3-sulfate) in galactosylceramide; thus, the defect leads to the accumulation of sulfatide. BMP stimulates its activity, it depends on sabotinov.

It is a lysosomal storage disease of accumulation, which is the common point in the family of leukodystrophies. Leukodystrophy effect on growth and/or development of the myelin sheath, the fatty covering that acts as an insulator around nerve fibers in the Central and peripheral nervous systems.

Like many other genetic diseases that act on lipid metabolism, there are several forms of MLD, which are postneonatal, juvenile and adult:

• When postreinforcement form, which is the most common form of MLD affected children begin to experience difficulty walking after the first year of life. Symptoms include atrophy and muscle weakness, muscle rigidity, developmental delay, progressive loss of vision leading to blindness, convulsions, impaired swallowing, paralysis and dementia. Children can fall into a coma. Without treatment, most children with this form of MLD die to 5 years, often much sooner.

• Children with juvenile form of MLD (beginning in 3-10 years), as a rule, begin with poor school performance, mental disorders and dementia, and then again what are the symptoms similar to postreinforcement shape, but with a slower progression. Age of death varies, but usually within 10-15 years from the onset of symptoms.

• The adult form usually begins after 16 years as a mental disorder or progressive dementia. MLD with early in adulthood progresses more slowly than postreinforcement and juvenile forms with delayed for a decade or more.

In rare cases, the body can compensate for the deficit, and the individual does not demonstrate the symptoms.

There is no cure for MLD and standardized treatment, this deadly disease. Children with advanced juvenile or Mature early and patients with postneonatal form, manifesting symptoms, get treatment, limited by alleviating pain and symptoms. Patients with symptomatic postreinforcement MLD, as well as with juvenile and adult MLD that are predictive or demonstrate moderate to medium symptoms, have the choice of bone marrow transplantation (including stem cells), which is under investigation.

In one of the embodiments the present invention relates to a method for treatment of metachromatic leukodystrophy through the modulation of the enzymatic activity of Ukrainian A.

p> The deficit sabotinov

The deficit prosaposin/sabotinov leads to severe neurological deficits in humans and mice.

Patients with tochkovymi mutations in saposin A, B, and C are the phenotypes of Krabbe disease, metachromatic leukodystrophy and Gaucher disease, which suggests that their primary substrates in vivo are galactosylceramide, sulfatide and glucosylceramide respectively.

Atypical Krabbe disease due to a deficiency saposin A: hereditary biochemical disturbance, leading to neurological regression for several months after birth. Usually death occurs in the first few years of life. Violation similar with Krabbe disease, but different genetic cause of the biochemical defect. Krabbe disease involves a defect in the gene galactocerebrosidase, while atypical Krabbe disease involves a defect in the gene for prosaposin that cause deficiency saposin A.

Saposin B, formerly known as SAP-1 and activator sulfatide, stimulates the hydrolysis of a wide range of substrates, including cerebrospinal, GM1 of ganglioside and globotriaosylceramide, Ukrainian, acidic beta-galactosidase and alpha-galactosidase, respectively. The deficit saposin B the man, described as an autosomal recessive trait, results in accumulation in tissues of cerebr is sidesurface and clinical, similar to the metachromatic leukodystrophy (metachromatic leukodystrophy deficit activator), albeit with the normal activity of Ukrainian. The deficit saposin B is a heterogeneous disease with a spectrum similar to the metachromatic leukodystrophy.

Saposin (SAP) C is required for the degradation of glucosylceramide, and its deficiency leads to various forms of Gaucher disease; nonaromatizable Gaucher disease due to a deficiency of SAP-C. patients see very high levels of activity chitotriosidase, the chemokine CCL18, the increased concentration of glucosylceramide in plasma and normal activity of β-glucosidase in skin fibroblasts. Identified a missense mutation p.L349P localized in the domain of SAP-C, and another mutation, p.M1L, localized in the initiator codon of the protein-predshestvennika of prosaposin.

Some patients with nonaromatizable Gaucher disease have identified mutations in saposin C, and saposin D.

The combined deficit of sabotinov C and D in mice leads to neuropathic phenotype with accumulation glucosylceramide and alpha hydroxycarbamide.

In mice deficit saposin D is associated with the accumulation of ceramide, partial loss of Purkinje cells and dysfunction of the urinary system. This phenotype mimics embryonic mortality, which demonstrate mouse with full Def is citom sour ceramidase, enzyme associated with saposiem D.

People have known for two mutations, which lead to complete inactivation of all four sabotinov and prosaposin. A General lack of sabotinov is a devastating disease with involvement of multiple organs and multiple sphingolipids. Reported a combined deficit of sabotinov (or deficit prosaposin) in the case of severe neurovisceral dystrophy, caused the death of a newborn. In the urine was increased many sphingolipids, with the greatest increase in globotriaosylceramide. Identified a new mutation in the gene PSAP, being homozygous for the mutation of the acceptor site of splicing two pairs above codon 10. This mutation leads to a premature stop-codon and the formation of low levels of the transcript.

In one of the embodiments the present invention relates to a method of treating deficiency sabotinov. This deficit of sabotinov can be selected from the group consisting of deficit saposin A deficit saposin B, deficiency saposin C deficiency saposin D and the combined deficit sabotinov (or deficit prosaposin).

Residual enzymatic activity

Lysosomal storage disorders, as described above in this document are caused by a defective enzyme. The specified defective enzyme may not have residual activity, or you may have some of the which the residual activity.

In this document the residual enzymatic activity means that although the enzyme is defective, for example, that caused by a mutation, the enzyme activity is not completely disappeared, but rather reduced to a pathological level.

In one aspect the present invention relates to bioactive tools for use in the treatment of lysosomal storage diseases and the treatment of individuals with lysosomal storage disease accumulation.

In one of the embodiments of the present invention lysosomal disease accumulation, which is treated according to the present invention, is characterized as having a residual enzymatic activity of the defective enzyme involved in the pathology of the disease.

In one of the embodiments specified residual enzymatic activity is in the range from 0.1% to 50%, such as in the range from 0.1 to 1%, for example, from 1%to 2%, such as from 2%to 3%, for example, from 3 to 4%, such as from 4 to 5%, for example, from 5 to 6%, such as from 6 to 7%, for example, from 7 to 8%, such as from 8 to 9%, for example, from 9 to 10%such as from 10%to 11%, for example, from 11 to 12%, such as from 12 to 13%, for example, from 13 to 14%, such as from 14 to 15%, for example, from 15 to 20%, such as from 20 to 25%, for example, from 25%to 30%, such as from 30%to 35%, for example, from 35 to 40%, such as from 40%to 45%, for example, in the range from 45 to 50% residual enzymatic activity./p>

Modern methods of treatment LSD

There is no cure lysosomal storage diseases, and treatment of the majority is symptomatic, although with some success performed bone marrow transplantation and replacement enzyme therapy (ERT). In addition, for a number of these diseases in specialized centers performed the transplantation of umbilical cord blood. However, transplantation therapy is accompanied by a large number of side effects and often causes complications in patients. In addition, at the present time for some of these diseases are considering therapy for the reduction of the substrate, the method used to reduce the accumulation of material.

For most lysosomal storage diseases remains a large unmet need for effective treatment method.

For a number of lysosomal storage diseases was developed substitution therapy enzyme, and for the treatment of Gaucher disease on the market for several years there is zeraim (Cerezyme®). The defective enzyme, glucocerebrosidase received recombinant techniques and was administered by intravenous infusion over several hours. Treatment is not a cure, and patients require lifelong treatment to stop the progression of the disease. Some of the symptoms you can improve pore the STV ERT.

However, for most LSD has not yet developed effective ERT. This can happen due to the fact that the active enzyme is a difficult task due to complex subunit structure of defective enzymes. In fact, the enzymes may receive incorrect to fold.

For those of LSD, ERT, there are obstacles that make the treatment less desirable. First, ERT is a very expensive form of therapy, which is a financial burden to society, making it unavailable for some patients. In addition, ERT specifically aimed at only one disease. Report some side effects of Cerezyme (Cerezyme®), including the development of the immune response, nausea, vomiting, abdominal pain, diarrhea, rash, weakness, headache, fever, dizziness, chills, back pain, palpitations, and symptoms suggestive of allergic reaction.

Thus, the disclosure of the present invention provides a new and innovative method for the treatment of lysosomal storage diseases. In particular, this refers to those diseases that are not developed an effective therapy for those who can benefit from less expensive treatment, and those who may benefit from combination therapy, comprising the bioactive is the tool of the present invention.

In this paper the method according to the present invention relates to a method of treatment, conduct which is significantly cheaper than ERT and which targets more than one lysosomal disease accumulation.

Molecular chaperones or heat shock proteins, described later in this document, because the authors found that the interaction between heat shock protein 70 and lysosomal BMP as described above in this document forms the basis for the modulation of lysosomal enzyme activity and treatment of lysosomal storage diseases according to the present invention.

Molecular chaperones

Spending a large amount of energy first transcription and then translation of the genetic code of the DNA, the cell eventually synthesizes a polypeptide whose function is probably required at this stage of life cells. However, to achieve full functional protein needed to overcome some of the final barriers - one of them is the correct folding of the primary polypeptide chain. Evolutionary requirements achieve proper folding are evident not only a huge waste of energy for the synthesis of peptide without the correct conformation and, consequently, the functions, but also the aggregation of such proteins in the lumen of the cells can be harmful to cells. This aggregation in dei is titelliste is a very likely outcome, considering the intracellular environment of high concentration of protein, therefore, is not a surprise that a complex and delicate mechanism of proteins exists to help the folding of the protein, allowing the functional state of the protein is maintained in these conditions. The data of proteins collectively referred to as molecular chaperones, as well as their counterparts in humans, they prevent unwanted interaction between their immature clients.

Molecular chaperones find all compartments of the cell, where it undergoes a conformational rearrangement of proteins, although protein synthesis is the main source nevernude peptides in the cell, the problem of cells, which occurs due to high temperature or other incentives that can make proteins structurally unstable and therefore prone to improper folding and aggregation, occurs with a specific cell response involving the production of protective proteins. This response is a phenomenon observed in each type of cells ranging from prokaryotes to eukaryotes and denoted as a response to heat shock or stress. Induced this response proteins known as heat shock proteins (HSP), there are several of their collections. Data collection was from a related sequence, structure and function of proteins, the WTO as chaperones from different families vary considerably in structure, and function in the cell. The main example of a family of chaperones are proteins Hsp70, which constitute the Central part of the pervasive system of chaperones present in most compartments of eukaryotic cells, eubacteria and many archebacteria. Recently, in addition to perform the function of a chaperone, this family was linked with other aspects of cellular homeostasis - most significantly through its antiapoptotic properties, their function in the immune system and the apparent dependence of malignant cells from positive regulation of Hsp70.

The family of heat shock proteins 70

Proteins Hsp70 is involved in a wide range of cellular processes, including protein folding and degradation of unstable cellular proteins, as well as the implementation zitoprotectivei functions. The overall function of Hsp70 in these processes, apparently, is the linking of short hydrophobic segments in a partially folded polypeptides, thereby facilitating correct folding and prevent aggregation. In eukaryotic chaperones Hsp70 interact in vivo with different classes of proteins that perform the function of regulation of critical stages of their functional cycle; among them, the protein of the J-domain of Hsp40 family. In addition, identified additional protein partners, some of which bind Hsp70 with other the systems chaperones, systems such as Hsp90.

Members of the Hsp70 family of man

Some of the important functions attributed to molecular chaperones, include the import of proteins into cell compartments, the folding of proteins in the cytosol, endoplasmic reticulum and mitochondria, preventing protein aggregation and refolding of incorrectly folded proteins. Currently, the family of Hsp70 person includes 10 members, encoded by different genes, and this section aims to provide an overview of the members of this family in terms of features, profiles, expression and homology. There is some confusion in the nomenclature of the various members of the Hsp70 family of man, although the General guidelines set out in Tavaria et al., that provides a logical link between the names of the loci, genes and proteins. However, because there is still some interspecific confusion, genes and proteins Hsp70 describe in this document the name of their loci. The name of Hsp70 may include two inducible members of the Hsp70 family with the names of the loci HSPA1A and HSPA1Β or to the whole family of Hsp70 in General, as can be seen from the context of the document. However, as used in the present invention, Hsp70 aims to designation of any of the two inducible members of the Hsp70 family with the names of the loci HSPA1A and HSPA1B.

HspA1A and Hsp1B

Transcribed from genes HSPA1A and HSPA1Β genes are two heat W is K/stress-inducible Hsp70 genes, and a large part of the literature considers Hsp70 person to proteins encoded by these two genes. These genes give rise to proteins containing 641 amino acid that has 99% homology with each other and which is first cloned and characterized members of the Hsp70 family. Genes linked to the MHC complex class III on 6p21.3, do not have introns, are the promoter regions containing the HSE, which allows them to bind HSF and to induce transcription in response to a variety of cell damage.

The protein sequence for heat shock protein 1A [Homo sapiens (HSPA1A) with molecular weight of 70 kDa (SEQ ID NO:1) (Sample no. NM_005345.5):

MAKAAAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLIGDAAKNQVALNPQNTVFDAKRLIGRKFGDPVVQSDMKHWPFQVINDGDKPKVQVSYKGETKAFYPEEISSMVLTKMKEIAEAYLGYPVTNAVITVPAYFNDSQRQATDGVIAGLNVLRIINEPTAAAIAYGLDRTGKGERNVLIFDLGGGTFDVSILTIDDGIFEVKATAGDTHLGGEDFDNRLVNHFVEEFKRKHKKDISQNKRAVRRLRTACERAKRTLSSSTQASLEIDSLFEGIDFYTSITRARFEELCSDLFRSTLEPVEKALRDAKLDKAQIHDLVLVGGSTRIPKVQKLLQDFFNGRDLNKSINPDEAVAYGAAVQAAILMGDKSENVQDLLLLDVAPLSLGLETAGGVMTALIKRNSTIPTKQTQIFTTYSDNQPGVLIQVYEGERAMTKDNNLLGRFELSGIPPAPRGVPQIEVTFDIDANGILNVTATDKSTGKANKITITNDKGRLSKEEIERMVQEAEKYKAEDEVQRERVSAKNALESYAFNMKSAVEDEGLKGKISEADKKKVLDKCQEVISWLDANTLAEKDEFEHKRKELEQVCNPIISGLYQGAGGPGPGGFGAQGPKGGSGSGPTIEEVD

The sequence of nucleic acid (DNA) for heat shock protein 1A [Homo sapiens (HSPA1A) with molecular weight of 70 kDa (SEQ ID NO:2) (Sample no. NM_005345.5):

1 ataaaagccc aggggcaagc ggtccggata acggctagcc tgaggagctg ctgcgacagt

61 ccactacctt tttcgagagt gactcccgtt gtcccaaggc ttcccagagc gaacctgtgc

121 ggctgcaggc accggcgcgt cgagtttccg gcgtccggaa ggaccgagct cttctcgcgg

181 atccagtgtt ccgtttccag cccccaatct cagagcggag ccgacagaga gcagggaacc

241 ggcatggcca aagccgcgc gatcggcatc gacctgggca ccacctactc ctgcgtgggg

301 gtgttccaac acggcaaggt ggagatcatc gccaacgacc agggcaaccg caccaccccc

361 agctacgtgg ccttcacgga caccgagcgg ctcatcgggg atgcggccaa gaaccaggtg

421 gcgctgaacc cgcagaacac cgtgtttgac gcgaagcggc tgattggccg caagttcggc

481 gacccggtgg tgcagtcgga catgaagcac tggcctttcc aggtgatcaa cgacggagac

541 aagcccaagg tgcaggtgag ctacaagggg gagaccaagg cattctaccc cgaggagatc

601 tcgtccatgg tgctgaccaa gatgaaggag atcgccgagg cgtacctggg ctacccggtg

661 accaacgcgg tgatcaccgt gccggcctac ttcaacgact cgcagcgcca ggccaccaag

721 gatgcgggtg tgatcgcggg gctcaacgtg ctgcggatca tcaacgagcc cacggccgcc

781 gccatcgcct acggcctgga cagaacgggc aagggggagc gcaacgtgct catctttgac

841 ctgggcgggg gcaccttcga cgtgtccatc ctgacgatcg acgacggcat cttcgaggtg

901 aaggccacgg ccggggacac ccacctgggt ggggaggact ttgacaacag gctggtgaac

961 cacttcgtgg aggagttcaa gagaaaacac aagaaggaca tcagccagaa caagcgagcc

1021 gtgaggcggc tgcgcaccgc ctgcgagagg gccaagagga ccctgtcgtc cagcacccag

1081 gccagcctgg agatcgactc cctgtttgag ggcatcgact tctacacgtc catcaccagg

1141 gcgaggttcg aggagctgtg ctccgacctg ttccgaagca ccctggagcc cgtggagaag

1201 gctctgcgcg acgccaagct ggacaaggcc cagattcacg acctggtcct ggtcgggggc

1261 tccacccgca tccccaaggt gcagaagctg ctgcaggact tcttcaacgg gcgcgacctg

1321 aacaagagca tcaaccccga cgaggctgtg gcctacgggg cggcggtgca ggcggccatc

1381 ctgatggggg acaagtccga gaacgtgcag gacctgctgc tgctggacgt ggctcccctg

1441 tcgctggggc tggagacggc cggaggcgtg atgactgccc tgatcaagcg caactccacc

1501 atccccacca agcagacgca gatcttcacc acctactccg acaaccaacc cggggtgctg

1561 atccaggtgt acgagggcga gagggccatg acgaaagaca acaatctgtt ggggcgcttc

1621 gagctgagcg gcatccctcc ggcccccagg ggcgtgcccc agatcgaggt gaccttcgac

1681 atcgatgcca acggcatcct gaacgtcacg gccacggaca agagcaccgg caaggccaac

1741 aagatcacca tcaccaacga caagggccgc ctgagcaagg aggagatcga gcgcatggtg

1801 caggaggcgg agaagtacaa agcggaggac gaggtgcagc gcgagagggt gtcagccaag

1861 aacgccctgg agtcctacgc cttcaacatg aagagcgccg tggaggatga ggggctcaag

1921 ggcaagatca gcgaggcgga caagaagaag gtgctggaca agtgtcaaga ggtcatctcg

1981 tggctggacg ccaacacctt ggccgagaag gacgagtttg agcacaagag gaaggagctg

2041 gagcaggtgt gtaaccccat catcagcgga ctgtaccagg gtgccggtgg tcccgggcct

2101 gggggcttcg gggctcaggg tcccaagggagggtctgggt caggccccac cattgaggag

2161 gtagattagg ggcctttcca agattgctgt ttttgttttg gagcttcaag actttgcatt

2221 tcctagtatt tctgtttgtc agttctcaat ttcctgtgtt tgcaatgttg aaattttttg

2281 gtgaagtact gaacttgctt tttttccggt ttctacatgc agagatgaat ttatactgcc

2341 atcttacgac tatttcttct ttttaataca cttaactcag gccatttttt aagttggtta

2401 cttcaaagta aataaacttt aaaattcaaa aaaaaaaaaa aaaaa

The protein sequence for heat shock protein 1B Homo sapiens (HSPA1B) with molecular weight of 70 kDa (SEQ ID N0:3) (Sample no: NM_005346):

MAKAAAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLIGDAAKNQVALNPQNTVFDAKRLIGRKFGDPVVQSDMKHWPFQVINDGDKPKVQVSYKGETKAFYPEEISSMVLTKMKEIAEAYLGYPVTNAVITVPAYFNDSQRQATDGVIAGLNVLRIINEPTAAAIAYGLDRTGKGERNVLIFDLGGGTFDVSILTIDDGIFEVKATAGDTHLGGEDFDNRLVNHFVEEFKRKHKKDISQNKRAVRRLRTACERAKRTLSSSTQASLEIDSLFEGIDFYTSITRARFEELCSDLFRSTLEPVEKALRDAKLDKAQIHDLVLVGGSTRIPKVQKLLQDFFNGRDLNKSINPDEAVAYGAAVQAAILMGDKSENVQDLLLLDVAPLSLGLETAGGVMTALIKRNSTIPTKQTQIFTTYSDNQPGVLIQVYEGERAMTKDNNLLGRFELSGIPPAPRGVPQIEVTFDIDANGILNVTATDKSTGKANKITITNDKGRLSKEEIERMVQEAEKYKAEDEVQRERVSAKNALESYAFNMKSAVEDEGLKGKISEADKKKVLDKCQEVISWLDANTLAEKDEFEHKRKELEQVCNPIISGLYQGAGGPGPGGFGAQGPKGGSGSGPTIEEVD

The sequence of nucleic acid (DNA) for heat shock protein 1B Homo sapiens (HSPA1B) with molecular weight of 70 kDa (SEQ ID N0:4) (Sample no: NM_005346):

1 ggaaaacggc cagcctgagg agctgctgcg agggtccgct tcgtctttcg agagtgactc

61 ccgcggtccc aaggctttcc agagcgaacc tgtgcggctg caggcaccgg cgtgttgagt

121 ttccggcgtt ccgaaggact gagctcttgt cgcggatccc gtccgccgtt tccagccccc

181 agtctcagag cggagcccac agagcagggc accggcatgg ccaaagccgc ggcgatcggc

241 atcgacctgg gcaccaccta ctcctgcgtg ggggtgttcc aacacggcaa ggtggagatc

301 atcgccaacg accagggcaa ccgcaccacc cccagctacg tggccttcac ggacaccgag

361 cggctcatcg gggatgcggc caagaaccag gtggcgctga acccgcagaa caccgtgttt

421 gacgcgaagc ggctgatcgg ccgcaagttc ggcgacccgg tggtgcagtc ggacatgaag

481 cactggcctt tccaggtgat caacgacgga gacaagccca aggtgcaggt gagctacaag

541 ggggagacca aggcattcta ccccgaggag atctcgtcca tggtgctgac caagatgaag

601 gagatcgccg aggcgtacct gggctacccg gtgaccaacg cggtgatcac cgtgcggcc

661 tacttcaacg actcgcagcg ccaggccacc aaggatgcgg gtgtgatcgc ggggctcaac

721 gtgctgcgga tcatcaacga gcccacggcc gccgccatcg cctacggcct ggacagaacg

781 ggcaaggggg agcgcaacgt gctcatcttt gacctgggcg ggggcacctt cgacgtgtcc

841 atcctgacga tcgacgacgg catcttcgag gtgaaggcca cggccgggga cacccacctg

901 ggtggggagg actttgacaa caggctggtg aaccacttcg tggaggagtt caagagaaaa

961 cacaagaagg acatcagcca gaacaagcga gccgtgaggc ggctgcgcac cgcctgcgag

1021 agggccaaga ggaccctgtc gtccagcacc caggccagcc tggagatcga ctccctgttt

1081 gagggcatcg acttctacac gtccatcacc agggcgaggt tcgaggagct gtgctccgac

1141 ctgttccgaa gcaccctgga gcccgtggag aaggctctgc gcgacgccaa gctggacaag

1201 gcccagattc acgacctggt cctggtcggg ggctccaccc gcatccccaa ggtgcagaag

1261 ctgctgcagg acttcttcaa cgggcgcgac ctgaacaaga gcatcaaccc cgacgaggct

1321 gtggcctacg gggcggcggt gcaggcggcc atcctgatgg gggacaagtc cgagaacgtg

1381 caggacctgc tgctgctgga cgtggctccc ctgtcgctgg ggctggagac ggccggaggc

1441 gtgatgactg ccctgatcaa gcgcaactcc accatcccca ccaagcagac gcagatcttc

1501 accacctact ccgacaacca acccggggtg ctgatccagg tgtacgaggg cgagagggcc

1561 atgacgaaag acaacaatct gttggggcgc ttcgagctga gcggcatccc tccggccccc

1621 aggggcgtgc cccagatcga ggtgaccttc gacatcgatg ccaacggcat cctgaacgtc

1681 acggccacgg acaagagcac cggcaaggcc aacaagatca ccatcaccaa cgacaagggc

1741 cgcctgagca aggaggagat cgagcgcatg gtgcaggagg cggagaagta caaagcggag

1801 gacgaggtgc agcgcgagag ggtgtcagcc aagaacgccc tggagtccta cgccttcaac

1861 atgaagagcg ccgtggagga tgaggggctc aagggcaaga tcagcgaggc ggacaagaag

1921 aaggttctgg acaagtgtca agaggtcatc tcgtggctgg acgccaacac cttggccgag

1981 aaggacgagt ttgagcacaa gaggaaggag ctggagcagg tgtgtaaccc catcatcagc

2041 ggactgtacc agggtgccgg tggtcccggg cctggcggct tcggggctca gggtcccaag

2101 ggagggtctg ggtcaggccc taccattgag gaggtggatt aggggccttt gttctttagt

2161 atgtttgtct ttgaggtgga ctgttgggac tcaaggactt tgctgctgtt ttcctatgtc

2221 atttctgctt cagctctttg ctgcttcact tctttgtaaa gttgtaacct gatggtaatt

2281 agctggcttc attatttttg tagtacaacc gatatgttca ttagaattct ttgcatttaa

2341 tgttgatact gtaagggtgt ttcgttccct ttaaatgaat caacactgcc accttctgta

2401 cgagtttgtt tgtttttttt tttttttttt ttttttgctt ggcgaaaaca ctacaaaggc

2461 tgggaatgta tgtttttata attttttat ttaaatatga aaaataaaat gttaaacttt

2521 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a

HspAIL and HspA2

Two members of the Hsp70 family called "genes chauvinists"because the male sex cells maintain their expression with a strong bias. HspA1L gene is constitutively expressed betinternet a member of the Hsp70 family, localized in 4 TPN towards the telomeres relative to the locus of HSPA1A in the same complex MHC class III on chromosome 6. It is expressed in a small number of before and after heat shock, but with the expression profile of contributing tests on mice, rats and humans with the protein of 641 amino acids, which is 90% homologous HspA1A. HspA2 gene was first isolated from the genomic library mouse and later showed that he constitutively expressed, albeit at low levels in various tissues of the human body, including skeletal muscle, ovary, small intestine, large intestine, brain, placenta and kidney, but is highly expressed in the testes. Its expression, or rather its lack, is associated with abnormal spermatogenesis man, and the male mouse hspA2(-/-)are sterile. The gene is localized on chromosome 14, gives rise to a protein of 639 amino acids 84% homology with HspA1A, although the exact localization is the subject of discussion, as in two articles represent different positions of loci 14q24.1 against 14q22.

HspA6 and HspA7

Genes hspA6 and hspA7 are induced thermal W is whom members of the Hsp70 family, for which there are no analogues in mice. They contain HSE in promotor regions and are bisantrene. They colocalize on chromosome 1 and have each other 94% homology to the sequence of nucleotides. However, only HspA6 is functional, while gene hspA7 contains a single nucleotide insertion, leading to the formation of a premature stop codon at position +1324. HspA6 protein has a length of 643 amino acids and shows 77% homology with HspA1A and HspA1B.

HspA5 and HspA9

Genes hspA5 and hspA9 are the two compartment-specific members of the Hsp70 family. Protein HspA5 length of 655 amino acids localized in the endoplasmic reticulum (ER) and facilitates the folding and transport of newly synthesized proteins in their compartment. Protein is 64% homologous HspA1A gene localized on 9q34. Protein HspA9 length of 679 amino acids localized in the mitochondria, where it facilitates the folding of proteins after their transport across the mitochondrial membrane. HspA9 localized on 5q31.1, protein is 52% homologous HspA1A.

HspA8

Like a member of the Hsp70 known as Hsc70, is encoded by gene called hspA8, 11q24, giving rise to a protein of 646 amino acids 86% homology with HspA1A, and is expressed constitutively in all tissues and cell lines. Protein is similar to Hsp70 on their functions in the cell, providing the required support in normal circumstances, N. which he is also credited role in "the strip" covered clatrina vesicles, and mediated by chaperones autophagy.

HspA3 and HspA4

This document does not discuss, because there are doubts as to whether there is HSPA3, and most likely, HSPA4 is a member of the Hsp110 family to the present time nothing is known about him, except that it is localized on 5q31.1-2.

Table 1
The list of genes of the Hsp70 family of man
LocusUsed in this document the name of the Gene/ProteinPosition% homology with HSPA1AAlternative names
HSPA1AhspA1A/HspA1A (Hsp70)6p23.1100Hsp70; Hsp72; Hsp70-1
HSPA1BhspA1B/HspA1B (Hsp70)6p23.199Hsp70; Hsp72; Hsp70-2
HSPA1LhspA1L/HspA1L6p23.190Hsp70-Hom; Hsp70t
HSPA2hspA2/HspA214q24.184 Hsp70-3
HSPA4hspA4/HspA45q31.131Hsp70RY; APG-2
HSPA5hspA5/HspA59q3464BiP; GRP78
HSPA6hspA6/HspA61q84Hsp70-6; Hsp70B'
HSPA7hspA7/HspA71q-Hsp70-7; Hsp70B
HSPA8hspA8/HspA8 (Hsc70)11q2486Hsc70; Hsp73
HSPA9hspA9/HspA95q31.152GRP75; PBP74; mtHsp75; mortalin; mot-2
Genes are listed by name locus used in this document the name, localization on the chromosome (position), homology in amino acid sequence with HspA1A and alternative names often cited in literature

Regulation of transcription of Hsp70

Genomic ft-p is hinting promoter Hsp70 person revealed, that heat shock/stress induces rapid binding of transcription factors heat shock (HSF) with a scope covering the sequence nGAAn called heat shock elements (HSE). Under normal conditions, Hsp70 is associated with HSF, which are located in the cytosol, but when the stress HSF separated from Hsp70 and rebuild homotrimer conformation after phosphorylation of PKC or other serine/ser / thr kinases. The HSF trimers fall into the nucleus, where they bind HSE localized in the promoter region of genes Hsp70, and then fosfauriliruyutza kinases HSF.

In this time, people have described three HSF (HSF1, HSF2 and HSF4). HSF1 is the main transcription factor activated in the most stressful conditions and are responsible for a wide range of incentives, which can be divided into physiological (i.e. cell division, hormone stimulation), pathological (i.e. infection, fever, inflammation, malignant neoplasm) and environment conditions (i.e. heat shock, heavy metals, ethanol). HSF2 only responds to geminus, while HSF4 preferably is expressed in the human heart, pancreas, brain and skeletal muscle, has no c-terminal hypoponera repeat distributed HSF all vertebrates, and acts as a repressor of the expression of HSP. Regulation of Hsp70 gene responsible for the synthesis of constitutively Express the alignment of Hsp70 (Hsc70), not well researched, but it seems that HSF is not involved in it.

Although HSF are the most significant factors regulating the expression of the HSP, it is shown that other transcription factors have the same ability. It is shown that the specific factors that bind to the CCAAT-box (CBF), induce the transcription of Hsp70, the tumor suppressor p53 can repress transcription by binding to the promoter region of Hsp70 and by neutralization with CBF, and protein 1 that communicates with the factors heat shock (HSBP1), counteracts the HSF and, thus, reduces the transcription of Hsp70.

Structural and functional properties of Hsp70

Structure and functions of Hsp70 is the most studied for Hsp70 of eubacteria, DnaK, it Hsp40 co-chaperone DnaJ and the exchange factor GrpE nucleotide. However, as a rule, the mechanism is considered as similar in eukaryotes, although evidence suggests about the separation of GrpE. This section focuses on the system of eukaryotic Hsp70, but also includes comments on the system of eubacteria where appropriate.

Hsp70 has two functional parts - N-terminal ATPase domain and a smaller C-terminal peptide-binding domain. ATPase domain contains two subdomains separated by a gap containing a binding site nucleotide, which determines the peptide-binding properties of the C-terminal domain. When ATP is points the user, the peptide substrate binds and dissociates rapidly, albeit with low affinity, in the case when no nucleotide or ADP is not associated with the N-terminal domain, the ratio of the binding and dissociation of the peptide decreases and increases the affinity. Thus, the hydrolysis of ATP acts as a molecular switch between two States Hsp70, cycles which regulate protein J-domain of Hsp40 family in eukaryotes and DnaJ and GrpE in eubacteria. N-terminal J-domain of Hsp40 binds to Hsp70, accelerating the hydrolysis of ATP and, thus, contributing to the capture of the peptide, whereas the C-terminal part of Hsp40 functions as a chaperone by recognition of hydrophobic peptides, resulting Hsp70 is involved in the primary polypeptide chains. It should be noted that molecular chaperones do not provide specific steric information for folding of the bound protein, but rather inhibit unproductive interaction, thus allowing the protein to fold into its native structure more efficiently.

In eubacteria GrpE induces the release of ADP from DnaK (bacterial Hsp70), while eukaryotic Hsp70 proteins such factor seems to be optional due to the fact that the rate-limiting stage in this cycle ATPase is not the dissociation of the bound ADP and ATP hydrolysis. However, additional proteins are used for re is ulali functioning of Hsp70 in eukaryotes; homoallylamines protein Hip (interacting with Hsp70 protein) performs the function of a positive regulator by stabilizing the ADP-bound state of Hsp70, while the protein to bind to carbonsilicon Hsp70 (CHIP), and is associated with Bcl-2 alunogen 1 (Bag-1) have inhibitory action - CHIP through inhibition of ATPase activity of Hsp70 and Bag-1 by anti readingbuy activity of Hsp70. Further cooperation provided with two Hsp40 proteins of human Hdj1 and Hdj2, which shows that in addition to their functioning as Hsp 40 (described above) they facilitate the mating of Hsp70 and Hsp90 by Hop (Hsp-organizing protein), adaptorname protein, which physically connects the two chaperones their domain tetratricopeptide repeats (TPR), which connect the long C-terminal sequences of Hsp70 and Hsp90, respectively. It was recently shown that some of the above proteins are regulators of migration negativnyh or irreversibly incorrectly folded proteins and chaperones in ubiquitin-proteosome system. Protein CHIP in addition to a negative regulatory role for Hsp70 is able to communicate with Hsp90 via the N-terminal TPR domain and aims to Hsp90 substrates for degradation by the C-terminal domain of ubiquitinate, but also able to functionally cooperate with BAG-1, which binds to Hsp70 (as well as proteo the wash). These data provide a link between the mechanisms that integrate the folding with the participation of chaperones and proteolytic degradation, the two main components of the quality control of proteins in the cytosol.

Cytoprotective by Hsp70

In addition to its anti-apoptotic abilities and, as a consequence, functioning as a molecular chaperone, i.e. facilitating the folding of proteins under denaturing conditions, Hsp70 is able to act on the survival of cells of various other ways, including the protection of the functioning of mitochondria after damage due to ischemia-reperfusion, block activation of stress N-terminal c-jun kinase (JNK) after stimulation of primary fibroblasts TNF, and it seems that the complex of Hsp70/Bag-1 regulates cell growth and mitogens in conditions of cellular stress. In addition, the ability of Hsp70 to protect the cells from cell death induced by several stimuli, such as TNF, TRAIL, oxidative stress, UV radiation and anticancer drugs, i.e. doxorubicin, etoposide and Taxol, emphasizes its antiapoptotic properties. In the end, the article also provides more direct evidence of interaction between Hsp70 and apoptosis induction, since it was shown that Hsp70 prevents factor induction of apoptosis (AIF), and also affects the antiapoptotic function after CAS is the basics-3.

Recent evidence also suggests that the potential cytoprotective action of Hsp70 due to the stabilization of lysosomal membranes. In proof of this, the depletion of Hsp70 starts early permeability of the lysosomal membrane and is mediated by cathepsins death of malignant cells, and exogenous Hsp70 inhibits the destabilization of lysosomes induced by various stress. In addition, mice with deficiency of Hsp70 suffer from pancreatitis caused by deficiency of lysosomal proteases into the cytosol. All these phenomena highlight the role of Hsp70 as an important regulator of PCD and, therefore, the factor of survival.

Hsp70 in malignant tumors

Hsp70 often sverkhekspressiya in malignant human tumors, and its expression correlates with poor prognosis for tumors of the breast and endometrium. In accordance with this Hsp70 increases the carcinogenic potential of cells of rodents implanted immuno-compromised or isogenic animals.

In addition, the role of Hsp70 as an important factor in the survival of malignant cells is grounded in article Wei et al., who conducted the first study of the depletion of Hsp70 in cancer cells. The results showed that when the expression of Hsp70 in various malignant cell lines ingibirovany by applying antimicrob the x oligomers, induced inhibition of cell proliferation followed by apoptosis. This work was confirmed by a series of experiments in which is mediated by adenovirus depletion of Hsp70 was launched specific lysosomal program of destruction of malignant cells.

In addition, in vivo studies using orthotopic xenografts of glioblastoma and breast carcinoma, as well as subcutaneous xenografts carcinoma of the colon in mice immunocompromised demonstrate protivolokatsionnaya potential depletion of Hsp70, because the tumors of mice treated with local impact of the above adenoviral constructs showed massive apoptosis-like cell death and the involvement of macrophages. These studies clearly demonstrate the dependence of certain tumors from the presence of Hsp70, although other research suggests that the cytotoxicity observed in the cell culture after mediated by adenovirus depletion of Hsp70 caused by a combination of mediated virus cellular stress and negative regulation of Hsp70. Despite this contradiction, induced depletion of Hsp70 cytotoxicity in cell culture does not depend on caspases, since neither the overexpression of Bcl-2 or pharmacological inhibition of caspases can't save cells. Rather, inducing the death of what sobytiami are running LMP and the release of cathepsins into the cytosol, because inhibition of cysteine cathepsins gives significant cytoprotection. In addition, in mice, the depletion of Hsp70 in the above-mentioned tumor xenografts leads to the release of cathepsins and tumor cell death.

As noted earlier, one of the cytoprotective mechanisms of Hsp70, which can be applied to malignant cells, is moving Hsp70 in endosomal compartment, where it performs the membrane-protective function. This move may not only be directed by the need to protect the lysosomal membrane, because studies show that more than 50% of tumors showing localization of Hsp70 on the surface of the cytoplasmic membrane area, which is directly connected with endolysosomal the compartment by means of endocytosis and secretion events, as described above. Exposed on the surface of Hsp70 is a unique epitope, which can act as a structure recognition for natural killer cells (NK), stimulating their proliferation and cytolytic activity. It is shown that NK cells activated this peptide sequence of Hsp70, inhibit tumor growth in mice with severe combined immunodeficiency (SCID), a possible mechanism for this could be that Hsp70 is associated with the surface of the cells, mediates apoptosis by what redstem specific binding and capture granzyme D regardless of perforin.

As described above, endolysosomal membrane and the cytoplasmic membrane is constantly used interchangeably. Thus, the presence of Hsp70 on the surface of malignant cells may be a "failed" result of two events that contribute to the progression of the tumor; the secretion of cathepsins, which promotes invasion and angiogenesis, and localization of Hsp70 on lysosomal membranes, thereby preventing accidental release of cysteine cathepsins into the cytosol and subsequent death of the cell.

Extracellular Hsp70

As stated in the previous paragraphs, the intracellular function of Hsp70 is important for the appropriate homeostasis of the cell, not least in the face of harmful challenges. However, an interesting role and extracellular Hsp70 (eHsp70), especially when it comes to immune and inflammatory responses, which, in addition, can play an important role in the elimination of malignant cells. In addition, involvement in General physiological adaptation to stress and protection against cell damage are also developing themes for eHsp70.

Extracellular Hsp70 and neuroprotection

The first proof of the existence of eHsp70 appeared in studies on giant AXONE squid, which showed that the temperature increase induces a number of heat shock proteins in glial sheath surrounding the axon,which were transferred to the axon. These data were soon reproduced in cultured embryonic cells of rats, and that it is important, at this stage, there was evidence of non-classical pathway of endocytosis, which is responsible for the release of Hsp70, while neither monensin or colchicine, an inhibitor of the classical pathway of secretion, could not block the secretion of Hsp70. After this publication, other articles provide examples of Hsp release glia and capture neurons in different systems of animal models, such as frogs, crayfish and rats. Support the role of glia as a source of eHsp70 people ensured the study of cultured cells in human glioblastoma. This study showed that under controlled conditions the cells release ~10 PG Hsp70 per million cells in the environment over a period of time of 24 hours. This release was increased in 2.5-5 times, when applied 20 min heat shock at the beginning of the time period. Importantly, this study also showed that the release of eHsp70 was higher than what can be detected by cell death. These data confirm initially put forward the hypothesis proposed by Tytell et al., that glial release of Hsp can be by supporting the functioning of the neuron during metabolic stress.

Evidence in vivo that eHsp70 has a neuroprotective function in acute when Resse, appeared in numerous studies. The study Tidwell et al. found that eHsp70 able to reduce the amount of cell death of motor neurons after axotomy when eHsp70 was made by gel sponge after axotomy. In the same study, the increased survival of sensory neurons in the ganglion dorsal root was also observed after administration of Hsp70, although this depended on a slightly higher doses of Hsp70 than motor neurons. In addition, it is shown that eHsp70 protects motor neurons otherwise destined to die during embryonic development of the chick, and protects motor neurons isolated from the spinal cord of the chick in the absence of trophic factors. The protective role of eHsp70 in vivo was also described light damage to the retina. In this study, Yu et al. after exposure to inducing damage to light was injected into the vitreous body of a solution of recombinant Hsp70 and Hsc70 in the dose, which was previously described as causing extensive degeneration of photoreceptors. Interestingly, the presence of a mixture of eHsp70 in the chamber vitreous of the right eye results in a significantly greater survival of photoreceptors of the retina. In addition, evaluation of capture-labeled fluorescein Hsc/Hsp70 shows that it is present in the retina after 6 hours after injection. It is shown that extracellular Hsp70 introduced intra who azalina, prevents the inevitable consequences of stress in rats, and recently described that entered administered intraperitoneally with recombinant human Hsp70 effective in increasing life expectancy, delaying the start of symptoms, preserving motor function and extending the survival of motor neurons in a model of amyotrophic lateral sclerosis mice. Additional work in vitro with the use of Hsp70 or a mixture of Hsc/Hsp70 in neurons, in addition, showed that eHsp70 may enhance the stability of the nerve cells to stress and to reduce polyglutamine toxicity and aggregation.

Extracellular Hsp70 and immunity

In addition to the role in cytoprotective found that associate with the cytoplasmic membrane and the free system eHsp70 play a role in immunity. Considering one of the main functions of Hsp70 as an intracellular chaperone proteins, perhaps it is not surprising that he may be involved in the binding of immunogenic peptides and help in their presentation through molecules major histocompatibility complex (MHC) class I. in Addition, it is shown that isolated from tumor eHsp70 acts as a chaperone for immunogenic peptides and selectively binds to antigen presenting cells (APC). After receptor-mediated endocytosis of the complex of Hsp70-peptide prezentuetsya on the MHC molecules of class I, resulting in citato the classical response of T cells. In addition, when acting as a chaperone for their own antigens Hsp70 is also able to bind microbial peptides and neetilirovannye CpG motifs in DNA of bacteria.

In addition to the role as antigen-presenting chaperone eHsp70 also involved in the stimulation of innate immunity. While it is shown that several types of cells releases Hsp70, also shows that eHsp70 binds to several receptors on different subpopulations of cells, including natural killer cells (NK), macrophages, monocytes and dendritic cells. Involved in recognition of eHsp70 receptors mainly include receptors pattern recognition (PR) and consist of multiple receptors from different families of receptors, such as toll-like receptors (TLR), scavenger receptors and lectins type C. After binding of the receptor eHsp70 can cause extensive cytokine response, including the release of proinflammatory cytokines, such as TNFα, IL-1β, IL-12, IL-6 and GM-CSF, a process that is triggered by the movement of NF-κB into the nucleus, suggesting the action of eHsp70 as a cytokine, which also leads to the suggestion to create eHsp70 the term chaperonin to better describe the unique features of eHsp70 as a chaperone and cytokine.

Most of the work in vivo on the role of eHsp70 in immunity conducted in rodent models. For example, increasing the concentration of eHsp70 in response to the shock is the first impact on the tail associated with a reduction in inflammation and a faster healing time after subcutaneous injection of E.Coli. In addition, the introduction of in vivo Hsp70 mouse accelerates delayed wound, property, probably because of phagocytosis by macrophages foreign substances in the wound.

There is not enough evidence immunomodulatory role of Hsp70 in humans, but research shows the relation with the increase of eHsp70 and prognosis/outcome of brain injury, although it also shows the opposite. However, it is also known that the concentration of eHsp70 decline in old age, suggesting age-related reduction of ability to perform a full response to stress, which, among other things, can cause increased morbidity and mortality observed during aging, although it remains very controversial.

The release of Hsp70

In addition to the data showing the transfer of eHsp70 between neighboring cells, such as model glia/axon, in several articles confirmed the presence of free eHsp70 in the bloodstream. To present in this compartment Hsp70 is a necessary release from the body/cells. As a rule, consider the two main ways of achieving this. One is the passive way in which the discovery of eHsp70 in peripheral blood flow is caused by the release of intracellular aggregate Hsp70 due Lissa or cell death. An alternative, or perhaps additional the but Hsp70 is actively released through a nonclassical pathway of endocytosis.

Suggest that Hsp70 together with other heat shock proteins are released only in pathological conditions, leading to necrotic death, but not when the programmed cell death. No doubt, severe trauma and pathological conditions, leading to necrosis, can lead to the release of Hsp70 in the bloodstream. This is convincingly confirmed and logically expected. However, recent studies have shown that Hsp70 can be released from intact cells by active mechanisms, and that the level of incentives determines the way of liberation. Convincing proof necroticism release of Hsp70 is also presented in the study induced by exercise release eHsp70 in the peripheral blood. Depending on the way the exercises (the higher the exercise, the greater the release) in peripheral blood flow can be defined substantially increase eHsp70 and it is important that unknown studies that reported a direct correlation between eHsp70 and markers of muscle injury. What eHsp70 may be released regardless of the damage of cells and tissues, among other things elegantly demonstrated Fleshner with co-workers, who showed that psychological stress, such as fear of the predator and the shock can call the to the stress-induced release of eHsp70, the process, which is assumed to depend on catecholamine transmission signal.

The way in which Hsp70 leaves the cell, it is still unclear, not least because of the fact that Hsp70 does not contain any classical peptide leader sequence, which may direct him to the secretion. In addition, since the classical secretion discussed above, suggest that there must be an alternative mechanism of secretion of eHsp70. Demonstrated that eHsp70 may be released in vesicles, characterized as actsoma, but also presented evidence that eHsp70 may be released as a free eHsp70, as in cellular systems and in vivo. Assume that the release of eHsp70 necessary lipid rafts, although this is subject to discussion. In addition, it is shown that functional lysosomal compartment required for the release of eHsp70 and that this release is accompanied by the presence of lysosomal protein markers on the cell surface, suggesting that secretion depends on the fusion of cytoplasmic and lysosomal membranes. Regardless of whether the release by eksasol or directly from lysosomes, it is interesting to note that, apparently, all methods of release involved some type of secretory MVB/late endosomal/Lisa analnogo compartment.

Because catecholamines via α1-adrenergic receptor can lead to the release of intracellular calcium and because assume that the same release of calcium causes the exocytosis of actcom, multivesicular cells and lysosomes, the modern hypothesis is that during periods of stress, increased noradrenaline, acting via α1-adrenergic receptors leads to release of calcium inside the cell and the subsequent release of Hsp70 in actsoma.

The bioactive agent according to the present invention

In one of the embodiments the present invention relates to the modulation of enzyme activity, where the specified enzyme interacts with BMP, through the application of bioactive means capable of increasing the intracellular concentration and/or activity of Hsp70.

Modulation of the enzymatic activity of the present invention can be obtained by providing one of the following classes of compounds and therapies that increase the intracellular concentration and/or activity of Hsp70:

- Hsp70, or a functional fragment or variant

- Inductors or conductor Hsp70

▪ Low molecular weight drugs, such as bioklima and arimoclomol

▪ Fluidization membrane, such as benzyl alcohol

▪ Sublethal thermal therapy (≤42°C) or hyperte the MIA

▪ Some medicines from the group of anti-inflammatory and anticancer drugs

▪ Cellular stress

▪ Reactive oxygen species (ROS)

▪ Epinephrine, norepinephrine

▪ UV radiation

▪ Radiation therapy

Thus, the bioactive agent of the present invention is any agent, reagent or a compound that increases the intracellular concentration and/or activity of Hsp70; and includes himself HSP70, or a functional fragment or variant and any inductor or conductor Hsp70 is known to the person skilled in the art, where the aforementioned bioactive agent capable of modulating the activity of an enzyme that interacts with BMP.

It follows that the bioactive agent may increase the intracellular concentration and/or activity of Hsp70 directly or indirectly.

In one of the embodiments the bioactive agent of the present invention is Hsp70, or a functional fragment or variant.

In another embodiment, the bioactive agent of the present invention is an inductor or coinduction Hsp70.

In one embodiment, the implementation of the bioactive agent according to the present invention contains a combination of Hsp70, or a functional fragment or variant and the inductor or conductor Hsp70.

Aspect according to the present invention is the provision of bioactive funds capable of increasing the intracellular concentration and/or activity of Hsp70 for use as a medicine.

An additional aspect of the present invention is the provision of bioactive means capable of increasing the intracellular concentration and/or activity of Hsp70 for use in the treatment of lysosomal storage diseases.

An additional aspect of the present invention is the provision of bioactive means capable of increasing the intracellular concentration and/or activity of Hsp70 for use as a medicament or for use in the treatment of lysosomal storage diseases.

In one embodiment, the implementation of the said treatment may be prophylactic, healing or soothing. In one particular embodiment, said treatment is prophylactic. In another embodiment, said treatment is healing. In an additional embodiment, said treatment is softening.

In one of the embodiments specified lysosomal disease accumulation selected from the group consisting of disease, Niemann-pick, Farber disease, Krabbe disease, Fabry disease, Gaucher disease, metachromatic leukodystrophy, sialidase and deficit sabotinov.

In a specific embodiment, implemented the program specified lysosomal disease accumulation is Niemann-pick disease type A or B. In another specific embodiment, the specified lysosomal disease accumulation is a disease Farber. In another specific embodiment, the specified lysosomal disease accumulation is Krabbe disease. In another specific embodiment, the specified lysosomal disease accumulation is the metachromatic leukodystrophy. In another specific embodiment, the specified lysosomal disease accumulation is sialidosis. In another specific embodiment, the specified lysosomal disease accumulation is Fabry disease. In another specific embodiment, the specified lysosomal disease accumulation is Gaucher disease. In another specific embodiment, the specified lysosomal disease accumulation is deficient sabotinov.

An aspect of the present invention is the provision of bioactive means capable of increasing the intracellular concentration and/or activity of Hsp70 for use in the treatment of lysosomal storage diseases, where these lysosomal storage disorders are one, such as two, for example three, such as four, for example, five, such as six, for example, a family of disorders selected from the group consisting of disease, Niemann-pick, Farber disease, Krabbe disease, illness Fab and, Gaucher's disease, metachromatic leukodystrophy, sialidase and deficit sabotinov.

It follows that the bioactive agent according to the present invention can be used for the treatment of a subgroup of lysosomal storage diseases selected from the group consisting of disease, Niemann-pick, Farber disease, Krabbe disease, Fabry disease, Gaucher disease, metachromatic leukodystrophy, sialidase and deficit sabotinov.

In one particular embodiment, the bioactive agent of the present invention can be used to treat the disease Niemann-pick type A and Β and Farber disease.

In one embodiment, the implementation of the bioactive agent according to the present invention contains a combination of Hsp70, or a functional fragment or variant and a substance that increases the interaction between Hsp70 and BMP.

Another additional aspect of the present invention is the provision of bio-active means capable of increasing the intracellular concentration and/or activity of Hsp70 for the manufacture of a medicinal product for the treatment of lysosomal storage diseases.

The bioactive agent - Hsp70, or a functional fragment or variant

In one of the embodiments the present invention relates to the modulation of enzyme activity, where the specified enzyme vzaimode is istue with BMP, through the use of Hsp70, or a functional fragment or variant.

An aspect of the present invention is the provision of Hsp70, or a functional fragment or variant for use as a medicine.

An additional aspect of the present invention is the provision of Hsp70, or a functional fragment or variant for use in the treatment of lysosomal storage diseases.

Another additional aspect of the present invention is the provision of application Hsp70, or a functional fragment or variant for the manufacture of a medicinal product for the treatment of lysosomal storage diseases.

In one of the embodiments specified lysosomal disease accumulation selected from the group consisting of disease, Niemann-pick, Farber disease, Krabbe disease, Fabry disease, Gaucher disease, metachromatic leukodystrophy, sialidase and deficit sabotinov.

It should be understood that Hsp70, or a functional fragment or variant of the present invention may be any natural or synthetic product, and can be obtained by any conventional method known to the expert in this field.

In one embodiment, the implementation of Hsp70, or a functional fragment or variant is derived from natural IP the student. Specified natural source can be any plant, animal, or bacteria that Express, or they can induce the expression of Hsp70 in a form suitable for introduction to the needy in the individual.

However, in the preferred embodiment, Hsp70, or a functional fragment or variant are synthetically. Hence, Hsp70, or a functional fragment or variant can in one of the preferred embodiments can be a recombinant protein, thus, the conventional method is marked as rHsp70.

Hsp70 of the present invention, synthetic or natural, may be a sequence derived from any suitable species of plants, animals or bacteria. In one of the embodiments specified rHsp70 is obtained from a mammal. Specified mammal can be selected from the group consisting of human (homo sapiens), mouse (mus musculus), cow, dog, rat, ferret, pig, sheep and monkeys. In another embodiment, the specified rHsp70 derived from bacteria.

Hsp70 partially characterized as having a very high degree of interspecific conservative sequence, thus possibly allowing Hsp70 derived from the same species, be applied to other types without calling damaging immune resp is the same.

In one specific embodiment, the specified rHsp70 has a sequence derived from the human Hsp70.

In one specific embodiment, the specified rHsp70 has a sequence derived from more than one species. Specified Hsp70, or a functional fragment or variant may thus, in one embodiment, the implementation to be a Chimera.

Recombinant protein is a protein derived from recombinant DNA. Recombinant DNA is a form of DNA that does not exist in nature, which is created by combining DNA sequences that do not normally exist together. In terms of genetic modification, recombinant DNA get by adding the relevant DNA into an existing DNA of an organism, such as bacterial plasmids, encoding different traits for a specific purpose. This is different from genetic recombination that this is not the result of processes in the cell, and designed by human beings.

In one embodiment, the implementation of Hsp70 according to the present invention has 100% homology with Hsp70 protein of the wild type. In another embodiment, Hsp70 according to the present invention has less than 100% homology with Hsp70 protein wild type, such as from about 99.9 to 95% homology, for example, from 95 to 90% homology, such as from 9 to 85% homology, for example, from 85 to 80% homology, such as from 80 to 75% homology, for example, from 75 to 60% homology with the wild-type protein. Regardless of the degree of homology with any variant of Hsp70, preserving its ability to modulate the enzymatic activity of the enzyme that binds to BMP, is in the scope of this invention.

In one of the embodiments the bioactive agent is Hsp70. In one of the embodiments specified is full-length Hsp70 Hsp70.

Also, the embodiment is the provision of a functional fragment or variant of Hsp70. As defined herein, a functional fragment or variant is any fragment of Hsp70 with the desired function, which in terms of the present invention is the ability to modulate the enzymatic activity of the enzyme, where the specified enzyme interacts with BMP.

In one of the embodiments the bioactive agent is a functional fragment or variant of Hsp70.

In one of the embodiments the bioactive agent is a functional fragment or variant of Hsp70 in which Hsp70 modify through deletions (deletions), add (additions) or replacement (replacement) Hsp70 wild-type.

Protein Hsp70 wild-type and has a total length of 641 amino acid. Assume that a fragment of Hsp70 is one of the embodiments includes any fragment with a total length of less than wild-type protein in 641 amino acid, such as less than 625 amino acids, for example, less than 600 amino acids, such as less than 575 amino acids, for example, less than 550 amino acids, such as less than 525 amino acids, for example, less than 500 amino acids, such as less than 475 amino acids, for example, less than 450 amino acids, such as less than 425 amino acids, for example, less than 400 amino acids, such as less than 375 amino acids, for example, less than 350 amino acids, such as less than 325 amino acids, for example less than 300 amino acids, such as less than 275 amino acids, for example, less than 250 amino acids, such as less than 225 amino acids, for example, less than 200 amino acids, such as less than 175 amino acids, for example, less than 150 amino acids, such as less than 125 amino acids, for example, less than 100 amino acids, such as less than 75 amino acids, for example, less than 50 amino acids, such as less than 25 amino acids.

Protein Hsp70 wild-type and has a total length of 641 amino acid. Assume that a fragment of Hsp70 in one of the embodiments includes any fragment with a total length of more than 10 amino acids, such as more than 25 amino acids, for example, more than 50 amino acids, such as more than 75 amino acids, for example, more than 100 amino acids, such as more than 125 amino acids, for example, more than 150 amino is the slot, such as more than 175 amino acids, for example, more than 200 amino acids, such as more than 225 amino acids, for example, more than 250 amino acids, such as more than 275 amino acids, for example, more than 300 amino acids, such as more than 325 amino acids, for example, more than 350 amino acids, such as more than 375 amino acids, for example, more than 400 amino acids, such as more than 425 amino acids, for example, more than 450 amino acids, such as more than 475 amino acids, for example, more than 500 amino acids, such as more than 525 amino acids, for example, more than 550 amino acids, such as more than 575 amino acids, for example, more than 600 amino acids, such as more than 625 amino acids.

It follows that the total length of the Hsp70 fragment of the present invention may in one of the embodiments to be in the range from 5 to 25 amino acids, such as from 25 to 50 amino acids, for example, from 50 to 75 amino acids, such as from 75 to 100 amino acids, for example, from 100 to 125 amino acids, such as from 125 to 150 amino acids, for example, from 150 to 175 amino acids, such as 175 to 200 amino acids, for example, from 200 to 225 amino acids, such as from 225 to 250 amino acids, for example, from 250 to 275 amino acids, such as from 275 to 300 amino acids, for example, from 300 to 325 amino acids, such as from 325 to 350 amino acids, for example, from 350 to 375 amino acids, such as from 375 to 400 aminoxy the lot, for example, from 400 to 425 amino acids, such as from 425 to 450 amino acids, for example, from 450 to 475 amino acids, such as from 475 to 500 amino acids, for example, from 500 to 525 amino acids, such as from about 525 to 550 amino acids, for example, from 550 to 575 amino acids, such as from 575 to 600 amino acids, for example, from 600 to 625 amino acids, such as from 625 to 640 amino acids.

In one specific embodiment, the fragment or variant of Hsp70 contains all or part of the ATPase domain of Hsp70. It follows that the fragment or variant of Hsp70 according to the present invention in one of the embodiments contains all or part of the amino acid at position 30 to 382.

In another specific embodiment, the fragment or variant of Hsp70 contains tryptophan at position 90 the amino acid ATPase domain of Hsp70.

A fragment of Hsp70 may be a truncated version of the protein of wild type, which means that it is a shorter version. The fragment may be truncated by shortening the protein with aminocore and carboxyl end, or it may be truncated by deletion of one or more internal regions of the protein of any size.

The fragment or variant of Hsp70 may in one of the embodiments to have 100% homology with the wild-type protein. In another embodiment, the fragment or variant of Hsp70 may also be a variant of Hsp70 to the th has less than 100% homology with the wild-type protein, such as from about 99.9 to 95% homology, for example, from 95 to 90% homology, such as from 90 to 85% homology, for example, from 85 to 80% homology, such as from 80 to 75% homology, for example, from 75 to 60% homology with the protein of the wild type.

It should be understood that any fragment or variant of Hsp70, which retains its ability to modulate the activity of the lysosomal enzyme is in the scope of this invention.

It should be understood that any fragment or variant of Hsp70, which retains its ability to interact with the BMP is in the scope of this invention.

It should be understood that the specific quantitative effect of a functional fragment or variant may differ from the effect of a full-sized molecules. In some cases, a functional fragment or variant may in fact be more effective than a full-sized molecule. In addition, the use of fragments instead of full-sized molecules may have an advantage due to the smaller size of the fragments.

In one embodiment, the functional fragment or variant of Hsp70 may be a variant of Hsp70 in which one or more amino acids are replaced. Specified replacement (s) may be equivalent or conservative substitution (replacement), or equivalent or non-conservative substitution (replacement).

One option is to implement replaced from 0.1 to 1% of the amino acid residues of the wild-type Hsp70, such as from 1 to 2% of amino acid residues, for example, from 2 to 3% of amino acid residues, such as from 3 to 4% of amino acid residues, for example, from 4 to 5% of amino acid residues, such as from 5 to 10% of amino acid residues, for example, from 10 to 15% of amino acid residues, such as from 15 to 20% of amino acid residues, for example from 20 to 30% of amino acid residues, such as from 30 to 40% of amino acid residues, for example, from 40 to 50% of amino acid residues, such as from 50 to 60% of amino acid residues, for example, from 60 to 70% of amino acid residues, such as from 70 to 80% of amino acid residues, for example, from 80 to 90% of amino acid residues, such as from 90 to 100% of amino acid residues.

In one of the embodiments substituted from 1 to 5 amino acid residues of the wild-type Hsp70, such as from 5 to 10 amino acid residues, for example, from 10 to 15 amino acid residues, such as from 15 to 20 amino acid residues, for example from 20 to 30 amino acid residues, such as from 30 to 40 amino acid residues, for example, from 40 to 50 amino acid residues, such as from 50 to 75 amino acid residues, for example, from 75 to 100 amino acid residues, such as from 100 to 150 amino acid residues, for example, from 150 to 200 amino acid residues, such as from 200 to 300 amino acid residues, for example, from 300 to 400 amino acid residues, such as the t 400 to 500 amino acid residues.

In one embodiment, the functional fragment or variant of Hsp70 is fused protein. In one embodiment, the implementation of the specified functional fragment or variant of Hsp70 fused with the label.

The advantages of using Hsp70, or a functional fragment or variant

As described earlier in this document, there is no cure lysosomal storage diseases, and treatment is mostly symptomatic, except for replacement therapy enzyme (ERT) for Gaucher disease and Fabry disease. As indicated, ERT is a very expensive form of therapy, which is effective for only one disease.

As is known to the authors, so far have not been successful attempts to provide ERT for other lysosomal storage diseases associated with the accumulation of lipids, thus, currently remains a large unmet need for effective and specific treatment for LSD data.

Introduction to the needy in this individual Hsp70, or a functional fragment or variant has a number of advantages compared with conventional methods of treating lysosomal storage diseases.

First, the preparation of recombinant protein, such as rHsp70 or its functional fragment or variant with application of modern who's technology is simple and direct way of obtaining a significant quantity of rHsp70 or its functional fragment or variant. Conventional methods of production of recombinant enzymes are well known specialist in this field.

In addition, the preparation of recombinant protein, such as rHsp70, its functional fragment or variant, is a cheap way to get a significant quantity of rHsp70 or its functional fragment or variant. In comparison with obtaining enzymes for ERT, the cost is greatly reduced.

In addition, the use of Hsp70, or a functional fragment or variant can be used to treat more than one specific lysosomal storage disorders. This also applies to inductors and conductorof Hsp70 according to the present invention. In fact bioactive agent capable of increasing the intracellular concentration and/or activity of Hsp70, can be used to treat any lysosomal storage disorders, which can be reversed through the modulation of enzymatic activity involved a defective enzyme, where the specified enzyme interacts with BMP.

In the end, because Hsp70 is present endogenous molecule, i.e. a molecule that originates from an organism, tissue or cell, it is expected that the introduction of Hsp70, or a functional fragment or variant does not cause or causes very low immune response. This is the main advantage of the LC. facilitates treatment and reduces the potential side effects when administered to an individual.

Ectopic expression of Hsp70

In one embodiment, the implementation of Hsp70, or a functional fragment or variant may be expressed by the vector. Thus, in one of the embodiments the invention relates to a vector, codereuse Hsp70, or a functional fragment or variant.

In one of the embodiments of the present invention Hsp70, or a functional fragment or variant can be entered in need of this individual in the form of a vector.

The vector used for ekspressirovali Hsp70, or a functional fragment or variant can be selected from the group consisting of viral vectors (retrovirus and adenovirus) or non-viral vectors (plasmids, cosmid, bacteriophages).

In one embodiment, the implementation of the specified vector contains one or more stations start replication, marker selection, and one or more recognition sites of the restriction endonucleases. In another embodiment, the specified vector is functionally linked to regulatory sequences that control transcription of the specified Hsp70, or a functional fragment or variant in a suitable cell host.

In one of the embodiments the present and the finding relates to a method for Hsp70, or a functional fragment or variant, as described in this document; the specified method including the steps of providing a vector encoding specified Hsp70, or a functional fragment or variant, and ekspressirovali specified vector in vitro or in vivo in a suitable organism, the host, and thus, getting Hsp70, or a functional fragment or variant.

The invention additionally relates to the selection of recombinant or transgenic cell host containing the vector encoding Hsp70, or a functional fragment or variant of the present invention.

The invention also relates to a method for obtaining recombinant or transgenic host cell comprising the steps of providing a vector encoding Hsp70, or a functional fragment or variant of the entry into force of this vector into the specified recombinant or transgenic cell host and also not necessarily, ekspressirovali specified vector in the indicated recombinant or transgenic cell host with obtaining, thus, recombinant or transgenic host cell producing the specified Hsp70, or a functional fragment or variant.

In another embodiment, the present invention relates to transgenic organism of a mammal containing the above-described cell-master.

In an additional embodiment, rashenny the body of a mammal, containing recombinant or transgenic cell host according to the present invention is not owned by the person.

Transgenic cell host can be selected from the group consisting of a host cell of a mammal, plant, bacteria, yeast or fungi.

To improve the delivery of DNA into the cell you want to protect DNA from damage and to facilitate its entrance into the cell. Created lipoplex and polyplex, have the ability to protect DNA from undesirable degradation during the transfection. Plasmid DNA may be coated with lipids in an organized structure, similar to the micelle or liposome. When organized structure forms a complex with DNA, it is called lipoplex. For forming liposomes can be used three types of lipids: anionic (negatively charged), neutral, or cationic (positively charged). Complexes of polymers with DNA called polyplexes. Most of polyplexes consists of cationic polymers, and obtaining them is governed by ionic interactions.

In one embodiment, the implementation of a vector containing Hsp70, or a functional fragment or variant can be used for gene therapy. Gene therapy is the introduction of the gene into the cells and tissues of an individual for the treatment of diseases, such as hereditary disease in which the remote mutant allele replace functional.

In another embodiment, Hsp70, or a functional fragment or variant can be entered as bare DNA. This is the simplest form of non-viral transfection. Shipping bare DNA can be performed by electroporation, sonoporation or "gene gun"that shoots covered with gold particles with DNA in the cell with gas under high pressure.

The bioactive agent - inductors and conductor Hsp70

In one of the embodiments the present invention relates to the modulation of enzyme activity, where the specified enzyme interacts with the BMP through the use of inductors or conductorof Hsp70.

Inducer of Hsp70 is the connection, which itself may enhance the expression of the Hsp70 gene and protein expression without concomitant stress.

Coinduction Hsp70 is a connection that can not enhance the expression of the Hsp70 gene and protein expression without concomitant (moderate) stress, but stress-induced increased levels of Hsp70 is also enlarged or increased their presence.

An aspect of the present invention is the provision of an inductor or conductor Hsp70 for use as a medicine.

An additional aspect of the present invention is the provision of an inductor or conductor Hsp70 DL the application in the treatment of lysosomal storage diseases.

Another additional aspect of the present invention is to provide use of an inductor or conductor Hsp70 for the manufacture of a medicinal product for the treatment of lysosomal storage diseases.

In one of the embodiments specified lysosomal disease accumulation selected from the group consisting of disease, Niemann-pick, Farber disease, Krabbe disease, Fabry disease, Gaucher disease, metachromatic leukodystrophy, sialidase and deficit sabotinov.

In a specific embodiment, the specified lysosomal disease accumulation is Niemann-pick disease type A or B. In another specific embodiment, the specified lysosomal disease accumulation is a disease Farber. In another specific embodiment, the specified lysosomal disease accumulation is Krabbe disease. In another specific embodiment, the specified lysosomal disease accumulation is the metachromatic leukodystrophy. In another specific embodiment, the specified lysosomal disease accumulation is sialidosis. In another specific embodiment, the specified lysosomal disease accumulation is Fabry disease. In another specific embodiment, the specified lysosomal disease accumulation is Gaucher disease. In yet another spiral is Mr. embodiment, the specified lysosomal disease accumulation is a deficit saposin.

In one of the embodiments the bioactive agent of the present invention is an inductor or coinduction Hsp70. In a particular embodiment, the bioactive agent of the present invention is an inducer of Hsp70. In another specific embodiment, the bioactive agent of the present invention is conductora Hsp70.

Low molecular weight drugs - derivatives of hydroxylamine

In one of the embodiments the bioactive agent of the present invention is conductora Hsp70. In an additional embodiment, the specified conductor Hsp70 is a small molecule drug.

In a specific embodiment, conductor Hsp70 of the present invention is a derivative of hydroxylamine. Specified derivative of hydroxylamine in an additional embodiment, is chosen from the group of bioclave (BRLP-42), arimoclomol (BRX-220), BRX-345 and BGP-15.

In a specific embodiment, the specified derivative of hydroxylamine is arimoclomol (BRX-220).

Bioklima ([2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine-carboxymethyl-chloride maleate) is a non-toxic compound, which was originally developed for the treatment of diabetic complications such as neuropathy. It is shown that bioklima improves survived the tion of cells in the experimental conditions of stress, partly by increasing intracellular heat shock proteins (HSP), including Hsp70, through the activation of HSF-1. It is shown that bioklima has the ability coinductive Hsp70 in the absence of nevernude proteins and that bioklima interacts and enhances the flow of negatively charged membrane lipids. BRX-345 is a structural analogue of bioclave with, to some extent, less ability to induce HSP.

Arimoclomol (BRX-220) is an analogue of bioclave, which also interacts and enhances the response to heat shock. Currently arimoclomol undergoing clinical trials for the treatment of ALS (amyotrophic lateral sclerosis); progressive neurodegenerative disorders. Arimoclomol owned by CytRx Corporation.

Thus, an aspect of the present invention is the provision of conductor Hsp70 derived hydroxylamine for use in the treatment of lysosomal storage diseases.

Another additional aspect of the present invention is the provision of applications conductor Hsp70 hydroxylamine derivative for the manufacture of a medicinal product for the treatment of lysosomal storage diseases.

In one of the embodiments specified lysosomal disease accumulation selected from the group consisting of disease, Niemann-pick, illness and Farber, Krabbe disease, Fabry disease, Gaucher disease, metachromatic leukodystrophy, sialidase and deficit sabotinov.

Membrane fluidization

In one of the embodiments the bioactive agent of the present invention is an inducer of Hsp70. In an additional embodiment, the specified inducer of Hsp70 membrane is fluidization.

Treatment membrane fluidization also can be described as lipid therapy.

In a specific embodiment, the Hsp70 inducer of the present invention is a membrane fluidization selected from the group of benzyl alcohol, heptanol, AL721, docosahexaenoic acid, aliphatic alcohols, olejowego alcohol, dimethylaminoethanol, A2C, farnesol and anesthetics, such as lidocaine, ropivacaine, bupivacaine and mepivacaine, as well as other well-known specialist in this field.

In addition to the denaturation part of a cell's proteins during heat shock (proteotoxicity) it is also assumed that the change in the fluidity of cell membranes is the sensor that triggers the response to heat shock and induces HSP. In fact, chemically induced rearrangement of the membrane, similar to the induced heat to the liquefaction of the cytoplasmic membrane, is able to activate HSP without causing denaturation of the protein.

The fluidity of member the us refers to the viscosity of the lipid bilayer of the cell membrane. Membrane phospholipids combine fatty acids of different lengths and saturation.

Membrane fluidization act through the inclusion between membrane lipids, thus inducyruya the effect of de-serializing by weakening the van der Waals interactions between the acyl chains of lipids.

Thus, an aspect of the present invention is the provision of a membrane fluidizer selected from the group of benzyl alcohol, heptanol, AL721, docosahexaenoic acid, aliphatic alcohols, olejowego alcohol, dimethylaminoethanol, A2C, farnesol and anesthetics, such as lidocaine, ropivacaine, bupivacaine and mepivacaine, as well as other well-known specialist in this field, for use in the treatment of lysosomal storage diseases.

Another additional aspect of the present invention is the use of membrane fluidizer selected from the group of benzyl alcohol, heptanol, AL721, docosahexaenoic acid, aliphatic alcohols, olejowego alcohol, dimethylaminoethanol, A2C, farnesol and anesthetics, such as lidocaine, ropivacaine, bupivacaine and mepivacaine, as well as other well-known specialist in this field, for the manufacture of a medicinal product for the treatment of lysosomal storage diseases.

In one of the embodiments listed the th lysosomal disease accumulation selected from the group consisting of disease, Niemann-pick, Farber disease, Krabbe disease, Fabry disease, Gaucher disease, metachromatic leukodystrophy, sialidase and deficit sabotinov.

Other methods of induction of Hsp70

Provides that any method of induction of Hsp70 expression included in the scope of the present invention, some of them are described later in this document.

The increased temperature of the individual is a potential inducer of HSP, including Hsp70, and such sublethal heat therapy is an aspect of the present invention. In one embodiment, the implementation of sublethal heat therapy include increased temperature of the individual to an internal temperature of approximately 38°C, such as about 39°C, for example, approximately 40°C, such as about 41°C, for example, approximately 42°C, such as about 43°C.

Thus, an aspect of the present invention is the provision of sublethal heat treatment for use in the treatment of lysosomal storage diseases.

Psychological stress, such as fear of predator and shock, can cause stress-induced release of eHsp70, process, presumably dependent catecholamine transmission signal. In addition, adrenaline and noradrenaline can cause the release of Hsp70.

It is shown that the following compounds of the Indus is zeroout (or conducerea) HSP, including Hsp70: membrane-interacting compound alkyldithiophosphate edelfosine (ET-18-OCH3 or 1-octadecyl-2-methyl-rat-glycero-3-phosphocholine); anti-inflammatory drugs, including cyclo-oxygenase inhibitors 1/2, such as celecoxib and rofecoksib, as well as NSAIDs, such as acetylsalicylic acid, sodium salicylate and indomethacin; prostaglandins PGA1, PGj2 and 2-cyclopenten-1-he; agonists gamma receptor-activated proliferation peroxisome; interacting with tubulin anti-cancer tools, including vincristine and paclitaxel; an insulin sensitizer pioglitazone; antitumor tools such as carboplatin, doxorubicin, fludarabine, ifosfamide and cytarabine; inhibitors of Hsp90 geldanamycin, 17-AAG, 17-DMAG, radicial, herbimycin-A and arachidonic acid; the proteosome inhibitors MG132 and lactacystin; inhibitors of serine proteases DCIC, TLCK and TPCK; antiulcer drug geranylgeranylation (GGA), rebamipide, carbenoxolone and polaprezinc (zinc L-carnosine); heavy metals (zinc and tin); anti-inflammatory drug dexamethasone; cocaine; nicotine; alcohol; agonists alpha-adrenergic receptors; cyclopentenone prostanoids; and vegetable medicines paeoniflorin, glycyrrhizin, celastrol, dihydrocholesterol, dihydrocholesterol diacetate and curcumin.

Thus the om, an aspect of the present invention is the provision of compounds selected from the group edelfosine (ET-18-OCH3 or 1-octadecyl-2-methyl-rat-glycero-3-phosphocholine), celecoxib, rofecoksib, acetylsalicylic acid, sodium salicylate, indomethacin, PGA1, PGj2, 2-cyclopenten-1-he agonists gamma receptor-activated proliferation peroxisome, vincristine, paclitaxel, pioglitazone, carboplatin, doxorubicin, fludarabine, ifosfamide, cytarabine, geldanamycin, 17-AAG, 17-DMAG, radicial, herbimycin-A, arachidonic acid, MG132, lactacystin, DCIC, TLCK, TPCK, geranylgeranylation (GGA), rebamipide, carbenoxolone, polaprezinc (zinc L-carnosine), dexamethasone, cocaine, nicotine, alcohol, agonists alpha-adrenergic receptors, cyclopentenone prostanoids, paeoniflorin, glycyrrhizin, celastrol, dihydrocholesterol, dihydrocholesterol diacetate and curcumin, as well as other HSP inducers known to the person skilled in the art, for use in the treatment of lysosomal storage diseases.

The pharmaceutical composition according to the present invention

The present invention relates to the modulation of enzyme activity, where the specified enzyme interacts with BMP, through the application of bioactive means to increase the concentration and/or activity of Hsp70, thus benefiting patients suffering from lysosomal storage diseases.

<> Because you can enter the bioactive agent according to the present invention as the raw chemical, it is preferable to present it as a pharmaceutical composition. Thus, the present invention additionally relates to pharmaceutical compositions for medical use, which contains the bioactive agent according to the present invention or its pharmaceutically acceptable salt as described herein and a pharmaceutically acceptable carrier.

An aspect of the present invention is the provision of compositions, such as pharmaceutical composition comprising a bioactive agent specified in the present document, which you can enter the needy in the individual.

In one of the embodiments the invention relates to compositions containing bioactive agent of the present invention. The composition, as described herein, in one of the embodiments can be combined with a physiologically acceptable carrier. The composition, as described herein, in one of the embodiments can be combined with a pharmaceutically acceptable carrier.

Pharmaceutical compositions containing the bioactive agent of the present invention, can be obtained by using common methods, such as opisanoj Remington: The Science and Practice of Pharmacy 1995, edited by E.W. Martin, Mack Publishing Company, 19th edition, Easton, Pa.

Bioactive means of the present invention can be for parenteral administration and can be represented as a single dose in ampoules, pre-typed syringes, small volume injections or in packages of drugs for multiple reception with the addition of preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous excipients, carriers, diluents or solvents, including aqueous solutions of mineral salts or other water soluble molecules, propylene glycol, polyethylene glycol, vegetable oils, animal oils, synthetic oils, and injectable organic esters, and can contain excipients, such as preservatives, moisturizing, emulsifying or suspendida, stabilizing and/or dispersing tools, dyes, buffers, thickeners, soljubilizatory etc. Alternative active ingredient may be a powder, obtained by aseptic isolation from solid or by lyophilization from the solution for preparation before use with a suitable vehicle, e.g. sterile, pyrogen-free water.

Pharmaceutically acceptable salt supplements, if you can get them, also included in the scope of nastoyascheevremya as specific hydrated forms of the salt. These salts are those which are acceptable in the field of pharmaceutical applications. Thus, this means that salt retains the biological activity of the parent compound, and salt has no adverse or harmful effects when applied and used in the treatment of diseases.

Pharmaceutically acceptable salts get accepted way. If the original connection is the basis, it is treated with an excess of an organic or inorganic acid in a suitable solvent. If the original connection is acid, it is treated with an inorganic or organic base in a suitable solvent.

You can apply any suitable composition of the bioactive means of the present invention, well-known specialist in this field.

In one embodiment, the implementation of Hsp70, or a functional fragment or variant consists in the biodegradable microsphere, such as a liposome.

Introduction

You can apply any suitable route of administration for delivery to a mammal, preferably a human, an effective amount of bioactive means of the present invention, where the specified bioactive agent may be Hsp70, or a functional fragment or variant.

Introduction to the needy in the individual bioactivities or pharmaceutical compositions can occur through three main shipping way: 1) local (drawing on the surface of the body, such as skin or mucous membranes); 2) enteral (via gastrointestinal or digestive tract) and 3) parenteral (in other ways than the gastrointestinal or digestive tract).

Local introduction includes cutaneous (applied to the skin), inhalation, enema, eye drops (conjunctiva), ear drops, intranasal path and vaginal administration.

Enteral introduction is any form of introduction, which involves any part of the gastrointestinal tract and includes oral administration (by mouth, for example, tablets, capsules, or drops), vnutriuretralnami (for example, suppositories or enema) introduction, in addition to gastric or duodenal probe for feeding.

Parenteral delivery, such as injection or infusion is effective for delivery of bioactive tools to target or the introduction of drugs into the bloodstream and includes intravenous (into a vein), intraarterial (into an artery), intramuscular (into the muscle), intracardiac (into the heart), subcutaneous (under the skin), intraosseous (in bone marrow), intradermal (into the skin), intrathecal or intraspinal (in the spinal canal), intraperitoneally (in the abdominal cavity), transdermal (diffusion through the intact skin), transmucosal (diffusion through a mucous membrane, for example, insufflate (inhalation), sublingual the second, buccal and vaginal suppositories), inhalation, epidural (epidural space) and the introduction into the vitreous body in the eye). Sublingual introduction (under the tongue) is also a form of parenteral administration, whereby the bioactive funds diffuse into the bloodstream through the tissue of the mucous membrane under the tongue. The bioactive agent according to the present invention it is possible to enter any parenteral delivery and, preferably, any of the above.

Parenteral delivery has the advantage of allowing you to avoid degradation in the gastrointestinal tract associated with enteral delivery.

Parenteral delivery has the additional advantage of avoiding presystemic metabolism associated with enteral delivery because it allows the compounds to be absorbed directly into the systemic blood flow.

Presystemic metabolism is a phenomenon of drug metabolism, in which the concentration of the drug is greatly reduced before it reaches the systemic blood flow. This is part of the medicinal product, lost in the absorption process, which usually refers to the liver and gut wall.

After swallowing medicines it is absorbed by the digestive system is the first and enters the portal system of the liver, passes through the portal vein to the liver before it reaches the rest of the body. The liver metabolizes many drugs, sometimes to such an extent that only a small amount of active drug released from the liver to the rest of the circulatory system. Thus, this first passage through the liver significantly reduces the bioavailability of the drug.

Four primary systems that influence the effect of the first pass of the medicinal product, are the enzymes of the lumen of the gastrointestinal tract, enzymes of the intestine, bacterial enzymes and liver enzymes.

Suitable dosage forms for such an introduction can be obtained by using common methods. Suitable dosage forms for administration by inhalation, such as aerosol composition or dosing inhaler, can be obtained by using common methods.

In one embodiment, the implementation of a specific method for introducing a bioactive means of the present invention is parenteral administration.

In one embodiment, the implementation of a particular method of parenteral administration of bioactive means of the present invention is intravenous, subcutaneous, intramuscular, intraarterial, subcutaneous or intraperitoneally injection.

In the nome of options for the implementation of a particular method of parenteral administration of bioactive means of the present invention is by inhalation.

In one embodiment, the implementation of a particular method of parenteral administration of bioactive means of the present invention is intravenous infusion.

Intravenous infusion according to the present invention in one of the embodiments may occur during the period of time from 10 minutes to 20 minutes, such as from 20 to 30 minutes, for example, from 30 to 40 minutes, such as from 40 to 50 minutes, for example, from 50 to 60 minutes, such as from 60 to 90 minutes, for example, from 90 to 120 minutes, such as from 2 hours to 3 hours, for example, from 3 to 4 hours, such as from 4 to 5 hours, for example, from 5 up to 6 hours, such as from 6 to 7 hours, for example, from 7 to 8 hours.

In a specific embodiment, the method of parenteral administration of bioactive means of the present invention is transmucosally delivery. Specified transmucosally shipping is in one of the embodiments sublingual delivery, in another embodiment, the specified transmucosally shipping is buccal delivery and in yet another embodiment, the specified transmucosally shipping is insufflate or intranasal delivery.

Dosage forms include tablets, lozenges, colloidal solutions, suspensions, solutions, capsules, creams, ointments, emulsions, gels, lotions, pastes, aerosols, or other forms known is fair in this field.

The effective dose of the applied active ingredient may vary depending on the specific applied compositions, method of administration, the treated condition and the severity of the treated condition. Such a dose can easily install specialist in this field.

In one of the embodiments the bioactive agent of the present invention is administered in a daily dose of from about 1 micrograms to about 100 milligrams per kilogram of animal body weight, giving as a single daily dose or as a divided dose or in the form of slow release. The dosage can be set in this range or even outside this range for optimal therapeutic response.

In one of the embodiments the bioactive agent of the present invention is administered in a dose from about 1 μg to about 10 μg per kg body weight, such as from about 10 μg to about 50 μg per kg of body weight, for example, from about 50 μg to about 100 μg per kg body weight, such as from about 100 μg to about 250 μg per kg of body weight, for example, from about 250 μg to about 500 μg per kg body weight, such as from about 500 μg to about 750 μg per kg of body weight for example, when listello from 750 μg to about 1000 μg per kg of body weight, such as from about 1 mg to about 10 mg per kg of body weight, for example, from about 10 mg to about 50 mg per kg of body weight, such as from about 50 mg to about 100 mg per kg of body weight.

This dose can be entered at certain time intervals and can be expressed in mg per kg of body weight per unit time. The specified unit of time in one of the embodiments may be minute, such as per hour, for example, a day, such as a week.

Combined treatment

An aspect of the present invention is the provision of bioactive means capable of increasing the intracellular concentration and/or activity of Hsp70 for use in the treatment of lysosomal storage diseases in combination with other treatment methods.

In one aspect the present invention relates to a method for the treatment of lysosomal storage disorders, including the introduction of bioactive means of the present invention in combination with at least one other treatment method.

Thus, in one embodiment, the implementation of the bioactive agent according to the present invention is administered to the needy in this individual, in combination with at least one other treatment method, such as conventional or known methods of treatment LSD.

It should be understood that bioact is the main means of the present invention is Hsp70 or its functional fragment, or option, or inducer or coinduction Hsp70.

Applying more than one method of treatment in combination may occur simultaneously or sequentially. Simultaneous introduction may contain two compounds in the same composition or may contain different compositions, or may be of one composition and one way of treatment, essentially carried out at the same time. Sequential introduction means that used more than one method of treatment at different points in time, such as the application of one method of treatment first and then the application of the second method of treatment. For optimum effect, the person skilled in the art can determine the time interval for applying more than one method of treatment, and he may in one of the embodiments can be from 30 minutes to 72 hours.

Treatment in the form of chemical compounds can be used together or separately, each in its most effective dose. Introduction more than one connection can have a synergistic effect, thus effectively reducing the required dose of the drug.

In one of the embodiments the bioactive agent of the present invention is administered to the needy in this individual in combination with a substitution enzyme therapy (ERT). Specified ERT is one of the embodiments can be selected from the group consisting of Cerezyme (Cerezyme®) (imiglucerase for injection), miglustat, fabrazyme (Fabrazyme®) (agalsidase beta) and replagal (agalsidase alpha).

In one of the embodiments the bioactive agent of the present invention is administered to an individual with Gaucher disease in combination with terazima (Cerezyme®) (imiglucerase for injection) or miglustat.

In another embodiment, the bioactive agent of the present invention is administered to an individual with Fabry disease in combination with fabrazyme (Fabrazyme®) (agalsidase beta) or replagal (agalsidase alpha).

In another embodiment, the bioactive agent of the present invention is administered to the needy in this individual, in combination with pain relievers.

In yet another embodiment, the bioactive agent of the present invention is administered to the needy in this individual, in combination with a corticosteroid.

The bioactive agent according to the present invention in one of the embodiments can be entered in need of this individual in combination with transplantation, such as bone marrow transplantation, transplantation of cord blood or stem cell transplantation.

The bioactive agent according to the present invention in another embodiment, it is possible to enter to the needy in this individual in combination with a therapy to decrease the value of the substrate.

In another embodiment, the bioactive agent of the present invention is administered to the needy in this individual in combination with symptomatic and supportive therapy such as physiotherapy.

Hsp70 increases the capture compounds

In addition, the authors of the present invention showed that Hsp70 increases the capture of other molecules via endocytosis (Fig. 16). This increased seizure can occur independently of Hsp70 by passive mechanisms that allow the connection easier to be captured by the cell in the presence of Hsp70, or it can occur depending on Hsp70 through direct binding to Hsp70.

The ability of Hsp70 to increase the capture compounds cage is an advantage in that it allows Hsp70, or a functional fragment or variant to be introduced into the cells to easily liable to capture by the cell.

In addition, the ability of Hsp70 to increase the capture compounds cage is an advantage of combined treatment regimens, because the presence of Hsp70 can enhance the capture of Hsp70 and connections introduced in combination with Hsp70.

As for the combined treatment, where one connection is the enzyme for ERT and the other is Hsp70 or its functional fragment, or variant, it can help effectively reduce the quantity of enzyme for ERT, it is possible to achieve efficient intracellular doses. This is appropriate, since ERT is very expensive.

In the situation in which the bioactive agent according to the present invention contains a combination of Hsp70, or a functional fragment or variant and the inductor or conductor Hsp70, the presence of Hsp70 may, therefore, increase the capture of the specified inductor or conductor Hsp70.

The modulation of the enzymatic activity of the enzyme

The present invention relates in one aspect to a modulation of the enzymatic activity. The specified enzyme may be an enzyme involved in the catabolism of lysosomal substances. And this modulation may result from the interaction between Hsp70 and BMP.

Thus, the authors of the present invention has described the interaction between Hsp70 and BMP, where Hsp70 interacts or binds to BMP with a certain affinity. Molecule, with "affinity" to the molecule X means herein that a molecule with affinity to the molecule X is associated with a molecule X to a specific defined number of specific conditions, but not necessarily defined) is not associated with other different molecules (for which it has no affinity) to the same extent in identical conditions. One of the measures describe the affinity of a molecule to another molecule is the dissociation constant, Kd. The smaller the Kd, the stronger the affinity. Design the antes dissociation can be defined well-known in this field means, such as the analysis of surface plasmon resonance. In this document it is preferable that the molecule with the "affinity" to another molecule X had a Kd for a given molecule X which is less than 100 mm, such as less than 10 mm, for example less than 5 mm, such as less than 1 mm, for example less than 0.1 mm, such as less than 0.01 mm, for example less than 1 μm, such as less than 100 nm, for example, less than 10 nm, such as less than 1 nm, for example, less than 100 PM, such as less than 10 PM, for example, less than 1 PM. In addition, in this document it is preferable that the molecule, which "has no affinity to the molecule X, has a dissociation constant, Kd, in relation to the binding molecules of X, which is at least 10 times higher, such as at least 20 times higher, for example at least 30 times higher, such as at least 40 times higher, for example at least 50 times higher, such as at least 60 times higher, for example, at least 70 times higher, such as at least 80 times higher, for example at least 90 times higher, such as at least 100 times higher than the Kd of binding (molecule X) of a molecule that has affinity to the molecule X. the Most preferred is at least a tenfold difference Kd between those molecules that are considered to have affinity, and those who and, which are regarded as not having affinity to the molecule X.

An aspect of the present invention is to provide methods of modulating the enzymatic activity of the enzyme, where the specified enzyme interacts with BMP (bis(monoacylglycero)phosphate), specified the method comprises the steps:

i) the introduction of bioactive means capable of increasing the intracellular concentration and/or activity of Hsp70;

ii) the assumptions of the interaction between BMP and Hsp70, and

iii) modulation of the enzymatic activity of an enzyme that interacts with BMP.

This interaction is in one of the embodiments may be direct or specified interaction in another embodiment, may be indirect.

In one of the embodiments the present invention relates to a method of modulating the enzymatic activity of the enzyme, where the specified enzyme interacts with BMP (bis(monoacylglycero)phosphate), specified the method comprises the steps:

i) the introduction of bioactive means of the present invention;

ii) the assumptions of the interaction between BMP and Hsp70, and

iii) modulation of the enzymatic activity of an enzyme that interacts with BMP.

In one of the embodiments specified Hsp70 forms a covalent or non-covalent complex with BMP.

In one embodiment, the implementation of the specified BMP vzaimode is istue with saposiem. In an additional embodiment, the specified saposin can be selected from the group consisting of saposin A, saposin B, saposin C and saposin D.

In an additional embodiment, the specified enzyme selected from the group consisting of sphingomyelinase, sour sphingomyelinase (aSMa), sour ceramidase, beta galactosylceramidase, alpha-galactosidase, beta-galactosidase, glucosylceramidase, sialidase and Ukrainian.

In one specific embodiment, the modulation of enzyme activity is the increase of the enzymatic activity.

In one embodiment, the implementation of this increase of the enzymatic activity is increased in the range from 1 to 5%, such as in the range from 5 to 10%, for example, in the range from 10 to 15%, such as in the range from 15 to 20%, for example, in the range from 20 to 25%, such as in the range from 25%to 30%, for example, in the range from 30 to 35%, such as in the range from 35 to 40%, for example, in the range from 40 to 45%, such as in the range from 45 to 50%, for example, in the range from 50 to 60%, such as in the range from 60 to 70%, for example, in the range from 70 to 80%, such as in the range from 80 to 90%, for example, in the range from 90 to 100%, such as in the range from 100 to 120%, for example, in the range from 120 to 140%, such as in the range from 140 to 160%, for example, in the range from 160 to 180%so as in the range from 180 to 200%, for example, in the range of the zone from 200 to 250%, such as in the range from 250 to 300%, for example, in the range from 300 to 400%, such as in the range from 400 to 500%, for example, in the range from 500 to 750%, such as in the range from 750 to 1000%, for example, in the range from 1000 to 1500%, such as in the range from 1500 to 2000%, for example, in the range from 2000 to 5000%.

In another aspect, the present invention relates to a method of identifying binding partners for complex Hsp70-BMP, comprising the steps of allocating a specified complex Hsp70-BMP and allocation of these binding partners. In one embodiment, the implementation of the specific binding partner is an agonist. In another embodiment, the specified binding partner is an antagonist.

In another aspect, the present invention relates to a complex of Hsp70-BMP and its application to pharmaceuticals, such as for the treatment of lysosomal storage disorders.

In one of the embodiments the present invention relates to an antibody that specifically recognizes a complex of Hsp70-BMP.

The method of treatment

In one aspect the present invention relates to a method of treatment need in the individual.

Thus, an aspect of the present invention is the provision of a method for the treatment of lysosomal storage disorders, containing the introduction bioactive means of the present invention nordaus the Musya in the individual.

Hence, in one embodiment, the implementation of the said treatment may be prophylactic, healing or soothing. In one particular embodiment, said treatment is prophylactic. In another embodiment, said treatment is healing. In an additional embodiment, said treatment is softening.

The bioactive agent used in the present invention, in one embodiment, the implementation can be as a pharmaceutical composition.

In one of the embodiments specified lysosomal disease accumulation selected from the group consisting of disease, Niemann-pick, Farber disease, Krabbe disease, Fabry disease, Gaucher disease, metachromatic leukodystrophy, sialidase and deficit sabotinov.

In a specific embodiment, the specified lysosomal disease accumulation is Niemann-pick disease type A or B. In another specific embodiment, the specified lysosomal disease accumulation is a disease Farber. In another specific embodiment, the specified lysosomal disease accumulation is Krabbe disease. In another specific embodiment, the specified lysosomal disease accumulation is the metachromatic leukodystrophy. In another particular variant is NTE implementation specified lysosomal disease accumulation is sialidosis. In another specific embodiment, the specified lysosomal disease accumulation is Fabry disease. In another specific embodiment, the specified lysosomal disease accumulation is Gaucher disease. In another specific embodiment, the specified lysosomal disease accumulation is deficient sabotinov.

In one of the embodiments specified lysosomal storage disease characterized by an increased intracellular accumulation of sphingolipid.

In one embodiment, the implementation of said treatment reduces the intracellular accumulation of substances in need of this individual. Specified substance may be a substance which is normally degraded in the lysosomes. In one embodiment, the implementation of the specified substance is sphingolipids.

In one embodiment, implementation of the treatment according to the present invention reduces the intracellular accumulation degraded in the lysosomes of substances, such as sphingolipid to less than 100% of the accumulated amount, such as less than 90% of the accumulated amount, for example less than 80% of the accumulated amount, such as less than 70% of the accumulated amount, for example, less than 60% of the accumulated amount, such as less than 50% of the accumulated amount, for example, less than 40% of the accumulated amount, such as less than che is 30% of the accumulated amount, for example, less than 20% of the accumulated amount, such as less than 10% of the accumulated amount, for example, less than 5% of the accumulated amount.

In one embodiment, implementation of the treatment according to the present invention reduces the intracellular accumulation of sphingolipid on at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, for example at least 35%, such as at least 40%, for example at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, e.g. at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, e.g. at least 95%, such as at least 100%.

In one embodiment, the implementation of the specified accumulated sphingolipid selected from the group consisting of sphingomyelin, ceramide, galactosylceramide, globotriaosylceramide, glucosylceramide, GM3 and sulfatide.

The degree of decrease in the intracellular concentration degraded in the lysosomes of substances, such as sphingolipid may depend on factors such as the form of administration, dosing schedules, etc.

In one embodiment, the implementation of said treatment Uwe is icepay lifetime specified in need thereof of an individual.

It follows that the duration of life in one of the embodiments may increase the time from 6 months to 1 year, such as from 1 to 2 years, for example, from 2 to 3 years, such as from 3 to 4 years, for example, from 4 to 5 years, such as from 5 to 6 years, for example, from 6 to 7 years, such as from 7 to 8 years, for example, from 8 to 9 years, such as from 9 to 10 years, for example, from 10 to 12, such as from 12 to 14 years, for example, from 14 to 16, such as from 16 to 18 years, for example, from 18 to 20 years, such as from 20 to 25 years, for example, from 25 to 30 years, such as from 30 to 40 years, for example, from 40 to 50 years, such as from 50 to 60 years, for example, from 60 to 70 years, such as from 70 to 80 years, for example, from 80 to 90 years, this as from 90 to 100 years.

In one embodiment, the implementation of the life expectancy increases by at least 6 months, such as at least 1 year, such as at least 2 years, for example, 3 years, such as at least 4 years, for example, 5 years, such as at least 6 years, for example, 7 years, such as at least 8 years, for example, age 9, such as at least 10 years, for example, 12 years, such as at least 14 years, for example, age 16, such as at least 18 years of age, for example, 20 years, such as at least 25 years, for example, 30 years, such as at least 40 years, 50 years, such as at least 60 years, for example, 70 years, such as at least 0 years for example, 90 years, such as at least 100 years.

An aspect of the present invention is the provision of a method of increasing the life expectancy of a patient with a lysosomal storage disease of accumulation, where the method includes the introduction of bioactive means of the present invention to the needy in the individual.

In one of the embodiments the present invention relates to a method of increasing the life expectancy of a patient with a lysosomal storage disease of accumulation, where the method includes the introduction of bioactive means of the present invention to the needy in this individual, where the specified duration of life increases with time from 6 months to 1 year, such as from 1 to 2 years, for example, from 2 to 3 years, such as from 3 to 4 years, for example, from 4 to 5 years, such as from 5 to 6 years, for example, from 6 to 7 years, such as from 7 to 8 years, for example, from 8 to 9 years, such as from 9 to 10 years, for example, from 10 to 12 years, such as from 12 to 14 years, for example, from 14 to 16, such as from 16 to 18 years, for example, from 18 to 20 years, such as from 20 to 25 years, for example, from 25 to 30 years, such as from 30 to 40 years, for example, from 40 to 50 years, such as from 50 to 60 years old for example, from 60 to 70 years, such as from 70 to 80 years, for example, from 80 to 90 years, such as from 90 to 100 years.

In one of the embodiments the present invention relates to a method took the ing life expectancy of a patient with a lysosomal storage disease accumulation, where this method includes the introduction of bioactive means of the present invention to the needy in this individual, where the life expectancy is increased by at least 6 months, such as at least 1 year, such as at least 2 years, for example, 3 years, such as at least 4 years, for example, 5 years, such as at least 6 years, for example, 7 years, such as at least 8 years, for example, age 9, such as at least 10 years, for example, 12 years, such as at least 14 years, for example, age 16, such as at least 18 years of age, for example, 20 years, such as at least 25 years, for example, 30 years, such as at least 40 years, 50 years, such as at least 60 years, for example, 70 years, such as at least 80 years, for example of 90 years, such as at least 100 years.

EXAMPLES

Example 1: the Interaction between Hsp70 and bis(monoacylglycero)phosphate complementary mechanism stabilizes and promotes the survival of cells

Summary

The permeability of the lysosomal membrane is an evolutionary conservative characteristic stress-induced cell death. In this paper the authors show that the major stress-inducible heat shock protein 70 (Hsp70) enhances the survival of cells through the stabilization of lysosomes via a pH-dependent high-affinity binding to e is dolicocephaly anionic phospholipid bis(monoacylglycero)phosphate (BMP; also referred to as osobistewymiana acid). Positively charged ATPase domain of Hsp70 is responsible for binding, but for the effective stabilization of lysosomes also requires the substrate-binding domain. It is important that you obtain the cytoprotective action by delivering the endocytosis of recombinant Hsp70 and specifically to reverse the extracellular antibodies to BMP or inhibitors of Hsp70. Thus, this interaction protein-lipid opens exciting opportunities for the development of cytoprotective and cytotoxic lysosome-specific therapies for the treatment of degenerative diseases and malignant tumors, respectively.

Introduction

Complementary mechanism are highly dynamic by cytosolic organelles that take membrane transport from the biosynthetic network (TRANS-Golgi), endocytotic, phagocytose and autopaging ways. They contain more than 50 acid hydrolases, which can process all major macromolecules of cells prior to their degradation products available for metabolic recycling. Recently revealed that in addition to their catabolic functions "homemaking", lysosomal proteases, cathepsins are important effectors in the evolutionary conservative programs of cell death induced by, for example, receptors death when the family receptor tumor necrosis factor, hypoxia, oxidative stress, osmotic stress, heat and anticancer drugs. Cathepsin-dependent cell death is characterized early by the permeability of the lysosomal membrane and the subsequent moving of cathepsins into the cytosol, where they can initiate the caspase-dependent and-independent pathways of cell death. Thus, the integrity of the lysosomal membrane acts as an important regulator of the survival of cells under different conditions of stress. While the report that inhibitors of cytosolic cysteine proteases protect against induced by cathepsins cell damage in mammalian cells, as well as in the nematode Caenorhabditis elegans, the mechanism by which cells regulate the stability of lysosomal membranes, remains largely unclear. However, the recent indirect evidence suggests that the potential cytoprotective action of the basic stress-inducible Hsp70 is due to the stabilization of lysosomal membranes. Depletion of Hsp70 starts early permeability of the lysosomal membrane and cathepsin-mediated destruction of malignant cells, and exogenous Hsp70 inhibits lysosomal destabilization induced by various stress. In addition, mice with deficiency of Hsp70 suffer from pancreatitis caused by Pronkin is the venue of lysosomal proteases into the cytosol.

The molecular mechanism underlying lysosomal-protective potential of Hsp70 remains difficult to understand, but the key to this mechanism of action may be associated with stress and malignant tumor of moving a small portion of Hsp70 in endolysosomal compartment. The main purpose of this study is to determine whether in fact lysosomal localization key for the cytoprotective action of Hsp70. It is noteworthy that in the present document, the data indicate that Hsp70 with high affinity associated with lysosome-specific lipid BMP, and this interaction protein-lipid stabilizes complementary mechanism. It is important that this cytoprotective mechanism can be applied through extracellular introduction of the Hsp70 or compounds that counteract binding of Hsp70-BMP or specific functioning of Hsp70 in the lysosomal compartment.

Results and discussion

To study whether lysosomal localization key for the cytoprotective action of Hsp70, the authors present invention was obtained recombinant Hsp70 (rHsp70) and used the advantage of the cellular mechanism of endocytosis for tagging rHsp70 in the lumen of lysosomes. Immunocytochemical analysis of cell osteosarcoma U-2-OS, incubated with labeled with Alexa Fluor 488 rHsp70 the neck is Lyali clear colocalization subjected to endocytosis rHsp70 late endosomal and lysosomal proteins markers (lysosome-associated membrane proteins 1 and 2 and lysosomal integral membrane protein-1 (LIMP-1)) and endolysosomal-specific lipid (BMP), while not watched colocalization with markers of endoplasmic reticulum (Ca2+-Endoplasmic reticulum ATPase (SERCA)), Golgi apparatus (golgin-97) or mitochondria (cytochrome C (cyt c)). Also watched lysosomal localization in living cells, where subjected to endocytosis rHsp70 was colocalizes with Lysotracker® Red, but not with Mitotracker® Red. To determine the number subjected to endocytosis Hsp70 was calculated fluorescence signal from the loaded rHsp70* cells and found that, on average, 70 ng rHsp70* captured 1*105cells. To determine whether localized rHsp70* only in the lumen, or it is directly connected with endolysosomal membranes were subfractional loaded rHsp70* cells U-2-OS and measured the number of rHsp70*present in the light membrane fraction (LMF) (cell organelles, including early and late in endosome and complementary mechanism). Freeze-chipping organelles in LMF through repeated cycles of freezing/thawing in liquid nitrogen resulted in a total release of cathepsin Β in the supernatant, while protein lysosomal membranes LAMP-2 remained in sedimentary destroyed the membrane fraction. Using quantitative analysis subjected to endocytosis rHsp70* revealed that approximately 1/3 of the total rHsp70* remained in the sediment that convincingly p is well positioned to assume, he is connected with endolysosomal membranes. To assess whether subjected to endocytosis rHsp70 to stabilize lysosomal membranes, cells were loaded with acridine orange, metachromatic weak base that accumulates in acidic compartment of the cell, i.e. late endosomes and lysosomes, and makes them sensitive to photo-oxidation after exposure to blue light (Brunk et al., 1997; Nylandsted et al., 2004). Photo-oxidation leads to loss of lysosomal pH gradient and the penetration of acridine orange in the cytosol. It is easy to visualize and count as acridine orange shows a red fluorescence when concentrated in the acidic compartment, and green fluorescence when in the lower concentration is present in the cytosol. It is noteworthy that undergo endocytosis rHsp70 complementary mechanism protects against induced by blue light photo-oxidation, whereas in cells loaded with recombinant Hsc70 and Hsp70-2, which are 86% and 84% amino acid sequence homology with Hsp70, respectively, the protection was not observed. In addition, specific for Hsp70 small interfering RNA (siRNA) did complementary mechanism of cells U-2-OS sensitive to photo-oxidation, and this effect was completely reversible subjected to endocytosis rHsp70, appropriately showing that cytoprotective the first action of endogenous Hsp70 is mediated rather small fractions of proteins, localized in the lumen of lysosomes, what a great combination, present in the cytosol. Shown above effective capture of Hsp70 by endocytosis and stabilization of lysosomes can explain the recently reported amazing neuroprotective effects of extracellular Hsp70 entered into the injury site after various treatments, for which it is known that they run the lysosomal pathway of cell death, i.e. light damage to the retina and axotomy the sciatic nerve.

To study whether the protective effect of Hsp70 consequence of direct binding of Hsp70 with lysosomal membranes, the authors investigated its interaction with Palmitoyl-oleoyl-phosphatidylcholine (POPC) large single-layer vesicles (LUV), containing a variety of membrane-associated anionic lipids, i.e. Palmitoyl-oleoyl-phosphatidylserine (POPS; primarily in the inner layer of the cytoplasmic membrane), cardiolipin (primary mitochondrial) and BMP (primarily in late endosomes and lysosomes). Given the increasingly acidic environment endolysosomal of comparment after maturation of lysosomes compared the interaction of protein-lipid neutral (pH of 7.4) and acidic (pH of 6.0) conditions. At a pH of 7.4 rHsp70 causes a small relative change in the light scattering by POPC liposomes at an angle of 90°, showing the b is very weak binding to Balaam POPC. As previously reported for POPS, all negatively charged lipids increase the binding of rHsp70 with liposomes at neutral pH. This increase was approximately 4-fold, regardless of negatively charged lipids or charge density on the surface of liposomes (POPS has a net charge of -1, and cardiolipin and BMP have a net charge of -2). It is noteworthy that the decrease in pH from 7.4 to 6.0 essentially changes the profile link rHsp70 with lipids. Due to the fact that the binding POPS only slightly increased during the acidification, the binding of BMP nearly 20 stronger at acidic pH compared to neutral pH. pH-dependent high-affinity binding of Hsp70 with BMP confirmed by independent number of BIAcore experiments.

To study whether the pH-dependent high-affinity interaction between Hsp70 and BMP observed in vitro, mediated Hsp70 stabilization of lysosomes in living cells, the authors noted the cellular BMP, loading endolysosomal compartment cells U-2-OS antibodies to BMP, as described previously (Kobayashi et al., 1998). Interestingly, antibodies to BMP effectively inhibit the ability of rHsp70 to show protection against induced photooxidation of lysosomal permeability. Even more importantly, antibodies to BMP make cell osteosarcoma U-2-OS significantly more sensitive to this drug called cisplatin, which induces early permeability Liz is somolinos membrane in cells U-2-OS, and the other sensitive to this drug called cisplatin cell lines used in this study. Thus, the cells of carcinoma of the prostate PC-3 and DU-145 were also significantly more sensitive to cisplatin-indutsiruemoi cell death after treatment with antibodies to BMP.

Confirming that lysosomal interaction of Hsp70-BMP is important for the cytoprotective action of Hsp70, the authors then investigated which parts of Hsp70 protein is responsible for binding to the lipid. To determine this, we measured the shift of the fluorescence of tryptophan (W90 and W580) after coupling with rHsp70 BMP-containing liposomes at pH of 6.0. The authors received the mutant proteins rHsp70 with deletions of two major functional domains of the protein, i.e. amino-terminal ATPase domain (rHsp70-ΔATP; deletion of amino acids 119-426) and carboxy-terminal peptide-binding domain (rHsp70-ΔPBD; deletion of amino acids 437-617). Signal loss in the relative intensity of the peak fluorescence for Hsp70-ΔATP testified that the ATPase domain is required for high-affinity binding of Hsp70 with Balaam POPC/BMP. Then two tryptophan in Hsp70 was replaced by phenylalanine (W90F and W580F) to study what tryptophan is responsible for binding lipid and a shift of the fluorescence. The decrease in signal rHsp70-W90F with the lack of tryptophan in the ATPase domain (rHsp70-W90F) and unmodified signal rHsp70-W580F with the lack of tryptophan in the peptide-binding domain suggests, the tryptophan at position 90 docked with the lipid layer. As used above method measures only the relative shift of the fluorescence after insertion of a tryptophan in the lipophilic environment, the authors also analyzed the binding of lipids rHsp70 and its mutants are more quantitative way using the BIAcore system 2000 with immobilized BMP-containing LUV on the surface of sensor chip L1 at pH 4.5. And rHsp70, and rHsp70-ΔPBD demonstrate a strong interaction with BMP, while linking rHsp70-ΔATP significantly reduced, suggesting that Hsp70 interacts with BMP mainly through its ATPase domain. Surprisingly, the mutants by tryptophan demonstrate a convincing difference in their ability to interact with BMP. While the mutant rHsp70-W580F has essentially the same profile of interaction rHsp70, binding mutant rHsp70-W90F greatly reduced. Because rHsp70-W90F accordingly minimized, which were analyzed by circular dichroism in the near and far UV region, and is able to minimize the luciferase and hydrolyze ATP, mutation W90F specifically terminates the interaction between Hsp70 and BMP format, thus preserving the structural and functional aspects of the chaperone Hsp70. Thus, the mutant rHsp70-W90F unexpectedly gave us an invaluable tool for further research in delaet whether direct interaction between Hsp70 and BMP Hsp70 his lysosomal-protective properties. In fact, the mutant rHsp70-W90F completely loses its ability to protect the lysosomal membrane from photooxidation and cells from cisplatin-induced lysosomal cell death, whereas the mutant rHsp70-W580F shows the same efficiency as wild-type protein. Also the mutant rHsp70-ΔPBD that demonstrates unmodified ability to bind rich BMP membrane loses its ability to protect against photo-oxidation and cisplatin. These data demonstrate that binding of Hsp70 with BMP required, but it is not enough to protect the lysosomal membrane. In addition, intact carboxy-terminal peptide-binding domain is required for the stabilization of lysosomal membranes in living cells.

For a long time Hsp70 inhibitors were considered as interesting anticancer drugs. However, attention has focused on the inhibition of cytosolic Hsp70, and problems related to the delivery of medicines and lack of specificity among members of the Hsp70 family was a insurmountable barriers to the development of appropriate antagonists Hsp70. Having established that the binding of BMP and intact peptide-binding domain is required for the cytoprotective action of Hsp70, and confirming the potential of tagging interaction of Hsp70-BMP, then the authors investigated whether inhibitors of supernovae activity of Hsp70 also in order to counteract the protective effect endolysosomal Hsp70. It was performed by incubating cells with peptide-derived factor inducing apoptosis (ADD70), which inhibits supernovae the Hsp70 function by binding to its peptide-binding domain. It should be noted that this large peptide (388 amino acids) does not penetrate the cytoplasmic membrane, and, thus, provides another tool for specific tagging endolysosomal Hsp70. In particular, incubation of cells with peptide ADD70 completely blocks lysosomal-protective action subjected to endocytosis rHsp70 in cells U-2-OS. To study whether ADD70 also to counteract the cytoprotective action of Hsp70 cells themselves, the authors investigated its effect on cisplatin-induced cytotoxicity in transgenic for Hsp70 immortalized embryonic mouse fibroblasts (iMEF), in which transgenic Hsp70 causes almost complete resistance to cisplatin-induced cell death. It is noteworthy that the treatment ADD70 on transgenic Hsp70 iMEF effectively cancels Hsp70-mediated protective effect and makes them just as sensitive to this drug called cisplatin, as iMEF wild type. iMEF wild-type Express very low levels of Hsp70, and thus, the inability ADD70 in the future to make them sensitive to this drug called cisplatin confirms the idea that ADD70-mediated with sibilizare in fact is due to the inhibition of Hsp70. Similar to the treatment against BMP, processing ADD70 makes cell carcinoma of the prostate PC-3 and DU-145 sensitive to cisplatin-induced cytotoxicity.

Presented herein demonstrate that Hsp70 interacts directly with endolysosomal anionic lipid BMP, and that this interaction stabilizes endolysosomal membrane. Since the concentration of BMP increases in endocytotic vesicles during maturation of the endosome prior to the formation of multivesicular Taurus, late endosomes and lysosomes, pH regulation can be a method of targeting Hsp70 to BMP and complementary mechanism. Subdomains of Hsp70 significantly differ in their levels of significance, ATPase domain has pl by 1.72 units higher than the peptide-binding domain. This characteristic suggests that at acidic pH ATPase domain preferably positively charged, which may facilitate its interaction with anionic lipids. Because during endocytotic maturation pH decreases, increasing the positive charge and any anionic interaction even more amplified. Presented herein demonstrate the dependence of the interaction of the Hsp70-BMP from acidic pH, and ATPase domain confirms this theory. In addition, molecular modeling of the electrostatic surface of the ATPase domain of Hsp70 in the revealed, it forms almost wedge-shaped structure with a predominantly positive charge at the base of the wedge even at pH 7.0. Interestingly, W90 is this positively charged domain that may provide the key to the question of why the mutation Hsp70-W90F has such a profound impact on the ability of Hsp70 to interact with BMP and stabilize complementary mechanism. BMP is localized exclusively in the inner membrane endolysosomal compartment, where he supports the cleavage and release of lipids from lipid vesicles by acidic sphingomyelinase and proteins, activators of sphingolipids, giving rise to metabolites, such as ceramide and sphingosine-1-phosphate, which is involved in the destabilization of membranes and cell death. It should be noted that internal lysosomal membranes can be achieved by invagination of the outer membrane at the level of early and late endosomes, and thus, the corresponding vesicles is also likely to contain Hsp70. Thus, Hsp70 counteracts the role of BMP as a cofactor hydrolysis of sphingolipids and, thus, alters the lipid composition of lysosomes. To test this hypothesis, the authors are currently developing based on mass spectrometry technology for the quantitative analysis of metabolites of lysosomal sphingolipids.

The accumulated data suggest that increased e is pressia and altered transport of lysosomal proteases may form the Achilles ' heel of cancer cells through their sensitization to the permeability of the lysosomal membrane. Thus, the interaction of BMP-Hsp70 on endolysosomal membranes and the resulting stabilization endolysosomal compartment provides malignant cells protection, otherwise straight path to cell death. The molecular mechanism underlying this cytoprotective actions in real time opens exciting new possibilities for sensitization of malignant cells to the means, which induce the lysosomal pathway of cell death through specific inhibition of stabilizing complementary mechanism of Hsp70 function. On the contrary, the interaction between Hsp70 and BMP may provide new treatment strategies based on cytoprotective caused stabilizing complementary mechanism function introduced exogenous Hsp70, for such different damage as pancreatitis, damage to motor and sensory nerve induced by light damage to the retina.

Materials and methods

Cell culture and reagents

Cell lines human osteosarcoma U-2-OS were cultured in RPMI 1640 (Invitrogen) supplemented with 6% heat inactivating calf serum and penicillin-streptomycin. Received on transgenic Hsp70 and appropriate control iMEF and maintained as described previously (Nylandsted et al., 2004). All cells were grown at 37°C in an atmosphere of humidified air with 5% CO2that is not what has been many times tested and recognized negative for Mycoplasma.

Unless otherwise stated, all reagents were obtained from Sigma-Aldrich (Sigma-Aldrich Denmark A/S).

Recombinant proteins

Recombinant Hsp70 and its mutants were obtained using the vector system pET-16b (Novagen) with the induction of protein expression and subsequent Ni2+-affinity purification, optimized according to the Protocol of the manufacturer.

Tagging rHsp70 Alexa Fluor 488 was performed according to the manufacturer's Protocol (Molecular Probes).

Capture cells of recombinant proteins and antibodies:

Subconfluent cells were cultured in RPMI 1640 (Invitrogen) supplemented with 6% heat inactivating calf serum and penicillin-streptomycin. Recombinant proteins or lysates of reticulocytes was added directly to the medium to obtain the final concentration. Then cells were grown another 20 hours in the presence of protein/lysate.

The load cell antibody against BMP (LBPA) (6C4) was carried out using the techniques of this field.

Quantitative analysis subjected to endocytosis rHsp70* carried out by growing cells for 20 hours in the presence of rHsp70, after which cells were collected, washed 3 times in PBS and counted. To fully capture the cells used 1*105cells. Cells were literally by incubation for 30 min on ice in 100 μl of digitonin-PBS (200 μg/ml). Measured fluorescence on the readout device Spectramax Gemini (Molecular Devices). For lung membrane is fractions (LMF) collected a total of 10*10 6cells were washed 3 times in PBS and homogenized in the homogenizer of the downs, until the destruction of membranes has not reached 90%, which was determined by staining Trifanova blue. Then the cells were subjected to fractionation membrane, first removing the cytoplasmic membrane, the nucleus and heavy membrane fraction, after which it was collected LMF by centrifugation at 17000*g for 20 minutes Then LMF was divided into two - the first was taken as "full" LMF. The second fraction was freeze/thawed for 5 cycles in liquid nitrogen to break the membrane, and then centrifuged at 20000*g for 20 min to separate the membranes from the contents of the lumen. All cell after collecting was carried out at the max 4°C.

Analysis of the integrity of lysosomes and cell viability

Subconfluent cells U-2-OS, incubated with 2 μg/ml acridine orange for 15 min at 37°C, washed, irradiated and analyzed in a balanced salt Hanks solution with addition of 3% FCS. Cells for visualization of individual cells were selected from eight pre-defined areas in each well in transmitted light, after which the cells were immediately visualized and subjected to blue light arc mercury burner USH102 100 watts (Ushio electric)installed in the lamp U-ULS100HG (Olympus), for 20 seconds. Fluorescent microscopy was performed to invert the proofed microscope Olympus IX-70 x20 lens LCPIanFI with NA=0,40. The loss of lysosomal pH gradient quantitatively measured by counting the loss of the intense red color.

Apoptosis-like cell death was determined by staining cells 0.05 mg/ml hagstom 33342 (Molecular Probes) and counting cells with condensed nuclei in inverted fluorescent microscope (Olympus IX-70 (Filter U-MWU 330-385 nm). For each experiment we calculated the minimum of eight randomly selected areas. Cell viability was analyzed through analysis of the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), as described previously67. Necrotic cells were counted by flow cytometry with staining of the cells for 10 min at 37°C 2.5 μm SYTOX Green (Molecular probes) and then measured positively stained cells by the intensity of their fluorescence in the FL1 channel of a flow cytometer (FACSCalibur™; Becton Dickinson).

Cells were treated with cisplatin, as indicated, the cytosolic fraction was obtained by processing digitonin and determined the activity of cytosolic cysteine cathepsin (zFRa) and caspase-3-like (DEVDa) activity.

RNA interference

Used miRNAs included one focused on two genes encoding Hsp70 (HSPA1A and HSPA1B); 5'- GCCAUGACGAAAGACAACAAUCUGU-3' (Invitrogen) and control Hsp70 siRNA described previously. Oligofectamine (Invitrogen) was used as trasferire what it means.

Immunological analysis

Used primary antibodies included mouse monoclonal antibodies against Hsp70 (2H9; courtesy of Boris Margulis, Russian Academy of Sciences, St. Petersburg, Russia), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Biogenesis), BMP (6C4; (Kobayashi et al., 1998)), LIMP-1 (H5C6; developed by J. Thomas August, and James E.K. Hildreth and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, USA), cyt c (clone 6H2.B4, BD PharMingen), SERCA (IID8, Calbiochem) and Goldina-97 (CDF4, Molecular Probes). Proteins separated in 10% SDS-PAGE and transferred to nitrocellulose membrane, was determined using the indicated primary antibodies, suitable conjugated with horseradish peroxidase secondary antibodies from Dako, reagents for Western blotting ECL (Amersham) and device for reading a fluorescent image (LAS-1000Plus, Fujifilm).

Fluorescence spectra of tryptophan and scattering of light at an angle of 90° liposomes

Fluorescence spectra of tryptophan (RFI) and a scattering angle of 90° (RSI) was analyzed in HEPES buffer (20 mm HEPES, 0.1 mm EDTA, pH to 7.4 or 6.0, as indicated) using LUV containing these lipids, essentially as described previously. For RFI, LUV added 10 μm aliquots and spectra were recorded after 20 min stabilization period. For RSI recombinant proteins were added 0.12 nm aliquot.

Surface plasmon resonance (BIAcore)

To obtain LUV in org the organic solvent was dissolved a mixture of lipids, containing 10 mol.% sphingomyelin, 50 mol.% phosphatidylcholine, 20 mol.% cholesterol and 20 mol.% BMP, dried under a stream of argon gas and rehydratable buffer Tris/HCl (pH 7,4) (Kolzer et al., 2004). The mixture was frozen/thawed nine times in liquid nitrogen and then in an incubator at 37°C. After ultrasonic washing for 15 min the mixture 21 times was passed through a polycarbonate membrane with a pore diameter of 100 nm. Measurement of surface plasmon resonance was performed using the BIAcore system 2000 at 25°C. LUV (total lipid concentration 0.1 mm) was immobilized on the surface of sensor chip L1 (BIAcore) in PBS (load buffer). As a rolling buffer was used a buffer of sodium acetate (50 mm, pH 4.5). As a control directly on the surface of liposomes was injected acidic to sphingomyelinase (0.2 μm, 60 μl in the movable buffer). Received unit response between 4100RU-5250RU. Interesting protein was injected into a movable buffer at a flow rate of 20 μl/min at the indicated concentrations. After the introduction of the added phase dissociation 10 minutes

Molecular modeling

Analysis of the primary structure and molecular modeling was performed using software available on the Expert Protein Analysis System (EXPaSy), proteomics server of the Swiss Institute of Bioinformatics (http://expasy.org/). Molecular modeling was performed on the basis of the crystal structure of the ATPase domain of Hsp70 h the rights (pdb code: 1S3X) and substrate-binding domain of Hsc70 person (pdb code: 7HSC) using DeepView-Swiss PDB Viewer. The surface model was based on the Coulomb interaction at pH 7.0 using a solvent with a dielectric constant of 80 (H2O).

Statistical analysis

Statistical analysis was performed using bilateral paired t-test t-test to evaluate the null hypothesis. A basic level of statistical significance was defined as 5%, and all groups of data were tested using the criterion F on the comparability of their deviations. All statistical analysis was performed at least three independent experiments.

Discussion

The literature presents evidence that Hsp70 may be present on the cytoplasmic membranes of tumor cells, as well as endolysosomal system. In addition, it is known that Hsp70 can be released into the bloodstream during various stress-inducing events, most typically with fever, trauma and intensive physical exercises, most entertaining when psychological stress, although this work has been carried out mainly in the field of immunology. The presence of species of Hsp70 in endolysosomal the compartment also described for another member of the Hsp70 family; constitutively expressed Hsc70. The function of Hsc70 in this place actually gave the name of the process known as chaperone-mediated the autophagy.

However, from the literature, niceone is known about the molecular basis of the Association with cytoplasmic Hsp70 and endolysosomal membranes, which forced the authors to make this project.

Data from example 1 demonstrate that Hsp70 can interact with negatively charged membrane lipids, such as phosphatidylserine (PS), cardiolipin and bis(monoacylglycero)phosphate (BMP) at neutral pH. In the simulation of acidity, which can be expected in early endolysosomal system (pH 6,0), however, the profile of interaction varies greatly, and the affinity of Hsp70 for BMP becomes 20 times higher than at neutral pH and almost 9 higher than for PS. This interaction of the Hsp70-BMP was confirmed in a more elaborate system, BIAcore, in which pH is currently set at the level expected in late endosomes and lysosomes (pH 4.5), the main areas for the most part cellular BMP. Interestingly, well-known interacting with BMP protein, sour sphingomyelinase (aSMa), which uses the BMP as a cofactor, shows only half of affinity to BMP compared with Hsp70, demonstrating a high relative affinity of Hsp70 to BMP.

The interaction of Hsp70 with PS also described by others as possessing the interaction between Hsp70 mouse and sour glycosylamide, for which the interaction is dependent on N-terminal ATPase domain and in some cases also from the peptide-binding domain (PBD). However, unlike used in this document si the same time, these data were obtained in systems containing essentially only one lipid (90-100% and 100% pure lipid, respectively), probably not similar to any minimally complex lipid environment, which is expected in eukaryotic cells. However, the importance of the N-terminal region of Hsp70 to communicate with acidic lipids, as shown Harada et al., corresponds to the data of the applicants that the interaction of Hsp70 with BMP depends on its N-terminal ATPase domain. In addition, the authors showed that the tryptophan 90 (W90) Hsp70 is a critical amino acid, as its mutation significantly reduces the interaction of the Hsp70-BMP. A hypothetical model suggests that Hsp70 contains specific binding sites hydrophilic and hydrophobic parts of acidic glycolipids in the ATPase and peptide-binding domain (PBD).

Although this model may be applicable to bind Hsp70 with acidic glycolipids, authors should preferably offer a different model of interaction between Hsp70-BMP. Based on the data presented in this document: (I) the ability of PBD only a weaker interaction with the BMP; (II) the importance W90; (III) binding properties of the ATPase domain; and (IV) molecular modeling of the surface electrostatic potential of Hsp70, the authors suggested that Hsp70 interacts with BMP through electrostatically positively charged to vinoobraznyh subdomain at the base of the ATPase slit. As a conservative replacement W90 on phenylalanine significantly reduces the interaction of the Hsp70-BMP, not acting on readingbuy or ATPase activity of Hsp70, and because this single substitution of the amino acid does not change the electrostatic profile, it may be more reasonable to explain the interaction of Hsp70 with the more General anionic lipid motif is something between the two models. In this model, a positive surface charge facilitates electrostatic interactions, and specific residues, such as W90, can be involved in determining the specificity of binding of anionic lipid binding partners - in this case, BMP. Interestingly, this can potentially involve Hsp70 as a more General regulator of lipid homeostasis in the cell. In support of this, there are data demonstrating that the lipid cell membrane can act as the primary sensors of stress, such as fever and oxidative stress, and thus, the source inductors response to stress. In the presence of stress someone can say that the lipid of the cell membrane are key compartments, supporting homeostasis or actually modifying to run specific phenomena of the transmission signal in response to the call cell. The binding of Hsp70 with lipids, such as BMP, and the subsequent increase stability of Lisa amalnyj membranes and, possibly other cellular lipid phenomena may thus represent part of the overall cellular response to stress. In the case of a malignant tumor that could be the end of the most malignant tumors, but also from the perspective of a broader evolutionary perspective, coordinated response protein-lipid in the presence of cellular stress can achieve the goal.

Presented in this document, the data showing that only Hsp70, but not Hsc70 and Hsp70-2, can directly protect the lysosomal membrane, suggest that the basic stress-induced Hsp70, but not other kinds of Hsp70 may regulate potential lipid response to stress. However, it is also known that the depletion of Hsp70-2 also causes the permeability of the lysosomal membrane and cell death, although in this case the path is indirect, since it depends on LEDGF. However, the mechanism of action of LEDGF on lysosomal membranes remains unresolved.

To validate the importance of interaction between Hsp70-BMP in vivo, the authors noted BMP subjected to endocytosis antibodies and lysosomal Hsp70 by endocytosis AIF-derived polypeptide ADD70, otherwise the cell is not permeable. This confirms that the interaction between Hsp70 and BMP serves for the stabilization of lysosomal membranes, because the cells subsequently did significantly sensitive to the effects of direct the x-destructive lysosomal membrane incentives, as for LMP-inducing chemotherapeutic agent cisplatin, the profile of programmed cell death which is characterized as part of this project. It was previously shown that expression of ADD70 makes cancer cells sensitive to a variety of death stimuli and reduces and carcinogenesis carcinoma of the colon in rats and melanoma cells mouse in isogenic animals. The main difference between this approach and the approach presented in this paper is that the authors of the present invention sought to specifically mark lysosomal Hsp70 via endocytosis ADD70, while in previous studies for tagging more widely represented cytoplasmic Hsp70 was used cytosolic expression ADD70. The success of tagging endolysosomal interaction of Hsp70-BMP also provided specific crucial experiment for the idea about the marking of lysosomal compartments by means of endocytosis in methods of therapy, the concept, which may have broad therapeutic applications, because someone can imagine sensitization, for example, malignant cells to the means of inducing the lysosomal pathway of cell death through specific inhibition lysosome-stabilizing function of Hsp70. On the contrary, the interaction between Hsp70 and BMP may provide new treatment strategies, based the on cytoprotective, provided lysosome-stabilizing function of the introduced exogenous Hsp70 for such a variety of injuries, as pancreatitis, damage to motor and sensory nerve and sitoindosides damage to the retina. In fact, the concept of the mechanism of endocytosis for the introduction of specific cytotoxic compounds already used as delivery via endocytosis BH3 helix with a hydrocarbon crosslinks based on proapoptotic BH3-agonist interacting domain death Bid, which, as shown, induces apoptosis in leukemic cells. This process depends on the output of intact BH3 helix of the endocytotic compartment and activation of Bax and Bak to induce the release of cytochrome C and activates mitochondrial program of apoptosis. However, the mechanism of release of endocytotic system, unfortunately, is not considered in this work.

As shown herein, the interaction between Hsp70 and BMP depends on the ATPase domain of Hsp70. Interestingly, recent reports of the co-chaperone of Hsp70, Hsp70-binding protein 1 (HspBP1) can emphasize the importance of this positively charged region of Hsp70. Study of the crystal structure HspBP1, forming a complex with part of the ATPase domain of Hsp70 revealed that the interaction between them is mediated by a curved, fully α-SPIRA is inim layer in HspBP1, containing four Armadillo-like repeat. Concave surface of this curved layer covers the ledge II ATPase domain, the same ledge that forms the main part of electrostatically positively charged volume of the ATPase domain of Hsp70, which, according to the authors, mediates the interaction between Hsp70 and BMP. Further, the prospect of this is provided by another study, in which 14 of malignant cell lines were characterized in terms of their relative levels of Hsp70/HspBP1. In this other study was found that cell lines with a high molar ratio of HspBP1/Hsp70 more susceptible to anticancer drugs than lines with low and that overexpression of HspBP1 contributes to the permeability of the lysosomal membrane. Based on data communications and data presented in this example, someone may speak in favor of a model in which HspBP1 by binding to the positively charged region ATPase domain of Hsp70 destroys the interaction with BMP and, thus, its stabilizing effect on endolysosomal membrane, leading to increased sensitivity of LMP-inducing stimuli. For this reason, the domain HspBP1 with repetitions of Armadillo could potentially form the basis of rational drug design environments is TBA, as an argument in favor of ADD70. The effectiveness of such HspBP1-derived molecules easily test the systems described herein, and it is an interesting way to further the use of molecular mechanism described in this document.

As the authors show in this paper, Hsp70 binds to BMP with high affinity at acidic pH 4.5, even almost 2 times higher than in the case of "classical" partner binding of BMP sour sphingomyelinase (aSMa). Interestingly, BMP is stimulating cofactor for enzymatic hydrolysis not only sphingomyelin through aSMa, but mostly membrane-associated sphingolipids, because it also functions as a cofactor for protein activators of sphingolipids (SAP/saposin) A-D. Thus, the obvious question is whether the changes in any way Hsp70 by binding to BMP-binding properties aSMa and sabotinov, modifying these catabolism of membrane sphingolipids and glycosphingolipids and synthesis of downstream effector molecules such as ceramide and its metabolites, ceramide-1-phosphate, sphingosine and sphingosine-1-phosphate, all of which are involved in cell survival and death. In fact, the authors found that Hsp70 is capable of modulating the binding aSMa with containing BMP liposomes at pH 4.5, depending on the end of the ation of Hsp70. As you can see, low concentrations (3-150 nm) Hsp70 facilitate interaction aSMa with liposomes BMP-Hsp70, whereas higher concentrations of Hsp70 (300-1500 nm) have the opposite effect. Although our working concentration in the medium when added Hsp70 for endocytosis, was 300 nm, it is difficult on the basis of this estimate is entered vnutricinovialnoe concentration, and any conclusions about what may have Hsp70 on the activity of aSMase in vivo, remain theoretical. However, the staining on transgenic Hsp70 iMEF (Hsp70-TG) and iMEF wild-type (WT) with a monoclonal antibody against ceramide revealed that Hsp70 transgenic mice demonstrate a clear positive regulation of ceramide, which is present in the characteristic structure of the knots on the rope" on the periphery of the cells, as well as near the core. Further analysis carmignola iMEF through allocation of lipids and subsequent mass spectroscopy confirmed these data, because the total levels of ceramide from an average of 10.2 ng ceramide/mg protein for iMEF-WT to 14.9 ng/mg for transgenic for Hsp70 iMEF. In addition, the authors confirm that this effect can be attributed to the action of Hsp70, as the authors also profiled our loaded rHsp70 cells U-2-OS (i.e. 300 nm rHsp70 in complete medium for 24 hours, like all other experiments endocytosis Hsp70 presented in this document). Quantitative is the first analysis of ceramide in the loaded Hsp70 cells U-2-OS showed an increase in the total levels of ceramide from 2,99 ng ceramide/mg protein for control cells to 5,10 ng/mg for loaded Hsp70 cells (at the time of writing experiment held only once). However, taken together, they confirm the role of Hsp70 in the modulation of the levels of ceramide in the cells, although, of course, requires further validation. In addition, if these data can be confirmed, of course, a number of questions, such as compartmentalization of ceramide species, quantitative analysis of ceramide species (of which there are at least 50 different types of molecules), the profile analysis of the levels of ceramide in various stresses, the status of cell transformation, etc.

Interestingly, in previous study has considered one of these questions, which showed that heat shock (42,5ºC for 2 hours) causes an accumulation of ceramide in lymphocytes of acute leukemia Molt-4. This accumulation can be blocked by pharmacological inhibitors fumonisin B1 and mirocin, the latter of which refers to specific inhibitors of the synthesis of ceramide de novo, because it blocks the action of proteolytic palmitoyltransferase, the enzyme that initiates the synthesis de novo of new sphingolipids from the serine Palmitoyl-COA. Private mechanism of this increase in synthesis of ceramide de novo has been described in yeast, in which heat stress induces an acute influx of serine in the ER, which triggers the synthesis de novo. It is interesting to test whether it is possible to modulate the levels of ceramide observed after endocytosis rHsp70, Farmak is a logical inhibitors or observed increase is associated with catabolic pathways for the degradation of sphingolipids and stimulate this by binding of Hsp70 with BMP. Of course, you can also hypothesize about the model elements. In this model, the initial heat stress can lead to fluidization membrane, influx of serine rapid initiation of synthesis of sphingolipids de novo. Subsequently, the induction of Hsp70 due to thermal stress can lead to increased levels of Hsp70 in the cell, the interaction of Hsp70 with BMP, the increased activity aSMa, and possibly activity SAP, resulting in synthesis of ceramide catabolic way. This secondary response may Supplement the initial induction of de novo or may accept instead of it, because of the ongoing response de novo depends on the continued receipt of serine and Palmitoyl-KoA. However, it is necessary to test whether the protection of cells from the increase in the ceramide or perhaps provide it changed the levels of upstream and downstream metabolites.

At this stage it remains to answer some basic questions: How Hsp70 ends in the extracellular environment and inside endolysosomal compartment? Hsp70 is secreted and then is captured by endocytosis? or It is inside of lysosomes or more specialized secretory lysosomes, waiting for the releasing signal in the form of stress? And perhaps more importantly - What is the biological significance of the presence of Hsp70 in the extracellular environment?

Ho is I study presented in this work, unable to answer these complex questions, we can make some inferences. First, Hsp70 may undergo endocytosis in all tested in this project cell lines, indicating that a common way of recognition of extracellular Hsp70 (eHsp70). This corresponds to showing that eHsp70 can communicate with a number of receptors on different subpopulations of leukocytes. Involved in the recognition of extracellular Hsp70 (eHsp70) receptors mainly include receptors pattern recognition (PR) and consist of multiple receptors from different families of receptors, such as toll-like receptors (TLR), scavenger receptors and lectins type C. Since this project did not consider what the source mechanism of Hsp70 undergoes endocytosis (receptor-mediated, raft-dependent, clathrin-dependent and so on), you cannot specify whether PRR observed in our systems endocytosis eHsp70. However, 10-fold excess of its Hsp70 can't compete with capture AF488 labeled Hsp70 in cells U-2-OS and iMEF, and Vice versa, endocytosis was significantly enhanced in the presence of an excess of its Hsp70 that, to some extent, the evidence against saturated capturing mechanism.

Attention in the field of immunology was mainly focused on cytokine response and activation is born of immune protection, caused by binding of eHsp70 with PRR and, thus, not very much attention was paid to the effect of eHsp70 after binding of the receptor and initiate a signal.

The mechanism of release of Hsp70 in the extracellular environment and the effects of Hsp70, to some extent, already discussed in this document, although still lacking a satisfactory molecular understanding of fundamental mechanisms. However, there is growing evidence of the presence of Hsp70 in the circulatory system after stress, and accumulating data support the role of eHsp70, stress-induced or exogenous delivered, neuroprotection, as well as to stimulate a primary immune system protection. In relation to the release of Hsp70, the first proof of the transfer of Hsp70 from one cell to another has come from studies on the giant AXONE squid, and playback data results on cultured embryonic cells of rats was evidence that non-classical path exocytosis is responsible for the release of Hsp70.

Suggest that Hsp70 together with other heat shock proteins are released only in pathological conditions, leading to necrotic death, but not when programmed death. However, recent studies have shown that Hsp70 can be released from intact cells by active is the mechanisms and the level of incentives determines the way of liberation. It is important that there is no data about the studies that showed a direct correlation between eHsp70 and markers of muscle damage, although after exercise in peripheral blood flow, you can define a significant increase eHsp70. The most convincing and perhaps also the most interesting are open, showing that psychological stress, such as fear of predator and shock, can cause stress-induced release of eHsp70, a process that is assumed to depend on catecholamine transmission signal. This is particularly interesting, because catecholamines via α1-adrenergic receptor can lead to intracellular release of calcium and the release of calcium can cause exocytosis of actcom, multivesicular cells and lysosomes. For this reason, during periods of stress, increased noradrenaline, acting via α1-adrenergic receptors, may lead to the release of calcium inside the cells with the subsequent release of Hsp70 in actsoma. Proof of this hypothesis comes from evidence that eHsp70 may be released in vesicles characterized as actsoma, but also presents evidence that eHsp70 may be released as a free eHsp70, in cellular systems and in vivo. Also assume that the release of eHsp70 necessary lipid rafts, although it is also aspreva the tsya. In addition, it is shown that functional lysosomal compartment required for the release of eHsp70 and that this release is accompanied by the presence on the cell surface of lysosomal protein markers, suggesting that secretion depends on the fusion of cytoplasmic and lysosomal membranes.

Regardless of whether released Hsp70 in actsoma or as a free eHsp70, it is interesting to note that, obviously, some types of secretory MVB/late endosomal/lysosomal compartment involved in all methods of release. Based on these data and the results obtained in this paper the results, we can formulate a more comprehensive hypothesis about how Hsp70 penetrates from cytosol in the extracellular environment. However, the release of Hsp70-dependent increase of intracellular calcium, as it serves as a signal for exocytosis of endolysosomes. However, the presence of Hsp70 in this compartment depends on the interaction between Hsp70 and BMP, as described herein, since Hsp70 effectively aggregates on BMP-containing internal membranes in the late endosome/MVB/lysosome. Hsp70 can act in the later endosome and complementary mechanism through extracellular capture, such as endocytosis, as also described herein, or by invaginate outer membranes of early and late is their endosomes, as well as lysosomes, which capture intracellular Hsp70 and BMP near. Acidity compartment maintains strict preference localization of Hsp70 on BMP-containing membranes. After exocytosis some Hsp70 is still associated with the BMP-containing ectosomal, but the neutral pH found in the extracellular environment, but rather contributes to a significant shift in the equilibrium Hsp70-BMP towards more unrelated Hsp70, resulting in free, as well as associated with ectosomal Hsp70, which can then perform its extracellular functions.

Ultimately presented herein demonstrate that Hsp70 directly and pH-dependent interacts with endolysosomal anionic phospholipid BMP. The authors demonstrate that binding of Hsp70 with BMP mediated ATPase domain of Hsp70, involving tryptophan 90 and that this interaction leads to stabilization endolysosomal membrane, possibly by influencing the activity of other BMP-binding proteins. The authors also show that the explanation of this molecular mechanism opens exciting new possibilities for sensitization of malignant cells to the means of inducing the lysosomal pathway of cell death by specific inhibition lysosome-stabilizing function of Hsp70. On the contrary, the interaction between Hsp70 and BMP can provide but the s treatment strategies, based on cytoprotective caused lysosome-stabilizing function of the introduced exogenous Hsp70.

Example 2: the Interaction between Hsp70 and bis(monoacylglycero)phosphate acid activates sphingomyelinase, stabilizes lysosomal membranes and promotes the survival of cells

Heat shock protein 70 (Hsp70) is an evolutionary highly conserved molecular chaperone that promotes the survival of stressed cells by inhibiting the permeability of lysosomal membranes, characteristic stress-induced cell death. Clues to the molecular mechanism for this may lie in the recently described associated with stress and malignant tumors of the transfer of small amounts of Hsp70 in the lysosomal compartment. In this paper the authors show that Hsp70 stabilizes complementary mechanism by increasing the activity of the acidic sphingomyelinase (ASM), lysosomal lipase, hydrolysing sphingomyelin to ceramide and phosphorylcholine. In the acidic environment of Hsp70 with high affinity and specificity associated with endolysosomal anionic phospholipid bis(monoacylglycero)phosphate (BMP), an important cofactor for ASM, thus facilitating the binding of ASM with BMP and stimulating the activity of ASM. Inhibition of the interaction of Hsp70-BMP antibodies to BMP or a point heteroplasmy mutation (W90A) in Hsp70, as well as inhibition of the asset the spine of the ASM desipramine, effectively reverses Hsp70-mediated stabilization of lysosomes. It is noteworthy that reduced activity in ASM cells from patients with Niemann-pick disease A (NPDA), severe lysosomal storage disorders caused by mutations in the ASM gene, is also associated with a significant decrease in the stability of lysosomes, and this phenotype can be effectively corrected by restoring the activity of lysosomal ASM by treatment with recombinant Hsp70 or ASM. Taken together, these data open up exciting opportunities for the treatment of lysosomal storage diseases and malignant tumors is not transmitted by the cell connections that are fixed in the lumen of lysosomes via endocytotic delivery path.

Lysosomal proteases, cathepsins are important effectors in the evolutionary conservative programs of cell death induced by a large variety of stresses. Cathepsin-dependent cell death is characterized early by the permeability of the lysosomal membrane and the subsequent moving of cathepsins into the cytosol, where they can initiate the caspase-dependent and-independent pathways of cell death. To study whether lysosomal localization key to described the ability of Hsp70 to stabilize lysosomal membranes and to protect cells from stress-induced cell death, the authors took advantage of the advantages the STV cellular mechanism of endocytosis for tagging recombinant Hsp70 (rHsp70) in lysosomes. Immunocytochemical and biochemical analyses of cell osteosarcoma U-2-OS, incubated with labeled fluorochroman rHsp70, revealed effective capture rHsp70, its specific colocalization late endosomal and lysosomal markers and binding to lysosomal membranes (Fig. 5a, b and Fig. 9). Using visualization in real time to monitor the integrity of the lysosomal membrane (Fig. 5c), the authors showed that undergo endocytosis rHsp70 complementary mechanism protects from photo-oxidation (Fig. 5d). In addition, small interfering RNA (siRNA)specific for Hsp70, did complementary mechanism sensitive to photo-oxidation, and this effect was completely reversible subjected to endocytosis rHsp70, appropriately demonstrating that the protective effect of endogenous Hsp70 is mediated by a small fraction of the protein in the lumen of the lysosomes (Fig. 5e). Despite similar capture (data not shown)were observed stabilization of lysosomes with recombinant Hsc70 and Hsp70-2, which are 86% and 84% amino acid sequence homology with Hsp70, respectively (Fig. 5d).

The presence of Hsp70 in lysosomal membranes and its ability to withstand the hydrolytic lysosomal environment suggest that it is associated with lysosomal lipid membrane. Thus, the authors investigated the interaction of Hsp70 with Palmitoyl-oleoyl-phosphate is delmolino (POPC) large single-layer vesicles (LUV), contain various membrane-associated anionic lipids, i.e. Palmitoyl-oleoyl-phosphatidylserine (POPS; primarily in the cytoplasmic membrane), cardiolipin (primary mitochondrial) and BMP (primarily in late endosomes/lysosomes). Taking into account the increasingly acidic environment endolysosomal compartment after maturation of lysosomes, the authors compared the interaction of protein-lipid neutral (pH of 7.4) and acidic (pH of 6.0) conditions (Fig. 6a). At a pH of 7.4 rHsp70 causes a small relative change in light scattering at an angle of 90° in POPC liposomes, indicating very weak binding. As previously reported for POPS, all negatively charged lipids enhance the binding of rHsp70 with liposomes at neutral pH to about 4 times, regardless of the charge density on the surface of liposomes (in the range from -1 to -2) (Fig. 6a). It is noteworthy that the binding of BMP nearly 20 times stronger at acidic pH compared to neutral pH, while linking with POPS only slightly increased after acidification (Fig. 6a). The binding of Hsp70 to BMP with high affinity at acidic pH was confirmed in several independent experiments BIAcore (Fig. 6e and 7a). It is important that antibodies to BMP, delivered in endolysosomal compartment through endocytosis, effectively inhibit the ability of rHsp70 to stabilize complementary mechanism in living cells (Fig. 6b)and make the cells sensitive to this drug called cisplatin (Fig. 6c), an antitumor drug, inducing permeability of lysosomes.

To study how much of Hsp70 is responsible for binding to BMP, the authors measured the shift of the fluorescence of tryptophan docking rHsp70 and its mutants in BMP-containing liposomes. Signal loss in the relative intensity of the peak fluorescence of the mutant Hsp70 with the lack of amino acids 119-426 in the amino-terminal ATPase domain (rHsp70-ΔATP), but not with the lack of amino acids 437-617 carboxy-terminal peptide-binding domain (rHsp70-ΔPBD), suggests that the ATPase domain is required for high-affinity binding of Hsp70 with BMP (Fig. 6d). The authors then replaced the two tryptophan in Hsp70 on phenylalanine (W90F and W580F) and explored what tryptophanol is responsible for the shift of the fluorescence induced by lipid binding. The reduction of the signal only for rHsp70-W90F suggests that NH2-end of the protein binds to the lipid layer (Fig. 6d). A more quantitative analysis BIAcore interaction of BMP-rHsp70 confirmed that Hsp70 interacts with BMP mainly through its ATPase domain (Fig. 6e). Unexpectedly, mutation W90F specifically terminates the interaction between rHsp70 and BMP, while at the same time maintaining the structural (folding, analyzed by circular dichroism in the far and near UV region) and functional (folding of luciferase and ATP hydrolysis) aspects of Hsp70 ka the chaperone (Fig. 6e and data not shown). Thus, the mutant rHsp70-W90F provides authors an invaluable tool for further research is whether a direct interaction between Hsp70 and BMP lysosomal-protective properties. In fact, the mutant rHsp70-W90F completely loses its ability to protect the lysosomal membrane from photooxidation and cells from cisplatin-induced lysosomal cell death, whereas the mutant rHsp70-W580F shows the same protective effect as wild-type protein (Fig. 6f and g). It is important that the mutant Hsp70 undergoes endocytosis essentially as effectively as wild-type Hsp70 (data not shown). Thus, we have concluded that binding of Hsp70 with BMP is essential for lysosomal-stabilizing action of Hsp70.

Since the concentration of BMP in endocytotic vesicles increases with maturation of the endosome prior to the formation of lysosomes, regulation of pH may be the way Hsp70 targets the complementary mechanism. Calculate (PROTPARAM, proteomic server EXPaSy, Swiss Institute of Bioinformatics) revealed that the ATPase domain of Hsp70 has a theoretical pl by 1.72 units higher than the peptide-binding domain (6,62 against 4,9). This characteristic suggests that at acidic pH ATPase domain is predominantly positively charged, which may facilitate its interaction with anionic lipid is mi. Data showing the dependence of the interaction of the Hsp70-BMP from acidic pH and ATPase domain, supports this theory. In addition, molecular modeling of the electrostatic surface of the ATPase domain of Hsp70 revealed that it forms almost clinopodia structure with a predominantly positive charge at the base of the wedge containing W90, possibly explaining the profound impact of the mutation W90F on the ability of Hsp70 to interact with BMP and stabilize complementary mechanism (Fig. 6h).

BMP binds ASM with high affinity and stimulates its ability to hydrolyze sphingomyelin to ceramide and phosphorylcholine. The BIAcore analysis revealed that pretreatment BMP-containing LUV rHsp70 in subaqueously concentrations facilitates subsequent binding to ASM, while higher concentrations of rHsp70 demonstrate the inhibitory effect (Fig. 7a and 10). It is noteworthy that on transgenic Hsp70 embryonic mouse fibroblasts (Hsp70-MEF), protected from stress-induced lysosomal damage (Fig. 7e), demonstrating a significantly higher activity ASM than MEF wild-type (WT-MEF), and the processing of WT-MEF rHsp70 in cytoprotective concentration (300 nm) increases the activity of ASM to levels comparable with those in the Hsp70-MEF (Fig. 7b). To study whether ASM for lysosomal-stabilizing effect, the authors treated the cells with desipramine, a well-described is a diversified pharmacological inhibitor of ASM. The desipramine reduces the viability of MEF depending on dosage, and cell death associated with massive permeability of lysosomes, as shown by receipt of cathepsins into the cytosol (Fig. 7c and d). It is noteworthy that the desipramine-induced cell death and the permeability of lysosomes significantly reduced in Hsp70-MEF compared with WT-MEF. In addition, inhibition of ASM subconsciencely concentrations of desipramine reverses the stability of lysosomes to stress in Hsp70-MEF to the level of WT-MEF as evidence of accelerated loss of integrity of the lysosomal membrane after fotogaleria (Fig. 7e). In addition, lysosome-protective role supported by data showing that complementary mechanism in fibroblasts from patients with NPDA, fatal lysosomal storage disease accumulation, caused by mutations in the ASM gene, demonstrate extreme sensitivity to induced photooxidation damage (Fig. 8a). It is noteworthy that rHsp70 can also increase the enzymatic activity of endogenous mutant ASM, and simultaneously loaded rASM, in the patient's cells (Fig. 8b). Increased activity of ASM received by the loading of lysosomes rHsp70, rASM or their combination, correlate with their ability to stabilize and complementary mechanism to normalize the volume significantly increased lysosomal compartment in cells NPDA (Fig. 8b-d). It should be noted that similar to rsp70, rASM is also localized in the lysosomes (Fig. 8b).

Taken together these data suggest that the interaction of Hsp70-BMP stabilizes complementary mechanism through a mechanism involving rather the regulation of metabolism sphingomyelin than direct physical stabilization of the membrane. This indirect effect is confirmed by the fact that BMP is localized exclusively in the outer membrane endolysosomal compartment, where its main function is the maintenance of destruction and extraction of lipids or lipid vesicles by ASM and proteins, activators of sphingolipids. Interestingly, ASM-mediated increase of lysosomal concentration of ceramide modifies the steric conformation of lysosomal membranes and, thus, facilitates their fusion with other intracellular vesicles and cytoplasmic membrane. Thus, changes in the composition and volume of lysosomal membranes, as a result of ceramide-induced increase ability to merge, may participate in Hsp70-mediated increase the stability of lysosomes. On the other hand, various apoptotic stimuli induce the movement of ASM in another layer of the cytoplasmic membrane, where ceramide can form lipid microdomain that function as sites of activation of membrane-associated signaling molecules, is involved in apoptotic signaling. Thus, ceramide can have opposite effects on the survival of cells depending on, whether it is produced by inside lysosomes or on the plasma membrane. Above the molecular mechanism underlying the cytoprotective action of Hsp70, opens exciting new opportunities for sensitization of malignant cells to the means of inducing the lysosomal pathway of cell death by specific inhibition lysosome-stabilizing function of Hsp70. On the contrary, the ability of exogenously introduced rHsp70 alone or in combination with rASM can directly declare a new treatment of patients with NPD, possible methods of treatment, which is limited to gene therapy and stem cell therapy.

A summary of the ways

WT and Hsp70-MEF received, immortalization and maintained as described in this field. Fibroblasts NPDA person (83/24) were obtained from a skin biopsy 5-month-old patient with hepatosplenomegaly. Recombinant proteins were obtained using the vector system pET-16b Ni2+-affinity purification (Novagen) and were labeled Alexa Fluor 488 according to the manufacturer's Protocol (Molecular Probes). To analyze the integrity of lysosomes authors have developed a method of real time imaging of cells stained with acridine orange, metachromatic weak base that accumulates in acidic compartment glue the key, painting it red and making it sensitive to photooxidation. Induced by photo-oxidation loss of the pH gradient and the penetration of acridine orange in the cytosol of individual lysosomes visually counted as a "loss red dots in the cells U2-0-S and as the decline of the red and the increase in green fluorescence in fibroblasts through software Zeiss LSM DUO. Total and cytoplasmic (caused by digitonin) activity of cathepsins were measured in treated digitonin samples using probe zFR-AFC (Enzyme System Products), as described in this field. Fluorescence spectra of tryptophan and scattering of light by liposomes at an angle of 90° were analyzed in HEPES buffer (20 mm HEPES, 0.1 mm EDTA, pH as indicated), essentially as described in this field. Measurement of surface plasmon resonance was performed on immobilized LUV using system BIAcore 2000, as described in this field. Hsp70 siRNA (5'-GCCAUGACGAAAGACAACAAUCUGU-3') and control Hsp70 miRNAs were transfusional with oligofectamine (Invitrogen). Immunological analysis was performed according to standard protocols. Apoptosis-like cell death and permeability of lysosomal membranes were analyzed essentially as described in this field. The ASM activity was analyzed by Amplex Red Sphingomyelinase Assay Kit (A12220) from Molecular Probes described in this field modifications. Statistical analysis is conducted with the use of bilateral paired t-test, t-test, and all groups of data were tested on the comparability of their variance criterion F.

Ways

Cultivation of cells and reagents. Cells human osteosarcoma U-2-OS were cultured in RPMI 1640 (Invitrogen) supplemented with 6% heat inactivating calf serum and penicillin-streptomycin. Transgenic by Hsp70 corresponding control MEF received and maintained as described in this field. Primary fibroblasts NPDA person were grown on medium MEF with the addition of 1% sodium pyruvate, 1% HEPES, 1% L-glutamine. All cells were grown at 37°C in an atmosphere of humidified air with 5% CO2and repeatedly tested and recognized negative for Mycoplasma. Unless otherwise stated, all reagents were obtained from Sigma-Aldrich (Sigma-Aldrich Denmark A/S).

Analysis of the integrity of lysosomes. Subconfluent cells incubated with 2 μg/ml acridine orange for 15 min at 37°C, washed, irradiated and analyzed in a balanced salt Hanks solution with addition of 3% fetal calf serum. Cells for visualization of individual cells were selected from eight pre-defined areas in each well in transmitted light, after which the cells were immediately visualized and subjected to blue light arc mercury burner USH102 100 watts (Ushio electric)installed in the lamp U-ULS100HG (Olympus), for 20 seconds. Fluorescent Mick is oscope was performed on an inverted microscope (Olympus IX-70 x20 lens LCPIanFI with NA=0,40. The loss of lysosomal pH gradient quantitatively measured by counting the loss of the intense red staining. To work with larger the lysosomal compartment of various fibroblasts used in this study had developed a more advanced way. Cells for visualization of individual cells were selected from eight pre-defined areas in each well in transmitted light, after which the cells were immediately visualized and subjected to exposure to light 489 nm 100 MW diode laser, at the same time by laser scanning, making micrograph every 330 MS using confocal system Zeiss LSM LIVE DUO on the two channels separated by bandpass filters for 495-555 nm (green) and LP650 nm (red) light. Subsequently, the resulting slow write analyzed by integrated software Zeiss LSM DUO. Total and cytoplasmic (caused by digitonin) activity of cathepsins were measured in treated digitonin samples using probe zFR-AFC (Enzyme System Products), as described in this field.

Analysis of cell viability. Cell density was assessed by analysis of the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylethanone bromide (MTT, SIGMA-Aldrich), essentially as described in this field. Apoptosis-like cell death was evaluated by the of krasivaya cells 0.05 mg/ml hagstom 33342 (Molecular Probes) and counting cells with condensed nuclei on an inverted fluorescent microscope (Olympus IX-70 (filter U-MWU 330-385 nm). For each experiment we calculated the minimum of eight randomly selected areas.

Immunological analysis and microscopy. Used primary antibodies included mouse monoclonal antibodies against Hsp70 (2H9; courtesy of Boris Margulis, Russian Academy of Sciences, St. Petersburg, Russia), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Biogenesis), BMP (6C4), lysosomal integral membrane protein-1 (H5C6; developed by J. Thomas August, and James E.K. Hildreth and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, USA). Proteins separated in 10% SDS-PAGE and transferred to nitrocellulose membrane, was determined using the indicated primary antibodies, suitable conjugated with horseradish peroxidase secondary antibodies from Dako, reagents for Western blotting ECL (Amersham) and device for reading a fluorescent image (LAS-1000Plus, Fujifilm). For immunocytochemistry were used conjugated with Alexa Fluor®-576 or Alexa Fluor®-488 secondary antibodies. For visualization of lysosomal compartment in living cells was used Lysotracker Red®. Fluorescent pictures were obtained using a laser scanning microscope Zeiss Axiovert 100M. Quantitative analysis of Lysotracker and delayed entry to the integrity of lysosomes was carried out using the system Zeiss LSM LIVE DUO.

Fluorescence spectra of tryptophan and scattering l is poomani light at an angle of 90°. Fluorescence spectra of tryptophan (RFI) and the scattering of liposomes light at an angle of 90° (RSI) was analyzed in HEPES buffer (20 mm HEPES, 0.1 mm EDTA, pH to 7.4 or 6.0, as indicated) using LUV containing these lipids, essentially as described in this field. For RFI in 10 mm aliquots were added LUV and spectra were recorded after 20 min stabilization period. For RSI 0.12 nm aliquots were added to recombinant proteins.

Surface plasmon resonance (BIAcore). To obtain LUV mixture of lipids containing 10 mol.% sphingomyelin, 50 mol.% phosphatidylcholine, 20 mol.% cholesterol and 20 mol.% BMP, dissolved in organic solvents, and reduced in a stream of argon and rehydratable buffer Tris/HCI (pH of 7.4). The mixture was freeze-thawed nine times in liquid nitrogen and then in an incubator at 37°C. After ultrasonic washing for 15 min the mixture 21 times was passed through a polycarbonate membrane with a pore diameter of 100 nm. Measurement of surface plasmon resonance was performed using the BIAcore system 2000 at 25°C. LUV (total lipid concentration 0.1 mm) immobilizerpower on the surface of sensor chip L1 (BIAcore) in PBS (load buffer). As a rolling buffer was used a buffer of sodium acetate (50 mm, pH 4.5). As a control directly on the surface of liposomes was injected acidic to sphingomyelinase (0.2 μm, 60 μl in the movable buffer). Received unit response between 410RU-5250RU. Interesting protein was injected into a movable buffer at a flow rate of 20 μl/min at the indicated concentrations. When after rHsp70 should rASM, rASM was added to 180 seconds after 10 min of stage dissociation rHsp70 followed another 10-min stage of dissociation.

Molecular modeling. Analysis of the primary structure and molecular modeling was performed using software available on the Expert Protein Analysis System (EXPaSy), proteomics server of the Swiss Institute of Bioinformatics (http://expasy.org/). Molecular modeling was performed on the basis of the crystal structure of the ATPase domain of Hsp70 human (pdb code: 1S3X) and substrate-binding domain of Hsc70 person (pdb code: 7HSC) using DeepView-Swiss PDB Viewer. The surface model was based on the Coulomb interaction at pH 7.0 using a solvent with a dielectric constant of 80 (H2O).

The statistical analysis. Statistical analysis was performed using bilateral paired t-test t-test to evaluate the null hypothesis. A basic level of statistical significance was defined as 5% of all groups of data were tested using the criterion F on the comparability of their deviations. All statistical analysis was performed at least three independent experiments.

Example 3: Effect of benzyl alcohol on lysosomal storage disorders

In Examples 2 and 3 shows that Hs70 have lysosomal-stabilizing effect through interaction with BMP. For assessment does the same action when treating the cells with a chemical inducer of Hsp70, the fibroblasts of a patient with Niemann-pick disease type A (NPDA) was treated with low molecular weight inducer of Hsp70; benzyl alcohol (BA). The results are shown in Fig. 11. First, cells NPDA was treated with increasing doses of BA(0, 20, 30, 35, 40, 45 mm), literally and analyzed by Western blotting. The same amount of protein was added to each well. The expression of Hsp70 protein were evaluated for each condition, and showed that BA induces the expression of Hsp70, depending on the dosage (primary antibody: Stressgen SPA-810, specific for Hsp70). Then evaluated the stability of lysosomes NPDA Götz after treatment 40 mm BA using the same methods as described in example 2. Observed increased stability of lysosomes in response to BA. Ultimately, evaluated the cross-sectional area of lysosomes in cells NPDA Götz after treatment 40 mm BA using the same methods as described in example 2. Observed reduction in pathology.

1. The use of Hsp70, or a functional fragment or variant having from about 99.9 to 90% homology with the wild-type protein with retention of activity, where Hsp70, or a functional fragment or variant contains the ATPase domain of Hsp70, for the treatment of lysosomal storage disorders.

2. The use according to claim 1, where the specified Hsp70, or options the national fragment, or has 100% homology with Hsp70 protein wild-type.

3. The use according to claim 1, where the specified Hsp70, or a functional fragment or variant has from about 99.9 to 95% homology with the protein of the wild type.

4. The use according to claim 1, where the specified Hsp70, or a functional fragment or variant is a Hsp70 wild-type.

5. The use according to claim 1, where the specified Hsp70 is a full-Hsp70.

6. The use according to claim 1, where the specified Hsp70, or a functional fragment or variant contains a tryptophan at amino acid position 90 ATPase domain of Hsp70.

7. The use according to claim 1, where the specified lysosomal disease accumulation selected from the group consisting of disease, Niemann-pick, Farber disease, Krabbe disease, Fabry disease, Gaucher disease, sialidosis, metachromatic leukodystrophy and deficit sabotinov.

8. The use according to claim 1, where the specified lysosomal disease accumulation is Niemann-pick disease type a or type C.

9. The use according to claim 1, where the specified lysosomal disease accumulation is a Niemann-pick disease type C or type D.

10. The use according to claim 1, where the specified lysosomal disease accumulation is a disease of accumulation of lipids (or lipidosis).

11. The use of claim 10, where the disease is the accumulation of lipids selected from the group consisting of sphingolipidoses, gangliosidoses and Le is magistrati.

12. The use according to claim 1, where the specified lysosomal disease accumulation selected from the group consisting of mucopolysaccharidoses, disease, accumulation of glycoproteins (or glycoproteins) and mucolipidosis.

13. The use according to claim 1, where the specified lysosomal disease accumulation is characterized as having a residual enzymatic activity of the defective enzyme involved in the pathology of the disease.

14. Use item 13, where the specified residual enzymatic activity is in the range from 0.1% to 50%.

15. The use according to claim 1, where the specified Hsp70, or a functional fragment or variant is capable of interacting with BMP (bis(monoacylglycero)phosphate).

16. The use according to claim 1, where the specified Hsp70, or a functional fragment or variant is introduced in combination with at least one other method of therapy of lysosomal storage disorders.

17. The application of article 16, where at least one other method of therapy of lysosomal storage disorders selected from the group consisting of substitution therapy enzyme (ERT), therapy of the lower substrate, transplantation, corticosteroids, anesthetics, symptomatic and supportive therapy, inductors or conductorof Hsp70.

18. Use 17 where the specified replacement therapy enzyme selected from the group consisting of imiglucerase, miglo the Tata, agalsidase beta and agalsidase alpha.

19. The application 17, where the specified transplant is selected from the group consisting of bone marrow transplantation, transplantation of umbilical cord blood and stem cell transplant.

20. Drug representing Hsp70, or a functional fragment or variant having from about 99.9 to 90% homology with the wild-type protein with retention of activity, where Hsp70, or a functional fragment or variant contains the ATPase domain of Hsp70, for the treatment of lysosomal storage disorders.

21. The use of Hsp70, or a functional fragment or variant having from about 99.9 to 90% homology with the wild-type protein with retention of activity, where Hsp70, or a functional fragment or variant contains the ATPase domain of Hsp70, for the manufacture of a medicinal product for the treatment of lysosomal storage disorders.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: in order to obtain a biologically active substance after electric stimulation of chicken heads, blood is taken and incubated in the mode with voltage of 100-120 V, current force of 3-5 A during 3-5 seconds. Then, a serum is separated. A polypeptide fraction with molecular weight of 150-170 kilodalton is extracted from it. The obtained peptide fraction is lyophilised and sterilised by irradiation in a linear electron accelerator with a dose of 28-32 kGy. The invention can be used to obtain a remedy providing stimulation of proliferation of non-differentiated cells of human marrow, which can be used at treatment of oncohematological and autoimmune diseases.

EFFECT: preserving ability of non-differentiated cells of marrow to differentiation in a patient organism owing to excluding an inhibiting effect of an agent that stimulates proliferation; increasing a proliferation speed of non-differentiated cells of human marrow; reducing complications determined by immunogenic activity of the agent that stimulates proliferation, at use of an agent obtained by means of the proposed method; cheapening, simplifying the method owing to using available raw material.

2 tbl, 3 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and molecular genetics. There are disclosed the versions of disintegrin and their use in pharmacology. The version of disintegrin involves the recovered polypeptide which possesses αvβ3 receptor antagonist activity and substantially lower ability to block αIIbβ3 and/or α5β1 integrin receptor than wild-type disintegrin. The polypeptide is coded by a modified nucleotide sequence of disintegrin possessing lower ability to bind to αIIbβ3 and/or α5β1 integrin than wild-type disintegrin.

EFFECT: invention may be used for treating and/or preventing the αvβ3 integrin-associated diseases in a mammal, which include without limitation osteoporosis, bone tumor or cancer growth, angiogenesis-related tumor growth and metastasis, tumor metastasis in bone, malignancy-induced hypercalcemia, angiogenesis-related eye diseases, Paget's disease, rheumatoid arthritis, and osteoarthritis.

23 cl, 16 dwg, 1 tbl, 25 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to nicotinic acetylcholine receptor blocking biologically active peptides which can find application in biochemistry, biotechnology and medicine. The peptide azemyopsin has an amino acid sequence: H2N-Asp1-Asn2-Trp3-Trp4-Pro5-Lys6-Pro7-Pro8-His9-Gln10-Gly11 Pro12-Arg13-Pro14-Pro15-Arg16-Pro17-Arg18-Pro19-Lys20-Pro21-COX wherein X is a group OH or NH2, and has an ability to react selectively with nicotine muscarinic receptors. The peptide may be used an a base for preparing myorelaxants selectively blocking muscarinic receptors.

EFFECT: invention presents a new type of natural toxins reacting with nicotine receptors and containing no disulphide bonds.

2 cl, 5 dwg, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to applications of a fused polypeptide containing (i) MeCP2 protein and (ii) a protein transduction domain for preparing a drug for treating and/or preventing a developmentary neuropathy, and also to the pharmaceutical compositions containing such polypeptide, and a method of treating the developmentary neuropathy.

EFFECT: invention allows compensating MeCP2 function loss in damaged nerve cells.

11 cl, 8 dwg, 2 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: invention concerns a polypeptide exhibiting activity of telomerase reverse transcriptase (TERT), molecules of nucleic acid coding such polypeptide, a plasmid vector carrying said molecule of nucleic acid, an immortalised cell, transfected said molecule of nucleic acid, and applying such cell for producing an end substance.

EFFECT: invention allows creating the avian immortalised cell lines to be useable for making any end substance.

27 cl, 13 dwg, 9 ex

FIELD: medicine.

SUBSTANCE: invention may find application in methodology for creation of biologically active antibodies joined with live cell, address delivery of medicinal agents to target cells. Proposed method includes cloning of the second extracellular loop E2 Cx43, representing a fragment of connexin-43 Q173 -1208 with aminoacid sequence QWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTI, into two plasmid vectors pCBDQ and pHPMLQ. Then transformation of E.coli cells is carried out with produced plasmid DNAs. Then transformed cells are cultivated up to optical density of 0.7-0.9. Then plasmid expression 0.9-1.1 mM is induced with isopropyl thiogalactoside. Afterwards recombinant fused proteins and their release in presence of 7.9-8.1 M of urea by method of chromatography at Ni-NTA agarose.

EFFECT: proposed invention makes it possible to develop method to produce immunogenic recombinant extracellular fragment of rat connexin-43 in composition of highly immunogenic chimeric protein suitable to develop monoclonal antibodies.

2 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: substance of polypeptide nature with molecular weight 14350 Da, with N-end amino acid sequence, homologous phospholipase A2 of snake venom, and possessing properties of direct thrombin inhibitor of mixed type as well as antiproliferative action is separated of cobra venom Naja haje by three-stage liquid chromatography.

EFFECT: invention enables to produce selective direct thrombin inhibitor, possessing antiproliferative action.

1 tbl, 3 dwg, 10 ex

The invention relates to pharmacology, and in particular to means, inhibiting the transport of cationic amino acids, and reveals rabbit anticigarette induced against T-cell protein encoded by the genome of the IRU-2, and pharmaceutical composition comprising the specified anticigarette

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, more specifically to a pharmaceutical composition possessing antithrombotic, thrombolytic, immunomodulatory, anti-inflammatory action, normalising lipid and carbohydrate metabolism, more specifically to the pharmaceutical composition of the substance Pijavitum (hereinafter referred to Pijavitum) made from lyophilised medicinal leech. The above pharmaceutical composition is presented in the form of an enteric coated tablet.

EFFECT: coating prevents the active ingredients of Pijavitum from destruction under action of the enzymes and acid medium of the stomach.

FIELD: medicine.

SUBSTANCE: invention relates to a method of treating or reducing insulin resistance in susceptible warm-blooded animals, including people. Method includes introduction of a selective estrogen receptor modulator (SERM).

EFFECT: described is SERM combination with an amount of estrogen or a precursor of a sexual steroid hormone, selected from a group, consisting of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androst-5-en-3b,17b-diol and compounds, converted in vivo into one of the said precursors or estrogen.

2 cl, 13 ex, 13 tbl, 8 dwg

FIELD: chemistry, pharmacology.

SUBSTANCE: invention relates to medications, capable of inhibiting Na+/H+-exchange (NHE-exchangers, NHE inhibitors).

EFFECT: increased efficiency of inhibitors.

3 cl, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medication for treatment or prevention of disease, which developed on the basis of structural and/or functional, and/or compositional changes of lipids in cell membranes, selected from cancer, vascular diseases, inflammatory diseases, metabolic diseases, obesity and excessive body weight, neurological or neurodegenerative disorders, which represents compound of formula COOR1-CHR2-(CH2)a-(CH=CH-CH2)b-(CH2)c-CH3 (I) or its pharmaceutically acceptable salts and derivatives, selected from esters, ethers, alkyl, acyl, phosphate, sulfate, ethyl, methyl or propyl; in which a and c can have independent values from 0 to 7; b can have independent values from 2 to 7, where R1 is selected from the following radicals: H, Na, K, CH3O, CH3-CH2O and OPO(O-CH2-CH3)2, and R2 is selected from the following radicals: OH, OCH3, O-CH3COOH, CH3, Cl, CH2OH, OPO(O-CH2-CH3)2, NOH, F, HCOO and N(OCH2CH3)2.Invention also relates to application of formula (I) compound and pharmaceutical composition, which contains it.

EFFECT: medications, based on claimed compound, are more efficient than medications of preceding level of technology.

22 cl, 7 dwg, 16 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to compounds, represented by formula (I) , where X1 and X2 independently represent CH or N; ring U represents benzene ring, pyrazole ring, 1,2,4-oxadiazole ring, 1,2,4-thiadiazole ring, isothiazole ring, oxazole ring, pyridine ring, thiazole ring or thiophene ring, m represents integer number, which has values from 0 to 1; n represents integer number, which has values from 0 to 3; R1 represents hydroxygroup or C1-6 alkyl; R2 represents any of (1)-(3): (1) halogen atom; (2) hydroxygroup; (3) C1-6 alkyl or C1-6 alkoxy, each of which can independently contain any group, selected from group of substituents α; group of substituents α includes fluorine atom and hydroxygroup, or its pharmaceutically acceptable salt. Invention also relates to pharmaceutical composition, possessing inhibiting activity with respect to xanthenes oxidase, including formula (I) compound or its pharmaceutically acceptable salt as active ingredient.

EFFECT: derivative, which contains condensed ring structure, intended as means for prevention and treatment of disease, associated with abnormal level of uric acid in serum.

15 cl, 11 tbl, 126 ex

FIELD: medicine.

SUBSTANCE: method involves a combination of peloid applications and ultrasonic exposure. Peloid is applied on anterolateral thighs and an anterior abdominal wall. The applications are alternated every second day. Peloid is silt sulphide mud of Melkovodnenskiy deposit. The mud is applied in layer 10 mm thick at temperature 34-36°C. The applications are exposed to ultrasound at frequency 880 kHz in a continuous mode. The applications of the anterior abdominal wall are exposed to ultrasound at intensity 0.2-0.4 Wt/cm2 for 5 - 10 minutes. The anterolateral thighs are exposed to ultrasound at intensity 0.4-0.6 Wt/cm2, for 6-8 minutes for each thigh. Upon the completion of the procedure, the patient is covered with a hydrophobic tissue and left for 10 minutes. The therapeutic course is 10 procedures.

EFFECT: reducing obesity effectively by exposing on the hormonal activity of fat tissue, maintains the results obtained within three months.

2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to versions of a compound of formula where R1 is a hydrogen atom; R2 is a lower alkyl group; P is H; , where P1, P2 and P3 are identical or different and are selected from a hydrogen atom, a lower alkyl group and a C14-C22 alkenyl group substituted with a lower alkyl group; or where P1 is an alkenyl group, and each of P2 and P3 is a hydrogen atom; and Y is a C14-C22 alkenyl group with at least one double bond having a Z-configuration, and having a first double bond at the third carbon-carbon bond from the omega (ω)-end of the carbon chain, capable of lowering the level of triglycerides and cholesterol, a pharmaceutical or lipid composition based on the disclosed compounds, as well as use (versions) of the disclosed compounds.

EFFECT: high efficiency of using compounds.

32 cl, 6 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics, and aims at the prevention and treatment of hypovitaminosis and the normalisation of metabolism. The drug preparation contains vitamin A, vitamin D3, vitamin E, vitamin C; a selenium compound is presented by DAFS-25 in the following ratio of the ingredients in 1 l of the solution: vitamin A - 25.0-35.0 ml, vitamin D3 - 0.03-0.05 ml, vitamin E - 55.0-65.0 g, vitamin C - 90.0-110.0 g, DAFS-25 - 0.2-0.4 g, polysorbate-80 - 190.0-210.0 ml, 2-pyrrolidone 39.0-41.0 ml, distilled water - up to one litre.

EFFECT: using the declared invention enables increasing the immune status in poultry, normalising the antioxidant and detoxifying systems, improving the livability, egg production and meat production along with reducing the feed consumption per a unit of product.

3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to cardiology, and may be used for treating and preventing arterial hypertension with metabolic syndrome. That is ensured by adding the food ration with the functional foodstuff 'Samarskiy Zdorovyak' No 61 in a min daily dose of 33.3 g per one intake - with breakfast or lunch or dinner with underlying drug-induced therapy.

EFFECT: enabled treatment and prevention of arterial hypertension with metabolic syndrome

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to medicine. A pharmaceutical formulation for the treating diseases associated with endothelial dysfunction contains an active ingredient presented by a methyl pyridine derivative - 1.0-6.0 wt %; purine - 10.0-80.0 wt % and additive agents - the rest. The active substance is presented by compounds of a group: 3 -(N,N-dimethyl carbamoyloxy)-2-ethyl-6-methylpyridinium succinate, 3-methylpyridinium succinate, 2-ethyl-6-methyl-3-hydroxypyridinium hydrochloride, 6-trichloromethyl-2-chloropyridine (nitrapyrin), 2-ethyl-6-methyl-3-hydroxypyridine succinate. Purine is presented by inosine, adenosine, hypoxanthine. The pharmaceutical formulation may be presented in the form of injections, lyophilisate, solid capsules, tablets and suppositories.

EFFECT: formulation according to the invention provides creating the stable drug dosage form which considerably exceeds the existing analogues in pharmacodynamics activity on the endothelial dysfunction and toxicological properties.

4 cl, 4 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to the field of biotechnology. Claimed is a method of purification of a factor, contributing to wound healing, which represents a hepatocyte growth factor (HGF). All stages of purification are carried out in the presence of antithrombin III (AT-III). In accordance with the claimed method carried out are: defrosting of the frozen HGF-containing source and removal of sediment from the defrosted source. After that, the obtained solution, which contains a supernatant and AT-III, is brought in contact with a carrier for affinity chromatography on an immobilised heparin. Then, the solution is separated from the carrier for affinity chromatography. The carrier is brought in contact with a desorption buffer with ionic strength sufficient for HGF desorption. The desorption buffer, containing HGF, AT-III and histidine-rich glycoprotein (HRGP) is collected. Also claimed are wound-healing compositions, which contain HGF, AT-III and/or HRGP, purified by the claimed method.

EFFECT: inventions make it possible to increase step-by-step output of the hepatocyte growth factor, with the hepatocyte growth factor being concentrated in eluate in the presence of AT-III.

26 cl, 2 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine and can be used for modification of eating behaviour in a subject. For this purpose peripheral introduction of PYY in an amount, efficient for achieving physiological levels of PYY3-36 in blood, plasma and serum, determined after food intake, is performed; or performed is peripheral introduction of PYY agonist in an amount, efficient for imitation of physiological levels of PYY3-36 in blood, plasma or serum, determined after food intake with modification of eating behaviour, by reducing intake of calories, food consumption or appetite reduction or increase of energy consumption in the subject. Also claimed is application of PYY or its agonist as an active ingredient in production of medication.

EFFECT: group of inventions provides weight reduction in the subject due to reduction of intake of calories, appetite reduction or increase of energy consumption in the subject.

50 cl, 9 dwg, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to compositions and methods for prevention or treatment of renal fibrosis in an individual. Particularly, the method for prevention or treatment of renal fibrosis in an individual involves administering a composition containing a pharmaceutically effective amount of a vasoactive intestinal peptide (VIP) or one or more functional VIP fragments specified in VIP(10-28), VIP(4-12), VIP(4-16), VIP(4-20), VIP(4-24), VIP(6-10), VIP(6-12), VIP(6-16), VIP(6-20), VIP(6-24) or conventional substitutes thereof into the individual exposed to a risk of developing renal fibrosis or suffering from renal fibrosis.

EFFECT: using the group of invention enables preventing or delaying developing renal fibrosis in the individual more effectively.

15 cl, 4 dwg, 2 ex

Up!