Application of biotin-conjugated polypeptide for protein phosphorylating enzyme analysis

FIELD: medicine.

SUBSTANCE: there is offered a method to assess of enzyme's ability to the level of phosphorylation of polypeptide that implies a reaction of the analysed enzyme and a substratum presented with a biotin-conjugated fragment of 516 to 777 residues of a human insulin 1 receptor substratum (hIRS-1-p30), binding of the reaction product and immobilised streptavidin and detection of the level of phosphorylation by antibodies specific to the phosphorylated polypeptide residues.

EFFECT: according to the invention, the method allows identifying tyrosine and serine proteinases and can be taken as a basis of a test system for new modulators of their activity.

9 cl, 8 dwg, 4 ex

 

The invention relates to the use of the polypeptide to determine the ability of the enzyme to modulate the level of phosphorylation of the polypeptide. Other aspects of the invention relate to a method of determining such activity, as well as to a method of identifying substances which modify the ability of the enzyme.

Insulin is a protein hormone that affects a large number of ways of growth and metabolism due to the fact that it binds to the insulin receptor and thus activates its internal tyrosinekinase. This event leads to the phosphorylation of many proteins that can communicate with the insulin receptor (IR) on specific tyrosine residues. It thus phosphorylated proteins is also a family of proteins substrate of the insulin receptor (IRS).

The substrate of the insulin receptor 1 (IRS-1) is a cellular protein that can be phosphorylated by many kinases (trainstation or serine/travispacific protein kinase) on residues of tyrosine and/or serine and/or threonine. However, depending on the enzyme, probably specifically fosfauriliruyutza different residues tyrosine or serine/threonine. In addition to phosphorylation by tyrosinekinase, as, for example, in the case of the insulin receptor (White, 2002), the receptor for IGF-1 (White, 2002) or JAK 1/2 (Thirone et al., 1999), IRS-1, as is known, fosfauriliruetsa also serine/tyrosine kinases, for example, kinases from the family of CSWs (Schmitz-Peiffer, 2002), the complex of inhibitor-Kappa-b-kinase (Gao et al., 2002), C-Jun NH(2)-terminal kinase (JNK), Aguirre et al., 2000), protein kinase a (Sun et al., 1991), mitogen activated protein kinase (Mothe et al., 1996), protein kinase B (Paz et al., 1999), caseinline (Tanasijevic et al., 1993), glikogensintetazy-beta (Eldar-Finkelmann et al., 1997), AMR-activated kinase (Jacobsen et al., 2001) or phosphoinositol-3-kinase (Freund et al., 1995). The IRS molecules are key molecules in the path of signal transduction of insulin and plays a Central role in maintaining cellular functions such as growth, survival and metabolism. Phosphorylated IRS proteins thus serve as a “docking” proteins with multiple docking sites of the insulin receptor and the complex network of intracellular signaling molecules with the so-called 2 homologous domains of complex recognition signal (SRC) (SH2 domains). Activation of these Sh2-domain proteins in this activates specific signaling cascades, which again leads to activation of various effectors, which are located further down in the signaling cascade that ultimately results in mediating insulin signal in an extensive series of other intracellular signaling cascades (for review, see White, 2002).

The IRS refers to a group of phosphoproteins mass 160-185 KD is, which serve as a substrate of the insulin receptor. Famous four members of the family of the IRS (IRS-1, IRS-2, IRS-3 and IRS-4). They differ in tissue distribution, subcellular localization, specific development expression, the type of binding to the insulin receptor and the genus of SH2-proteins with which they interact. Four members of the family of the IRS on its primary protein structure is very similar: all have the amino(N)-limit pleckstrin-homology domain (PH domain)that binds to membrane phospholipids, phosphotyrosinespecific domain (PTB domain), carboxyl (C) the right end is attached to the PH domain and mediates the recognition sequence Asp-Pro-Glu of phosphotyrosine (NPEpY), which is localized in ikolomani region of the beta-subunit of the insulin receptor. Further, they have somewhat less strongly conservative C-terminal part, which has a different potential explanation of tyrosine phosphorylation, which may be contacted special containing SH2-domains proteins.

IRS-1 contains 21 possible phosphorylation site of tyrosine, some of which are localized in the levels of amino acid sequences, which may contact containing SH2-domain proteins. IRS-1, then, contains 30 potential sites of phosphorylation of serine/threonine motifs that m is able to be recognized by various kinases, such as, for example, kinases from the family of CSWs (Schmitz-Peiffer, 2002), the complex of inhibitor-Kappa-b-kinase (Gao et al., 2002), C-Jun NH(2)-terminal kinase (JNK), Aguirre et al., 2000), protein kinase a (Sun et al., 1991), mitogen activated protein kinase (Mothe et al., 1996), protein kinase B (Paz et al., 1999), caseinline (Tanasijevic et al., 1993), glikogensintetazy-beta (Eldar-Finkelmann et al., 1997), AMR-activated kinase (Jacobsen et al., 2001) or phosphoinositol-3-kinase (PI3-kinase, Freund et al., 1995). Inhibiting effects on the signaling pathway of insulin receptor can at least partially be attributed to the recently discovered role of phosphorylation of serine/threonine IRS-1, which is associated with the deterioration of interaction with the insulin receptor and/or a decrease in the tyrosine phosphorylation of IRS-1 and/or deterioration of the subsequent interaction with signaling proteins that can contact tyrosinosolvens IRS-1 (for review, see White, 2002). For various kinases, such as, for example, kinases from the family of CSWs (Schmitz-Peiffer, 2002), the complex of inhibitor-Kappa-b-kinase (Gao et al., 2002), C-Jun NH(2)-terminal kinase (JNK), Aguirre et al., 2000), protein kinase a (Sun et al., 1991), mitogen activated protein kinase (Mothe et al., 1996), protein kinase B (Paz et al., 1999), caseinline (Tanasijevic et al., 1993), glikogensintetazy-beta (Eldar-Finkelmann et al., 1997), AMR-activated kinase (Jacobsen et al., 2001) or phosphoinositol-3-kinase (PI3-kinase, Freund et al., 1995), still see the gli show that in vitro they directly phosphorylate IRS-1. In each case, increased kinase activity in intact cells inhibits the activity of the pathway of signal transduction of insulin. Further, in vitro phosphorylation of IRS-1 on serine residues/threonine, according to some studies, is in direct communication with the reduced tyrosine phosphorylation by insulin receptor (Le Marchand-Brustel, 1999)). The sequence of IRS-1, -2, -3 and -4 public. Encoding polynucleotide sequence and related protein sequences of these genes in the database at the NCBI nucleotide are numbered NM_005544 (IRS-1 hs), XM:007095 (IRS-2 hs), NM:03274 (IRS-3 rat), NM:003604 (IRS-4 hs). NCBI means the international information center for biotechnology (mailing address: National Center for Biotechnology Information, National Library of Medicine, Building 38A, Bethesda, MD 20894, USA; Web address: www.ncbi.nhm.nih.gov). Cloning of the IRS-1 gene is described, in particular, Araki et al., 1993 and Siemeister et al., 1996; cloning IRS-2, -3 and -4 described by Araki et al., 1994, Lavan et al., A and Lavan et al., 1997b.

To determine the ability and measuring the activity of various kinases in the case of phosphorylation of IRS-1 in the prior art there are known various methods, which are based either on radioactive detection methods (e.g., transfer from radioactive labeled phosphate on the substrate)or non-radioactive detection methods.

So, know the definition fosfauriliruet the Oia IRS-1 over the entire length IRS-1 protein, its fragments or peptides that have at least one site of phosphorylation, with the help of the way in which by incubation with radioactively labeled ATP and test kinase, depending on the ability of the kinase to fosforilirovanii IRS-1, radioactive phosphate residues are transferred to IRS-1. Then carry out a chromatographic or electrophoretic allocation of IRS-1 and detection of the amount transferred phosphate by flow scintillation or autoradiography (as described for full protein IRS-1 and glikogensintetazy-3-beta according to Eldar-Finkelman et al., 1997, for a fragment of IRS-1 (amino acids 516-777) and insulin receptor, IGF receptor-1 or recombinant kinase of the insulin receptor according Siemeister et al., 1995, or peptide IRS-1 (amino acids 601-616) using cell lysates, which contain activated protein kinase family of CSWs, according to De Fea et al., 1997). Further, according to Siemeister et al., 1995, the ability of fragments of IRS-1, for example, of a fragment of IRS-1 (amino acids 516-777) and insulin receptor to fosforilirovanii the receptor for IGF-1 or recombinant kinase of the insulin receptor by incubation with radioactive labeled ATP (adenosine-triphosphate), applying dropwise substrate to a positively charged membrane (nitrocellulose or similar material), washing and detection of the bound radioactively m is Canoga substrate using autoradiography or measurement of radiation.

Incubation of the biotinylated peptide IRS-1 (amino acids 601-616) with radioactively labeled ATP, application dropwise substrate on streptavidin coated membrane, washing and detection of the bound radioactively labeled substrate using autoradiography or measurement of radiation - this is another way to measure the ability of kinases to fosforilirovanii IRS-1 (see De Fea et al., 1997).

The disadvantage of the above-described radioactive methods of testing are obvious, as the handling of radioactivity makes a significant danger, is very expensive and thus not suitable in particular for high-performance methods (HTS-ways).

The disadvantage of the above are based on the use of short-chain peptides of the ways is that these peptides have a unfavorable kinetic constants (Vmax, Km) and to the same spatial structure of peptides is very different from the physiological enzyme substrates. This is evident, first, in a completely different conformation, so that there are no specific biological space that give specificity to the interaction of the enzyme-substrate, resulting in either a missing recognition (and, thus, the modification), or nonspecific recognition (and, thus, the modification), and to enom account come to incorrect results. In addition, the peptides because of their short chain have only one or a small number of sites of phosphorylation, so that research modifications by phosphorylation of a specific substrate by different enzymes require different peptides. Also the result is again a high value and only the relative applicability of the methods in the framework of the HTS.

The invention therefore consists in finding options for determining the activity of fosfauriliruetsa and/or dephosphorylated protein enzymes, which would not have the above disadvantages.

This problem is solved by applying polypeptide (Def GGs for peptide) to determine the ability of the enzyme and its functional fragment or a derivative thereof to modulate the level of phosphorylation of the polypeptide, wherein the polypeptide is biotinylated.

The invention is based on the results of studies in which it was unexpectedly found that in the case of polypeptides or proteins of full length does not occur any steric hindrance in relation to the binding of Biotin by streptavidin, biotinylation also not interferes with the test substrate phosphorylation by kinases.

The term “polypeptide” as part of the present invention involve the molecule, containing the Yu-related peptide bonded amino acids, which includes at least 50 linearly related in this way amino acids. Shorter molecules of this kind is known as peptides. The name “protein” refers to molecules that include at least one polypeptide chain, however, can also consist of several associated with each other or linked polypeptide chains. The term “protein” encompasses, therefore, the name “polypeptide”.

According to a preferred variant implementation of the various aspects of the present invention the polypeptide has a length of 50 amino acids and more preferably 50-300 amino acids.

According to a further preferred variant implementation of the various aspects of the invention the polypeptide has a mass of 1 kDa and more preferably 1-100 kDa and, particularly preferably, 10-50 kDa.

As a substrate of the enzyme understand any molecule that is suitable for modification by the enzyme. Natural substrates in the framework of the present invention are molecules that have a structure such that they are physiological or pathological environment in its natural state, and the ability to undergo modifications of the corresponding enzyme.

Modulation of the level of phosphorylation by the enzyme called type modification of the substrate through the om enzyme, in the case of which at least one phosphate group is transferred to the substrate or removed from it. Significant according to the present invention, the enzymes have therefore the ability to catalyze the and/or different reaction. They, thus, at least have the ability kinases and/or phosphatases, moreover, however, have other enzymatic properties (e.g. properties of proteases and so on). Different categories of enzymes and their properties are sufficiently known to the expert.

Functional fragment of the enzyme in this case is any fragment of the enzyme (hence, reduced correspondingly shortened in relation to existing natural form of the molecule), which still has the ability to modulate the level of phosphorylation of at least one polypeptide. The term “functional derivative” of the enzyme at this covers any type of modification of the enzyme in relation to existing in a natural condition form, which does not represent any shortened form, and derived enzyme still has the ability to modulate the level of phosphorylation of at least one polypeptide. The present invention relates also to the functional derivatives of the fragments of enzymes that can modulate the level of factorily the Finance of at least one polypeptide.

Determining the ability of the enzyme to modulate the level of phosphorylation of the polypeptide when this can be done both qualitatively and quantitatively (hence, in the form of measurements determining the number).

Proposed according to the invention, the application has the advantage thus achieved results on the basis of the length of the used substrates are more informative, as they encompass tertiary structure, which is more in line with the physiological data. Moreover, polypeptides used, in contrast to the known from the prior art peptides, have good kinetic constants (for example, in the case of IRS-1: Km is 19 μm, compared with peptides: >200 μm; see Siemeister et al., 1995) and in the analysis of substrates with multiple phosphorylation sites, you need only one substrate, with which you can define, for example, the ability of various enzymes.

The preferred embodiment of the invention relates to the application, which determines the ability of the enzyme to fosforilirovanii polypeptide.

Particularly suitable types of enzymes with kinase activity for the various aspects of the present invention are serine/threonine - or tyrosine kinase. Particularly suitable examples of kinases include, in private the tee, the insulin receptor, the IGF-1 receptors, trK-receptor, EGF-receptor, caseinline II, members of the family of protein kinase C, protein kinase/Akt, mitogen activated the protein kinase (MAP-kinase), GSK-3 beta, ERK 1/2, IKK-beta kinase, AMR-kinase, PI3-kinase, or JNK. Moreover, it is proposed according to the invention use is also suitable to measure the ability of the enzyme to dephosphorylate polypeptide.

Another aspect of the invention relates to a method for determining the ability of the enzyme and its functional fragment or a derivative thereof to modulate the level of phosphorylation of the biotinylated polypeptide. Suitable methods of determining the level of phosphorylation of biotinylated polypeptides include, for example, the methods, which are known to be suitable for determining the level of phosphorylation of peptides with a short chain. They are known to the expert.

According to a preferred variant of the proposed according to the invention the method is a method in which the ability of the enzyme and its functional fragment or derivative to fosforilirovanii polypeptide determined by carrying out the following stages:

an introduction to a contact of the enzyme or a functional fragment or derivative with the biotinylated polypeptide and the initiation of the reaction of phosphorylation in a suitable reaction is Oh mixture;

b) introduction to contact the reaction mixture with an associated media tool that is able to bind biotinylated polypeptide;

(C) determining the level of phosphorylation that is associated with the tool polypeptide.

Another preferred embodiment of the invention relates to a method for determining the ability of the enzyme and its functional fragment or derivative to dephosphorylate polypeptide by carrying out the following stages:

an introduction to a contact of the enzyme or a functional fragment or derivative with the biotinylated polypeptide, which has at least one phosphate residue, and the initiation of the reaction of phosphorylation in a suitable reaction mixture;

b) introduction to contact the reaction mixture with an associated media tool that is able to bind biotinylated polypeptide;

(C) determining the level of phosphorylation that is associated with the tool polypeptide.

The tool may be any type of molecule or supramolecular associations (e.g., body, or device), which is suitable for binding of the biotinylated polypeptide. Binding may occur with bitenova part or with the polypeptide, and the binding with the polypeptide preferably binding, depending on the level of phospho what helirovanie (for example, binding only in the phosphorylated or nefosfaurilirovanna status in relation to individual or multiple sites of phosphorylation). To preferred variants of implementation with regard to the funds are therefore streptavidin or specific to phosphorus antibodies (i.e. antibodies that can recognize the phosphorylation of specific residues in the polypeptide and specifically bind to a phosphorylated there polypeptide).

Used in the various aspects of the invention, the reaction mixture may be biochemical (i.e., in vitro) or cell type. The biochemical composition of mixtures depend on the “needs” of the studied enzyme, with suitable components and compositions, for example, ATP, buffer to establish the desired pH value of the environment and the desired salt concentration to ensure the activity of the enzyme, however, is known to a competent specialist. In the case of biochemical mixtures enzyme or polypeptide can be in the form of a recombinant and/or in the form of partially or completely purified from natural sources molecules and/or in the form of extracts from biological material, in particular extracts of cells or tissues.

Biological material, in particular, may include: cells, tissue or organ (such as, for example, brain, blood, liver, sales the NSV, kidney, heart, blood vessels), preferably cells of vertebrate animal, including humans, or cells from the cell culture. Used within the cells of the invention include all types of cells, e.g., eukaryotic or prokaryotic single-celled organisms (such as bacteria, for example E. coli or yeast, for example, S.pombe or s.cerevisiae), or cell lines derived from multicellular organisms (such as HeLa, COS, NIH 3T3, CHO etc), preferably mammalian cell lines. Cell tissue organ or system, vertebrate animal, including humans, can be obtained through the conventional technologies, as a blood sample, biopsy tissue or surgical technology. Obtaining such recombinant molecules, purification of naturally existing molecules from cells or tissue and obtaining extracts from cells or tissues sufficiently known to the expert (see also the examples below standard literature).

Cell system suitable for use in various aspects, also known specialist and preferably include isolated cells, which originally come from tissue systems (preferably, from vertebrates, particularly preferably from mammals and, in particular, from a person), especially predpochtitel what about in the form of cultured cell lines; they include, further, single-celled living organisms (eukaryotes or prokaryotes), such as yeast cells or bacteria, in particular in the form of cultured strains.

The media can be any molecules or supramolecular associations (as, for example, body or devices)that are suitable for removal from the reaction mixture peptide associated with them due to the interaction of the Biotin-streptavidin, or marking. Suitable devices are, for example, membranes, plates or body different form (denoted in this case as granules) of various materials, which are sufficiently known in the prior art. The media type will depend on the purpose of method (e.g., diagnostics, detection of biologically active substances or the detection of a new component interaction) and kind of detection, the choice of suitable carriers is within the competence of professional knowledge.

According to one option proposed in the invention of the method to the reaction mixture add radioactively labeled γ32P-ATP and determining the level of phosphorylation is carried out by measuring remaining on the carrier, preferably the membrane or plate, radioactivity after the implementation of at least one stage of leaching. Thus, what you can simply delete is not associated with the streptavidin components of the reaction mixture, including free radioactivity, so that the level of phosphorylation of the polypeptide can simply be defined using immobilizovannoi on the media radioactivity. As a means for binding the biotinylated polypeptide in this case particularly suitable streptavidin.

According to another preferred option proposed in the invention of the method to the reaction mixture antibody (BSP), which can specifically bind to a phosphorylated by the polypeptide. The antibody as in itself can be a tool, and can additionally be added to the tool (and it preferably is not specific to phosphorus antibodies and particularly preferably is a streptavidin). Determining the level of phosphorylation in this case is carried out by determining the number associated with the polypeptide antibodies. Suitable measures for labelling and detection antibodies are known to the specialist. So, on the one hand, it is possible to use suitable way labeled primary antibodies that are directly detected, or are suitable manner labeled secondary antibody directed against the FC portion (immunoglobulin fragment) primary antibodies, which increases the specificity of detection.

The term “antibodies is about” this includes both monoclonal antibodies and also polyclonal antisera, recombinante obtained antibodies and recombinante derived single-chain antibodies. Selecting and obtaining such antibodies are within the competence of professional knowledge, moreover, next, you must specify the following standard literature. Also suitable marking of such antibodies known in the art and include, for example, enzyme labeling, as CIP (calf intestinal phosphatase) or HRP (horseradish peroxidase), fluorochrome molecules, which when excited by exposure to light of a specific wavelength to generate the detected signal, as Texas red, SS3, FITC (fluorescein-isothiocyanate), or known fluorescent proteins. The selection of suitable markings is also consistent with professional knowledge. Suitable labelled or unlabelled primary and secondary antibodies, as well as receive them are known to the prior art, moreover, such antibodies are produced by different manufacturers. Primary and secondary antibodies are produced, for example, firms Becton Dickinson, Pharmacia or Santa Cruz Biotech.

According to a preferred variant of the above method of determining the number associated with the polypeptide antibodies is carried out by determining the amount of antibody remaining on the carrier preferably, the membrane or plate, after the implementation of at least one stage of washing.

According to another preferred option proposed in the invention method, associated with the tool carrier is a first carrier, which includes a first signal generator and binds the polypeptide with the second carrier, which comprises a second signal generator, and two signal generator capable of generating a detected signal, if they are in close proximity to each other, and determining the level of phosphorylation is implemented by definition, is generated if the detected signal. Preferably, in this context, the media represent the granules. Preferably, in this context, the tool is specific to phosphorus antibody. The media thus directly or indirectly may be associated with an antibody, preferably, indirectly through protein a, which is associated with the media. The second media when it directly or indirectly may be associated with a polypeptide, preferably indirectly through bitenova part of the biotinylated polypeptide; this is carried out preferably through associated with native streptavidin.

The signal generator may be any kind of agent or molecule that traveler fit(on) d the I generate a rectified signal; examples include fluorophores, which, after excitation due to the impact of energy emittiert light, which is detected directly or after amplification of the signal by using suitable means known in the art. The signal generator in the context of the present invention are chosen so that the signal is generated only when there is direct interaction tools (hence, preferably, specific to phosphorus antibodies with the polypeptide. Suitable carriers and signal generators are known (for example, in the form of ALPHAScreen™ or LANCE™, Perkin-Elmer Life Sciences; HTRF™, CIS Bio International). For signal generation decisive is the close proximity of the speakers to each other. So very unexpectedly it turned out that this kind of method is suitable for use in combination with the polypeptides, although they are clearly greater than those used in the prior art peptides.

The polypeptide within the various objects of the present invention thus preferably is a natural substrate of the enzyme, preferably neuroretinal length.

Particularly suitable polypeptides include any of the substrates of the kinase of the insulin receptor. Especially preferred if the polypeptide of the family of the substrates of the insulin receptor (IRS), preferably, IRS-1, -2, -3 or -4 and, particularly preferably, IRS-1 is whether its functional fragments or derivatives; therefore, fragments or derivatives (respectively, derived fragments)which are capable of phosphorylation using the insulin receptor. Further, preferably, when the IRS is a human IRS. Further, in the various aspects of the present invention, preferably the use of IRS-1, in particular, human IRS-1 with the sequence according to the identified sequence No. 1, particularly preferably human IRS-1, encoded by the sequence according to the identified sequence No. 2. The above polypeptides it is particularly suitable for determining the ability of the insulin receptor to fosforilirovanii. A preferred fragment of IRS-1 is a polypeptide with the amino acid sequence according to the identified sequence No. 3.

Various aspects of the invention can be applied in various fields. It is appropriate for their use, in particular, in the identification of substances that modify the ability of the enzyme or a functional fragment or a derivative thereof to modulate the level of phosphorylation of the polypeptide. Suitable analytical methods or systems, the so-called quantitative analyses, which measure the activity or concentration, respectively, the share of which in certain target molecules of the organism (so-called “targets”, in this case, the level of phosphorylation of the polypeptide), as parameters the effectiveness of potential biologically active substances known in the prior art. They can represent, for example, quantitative in vitro, therefore, quantitative biochemical analyses using a dedicated or partially dedicated components forming the reaction mixture, with which you can determine the effectiveness of potential biologically active substances. Moreover, it can also be a cellular test system (quantitative analysis), with which one can determine the activity of the protein target (hence, in this case the enzyme) and the effectiveness of potential biologically active substances in relation to the activity of this target molecules in the cellular environment.

Quantitative analysis with this is any type of analytical methods through which you can monitor the biological process. Usually regulate the flow of molecular processes and signaling cascades, part of the physiological pathways of metabolism and mechanisms of regulation, as well as pathological conditions found in cellular or biochemical systems. The pharmacological activity of the biologically active substance can then be identified by its ability oborudova the ü these paths and mechanisms.

For use in the detection of biologically active substances, in particular high-throughput screening of biologically active substances, quantitative analysis should be reproducible and preferably also standard, and reliable (hence, insensitive to external influences). Quantitative analysis should preferably be suitable for high throughput screening of chemicals for their ability to affect the activity of the target molecules. The type of quantitative analysis thus depends, in particular, the kind used to target molecules (for example, the exact type or rod main biochemical molecules, such as, for example, the polypeptide or polynucleotide) and “read out”, that is, the parameters which determine the activity of a target molecule.

In the prior art various types of quantitative analyses and for the most part well as test systems for them are produced by industrial manufacturers.

Suitable for determination of interaction between two components binding assays include, for example, radioisotope or fluorescent quantitative analyses, for example, the analysis of the polarization of fluorescence, test systems which are produced, for example, firms Panvera, Perkin-Elmer Life Sciences (NEN, LANCE™, AlphaScreen™) or CIS Bio International (HTRF™). Far the worse examples of quantitative analyses include cellular assays in which case cell line, upon request, stable (inducible or constitutive; chromosome or episome) or unstable expresses the recombinant protein. These analyses include, for example, analyses of the reporter gene, in which case define the regulation of a specific promoter or the regulation of the pathway of signal transduction or a member of the cascade of signal transduction activity of the reporter enzyme, the expression of which is under the control of an appropriate promoter. For this type of analysis it is necessary to generate a recombinant cell line that expresses a reporter gene under the control of a specific promoter, which itself examined or regulated investigational cascade of signal transduction. Suitable reporter enzyme known to the competent specialist and include luciferase fireflies, the Renilla luciferase (both are produced, for example, the firm Packard Reagents), β-galactosidase, etc. the Selection of suitable cell lines are known to the competent specialist and depends, in particular, on the purpose of analysis or “read out”. Typically, this cell line, which can easily be cultivated and transferout, such as, for example, HeLa, COS, CHO or NIH-3T3 cells.

To determine the phosphorylation of the protein, respectively, kinase activity is suitable, for example, the analysis of polarization fluoresc is ncii, for example, test system which is manufactured by a company Panvera; analysis by the method of homogeneous fluorescence with time resolution (HTRF™, Cis Bio International) or LANCE™ (Perkin-Elmer Life Sciences) or analysis by the method of homogeneous enhanced proximal luminescence (ALPHAScreen™ company Perkin-Elmer Life Sciences).

Particularly preferred definition, in the framework of the present invention, kinase activity using the test system ALPHAScreen™ company Perkin-Elmer Life Sciences carried out, for example, that using the studied kinases phosphorylate biotinylated peptide in the biochemical mixture in the presence of ATP. Phosphorylated peptide is then associated through specific to phosphorus antibodies, which is associated with conjugated with protein a or equipped with suitable secondary antibodies granules acceptor. In the same mixture are associated with streptavidin granules donor contact bitenova part of the peptide. By binding with the peptide pellets acceptor and donor are in close proximity, which causes a cascade of chemical reactions that generate very amplified detected signal luminescence: by laser excitation in the granule donor is excited by light sensitivity, and oxygen environment is transferred to the singlet state. Singlet oxygen then diffuses into the Grand prize is Lou acceptor, where it excites dioxanone derived, which, therefore, causes chemiluminescence with a wavelength of 370 nm, which, for its part, excites other fluorophores in the granule acceptor, luminescence light with wavelengths from 520 nm to 620 nm. Since the excitation of fluorophores singlet oxygen occurs only when the granules of the donor and acceptor are in close proximity, only then generate detected signals.

Other types of analyses and other types of “read outs” also sufficiently known to the expert.

It is particularly preferably used in the form of high-performance methods (HTS, high-throughput screening), through which you can analyze for a very short time a large number of substances.

Depending on the problem formulation, modification, modulation, when this might indicate the activation or inhibition of modulating the enzyme. The type of modification includes any possible influences that ultimately affect catalyzed by the enzyme level of phosphorylation of the polypeptide, as a modification of the interaction of substrate-enzyme or modification of catalytic activity of the enzyme, however, also preferably, in the case of analysis using cell reaction mixtures) modification of the expression of fermenta etc.

Another aspect of the invention relates to a method of identifying substances which modify the ability of the enzyme or a functional fragment or a derivative thereof to modulate the degree of phosphorylation of the polypeptide, by carrying out the following stages:

a) determining the ability of the enzyme or its functional fragment, or a derivative thereof to modulate the level of phosphorylation of the polypeptide according to the proposed invention is the above method, and the reaction mixture does not add the test substance;

b) determining the ability of the enzyme or its functional fragment, or a derivative thereof to modulate the level of phosphorylation of the polypeptide according to the above-described method according to the invention, and the reaction mixture was added the test substance;

(C) comparison of the ability of p. (a) with such under item (b).

Proposed according to the invention, the methods thus particularly suitable for the identification of pharmacologically active substances for the treatment of non-insulin-dependent diabetes (NIDDM), Oncology (IGFRK) or for the treatment of inflammatory processes (IKK-kinase).

The invention is explained more below by using different figures and examples, which do not limit the scope of protection of the invention.

Example 1

Used a fragment of IRS-1

To demonstrate the capabilities of the bio is to Interoute polypeptide substrate of the signaling pathway of insulin and use it to offer according to the invention of applications and methods, was chosen large fragment length 262 amino acids of the human IRS-1 (AA-AA), which contains the Central potential sites of tyrosine phosphorylation (in bold), as well as serine (underlined) (Siemeister et al., J. Biol. Chem., 1995). The fragment contains five potential sites of tyrosine phosphorylation, which are underlined in the figure 3 and are presented below along with their motif in bold:

Serine 612, 632, 662, and 731, which represent four potential phosphorylation site sericinus in YMXMSP-motives, localized near the sites of tyrosine phosphorylation of the insulin receptor, which is located in the binding sites for SH2 domains. Mutation of these serine residues to alanine leads to an increase mediated IRS-1 activity phosphatidylinositides (PI3K) (Mothe et al., 1996), which indicates that it was given inhibitory function. However, we cannot exclude that there are other sites of serine phosphorylation, which at present still unknown.

Example 2

Cloning and biotinylation hlRS-1-p30

To study the domains with a length of 262 amino acids D516-P777 (hlRS-1-p30) human IRS-1 is first expressed in E. coli as described Siemeister et al., 1995. Expressing vectors were obtained by the insertion of the floor of the nucleotide sequence according to the identified sequence No. 10 (cDNA sequence hlRS-1-p30) in plasmid pET3d (issued under the registration number 69421 company Novagen) in the usual ways. For this first “empty” vector was restrictively using enzymes NcoI (manufactured by a company Roche Diagnostics GmbH, Mannheim, under registration number 835315) and BamHI (manufactured by a company Roche Diagnostics GmbH, Mannheim, under registration number 656275) under standard conditions and purified using a rotating columns (manufactured by the firm Quiagen, Hilden, under registration number 28104).

Biotinylation carried out in accordance with the instructions of the commercial supplier of enzymes, Darmstadt, Germany, through the conventional technologies. The expression of the fragment of the insulin receptor hlRS-1-p30 was carried out as described Siemeister et al., 1995. To test whether expression was obtained protein extracts from E. coli (strain: E.coli BL21, supplied by the company Novagen under registration number 69451-3), were separated by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate in standard conditions (see, for example, the following standard literature) and received in standard conditions at colouring with a solution for staining with Coomassie (see, for example, the following standard literature). Cleaning hlRS-1-p30 was carried out according Siemeister and other Biotinylation hlRS-1-30 carried out enzymatically using transglutaminase.

Example 3

ALPHAScreen™: Phosphorylation of the biotinylated fragment RS-1 c using purified by affinity chromatography using the lectin wheat germ insulin receptor from rat liver.

In the experiment, the results of which are presented in the figure 4, purified by affinity chromatography using the lectin wheat germ the insulin receptor from rat liver (WGA-IR, SEQUACC room NP_058767 or manufactured by the company Sigma under the registration number 70543) were incubated with various concentrations of human insulin (e.g., manufactured by the company Sigma under the registration number I-9266) and 85 nm of the biotinylated fragment IRS for 10 minutes at 4°C in 50 mm Tris buffer, pH 7.4, 8 mm MgCl2, 2 mm MnCl2with further incubation after the addition of ATP (final concentration 50 μm) for 30 minutes at 30°C. the Reaction was then stopped by adding ethylenediaminetetraacetic acid to a final concentration of 20 mm and phosphorylation of IRS-1 was determined by using directly associated with a specific acceptor for p-Tyr antibodies (supplied by the company Perkin-Elmer Life Sciences under the registration number S), as is seen in figure 4 “Readout”. Using this method, you can determine the EC50 (concentration causing 50%effect) for insulin to 10 nm.

Example 4

ALPHAScreen™: Phosphorylation of the biotinylated fragment of IRS-1 through SW and recombinant kinase of the insulin receptor

ALPHAScreen™ company Perkin-Elmer Life Sciences enables the detection of interaction is between the phosphorylated fragment of IRS-1 and antibodies that recognize phosphorylated residues serine or tyrosine (p-Ser/p-Tyr antibodies). Biotinylated IRS-1 when bound to streptavidin-donor antibody using associated with the acceptor protein a or suitable associated with acceptor secondary antibodies. When there is an interaction, the acceptor reaches and remains in the immediate vicinity of the donor so that singlet oxygen atoms, which are generated by the donor, by diffusion can achieve chemiluminescent groups in the granule of the acceptor, resulting in the ultimate emission of the detected light.

The intensity of the light generated according to the above analysis and are presented as histograms in figure 5A and 5B (the so-called “Readout”), after incubation for 30 minutes of IRS-1 with protein kinase and ATP, and then adding p-Ser antibodies (supplied by company Biosource, Belgium, under the registration number 44-550) and further incubation for 120 minutes, detect by measuring using a Perkin-Elmer Fusion or AlphaQuest Instrument and quantify. The comparison of the generated light intensities in the presence and in the absence of the RCC presented in figure 11A. In the experiment, the result of which is presented in figure 11B, the recombinant kinase of the insulin receptor (IRK, amino acids 941-1343, NCBI, the access number NM_000208) enable the Ute by incubation with polylysine for 10 minutes at a temperature of 30°C in reaction buffer: 50 mm Tris-buffer, rn,4, 8 mm MgCl2, 50 μm ATP, and then add the substrate IRS to IRK followed by incubation for 30 minutes at 30°C. Phosphorylation of IRS-1 is determined by use directly associated with the specific acceptor for p-Tyr antibodies (supplied by the company Perkin-Elmer Life Sciences under the registration number S), resulting in a gain presented in figure 11B “Readout”.

The above studies for the first time it was possible to demonstrate that biotinylated polypeptides can be fosforilirovanii by kinases. This is demonstrated using the fragment hlRS-1 mass of 28 kDa, which is in biotinylating condition can be phosphorylated using sericinus δ and tyrosine kinase of the insulin receptor. Detection using specific to phosphorus antibodies also proved successful, and steric obstruction due to the amount of polypeptide in combination with biotinyl residue does not interfere with the reaction detection. This allows you to create, based on the principle of ALPHA-Screens™, a homogeneous system of analysis, which can be used to determine the level of phosphorylation of polypeptides using possible by biotinidase treatment technologies and detection. This principle of analysis, in this context, for the first time applied to a protein fragment size floor the peptide (or rather, 28 kDa). This allows you to perform advanced searches pharmacologically active substances that interact with a complex system of phosphorylation and dephosphorylation of cells (diagnosis-dependent phosphorylation of diseases), the identification of new protein kinases for specific polypeptides in large, even structurally intact physiological substrates, which significantly increases the specificity of fosforilirovanii underlying these studies, and thus, the information content of the received data. Used in this context, the analysis system “Readout” is not radioactive, and luminescent, which represents an advantage in terms of use in the methods of high-throughput screening (HTS). Presented in this context quantitative analysis, therefore, can be used for HTS of any enzymes, modulating the level of phosphorylation of polypeptides and proteins, such as kinases and phosphatases, to identify new kinds of biologically active substances or verification of known biologically active substances. Similarly, it is suitable for other ways above the ways of the search for new enzymes that phosphorylate specific polypeptides, for example, new fosfauriliruetsa IRS-1 kinases in a full cell lysates./p>

Description of figures

Figure 1

The protein sequence of IRS-1 to IRS-4 (identified by sequence No. 1-4). Access number sequences (NCBI database for proteins) four members of the family are: NM_005544 (IRS-1 hs);:M:007095 (IRS-2 hs); NM:032074 (IRS-3 rat); NM_003604 (IRS-4 hs).

Figure 2

Coding DNA sequence of IRS-1 to IRS-4 (identified by sequence No. 5-8). Access number sequences (NCBI database for nucleotides) four members of the family are: NM_005544 (IRS-1 hs);:M:007095 (IRS-2 hs); NM:032074 (IRS-3 rat); NM_003604 (IRS-4 hs).

Figure 3

Including 262 amino acid domain of the protein IRS-1 (hlRS-1-p30), which is used for this study. Serine 612, 632, 662, and 731 are underlined. Motives phosphorylation of tyrosine YXXM bold.

Figure 4

The results ALPHAScreens in the application of the insulin receptor, purified by affinity chromatography using the lectin wheat germ.

Figure 5

The Results ALPHAScreens™.

Figure 6

A visual representation of the interactions of insulin receptor, IRS-1 and sericinus.

Figure 7

A visual representation of the possible molecular mechanisms active in the inhibition of phosphorylation of serine.

Materials and methods

If not stated otherwise, the methods are known to the competent specialist standard methods and information about them can be obtained from the above literature, particularly the literature by standard methods (herein also referred to as the standard literature).

Reduction

For amino acids (in General, also abbreviated as AA or AS) use three - or one-letter codes (see also specified standard literature); nucleotides using commonly used single-letter abbreviations (see also specified standard literature).

1. The method of determining the ability of the enzyme to modulate the level of phosphorylation of the biotinylated polypeptide by tyrosine or serine using stages:
a) contacting the enzyme with biotinylated fragment 516-777 human IRS-1 (hIRS-1-P30) and the initiation of the reaction of phosphorylation in a suitable reaction mixture,
(b) contacting this reaction mixture was immobilized on the carrier means, which detects the ability to communicate with hIRS-1-P30,
c) determining the level of phosphorylation that is associated with this tool hIRS-1-P30,
wherein the reaction mixture is added an antibody that can specifically bind to a phosphorylated hIRS-1-P30, and determine the level of phosphorus is the stimulation is carried out by determining the number associated with hIRS-1-P30 antibodies.

2. The method according to claim 1, characterized in that the carrier is a membrane or plate.

3. The method according to claim 1, characterized in that the agent is streptavidin.

4. The method according to claim 3, characterized in that associated with immobilized by means of the media is the first media that contains the first signal generator, and the antibody is associated with a second carrier, which contains the second signal generator, both signal generator capable of generating a detected signal, if they are in close proximity to each other, and determining the level of phosphorylation is carried out by determining, generates a signal.

5. The method according to claim 1, characterized in that the enzyme is a receptor.

6. The method according to claim 1, characterized in that the enzyme is one of the following kinases or their functional fragment or derivative: insulin receptor, IGF receptor-1, trK-receptor, EGF-receptor, caseinline II, protein kinase C, protein kinase B/Akt, mitogen-activated protein kinase (MAP-kinase, GSK-3, ERK or JNK.

7. The method according to claim 1 for identifying substances that modify the ability of the enzyme to modulate the level of phosphorylation of the polypeptide.

8. The method of identifying substances which modify the ability of the enzyme or its functional fragments or derivatives of tolerovat the level of phosphorylation of the polypeptide, using stages:
a) determining the ability of the enzyme to modulate the level of phosphorylation of the polypeptide according to the method according to one of claims 1 to 4,
b) determining the ability of the enzyme to modulate the level of phosphorylation of the polypeptide according to the method according to one of claims 1 to 4, where the reaction mixture are added test substance, (C) comparison of the measurement stage a) with the result of the measurement stage b).

9. The method according to claim 7 for the identification of pharmacologically active substances for the treatment of insulin-dependent diabetes mellitus (NIDDM).



 

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