A dna molecule encoding a polypeptide capable of contact with the intracellular domain of the receptor fas ligand(fas-ic), a polypeptide, the method of its production, vector, mode of action of the ligand fas-r cells (options), the pharmaceutical composition (options), sposob isolation and identification of protein
The invention relates to biotechnology, in particular genetic engineering, and can be used to produce a protein capable of modulating the function of the Fas-receptor. The polypeptide able to bind with the intracellular domain of Fas-R, is obtained by culturing cells transformed by a vector containing a DNA sequence encoding the specified polypeptide. Modulation of action of the ligand Fas-R is performed with the use of the polypeptide or mRNA encoding the polypeptide. Pharmaceutical composition for modulation of the action of the ligand Fas-R on the cell contains the specified polypeptide or viral vector, its encoding. The invention allows the development of a therapeutic agent to modulate the cytotoxic activity of Fas-R. 9 AD. and 3 C.p. f-crystals, 8 ill., 1 PL.
The scope of the invention
This invention generally relates to the field of receptors belonging to the superfamily of receptors of the TNF/NGF, and control their biological functions. The superfamily of receptors of the TNF/NGF includes such receptors as P55 and P75 of tumor necrosis factor (TNF-R) and the receptor for FAS ligand (also called FAS/APOI or FAS-R, will be referred to as FAS-R) and others. Specifically dantrolene domain (IC) FAS-R (this intracellular domain was designated FAS-IC). This new protein is capable of modulating the function of FAS-R. in Addition, MORT-I is capable of self-and can activate its own cytotoxicity cells. The invention also concerns the receipt and use of the MORT-I.
It should be noted that the names HF-I (original designation) and MORT-I (designation used currently) both are used everywhere and refer to the same protein.
Background of the invention and the source data
The tumor necrosis factor (TNF-and Lymphotoxin (TNF-) (hereinafter TNF-and TNF-both will be denoted TNF) are multifunctional cytokines that provoke inflammation, synthesized mainly managername phagocytes, and providing a variety of effects on cells (Wallach, D. (1986) in: Interferon 7 (Ion Grosser, ed.), pp.83-122, Academic Press, London; and Beutler and Cerami (1987)). TNF-and TNF-exert their effects by binding to receptors on the cell surface. Some of these effects are good for the body: for example, they can kill tumor cells or cells infected with the virus, and to strengthen Protopopova and infectious agents, and in healing wounds. So, TNF can be used as an antitumor agent. In this case, it binds to receptors on the surface of tumor cells and thus initiate processes leading to the death of tumor cells. TNF can also be used as anti-infective tools.
However, as TNF-and TNF-also have harmful effects. There is evidence that excessive production of TNF-can play a major pathogenic role in several diseases. So, now it is known that the effect of TNF-first of all, on the vascular system is the main cause of symptoms of septic shock (Tracey et al., 1986). In some diseases, TNF can cause excessive weight loss (cachexia), inhibiting the activity of adipocytes and causing anorexia, and therefore he was called cajetina. He was also described as a mediator of tissue damage in rheumatic diseases (Beutler and Cerami, 1987) and as the primary mediator of the observed violations of the reactions of graft - versus-host (Piquet et al., 1987). In addition, it is known that TNF is involved in inflammation and many other diseases.
Initiation and/or modulation viseum the P55 and P75 TNF-R, which specifically bind both TNF-and TNF-. These two receptors are structurally distinct intracellular domains, which indicates that the difference coming from them signals (see Hohmann et al.; 1989; Engelmann et al., 1990; Brockhaus et al., 1990; Leotscher et al., 1990; Schall et al., 1990; Nophar et al., 1990; Smith et al., 1990; and Heller et ai., 1990). However, there is still need to identify cellular mechanisms, such as various proteins and other factors involved in the transmission of intracellular signals from the P55 and P75 TNF-R (PCT/US95/05854 and below in this paper, the material described new proteins, the ability to communicate with RS and p55IC). It is intracellular signals, which usually occur after binding of the ligand, that is, TNF (or), with the receptor responsible for the beginning of a cascade of reactions that ultimately lead to the observed response of the cells to TNF.
With regard to the cytotoxic action of TNF, the most studied in the present cells this action is triggered mainly by receptor P55 TNF-R Antibodies against the extracellular domain (domain, a ligand-binding) P55 TNF-R may themselves run cytotoxic effect (see Erapol generation process the generation of intracellular signals. The study of mutations (Brakebusch et al., 1992; Tartaglia et al., 1993) showed that the biological function of the P55 TNF-R depends on the integrity of its intracellular domain, and accordingly suggested that the initiation of intracellular signal leading to the cytotoxic effect of TNF, is carried out as a result of joining two or more intracellular domains of the P55 TNF-R. in Addition, TNF (and) is detected as homotrimer and, as suggested, induces intracellular signal through the P55 TNF-R, thanks to its ability to bind and form cross-links between molecules of the receptor, i.e., to carry out the aggregation of the receptor. In PCT/US95/05854, and in this paper the following describes how p55IC and p55DD can coassociativity and to induce a neutral ligand such effects on the cell, which provides TNF.
Another member of the superfamily of receptors of the TNF/NGF is the FAS receptor (FAS-R), which is also called the FAS antigen, protein cell surface, expressively in various tissues and detecting homology with a number of cell-surface receptors, including TNF-R, NGF-R and FAS-R mediates the death of cells by apoptosis (Itoh et al., 1991) and, apparently, serves as a factor negatively the kăđẫa own antigens. Also found that mutations in the gene FAS-R (lpr) cause lymphoproliferative disorder in mice, which corresponds to an autoimmune disease of human systemic lupus erythematosus (Watanabe-Fukunaga et al., 1992). Ligand FAS-R, apparently, is a molecule of the cell surface, which along with other cells are T-cells, killer cells (or cytotoxic T-lymphocytes CTLs). Therefore, when such CTL come into contact with cells bearing FAS-R, they are able to induce apoptosis of cells carrying FAS-R. Forth, were obtained monoclonal antibodies against FAS-R and these monoclonal antibodies were able to induce apoptosis of cells carrying FAS-R, including mouse cells transformed with cDNA that encodes FAS-R man (Itoh et al., 1991).
He also discovered that various other normal cells along with T-lymphocytes Express FAS-R on the surface and can be killed by running this receptor. Suppose that uncontrolled induction of this process of cell death contributes to tissue damage in certain diseases, such as destruction of liver cells in acute hepatitis. So finding ways to reduce the cytotoxic activity of FAS-R may have therapeutic value.
HIV, bear on the surface of the FAS-R, antibodies against FAS-R or FAS ligand-R can be used to trigger receptor-mediated FAS-R cytotoxic effects in these cells and thus serve as a means of combating such malignization cells or cells infected with the HIV virus (see Itoh et al., 1991). Therefore, the search for other ways to increase the cytotoxic activity of FAS-R may also have therapeutic value.
For a long time considered it necessary to find a way of modulation of the response of cells to TNF (or) and FAS ligand, for example, in such pathological cases, which are mentioned above, where there is increased expression of TNF and FAS ligand, it is desirable to suppress the cytotoxic effects induced by TNF or FAS ligand-R. In other cases, such as in healing wounds, it is desirable to increase the effect of TNF, or in the case of FAS-R, it is desirable to increase the effects mediated by the FAS-R, tumor cells or cells infected with the HIV virus.
The authors of this invention was used a number of approaches (see, for example, European Application EP 186833, EP 308378, EP 398327 and EP 412486) to regulate the harmful effects of TNF by inhibiting the binding of TNF to its receptors antic the internal domains of the receptors), competing with the receptor TNF-R on the cell surface for binding TNF. Further, given that the binding of TNF to receptors is necessary for the induction effects of TNF on the cell, the authors of this invention have applied the approach to modulate the effects of TNF, based on the modulation of the activity of TNF-Rs. Briefly EP 568925 (IL 101769) relates to a method of modulation transfer signal and/or cleavage within the TNF-Rs, resulting in peptides or other molecules can interact with the receptor or effector proteins involved in the interaction with the receptor, thus modulating the normal functioning of the TNF-R IN EP 568925 describes the structure and properties of various mutants of the P55 TNF-R with mutations in the extracellular, transmembrane and intracellular domains of the P55 TNF-R. in This way, in the above domains were identified region, which are essential to the functioning of the receptor, i.e., to bind ligand (TNF) and subsequent signal transduction and intracellular transfer of the signal that ultimately leads to the observed effect of TNF on the cell. Further, there is also described a number of approaches for separation and identification of proteins, peptides or other factors that are able to communicate with different region the activity of TNF-R. In EP 568925 was also outlined a number of approaches for isolation and cloning of DNA sequences encoding such proteins and peptides; construction of expression vectors for the synthesis of these proteins and peptides; generate antibodies or their fragments that interact with the TNF-R or with the above-mentioned proteins and peptides, linking different areas of TNF-R. However, in EP 568925 not listed actually existing proteins and peptides that bind to the intracellular domain of TNF-R (for example, the P55 TNF-R), not described approach using yeast of digiridoo for isolation and identification of such proteins and peptides, bind to the intracellular domain of TNF-R. Previously were not detected proteins or peptides that can bind the intracellular domain of FAS-R.
Thus, when you want to suppress the effect of TNF or FAS ligand-R, it is desirable to reduce the amount of TNF-R or FAS-P on the cell surface or their activity, while attempts to increase the effect of TNF or FAS ligand-P, it is desirable to increase the number of TNF-R or FAS-R or their activity. This purpose was defined and analyzed the nucleotide sequence of the promoters of the P55 TNF-R and P75 TNF-R and was discovered a number of key sequences, characteristic of R is the level of the promoter, i.e. suppression reader promoter to reduce the number of receptors and enhance reading with promoters to increase the number of receptors (see IL 104355 and IL 109633). Relevant studies relating to the control of the FAS-R at the level of the promoter of the gene FAS-R, not yet published.
Further, we should mention the following. It is known that receptors of the tumor necrosis factor (TNF) and structurally related receptors FAS-R run in the cells upon stimulation with their ligands, synthesized by leukocytes, destructive activity, which lead to the death of these cells, but the mechanisms of this run is still poorly understood. Studies of the mutations show that in the development of signals cytotoxicity receptors FAS-R and P55 TNF (p55-R) involves various areas of their intracellular domains (Brakebusch et al., 1992; Tartaglia et al., 1993; Itoh and Nagata, 1993). These areas (the"domain of death") have a similar sequence. "Domain of death" as FAS-R and p55-R tend to self. Their self-Association, apparently stimulates the aggregation of the receptors, which is required for initiation signals (see PCT/US95/05854, and Song et al., 1994; Wallach et al., 1994; Boldin et al., 1995), and a high level of expression of receptors can lead to the start signal, independent of ligand (PCT/US95/05 may regulate the action of ligands, belonging to the superfamily of TNF/NGF, such action is, what has the TNF or FAS ligand-R on the cell, Poreba processes generate intracellular signals. These processes are, apparently, are largely intracellular domains (ICs) receptors of the superfamily of receptors of the TNF/NGF, such as TNF-Rs, i.e., the intracellular domains of the P55 and P75 TNF-R (RS and RS respectively), and FAS-IC.
In accordance with this object of this invention to provide proteins, namely MORT-I analogs, fragments and derivatives, which are able to bind with the intracellular domain of FAS-R Suggest that these proteins are involved in the processes of transmission of intracellular signals initiated by the binding of FAS ligand to its receptor. Protein MORT-I, its analogs, fragments and derivatives of the present invention differ from the proteins that bind FAS-IC, described in the previously mentioned applications.
Another objective of the invention is to provide antagonists (e.g. antibodies) against these molecules that bind FAS-IC, i.e. against the protein MORT-I analogs, fragments and derivatives. These antagonists can be used to suppress propagation of signals, when such proteins that bind FAS-IC are positive signalmessage FAS-IC, are negative effectors (i.e., inhibit signals).
Another objective of the invention is to use protein MORT-I analogs, fragments and derivatives for the selection and characteristics of other proteins or factors, which may, for example, to participate in the final link in the process of signal transmission, and/or to highlight and identify other receptors involved in the initial stages of the process of signal transmission. These proteins, factors and receptors (e.g., other FAS-R or related receptors) could be in contact with the protein MORT-I, its analogs, fragments and derivatives and thus to take part in their operation.
In addition, the purpose of this invention is the use of the above protein MORT-I analogs, fragments and derivatives as antigens in obtaining polyclonal and/or monoclonal antibodies against them. These antibodies in turn can be used, for example, to clear a new protein MORT-I from other sources, for example from cell extracts or lines of transformed cells.
Further, these antibodies can be used for diagnostic purposes, for example for identifying disorders related to abnormal functioning of the economic structures, including protein MORT-I, its analogs, fragments and derivatives, and pharmaceutical compositions comprising the above antibodies or other antagonists.
Summary of invention
According to this invention we have discovered a new protein that is able to bind with the intracellular domain of FAS-R, This binding protein (FAS-IC, can act as a mediator or modulator of the action of the ligand FAS-R cells, Poreba or modulating the process of transmitting signals, which typically occurs after binding of the ligand FAS-R with the cell membrane.
This new protein identified as HFI or MORT-I (Mediator of Receptor Toxicityand in addition to its ability to specifically bind to a FAS-IC has other properties (see Example 1), for example, it has a region homologous to regions of the "death domain" (DD) receptors p55-TNF-R and FAS-R (p55-DD and FAS-DD) and therefore capable of self. MORT-I can also be activated in the own cell cytotoxicity activity, which may be related to its ability to self. Now also found that co-expression of the area in MORT-I (HFI), which contains a sequence homologous to the "domain of death" (MORT-DD, prisutstvie be expected on the basis of its ability to communicate with the "domain of death" FAS-IC. Next, in the same experimental conditions, it was found that co-expression of that section MORT-I, which does not contain the region MORT-IDD (N-terminal part of MORT-I, amino acids 1 to 117, "the head of MORT-I"), does not lead to the suppression of cell death induced by FAS, and in no way does not increase the cytotoxicity of the cells, induced by FAS.
In accordance with this invention provides a DNA sequence encoding a protein, which is designated here as MORT-I, its analogs or fragments, each of which is able to communicate and interact with the intracellular domain of the ligand to the FAS receptor (FAS-IC).
In particular, the present invention provides a DNA sequence from a group including:
(a) the cDNA sequence of the region that encodes a protein MORT-I;
(b) a DNA sequence capable of gibridizatsiya with cDNA of paragraph (a) under mild conditions. This sequence encodes a biologically active protein that binds the intracellular domain of FAS-R; and
(c) a DNA sequence which is obtained from DNA (a) and (b) as a result of the degeneracy of the genetic code. This sequence encodes a biologically active protein that binds the intracellular domain of FAS-R.
A typical example of sledovatelnot, is depicted in Fig.4.
The present invention also provides a protein MORT-I, its analogs, fragments or derivatives encoded by the above-mentioned sequences of this invention, and these proteins, its analogs, fragments and derivatives are able to bind or interact with the intracellular domain of FAS-R.
A typical example of the above-mentioned protein of this invention is a protein MORT-I have obtained from nucleotide sequences amino acid sequence depicted in Fig.4.
In the present invention are also vectors encoding a protein MORT-I, its analogs, fragments or derivatives of this invention which contain the above-mentioned DNA sequences of this invention, and these vectors can be expressed in appropriate eukaryotic or prokaryotic cells of the host; transformed eukaryotic or prokaryotic host cell containing such vatory; and a method of obtaining protein MORT-I analogs; appropriate conditions for expression of the indicated protein, its analogs, fragments or derivatives; conducting post transcriptional modification of the above protein required for obtaining the proteins, and provide the funds from the above transformed cells or from cell extracts of the above transformed cells.
In another section of this invention are also antibodies, or active derivatives or fragments of these antibodies against the protein MORT-I analogs, fragments and derivatives of the present invention.
In another section of the invention provides various examples of application of the above DNA sequences or proteins coded by them in accordance with this invention. Among them there are the following examples:
(i) method of modulation steps ligand FAS-R cells carrying FAS-R, including the processing of the above-mentioned cells by the protein MORT-I, its analogs, fragments or derivatives, or combinations thereof according to this invention, each of which is able to bind with the intracellular domain and modulate the activity of FAS-R. In this method, treatment of the cells includes an introduction to the specified cell protein MORT-I, its analogs, fragments or derivatives, or combinations thereof in a form suitable for intracellular introduction or introduction into the above cell DNA sequences encoding the above proteins, analogs, fragments or derivatives, or combinations thereof, in the composition of the appropriate expression vector carrying the above sequence, with the above vector can effectively which is expressed in said cell;
(ii) method of modulation steps ligand FAS-R cells, including treatment of these cells with protein MORT-I, its analogs, fragments or derivatives, each of which is able to bind with the intracellular domain and to modify the activity of FAS-R. the Above processing cells includes an introduction to the specified cell in the specified protein MORT-I, its analogs, fragments or derivatives in a form acceptable to their introduction into the cell, or the introduction in the specified cell of a DNA sequence that encodes this protein MORT-I, its analogs, fragments or derivatives in the composition of the corresponding vector, bearing these sequences, with the specified vector can effectively embed the specified sequence in the specified cell in such a way that said sequence is expressed in said cell;
(iii) method, such as in (ii) where the specified treatment of these cells is transfection of these cells with the recombinant vector of the virus of animals that includes the following steps:
(a) constructing a recombinant vector of the virus in animals carrying a sequence encoding a protein shell of the virus (ligand), which is able to bind to specific Retz is its analogues fragments and derivatives of this invention, which upon expression in a given cell is capable of modulating the activity specified FAS-R; and
(b) infection of the specified cell in the specified vector ();
(iv) the method of modification actions ligand FAS-R cells carrying FAS-R, including treatment of these cells with antibodies or their active derivatives or fragments according to this invention, with the specified cell treatment is appropriate composition containing these antibodies, active fragments or derivatives. When the protein MORT-I or part thereof is located on the cell surface, the composition is prepared in a form for use outside the cell, and when this protein is intracellular, the composition is prepared in the form for introduction into the cell;
(v) method of modulation effect of the ligand on cells carrying FAS-R, including the processing of these cells oligonucleotide sequence that encodes the antisense sequence of at least part of the sequence MORT-I of this invention, with the indicated oligonucleotide sequence capable of blocking MORT-I;
(vi) method, such as in (v), where the processing cells is transfection of these CL the private vector of the virus in animals, carrying a sequence encoding a protein shell of the virus (ligand), which is able to bind to specific receptor on the cell surface, the carrier FAS-R, and the second sequence, which is an oligonucleotide sequence that encodes the antisense sequence of at least part of the sequence MORT-I according to this invention, with the indicated oligonucleotide sequence capable of blocking the expression of protein MORT-I with the introduction of this virus into the specified cell; and
(b) infection of the specified cell in the specified vector ();
(vii) a method of treatment of tumor cells or cells infected with the HIV virus or other desired cells, including:
(a) construction of recombinant vector of the virus in animals carrying a sequence encoding a protein shell of the virus, which is able to bind with a receptor on the surface of tumor cells, or with a receptor on the surface of cells infected by the HIV virus, or receptor, which are desired cells, and a second sequence encoding one of the proteins MORT-I, its analogs, fragments and derivatives of this invention that when the expression in the specified ouhh; and
(b) infection with the indicated tumor cells; cells infected by the HIV virus; or any other cells of the specified vector ();
(viii) the method of modification actions ligand FAS-R cells, including application procedures using a ribozyme, in which the vector encoding the sequence of the ribozyme capable of interacting with a cellular mRNA sequence that encodes a protein MORT-I of this invention, is inserted in the specified cell in a manner that ensures the expression of the sequence of the ribozyme in the specified cell. In this method, when the expression specified sequence of the ribozyme she interacts with the specified cellular mRNA sequence and cleaves the sequence of mRNA, leading to the suppression of expression of the protein MORT-I in these cells;
(ix) the method listed above, where this protein MORT-I or the sequence encoding this protein MORT-I, includes at least the portion of the protein MORT-I, which specifically binds to FAS-IC, or at least that part of the sequence that encodes the protein site MORT-I specifically bind to the FAS-IC;
(x) method for isolation and identification of protein that can svyazyvayuschii cloning using PCR, in which the sequence or part thereof according to this invention are used as probes, communicating with portions of the cDNA or DNA segments from a library of genes that have at least partial homology with these probes. These related sequences are then amplified and cloned using the PCR procedure. The result was a clone encoding a protein that has at least partial homology with these sequences according to this invention.
The present invention also provides a pharmaceutical composition for modulation of the actions of the FAS ligand to a cell, comprising as active ingredient one of the following additives:
(i) protein MORT-I of this invention, its biologically active fragments, analogs, derivatives or mixture thereof; (ii) a recombinant vector virus animals, encoding a protein capable of binding a receptor on the cell surface, and encoding a protein MORT-I or biologically active fragments or analogs according to this invention; and (iii) oligonucleotide sequence encoding the antisense sequence of the sequence MORT-I of this invention where the specified oligonucleotide sequence can be Deuteronomy is, is the MORT-I is a special area that is associated with the FAS-IC, and another separate area, which is involved in self-MORT-I, and, accordingly, a separate area or part of the MORT-I can be independently used to identify other proteins, receptors, etc. that are able to communicate with MORT-I or FAS-R and can participate in the processes of transmission of intracellular signals, due to the MORT-I and FAS-R. Next, MORT-I may have other types of activity, associated with either of the above or other areas of MORT-I, or their combinations, such as enzymatic activity, related to the effects of MORT-I own the cytotoxicity of the cells. Thus, MORT-I can also be used for the specific identification of other proteins, peptides, etc. that can participate in the manifestation of such additional activities in MORT-I.
In the following detailed description of the present invention are also other aspects and applications.
It should be noted that when used herein, the terms "Modulation of the actions of the FAS ligand on the cell and Modulation steps MORT-I on cell" refers to processing in vitro and in vivo.
Brief description of drawings
Fig.1 a and b reproduce autoradiography autoradiogram of immunoprecipitated proteins from extracts of HeLa cells, transfected fused protein FLAG-NFI (FLAG-MORT-I) cDNA or luciferase (control) and immunoprecipitate performed using antibodies against FLAG; and
in Fig.1B shows autoradiogram characteristic gel, reflecting the interaction between HFI and FAS-IC in vitro using autoradiographical analysis of binding of metabolically labeled by /35S/-methionine HFI synthesized in transfected HeLa cells, in the form of protein, merged with oktapeptidom FLAG (FLAG-MORT-I), with GST fused protein GST-FAS-IC human and mouse (GST-huFAS-IC, GST-mFAS-IC) and fused protein GST-FAS-IC, in which FAS-IC contains a mutation replacing Ile on l in position 225 (GST-mFAS-IC 1225A). Proteins from HeLa cells, labeled by /35S/, including labeled protein FLAG-MORT-I was first extracted and then gave them to interact with various proteins GST and GST-FAS-IC (attached to glutathione beads) and then separated using SDS-PAGE. In all control experiments, the extracts from HeLa cells, transfected with a luciferase, interacted with GST and fused protein GST-FAS-IC and perform SDS-PAGE. Fig. 1A and 1B described in Example 1.
Fig.2A, b and C reproduce autoradiogram gels SDS-PAGE (10% acrylamide), in which he divided the various immunoprecipitate of travelbank in HeLa cells. the only protein HFI-FLAG (FLAG-MORT-I), protein HFI-FLAG and FAS-R man (FLAG-MORTI+FAS/APOI) or FAS-R man (FAS/APOI) (Fig.2A); or protein HFI-FLAG and p55-R man (FLAG-MORTI+p55-R) (Fig.2B); or protein HFI-FLAG and chimeric protein of FAS-R and P55 person, in which the extracellular domain of FAS-R was replaced by the corresponding region of the p55-R (FLAG-MORTI+Chimera p55-FAS only), or chimeric protein FAS-R, p55-R (Fig.2C). In all cases, the transfected cells were metabolically labeled by /35S/-cysteine (20 mccu/ml) and /35S/-methionine (40 mccu/ml) and protein was extracted. Protein extracts from different transfected cells were then immunoprecipitated different antibodies: anti-FLAG, anti-FAS, anti-P75-R and anti-P55-R (FLAGFAS,p75-R andp55-R in Fig.2A-C) and were divided by SDS-PAGE. In Fig.2A left visible protein bands corresponding to FAS-R (Fas/APOI) and HFI-FLAG (FLAG-MORTI); between Fig.2A and b shows the relative position of the standard markets molecular weight (D); and Fig.2C on the right are visible protein bands corresponding to p55R and the Chimera p55-FAS. Fig.2A-C described in Example 1.
Fig.3 reproduces the Northern-blotting, in which poly A+RNA (0.3 ág) from transfected HeLa cells were identified using cDNA probes HF-I, as described is obtained from this amino acid sequence HF-I, as described in Example 1, in which the "domain of death" is underlined as a possible site of the beginning of the broadcast, i.e., emphasize the methionine residue at position 49 (M, underlined with a solid line). The asterisk indicates the stop codon of translation (nucleotides 769-771). In the beginning and middle of each line given two numbers designating the relative position of the nucleotides and amino acids in this sequence, counting from the beginning of the sequence (5-end), where the first number indicates the nucleotide position, and the second amino acid.
Fig.5 represents the results of experiments on the determination of the C-end of MORT-I, where Fig.5 play radioautogram gel after SDS-PAGE (10% acrylamide), which divided the various fused protein products MORTI-FLAG expressed in HeLa cells and metabolically labeled by35S-cysteine and35S-methionine followed by immunoprecipitation or using monoclonal antibodies anti-FLAG (M2) (tracks 2, 4 and 6) or, in control, using antibody-P75 TNF-R (#9) (tracks 1, 3 and 5), as described in Example 1.
Fig.6 (a and b) plots do not depend on ligand start cytotoxic effects in cells transfected MORT-I, where the viability of the cells determined by the hair cells, expressing transfairusa DNA by measuring the amount of Sekretareva on Wednesday alkaline phosphatase placenta (Fig. 6B). HeLa cells were transferrable vectors encoding HFI (MORTI), FAS-IC man, the P55-IC person or luciferase (control). The expression of these vectors is controlled by tetracycline. In all cases, these cells were transferrable also drug cDNA coding for secreterial alkaline phosphatase, which allows to identify the effect of temporal expression of these proteins on cell viability. In Fig.6A and 6B bright bars represent the transfected cells growing in the presence of tetracycline (1 μg/ml to block expression), and the shaded bars represent the transfected cells growing in the absence of tetracycline. Fig.6A and described in Example 1.
Fig.7 provides a graphical representation of the effect of different sections of the protein MORT-I on the cytotoxic effects of cell mediated receptor FAS-R, as described in Example 1.
A detailed description of the invention
One aspect of this invention relates to a new protein MORT-I (HFI), which are able to bind with the intracellular domain of the receptor FAS-R, belonging to the superfamily of TNF/NGF, and therefore can be considered as a mediator or is Ivanyi of FAS ligand to FAS-R. Amino acid sequences and DNA sequences for MORT-I according to this invention are novel sequences; they are not included in the data Bank for DNA or amino acid sequences in GENEBANK or PROTEIN BANK.
Thus, the present invention describes a DNA sequence encoding a protein MORT-I, and protein MORT-I encoded by this DNA sequence.
In addition, the present invention describes DNA sequences encoding biologically active analogs, fragments and derivatives of the protein MORT-I, and analogs, fragments and derivatives encoded by these DNA sequences. The receipt of such analogs, fragments and derivatives is carried out using standard procedures (see, for example, Sambrook et al., 1989), which allows you to get the division, insertion or substitution of one or more codons in a DNA sequence that encodes a protein MORT-I, which results in analogues with compared to natural protein at least one modified amino acid. Acceptable are also analogs that retain at least the ability to bind to the intracellular domain of FAS-R, or analogs, which may mediate or any other binding, or the receptor, protein or other factor next link in the chain reaction, or not catalyze the reaction, depending on the signal. This way you can receive analogue, which has a so-called dominant-negative effect, namely analogue, which has a defect or by binding to FAS-IC or in a subsequent transmission signal after binding. Such analogs can be used, for example, to suppress the action of FAS ligand by competing with natural proteins that bind FAS-IC. In the same way it is possible to obtain a so-called dominant-positive counterparts, which can serve to enhance the effect of FAS ligand. They can have the same or better characteristics of the binding of FAS-IC and the same or better transmission characteristics of signals than the natural proteins that bind FAS-IC. In a manner analogous to that described for obtaining analogs of MORT-I, it is possible to obtain biologically active fragments R-I. Acceptable are such fragments MORT-I, which retain the ability to bind FAS-IC, or those that mediate or binding or enzymatic activity, as noted above. In accordance with this can be obtained fragments MORT-I, which have a dominant negative or dominant-positive effect as it Ballogou of this invention, namely, they are defined as areas MORT-I, taken from the full sequence MORT-I, and each such lot or fragment has the above desirable activity. In the same way can be obtained derivatives using standard modifications of the side groups of one or more amino acid residues of the protein MORT-I, its analogs or fragments, or by conjugation of the protein MORT-I, its analogs or fragments with another molecule, e.g. antibody, enzyme, receptor, etc., as is known in the art.
A new protein MORT-I, its analogs, fragments and derivatives have a number of possible applications.
(i) They can be used to simulate or enhance the function of ligand FAS-R in situations where it is desirable to enhance the effect of ligand FAS-R, for example with the use of antineoplastic, anti-inflammatory or antiviral (anti-HIV) antibodies, when it is desirable to obtain the cytotoxicity induced by FAS ligand-R. In this case, protein MORT-I, its analogs, fragments or derivatives that increase the effect of ligand FAS-R, i.e., cytotoxic effect, can be introduced into the cell using standard procedures known as perse. For example, since the protein MORT-I is intracellular, and must be entered what about the protein in these cells. One way to do this is to build a recombinant animal virus, for example, derived from cowpox, with DNA, which introduced the following two gene: a gene that encodes a ligand that binds to cell surface proteins expressed by the cells, such as protein gp120 of AID virus (HIV), which specifically binds to certain cells (CD 4 lymphocytes and related leukemia), or any other ligand that specifically binds to cells bearing FAS-R, so that the recombinant viral vector possessed the ability to bind to cells, bearing FAS-R; and the gene encoding the protein MORT-I. Thus, when expressi protein shell of the virus to bind to the surface of the cell, tumor cell, or another cell carrier FAS-R, will turn into the target of this virus, as a result of this protein MORT-1 will be introduced into these cells using virus and its expression in these cells, it will lead to enhanced activity of ligand FAS-R, leading to the death of tumor cells or other cells bearing FAS-R, you want to kill. Such a recombinant animal virus is constructed using standard procedures (see, for example, Sambrook et al., 1989). Another possibility consists in reservates it. Another possibility is the modification of the method described by Lin and others in the Journal of Biological Chemistry, Vol.270, No.24, pp.14255-14258, 1995.
(ii) They can be used to suppress the actions of ligand FAS-R, for example, in the case of tissue injury in septic shock, rejection of graft - versus-host, or acute hepatitis, when it is desirable to block intracellular signal induced upon binding of the ligand FAS-R FAS-R. In this case, for example, using standard procedures to enter into the cells of the oligonucleotide with antisense coding sequence of the protein MORT-1. It will effectively block the translation of mRNA encoding the protein of MORT-1 and, thus, to block its expression and lead to the suppression of the action of the ligand FAS-R. Such oligonucleotides can be introduced into cells using the above approach with the use of recombinant virus in which the second sequence, a part of this virus is the oligonucleotide sequence.
Another possibility is the use of antibodies against protein MORT-1, which inhibit its activity in the transmission of intracellular signals.
Another recently developed method of suppressing Denk, which is specifically cleaved RNA. You can create ribozymes that cleave specific RNA, for example mRNA, protein-coding MORT-1 of the present invention. Such ribozymes must have sequence-specific mRNA MORT-1, and possess the ability to interact with a complementary mechanism with subsequent cleavage of this mRNA. This leads to a decrease (or complete loss) of expressi protein MORT-1, and the reduction of the expression depends on the level of expression of the ribozyme to the target cells. For the introduction of ribozymes in the desired cells (e.g., bearing FAS-R) you can use any suitable vector, such as plasmid, vectors of animal viruses (retro-viruses), which are usually used for this purpose (see above (i), where the virus has as a second sequence of cDNA encoding the ribozyme with the selected sequence). (Reviews, methods, etc. concerning ribozymes, see Chen et al., 1992; Zhao and Pick, 1993; Shore et al., 1993; Joseph and Burke, 1993; Shimayama et al., 1993; Cantor et al., 1993; Barinaga, 1993; Crisell et al., 1993 and Koizumi et al., 1993).
(iii) They can be used for isolation, identification and cloning of other proteins that are able to communicate with them, such as other proteins involved in chain transfer processes vnutrimolekulyarnoi DNA can be used in the system of yeast dihybrid (see below). Example 1) in which the sequence for protein MORT-1 will be used as "bait" for the selection, cloning, and identification of cDNA or DNA library for genes other sequences ("victims"), encoding proteins that may contact with MORT-1. In the same way you can determine whether protein MORT-1 of the present invention to communicate with other cellular proteins, such as other receptors from superselect receptors TNF/NGF.
(iv) Protein MORT-1, its analogs, fragments or derivatives can also be used for isolation, identification and cloning of other proteins of the same class, i.e. such that are associated with the intracellular domain of FAS-R or functionally related receptors and are involved in the transmission of intracellular signals. For this purpose, can be used the above-mentioned system of yeast dihybrid or newly developed system based on the use of relaxed southern hybridization followed by cloning using PCR (Wilks et al., 1989). In the publication of Wilkes and others described the identification and cloning of suspected protein-tyrosinekinase with a soft southern hybridi is called, typical kinase. According to this invention, this approach can be applied by using a sequence that encodes a protein MORT-1, for isolation and cloning of related proteins that bind the intracellular domain of FAS-R.
(v) Another approach using protein MORT-1, its analogs, fragments or derivatives is to use them in methods of affinity chromatography for isolation and identification of other proteins or factors with which they are able to contact, such as other receptor related receptor FAS-R, or other proteins or factors involved in the transmission of intracellular signals. In this application, protein MORT-1, its analogs, fragments or derivatives of this invention each of them individually can be attached to the matrices for affinity chromatography. Then with these immobilized proteins incubated extracts, or selected proteins, or factors thought to be involved in the transmission of intracellular signals. After completing affinity chromatography these other proteins and factors that are associated with protein MORT-1, its analogs, fragments or derivatives of the present invention, can be eluted, to isolate and characterize.
(vii) MORT-1 can also be used as an indirect modulator of a number of other proteins because of its ability to communicate with other intracellular proteins (the so-called binding proteins R-1), which in turn directly bind to other intracellular proteins or intracellular domain of the transmembrane protein. An example of such a protein or the intracellular domain is a well-known TNF receptor P55, intracellular signals which modulate a number of other proteins that directly bind to the E. the Example 2), which binds to the intracellular domain of TNF receptor P55.
With the aim of modulating these other intracellular proteins or intracellular domains of transmembrane proteins protein MORT-1 can be entered in the cell with the help of some ways mentioned above (ii).
It should also be noted that the isolation, identification and characterization of protein R-1 of this invention can be performed using various well-known screening procedures. For example, one of these screening procedures, the procedure of yeast dihybrid set forth herein (Example 1), was used to identify protein MORT-1 of this invention. Similarly, as indicated above, and further, can be used in other procedures such as affinity chromatography, procedures, DNA hybridization and other known procedures for isolation, identification and characterization of protein R-1 of this invention or for separation, identification and characteristics of other proteins, factors, receptors, etc. that are able to bind with protein R-1 of this invention.
Further, it should also be emphasized, CTOC properties of protein R-1 is its ability to bind to FAS-IC, as well as the ability to self. R-1 can also activate supprimer 1), plot R-1, which binds to FAS-IC, differs from the plot R-1, which participates in the self. R-1 may also have other types of activity associated with the function of the different above mentioned areas molecules R-1, or any other parts of the molecule, or their various combinations. These other types of activity can be enzymatic or to treat the binding of other proteins (for example, proteins that bind R-1, or other receptors, factors and so on). Thus, R-1 can be used in the above methods of modulation steps ligand FAS-R or cellular effects mediated R-1, or it can be used to modulate other cellular processes signaling related to other receptors, factors, etc.
More specific in this aspect of the invention is the fact that the DNA molecule encoding R-1 or its mutant form (I.e., DNA encoding analogs or active fragments R-1) can be used in gene therapy (i.e., by the methods set forth above in (i) and (ii) to modulate the activity of FAS-R (or modulation or mediation of the actions of the FAS ligand on cells). In addition, since MORT-1 also has cytotoxic effect on the cells, these interacted MORT-1 cells (including application methods, described above in (i) and (ii)). In these applications for gene therapy MORT-1, its analogs and derivatives can be used in three ways:
(a) a protein R-1, its analogs, derivatives or active fragments that are able to communicate both with FAS-IC and MORT-1 (i.e., contain two regions MORT-1, one of which is involved in binding to the FAS-IC, and the other is involved in self-R-1), can be used for modulation effects associated with FAS-R, MORT-1;
(b) a portion of the protein MORT-1, analogs, derivatives and active fragments of this part of MORT-1, which are associated with FAS-IC, can be used for the induction of "dominant negative" effect on FAS-IC, i.e., to suppress the cellular effects mediated by the FAS-R, or can be used for induction "build options" action on FAS-IC, i.e., increasing the cellular effects mediated by the FAS-R; and
(c) the portion of the molecule MORT-1, analogs, derivatives and active sections of this part that are specifically associated with MORT-1, can be used for induction of a dominant negative effects on the MORT-1, i.e., either suppress or increase the cellular effects associated with MORT-1.
As set forth above in (vi), protein MORT-1 may be used to produce antibodies to MORT-1. These antibodies intital or their fragments relate specifically MORT-1.
As for these antibodies, the term "antibody" everywhere refers to polyclonal antibodies, monoclonal antibodies (mAb); chimeric antibodies; idiotype antibodies (anti-Id) against antibodies that can be labeled in soluble or bound form; as well as fragments thereof, obtained using known methods, such as enzymatic cleavage, peptide synthesis or recombinant technology, and others.
Polyclonal antibodies are heterogeneous populations of antibody molecules from the serum of animals immunized with the antigen. Monoclonal antibodies contain essentially homogeneous population of antibodies to these antigens, in which antibodies contain binding sites essentially to the same epitope. MAb can be obtained using methods known to experts in this field. See, for example, Kohler and Milstein, Nature, 256: 495-497 (1975); US Patent No. 4376110; Ausubel et al., eds., Harlow and Lane ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory (1988); and Colligan et al., eds.. Current Protocols in Immunology, Greene publishing Assoc. and Wiley Interscience, N. Y. (1992, 1993), here is the almost complete list of references on this issue. Such antibodies can refer to any class of immunoglobulins, including IgG, IgM, IgE, IgA, GILD, and any of their subclasses. Hibri the equipment production techniques mAd in vivo or in situ due to the high titer of antibodies synthesized using these methods.
Molecules chimeric antibodies consist of different parts originating from different animal species, for example, have a variable region of murine mAb and a constant region of human immunoglobulin. Chimeric antibodies are primarily used to reduce immunogenicity in their application and to increase the output when receiving, for example, when the output of murine mAb of hybrid of the above, but higher immunogenicity in humans, then the use of chimeric mAb man/mouse. Chimeric antibodies and methods for their preparation are known in the art (Cabilly et al., Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne et al., Nature 312:643-646 (1984); Cabilly et al., European Patent Application 125023 (published November 14, 1984); Neuberger et al., Nature 314: 268-270 (1985); Taniguchi et al., European Patent Application 171496 (published February 19, 1985); Morrison et al., European Patent Application 173494 (published March 5, 1986); Neuberger et al., PCT Application WO 8601533 (published March 13, 1986); Kudo et al., European Patent Application 184187 (published June 11, 1986); Sahagan et al., J. Immunol. 137:1066-1074 (1986); Robinson et al., International Patent Application No. WO 8702671 (published may 7, 1987); Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Sun et al., Proc. Natl. Acad. Sci USA 84:214-218 (1987); Better et al., Science 240:1041-1043 (1988); and Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, given above). This list will contain praktyczny unique determinants, usually located in antigennegative site of the antibody. Antibodies against Id can be obtained by immunization of animals of the same species and genotype (e.g., mice), as an animal that serves as a source of mAb, monoclonal antibody mAb against which prepare anti-Id. Immunized animal will recognize and respond to idiotypical determinants of antibodies taken for immunization, and to synthesize antibodies to these idiotypical determinants (anti-Id antibodies). See, for example, US Patent No. 4699880, which includes almost all of the links on this issue.
Antibodies anti-Id can also be used as an immunogen for the induction of immune responses in other animals, synthesizing the so-called anti-anti-Id antibodies. Anti-anti-Id antibodies may recognize epitopes that are identical epitopes of the original mAb, which induced anti-Id. Thus, using antibodies to idiotypical determinants mAb, it is possible to identify other clones expressing antibodies with the same specificity.
In accordance with this mAb raised against protein MORT-1, its analogs, fragments or derivatives of this invention can be used for the induction of anti-Id antibodies in suitable the FL hybrid, secreting anti-Id mAb. Further, anti-Ib mAb can bind to the carrier, for example, hemocyanin of fissurella (KLH) and used to immunize others BALB/c mice. Serum from these animals will contain anti-anti-Id antibodies that have the binding properties of the original mAb against an epitope of the above protein MORT-1, its analogs, fragments or derivatives thereof.
Thus, the anti-Id mAb have their own idiotypical epitopes, or "idiotopes" structurally similar to the identified epitopes, for example, as the GRB protein.
The term "antibody" refers to as intact molecules and fragments thereof, for example, Fab and F(ab)2that are capable of binding antigen. Fab and F(ab)2fragments that do not have Fc fragment of intact antibody, understandably faster out of the circulation and may have less tissue specificity than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).
You must understand that Fab and F(ab)2fragments and other fragments of the antibodies suitable for the present invention, can be used to detect and quantify protein MORT-1 using the methods, is assalone, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab)2fragments).
They say that antibody "capable of binding" a molecule if it is capable of specifically reacting with this molecule by binding the antibody. The term "epitope" refers to the plot of any molecule that can be recognized by this antibody and to contact them. Epitopes or antigenic determinants usually consist of chemically active groups on the surface molecules, such as amino acids or side chains of sugars, and have specific three-dimensional structure, as well as specific charge.
"Antigen" is a molecule or molecules that are capable of binding the antibody, and, in addition, is able to induce the animal antibody synthesis, the ability to communicate with the epitope of this antigen. The antigen may have one or more epitope. The above specific reaction means that the antigen will react highly selective manner with the appropriate antibody, but does not react with the multitude of other antibodies which can be evoked by other antigens.
Antibodies, including antibody fragments, necessary for the present invention, could is OK which Express MORT-1 of the present invention. This can be done using methods immunofluorescence analysis using antibodies labeled fluoresceine dyes (see below) using light microscopy, flow cytometry or fluorometry.
The antibodies (or fragments thereof), suitable for the present invention can be used for histological examination by immunofluorescence or immunoelectron microscopy, for example to detect in situ MORT-1 of the present invention. The in situ detection can be accomplished by obtaining a tissue sample from the patient and treatment of this sample labeled antibodies of the present invention. The biological sample or processed directly labeled antibody (or fragment) against this antigen, or a first corresponding unlabeled antibody, and then a second labeled antibody against the first antibody. When using this procedure, we can determine not only the presence of MORT-1, but also its distribution in the examined tissue. Using this invention, any expert will understand that any of a variety of histological methods (such as staining procedures) can be modified in relation to about the of ASCA, for example, a biological fluid, extract from a tissue, freshly isolated cells, such as lymphocytes or leukocytes, or cells, inkubiruemykh in culture in the presence of the required number of labeled antibody capable of identifying the protein MORT-1, and detecting the antibody by any of a number of well known methods.
The biological sample can be applied to a solid substrate or carrier such as nitrocellulose, or other solid substrate or media that have the ability to be immobilized cells, particles, cells or soluble proteins. The substrate or carrier can then be washed with an appropriate buffer, and then process labeled antibodies according to this invention, as described above. A rigid substrate or carrier can then be washed a second time with buffer to remove unbound antibodies. The amount of bound label on the substrate or carrier you may then find appropriate ways.
"Solid substrate", "solid-phase media", "hard substrate", "hard media", "substrate" or "carrier" are used to denote a substrate or carrier, capable of binding an antigen or antibody. Well-known substrates or carriers include glass, liability, Gabro and magnetites. For the purposes of this invention the natural media can be soluble to some extent or insoluble. The substrate material may have virtually any possible structural form, but such that its molecules have the ability to contact the antigen or antibody. Thus, the shape of the substrate or carrier can be spherical in form of balls, cylindrical with the inner surface of verified tube or the outer surface of the wand, or the surface may be flat, as in a leaf verified strips, etc. Prefer substrates or carriers in the form of balls of expanded polystyrene. Specialists in this field, perhaps there are other convenient media for binding of antibodies or antigens, or they can choose them by routine experimental verification.
Binding activity of the party of the antibodies of this invention, as noted above, can be determined using well known methods. The specialist is able to determine operative and optimal conditions for such tests, using routine experimental verification.
In these tests can be entered other steps, such as washing, stirring, shaking, filtering, etc., as adopted illo according to this invention, is the accession of the enzyme and the use of enzyme immunoassay (EIA). This enzyme then when interacting with an appropriate substrate will react with the substrate, and the resulting chemical reaction product can be detected, for example, using spectrophotometry, fluorometry or visually. The enzymes that are used for labelling of antibodies include, for example, malate dehydrogenase, nuclease from Staphylococcus, Delta-5-steroisomers, yeast alcoholdehydrogenase, alphaglucosidase, triosephosphate, horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphatedehydrogenase, glucoamylase, acetylcholinesterase and other enzymes.
Detection of antibodies can be performed using chronometrically methods based on detection of a chromogenic substrate of the enzyme. Detection can also be performed by visual comparison of the magnitude of the enzymatic reaction of the substrate with a standard sample prepared by a similar method.
The detection antibody can also be performed using other various methods of immunological analysis. For example, the immunological analysis (PIA). A good description of the PIA can be found in the book. Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T. S. et al., North Holland Publishing Company, NY (1978), specifically referring to the Chapter "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T., where there are appropriate links. The radioactive isotope can be registered using the-counters, acquired scintillation counter or by using autoradiography.
Antibodies of this invention can also mark fluorescein connection. Labeled fluorescent label antibodies when exposed to light of an appropriate wavelength can be detected by their fluorescence. The most commonly used compounds for fluorescent labels are fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
Antibodies can also mark fluorescamine metals, for example,152E or other of the series of lanthanides. These metals can be attached to the antibody using halirous metal groups, for example diethylenetriaminepentaacetic acid (ETP).
The antibody can be marked also by joining it chemiluminescense connection. The presence of antibodies, labeled chemiluminescent substance, it is possible to determine the TCI chemiluminescense compounds are luminal, isoluminol, hydrochloride acridin, imidazole, aromaticheski ester greeneway acid, diethyl ester of oxalic acid.
The antibody of the present invention can also mark the connection, capable of bioluminescence. Bioluminescence is a type of chemiluminescence found in biological systems, in which protein-catalyst enhances the reaction chemiluminescence. The presence of a bioluminescent protein is determined by the presence of luminescence. Important bioluminescent compounds as labels are luciferin, luciferase and akarin.
The antibody molecules of the present invention can be adapted for use in immunometric test, also known as "dvuhsimovyiy", or "sandwich"test. In a typical immunometric test a certain amount of unlabeled antibody (or fragment of antibody) is associated with a rigid substrate or carrier, and then add a certain amount of labeled antibodies that can detect and/or opredelit the number of ternary complex formed by the antibody in the solid phase, antigen, and labeled antibody.
Typical and preferred immunodeficiency test includes "early" test, in which antit the b antigen from the sample by formation of a binary complex of antibody-antigen in the solid phase. After the required incubation period, the solid substrate or carrier is washed to remove material liquid sample, including unreacted antigen, if any, and immersed in a solution containing an unknown quantity of labeled antibody (which function as signaling molecules"). After the second incubation period, in which the labeled antibodies form a complex with the antigen bound to a solid substrate or carrier unlabeled antibody, the solid substrate or carrier is washed a second time to remove the unreacted labeled antibody.
In another type of "sandwich"test, which may also be suitable for antigens of this invention are the so-called "simultaneous" and "reverse" tests. Lump-sum test includes a single stage incubation, as and antibody associated with the solid substrate or carrier and the labeled antibody are simultaneously added to the test sample. After completion of the incubation period, the solid substrate or carrier is washed to remove the residue of the liquid sample and unreacted labeled antibody. The presence of labeled antibody associated with the solid substrate or carrier, and then define, as it is done in "ranjitha sample, then, after the required incubation period, added unlabeled antibody associated with the solid substrate or carrier. After the second incubation period, the solid phase is washed with an appropriate way to remove residues of the test sample and unreacted labeled antibody. The definition of labeled antibodies, contacting the solid substrate or carrier is then carried out, as in "early" and "lump sum" tests.
MORT-1 of this invention can be obtained using standard procedures of recombinant technology (see, for example, Sambrook, et al., 1989), in which the appropriate eukaryotic or prokaryotic host cell transformed suitable eukaryotic or prokaryotic vectors containing sequences encoding these proteins. Accordingly, the invention also applies to such expression vectors and transformed cells of the host for the synthesis of proteins of this invention. As mentioned above, these proteins are also their biologically active analogs, fragments and derivatives, and in accordance with the number of vectors encoding them, include vectors encoding analogs and fragments of these proteins, and in the number of transformed cells-Ho is obtained by standard modifications of proteins, their analogues or fragments synthesized in transformed cells of the host.
This invention also concerns pharmaceutical compositions that include recombinant vectors of animal viruses encoding a protein MORT-1. These vectors encode the protein shell of a virus that can specifically bind to proteins of the surface of target cells (e.g. tumor cells) so that has been the introduction sequence to MORT-1 in the cell. Other aspects of this invention are given in the following examples.
Now, this invention will be described in more detail in the following examples, not limiting the scope of the invention, and the corresponding figures.
EXAMPLE 1: CLONING AND isolation of PROTEIN MORT-1, WHICH BINDS TO the INTRACELLULAR DOMAIN of FAS-R
(i) Screening in digibridge system and the test expression-galactosidase in digibridge
Separation of proteins interacting with the intracellular domain of FAS-R, used yeast digibeta system (Fields and Song, 1989; see also joint applications IL 109632, 112002 and 112692). In brief this digibeta system is a genetic analysis in yeast cells, allowing to detect stackable, such as GAL4, which has two different domain - domain DNA binding domain and activation. When the expression of these domains and their reunion restored formed GAL4 protein is able to connect with 5-district activating sequence, which in turn activates the promoter that controls the expression of gene signal, for example, lac Z or HIS3. The expression of these genes easily observed in cultured cells. In this system, the genes for two proteins whose interaction is investigated, cloned in two different expression vectors. In one expression vector sequence for one of these proteins is cloned along with the sequence for the domain DNA binding protein GAL4, which leads to the formation of a hybrid protein containing the domain of the GAL4 DNA binding. In another vector sequence for a different protein is cloned together with the activation domain GAL4. These two vectors together transform the host cell yeast strain having a gene signal l Z or HIS3-controlled 5-district GAL4 binding sites. Signaling gene will be expressed only in those transformed cells of the host (cotransformation) where both g the AU Z, host cell in which it is expressed, will be painted in blue color when added to a culture X-gl. Therefore, the presence of colonies of blue color indicates that the two investigated cloned protein is able to interact with each other.
Using this system of dihybrids, the intracellular domain of FAS-IC cloned separately into the vector pGBT9 (the raw sequence of the GAL4 DNA binding obtained in the CLONTECH, USA, see below) to obtain a protein with a domain of the GAL4 DNA binding. For cloning FAS-R in pGBT9 used the clone encoding the full-size cDNA sequence for FAS-R (see joint proposal, IL 111125) using standard procedures using different restrictus was cut section that encodes the intracellular domain of the IC. Then this clone was isolated and inserted into the vector pGBT9, cut the appropriate restrictedly in the field of multiple cloning sites (MCS). It should be noted that FAS-IC occupies the site in intact FAS-R from 175 to 319 residue, and it is this section of FAS-IC was inserted into the vector pGBT9 (see also IL 111125).
The above hybrid (chimeric) vector with cDNA from a library of genes in human cells HeLa, cloned in the vector pGADGH, not ectory - pGBT9 and pGADGH carrying the cDNA library of HeLa cells, as well as the strain of yeast were taken from Clontech Laboratories, Inc., USA, as an integral part of the system MATCHMAKER Two-Hybrid System, #PT1265-1).
In conjunction transfected yeast conducted selection on the ability to grow in an environment that does not contain histidine (Wednesday His-), with growing clones indicated the presence of positive transformants. Otsilindrovannye yeast clones were then tested for the ability to Express the gene lac Z, i.e., on the activity of the LAC Z in them. This was done by adding to the culture medium X-gal, which is under the-galactosidase enzyme encoded by the gene lac Z, decomposes with the formation of a blue-colored product.
Thus, the blue colonies indicate the activity of the gene in them l z activity of the gene lac Z requires that the activator GAL4 transcription was in active form in the transformed clones, namely that DNA binding domain encoded by the above-mentioned hybrid vector, properly connected with the activation domain GAL4 encoded by other hybrid vector. Such a connection is possible only if two proteins, merged respectively with different domains of GAL4, possessed the ability to consistently cooperation are colonies, which together transformed with the vector coding for FAS-IC, and a vector coding for the protein product of the human cells HeLa being able to contact FAS-IC.
DNA plasmids from above His+, LAC Z+colonies of yeast was isolated and by electroporation introduced into E. coli strain NV using standard procedures, and then transformants were selected Leu+and resistant to ampicillin, and these transformants are hybrid vector pGADGH, which has a coding sequence for AMRRand Leu2. Therefore, such transformants are clones carrying sequences encoding the identified new proteins that are able to communicate with FAS-IC. The DNA plasmid was then isolated from these transformed cells of E. coli and again tested as follows:
(a) by retransformation it together with the original hybrid plasmid that encodes FAS-R (hybrid pGTB9, carrier FAS-IS), yeast strain HF7, as described above. To control for co-transformations were used vectors carrying sequences encoding unnecessary protein, for example, age-Lamin, or only rstt together with a plasmid that encodes a binding protein (FAS-IC (i.e., MORT-1). Joint is the ed with the addition of different amounts of 3-aminotriazole; and
(C) by retransformation yeast cells by the host strain SFY526 DNA plasmid and the original hybrid plasmid with FAS-IC and control plasmids described in (a), with subsequent determination of the activity of the LAC Z+(efficiency-gal, i.e., the formation of blue colonies).
The results of these tests revealed that the nature of growth of the colonies in His environment-was identical to the nature of activity of the LAC Z as proven colored colonies, i.e. the colony His+had also and phenotype LAC Z+. Further, the activity of the LAC Z in liquid medium (preferred culturing conditions) were tested after transfection hybrid plasmids with the domain of the GAL4 DNA binding and activation domain in yeast cells SFY256 have the best inducibility LAC Z activator GAL4 transcription than yeast host cell HF-7.
Using the above procedures was identified, isolated and characterized a protein called HF-1 and now called MORT-1, "Mediator of Receptor-induced Toxicity".
It should also be mentioned that in some of the above tests the expression-galactosidase in digibridge system expression-g is equl contain cDNA inserts MORT-1. Selected thus cloned cDNA insert MORT-1 were sequenced using standard procedures for determining DNA sequences.
Amino acid sequence of MORT-1 received from the sequence of nucleotides in DNA. Amino acid residues in proteins were numbered, as is done in the database Swiss-Prot.
Mutants with deleteme were obtained using PCR, and point mutants using mutagenesis, oligonucleotide-directed (Current Protocols in Molec. Biol., 1994).
(ii) Induced the expression, metabolic labeling and immunoprecipitation proteins.
MORT-1, attached to the N-end to octapeptide FLAG (FLAG-HF-1; Eastman Kodak, New Haven, Ct., USA), Chimera (FAS-IC, FAS-R, p55-R, including extracellular domain p55R (amino acids 1 to 168), fused with the transmembrane and intracellular domain of FAS-R (amino acids 153 at 319), and the cDNA luciferase that serves to control, expressed in HeLa cells. Expression was performed using the expression vector controlled by the tetracycline, the clone of HeLa cells (HtTA-1), which is controlled by the tetracycline transactivator (Gossen and Bujard, 1992; as described in PCT/US95/05854; see also Boldin et al., 1995). Metabolic labelling with [35S]-methionine and [35S]-cysteine (DUPONT, Wilmington, De., USA and the Ame Dulbecco, not containing methionine and cysteine, but supplemented with 2% cialisbuynow fetal calf serum. Then the cells were literally in RIPA buffer (10 mm Tris-HCl, pH 7.5, 150 mm NCl, 1% NP-40, 1% desoxycholate, 0,1% SDS and 1 mm EDTA), and the lysate was pre-prosvetlili by incubation with ballast rabbit anticorodal (3 μl/ml) and balls Sepharose with G protein (Pharmacia, Uppsala, Sweden; 60 μl/ml). Immunoprecipitation performed by incubation for 1 h at 4°With 0.3 ml of lysate with mouse monoclonal antibodies (5 μl/sample) against octapeptide FLAG (M2; Eastman Kodak), pp 55-P (#18 and #20; (Engelmann et al., 1990)), or FAS-R (ZB4; Kamiya Southand Oaks, Ca., USA), or with the appropriate izotopicheskii murine antibody as a control, followed by incubation for 1 h with beads Sepharose, Laden G protein.
(iii) Binding of in vitro
Prepared glutathione S-transferase fused with Fas-IC wild-type or mutandis Fas-IC, and adsorbing it on beads with glutathione (as described in IL 109632, 111125, 112002, 112692; see also Boldin et al., 1995; Current protocols in molecular biology, 1994; Frangioni and Neel, 1993). The binding of metabolically labeled fused protein FLAG-HF-1 CST-Fas-IC was assessed by incubation of beads for 2 h at 4°With extracts of HeLa cells, metabolically labeled [35S]-methionine (60 MK Kiu/ml), the cat is Tola, 1 mm EDTA, 1 mm phenylmethylsulfonyl, 20 μg/ml of Aprotinin, 20 μg/ml leupeptin, 10 mm sodium fluoride, and 0.1 mm sodium Vanadate (1 ml per 5×105cells).
(iv) Analysis of cytotoxicity triggered induced expression of HF-1.
HF1 cDNA, Fas-IC, p55-IC and luciferase was inserted in controlled by the tetracycline expression vector and they were transferrable cells t-1 (cell line HeLa) (Grossen and Bujard, 1992), together with cDNA Sekretareva alkaline phosphatase placental placed under the control of the SV40 promoter (vector pSBC-2 ((Dirks et al., 1993)). Cell death was evaluated at 40 h after transfection in the test by the absorption cells neutral red (Wallach, 1984) or for a specific estimates of the death of those cells that Express transfected cDNA, by determining the amount of alkaline phosphatase from the placenta, Sekretareva in the growth environment on the 5th h of incubation.
In another series of experiments to analyze the field of protein MORT-1 (HF-1) involved in the binding to the FAS-IC, in HeLa cells, which contain controlled by the tetracycline transactivator (HtTA-1), induced a temporary expression of the following proteins using controlled by the tetracycline expression vector (pUHD 10-3): only FAS-R person; FAS-R and N-terminal part of R-1 (amingo the scope of its "domain of death" (amino acids 130 245, "MORT-100", see also IL 112742); FLAG-55.11 (amino acids 309-900 protein 55.11, merged its N-end with octapeptide FLAG, and is 55.11 protein specific binding P55-IC, see also IL 109632). 12 h after transfection cells were collected by trypsinization and again planted at a concentration of 30,000 cells/cell. After incubation for 24 h, cells were treated for 6 h with monoclonal antibodies against the extracellular domain of FAS-R (monoclonal antibody CH-11, Oncor) at various concentrations (0.001 to 10 μg/ml monoclonal antibody) in the presence of 10 g/ml of chicagocrime. After this was determined by cell viability test by the absorption of neutral red and the results were calculated as % of living cells with respect to cell number, inkubiruemykh with one cycloheximide (in the absence of monoclonal antibody CH-11 against FAS-R).
(v) Northern analysis and identification of sequences.
Poly And+RNA was isolated from HeLa cells (set Oligotex-dT mRNA, QIAGEN, Hilden, Germany). Northern analysis using as sample cDNA HF-1 was performed using a conventional method (as described in PCT/US95/05854; see also Boldin et al., 1995). The nucleotide sequence for MORT-1 (HF-1) was determined in both directions using the method of termination of dideoxy chain.
Application digibridge test to assess the binding specificity of this protein (MORT-1, "Mediator of Receptor-induced Toxicity") c Fas-IC and to identify specific areas in Fas-IC, with which he is associated, has led to the following results (table 1):
(a) this protein is associated with human and murine Fas-IC, but not associated with a number of other proteins tested, including three receptor family of receptors TNF/NGF (TNF receptors P55 and P75, and CD40);
(b) it is shown that mutation of a substitution at position 255 (Ile) in the "domain of death" FAS-R, blocking the transmission of signals both in vitro and in vivo (mutation 1prcg(Watanabe-Fukunaga et al., 1992; Itoh and Nagata, 1993)), also prevents binding to MORT-1 FAS-IC;
(c) the binding site MORT-1 FAS-R is in the domain of death" of this receptor, and
(d) MORT-1 binds with itself. In self and in linking HF-1 FAS-R involved different region of the protein. Fragment of MORT-1, corresponding to residues 1 to 117, associated with a full-sized molecule, MORT-1, but not related to the same fragment, nor with FAS-IC. Conversely, a fragment corresponding to residues 130 245, binds to FAS-R, but is not associated with MORT-1. In addition, from the results, the actu "domain of death" p55-R for self-P55-1C. Deletions on both sides of this "domain of death" does not affect their ability to self, but deletions within these "domain of death" affect the self-Association.
In the case of MORT-1 binding to MORT-1 FAS-IC also depends on a (full) "domain of death" FAS-R, the binding of FAS-R does not depend on areas outside of the "domain of death" FAS-R.
Table 1 shows the results of interaction between pairs of proteins, one of which is encoded by design rswt containing the code for the domain DNA-binding protein GAL4, and the other is encoded by design pGAD-CH containing code activation domain GAL4. The interaction of these proteins occurred in yeast cells, SF 526, together transfected with the indicated constructs, and were analyzed using the test expression-galactosidase on the filter. Among the structures of the DNA-binding domain comprises four designs Fas-IC man; four designs Fas-IC mice, including two full-sized design, having a mutation of substitution of Ile for Leu or Ile at l in position 225 (I225N and I225A respectively); and three designs HF-1 (R-1). All these designs are depicted on the left side of Table 1. In a number of designs activation domain includes three designs HF-1, p is Fas-IC man, moreover, the design part of the Fas-IC is the same as the above-mentioned structure of the DNA-binding domain. The intracellular domains of the receptor P55 TNF person (residues 206-426 p55-IC), CD40 person (residues 216-227) and receptor P75 human TNF (residues 287-461), and Lamin, cyclin D and "empty" Gal4 vector (pGBT9) served as negative controls in the form of structures of the DNA-binding domain. SNF-1 and SNF4 served as positive controls in the form of structures of the DNA-binding domain (SNF1) and activation domain (SNF4). "Empty" GAL4 vectors (pGADCH) also served as negative controls in the form of structures of the activation domain (more details of the p55-IC and P75-IC, see PCT/US95/05854). The symbols "++" and "+" indicate the development of a strong staining for 30 and 90 min of the test, respectively; and "-" indicates the absence of staining within 24 hours Combinations for which these symbols are not available, has not been tested.
The expression of molecules HF1 (MORT-1), fused to the N-end octapeptide FLAG (FLAG-HF-1), resulted in formation in HeLa cells proteins of four different sizes: about 27, 28, 32 and 34 kD. In Fig.1 (a and b) results showing interaction HF1 with Fas-IC in vitro. As indicated above in the description of Fig.1A and b, in Fig.1 reproduced control radioautogram of immunoprecipitate proteins of the and Fig.1B is reproduced radioautography, showing the interaction in vitro between HF1 and Fas-IC, where HF1 is in the form of labeled [35S]-methionine fused protein HF1-FLAG obtained from the extract transfection in HeLa cells, a Fas-IC - in the form of a fused protein between human and mouse GST-FAS-IC, one of which has a mutation substitution at position 225 in Fas-IC. All of these fused proteins GST-FAS-IC obtained in E. coli cells. Fused with GST proteins before interaction with extracts containing protein HF1-FLAG, attached to the balls of glutathione. After this interaction was performed SDS-PAGE. Thus, the interaction in vitro was evaluated using radioautography after SDS-PAGE by the binding of metabolically labeled protein HF1, synthesized in HeLa cells in the composition of the fused protein HF1 with octapeptide FLAG(FLAG-HF1), with GST fused with human or murine Fas-IC (GST-huFas-IC, GST-mFas-IC); or with GST fused with Fas-IC containing the mutation replacement Ile on l in position 225. As can be seen from Fig.1B, all four protein FLAG-HF1 find the ability to bind to Fas-IC during incubation with fused protein GST-Fas-IC. As in the test system of yeast dihybrid, HF1 is not associated with the fused protein GST-Fas-IC, containing site mutation replacement lprcg(I225A).
Proteins encoded by cDNA FLAG-HF1, also find the ability to bind to the intracellular domain of F the th domain of the p55-R (P55-FAS) in the co-expression of these receptors in HeLa cells. In Fig.2 (a, b, C) results showing interaction HF1 with FAS-IC in transfected HeLa cells, i.e. in vivo. As mentioned above, in the description of Fig.2 a, b, C, these figures are reproduced radioautography of precipitates of various transfected HeLa cells, which demonstrate the interaction in vivo and specific interaction between HF1 and FAS-IC cells, together transfected with constructs encoding these proteins. Thus, protein FLAG-HF1 expressively and metabolically were marked by [35-S]-cysteine (20 MK Kiu/ml) and [35S]-methionine (40 MK Kiu/ml), HeLa cells alone or together with Chimera FAS-R human FAS-R, in which astralloy domain of FAS-R replaced by the corresponding region of the p55-R man (p55-FAS), PII along with the p55-R as a negative control. Cross-immunoprecipitate HF1 together with expressed receptor was performed using the indicated antibodies (Fig.2A-C). As can be seen from Fig.2A-C, FLAG-HF1 able to bind with the intracellular domain of FAS-R, as well as with recombinant intracellular domain of FAS-R, p55-R, having the extracellular domain of the p55-R and the intracellular domain of FAS-R co-expression of these receptors in HeLa cells (see the middle of the track of Fig.2A ickleton also leads to precipitation together downregulation of FAS-R (Fig.2A) or co-expressed chimeras p55-FAS (Fig.2C). Conversely, immunoprecipitate of these receptors leads to joint precipitation of FLAG-HFl (Fig.2A and 2C).
Analysis by Northern hybridization using as a probe HF1 cDNA hybridization reveals only one transcript in HeLa cells. In Fig.3 shows Northern blotting, in which poly (A+PHK (0.3 ág) from transfected cells hybridized with cDNA HF1. The size of this transcript (about 1.8 kV) is close to the size of the HF1 cDNA (about 1702 nucleotides).
When the sequence analysis, it was found that this cDNA contains an open reading frame of approximately 250 amino acids. In Fig.4 shows the preliminary nucleotide sequence and derived from it the amino acid sequence HF1, in which the main part of the "domain of death" highlighted as a possible initial Met residue (position 49, bold underlined M), and marked the stop codon of translation (the asterisk under the codon in position 769-771). The main part of the "domain of death" reveals homology with known main parts of the "death domains" of the p55-R and FAS-R (P55 DD and FAS-DD). For a precise definition of the C-terminal part of the HF-1 and obtain accurate data on the N-terminal part of HF1 (starting balance Met) were performed the following experiments.
With the help of what predom FLAG (FLAG-HF1). When the expression of these constructs in HeLa cells expressed proteins metabolic marked using35S-cysteine and35S-methionine (see above about the same in Fig.5B). Molecules HF1-FLAG was coded following cDNA containing various areas with different code sections N 1:
i) cDNA of octapeptide FLAG attached to the 5-the end of the HF1 cDNA from which the cut nucleotides 1 through 145 (see Fig.4);
ii) the cDNA of octapeptide FLAG attached to the 5-end full-size cDNA HF1 (see design FLAG-HF1 above in Fig.1B);
iii) cDNA of octapeptide FLAG attached to the 5-the end of the HF1 cDNA from which the cut nucleotides 1 to 145, and nucleotides with 832 in 1701 (see Fig.2), and the codon GCC in position 142-144 changed to TCC in order to prevent the start of the broadcast in this site.
After the expression of the above-mentioned fused products HF1-FLAG held their immunoprecipitation, as described above, or using monoclonal antibodies anti-FLAG (M2) or control antibodies anti-75F-R (H9), and then the separation in SDS-PAGE (1% acrylamide) and radioautography. The results are shown in Fig.5, where the reproduced radioautogram separated above the slit Belko the DNA octapeptide FLAG joined 5-the end of the HF1 cDNA from which the cut nucleotides 1-145.
Tracks 3 and 4: protein HF1-FLAG encoded by cDNA of octapeptide FLAG attached to the 5-end of the cDNA full-HF1.
Tracks 5 and 5: protein HF1-FLAG encoded by cDNA attached to the 5-the end of the HF1 cDNA from which the cut nucleotides 1-145, and 832-1701, a GCC in position 142-144 replaced by TCC in order to prevent the start of the broadcast in this site.
Immunoprecipitation samples in lanes 2, 4 and 6 were performed with monoclonal antibodies anti-FLAG, and samples in lanes 1, 3 and 5 - antibodies anti-p75TNF-R.
From radioautography in Fig.5 shows the following. The identity of the dimensions of the products in lanes 2 and 4 confirms that the nucleotides 769-771 are the sites of translation termination for HF1, i.e., the codon is a stop signal, as shown by the asterisk in Fig.4. Further, the presence of a broad band, which actually contains two product broadcast (visible on the gel, but because of the strong labeling of merging into one big strip on radioautography) on track 6, shows that the presence of two additional products (higher molecular weight in sikuli, and the methionine residue at position 49 in the sequence HF-1 (see bold underlined M at position 49 the amino acid sequence of Fig.4). Thus, the above results confirm the correctness of the given amino acid sequence With late HF1 and serve as proof that the N-end HF-1 may be in position 49 of the sequence depicted in Fig.4.
Indeed, additional experiments on the expression HF-1 without attached to its 5-end of octapeptide FLAG was shown that the Met49serves as an effective site of translation initiation.
It should be recalled that the search in the database "Gene Bank" and "Protein Bank is not detected sequence corresponding to the sequence HF-1, is shown in Fig.4. Thus, HF1 is a new protein specific binding of FAS-IC.
High level of expression of the P55-IC starts cytotoxic effect (see IL 109632, 111125 and Boldin et al., 1995). Expression of Fas-IC in HeLa cells also leads to such effect, although smaller in size, which can only be detected using a sensitive test. In Fig.6A and In the charts for activity-dependent ligand heat expression HF1, Fas-IC man, the P55-IC man, or luciferase - in control, on the viability of HeLa cells was analyzed using the expression vector controlled by tetracycline. Cell viability was determined after 40 min after the introduction of the cDNA in the presence of (unpainted columns of Fig.6A and b) or absence (shaded bars in Fig.6A and b) tetracycline (1 μg/ml to block expression) together with the cDNA coding secreterial alkaline phosphatase placenta. Cell viability was determined either in the test by the absorption of the dye neutral red (Fig.6A), or when specific definition of viability of cells that Express transfairusa DNA by measuring the amount of alkaline phosphatase placenta, Sekretareva in growth medium (Fig.6B).
It Is Evident From Fig.6A and shows that the expression of HF1 in HeLa cells killed significantly more cells than the expression of FAS-IC. Cytotoxic effects of the p55-IC, FAS-IC and HF1, apparently, associated with areas of "domain of death", available at all of these proteins, the domains of death" which have a predisposition to self and may therefore provoke cytotoxic effects. The above-mentioned properties of HF1 (MORT-1); namely, the specific Association HF1 with a special area in FA is ti (lpr mutationcgwhich prevents the transmission of signals, blocks the binding of HF1, show that this protein plays a role in signal transmission and the start of cell death. This conclusion is indicated by the ability HF1 run effect of the own cell cytotoxicity. So, HF1 (MORT-1) can operate (i) as a modulator of self-FAS-R, thanks to its ability to bind to FAS-R as well as in self, or (ii) to serve as a place of joining of other proteins that participate in signal transduction-dependent FAS, i.e., HF1 may be protein "docking" and therefore to associate along with FAS-R other receptors, or (iii) is an integral part of another transmission system, which interacts with alarm system FAS-R.
For further analysis regions MORT-1 (HF1) involved in the binding of FAS-R and modulation of cellular effects (cytotoxicity), mediated by the FAS-R, were performed the above experiments using vectors that encode proteins MORT-1 ("the head of MORT-1, amino acids 10117 and MORT-1 dd, amino acids 130-245) (separately), with the vector coding for FAS-R, for co-transfection of HeLa cells. In these experiments, conducted the temporal expression of different proteins or combinations of proteins in the, tiraumea these proteins are controlled by the tetracycline expression vector pUHD 10-3. In the control transfected used vectors encoding only FAS-R, and vectors encoding the protein FLAG-55.11 (protein 55.11 represents part of a protein from amino acids 309 900, specifically linking the P55-IC, was merged (N-end) with oktapeptidom FLAG).
Transfected cells after appropriate incubation periods (see above (iv)) was treated with various concentrations of monoclonal antibodies anti-FAS-R (SN-11), which is specifically associated with the extracellular domain of FAS-R expressing cells. This binding of the antibody anti-FAS-R induces the aggregation of FAS-R on the cell surface (much the same as when the binding of the ligand FAS-R) and induces a chain of intracellular signals mediated by the FAS-IC, which ultimately leads to cell death (cytotoxicity cells, mediated by the FAS-R). Used concentrations of monoclonal antibodies anti-FAS-R (SN-11) 0.01 to 10 μg/ml, usually such concentration 0.005; of 0.05, 0.5 and 5 μg/ml Cells were treated with antibodies anti-FAS-R in the presence of 10 μg/ml cycloheximide.
The results of the above analysis is shown in graph form in Fig.7, in which the % of viable transfected cells is for each of the four groups of transfected cells. These groups transfected cells are marked by different symbols: (i) an unfilled squares represent cells, transferi-rowanne only control vector coding for a protein FLAG-55.11 ("55.11", negative control), no binding of FAS-IC; (ii) the filled squares represent cells transfected together with vectors encoding the FAS-IC, and vectors encoding the C-terminal part of MORT-1, amino acids 130-245, which contains a region homologous to the "death domain" (dd) MORT-1 ("fas+mort Idd"); (iii) the shaded triangles represent cells transfected only with the vector coding for FAS-R ("fas", positive control); and (iv) unfilled circles denote cells, together transfected with vectors encoding the FAS-R, and vectors encoding the N-terminal part of MORT-1, amino acids 1-117, "the head of MORT-1" ("fas-morthe").
From the results shown in Fig.7, it is seen that the expression of FAS-R transfected cells attached to the cell sensitivity to the cytotoxic action of antibodies anti-FAS-R (compare "fas" with "55.11"). Further, co-expression region MORT-1, which contains a region homologous to the "domain of death" and FAS-R ("fas+mort Idd"), strongly inhibits cell death induced by FAS (i.e., mediated by the FAS-R), as it followed the, co-expression of N-terminal part of MORT-1 and FAS-R ("fas+morthe") or no effect on cell death, mediated by the FAS-R, or only slightly increases the cytotoxicity (i.e., increases cell death).
Thus, the above results clearly show that the protein MORT-1 has two different areas involved in the binding of FAS-IC and the mediating domain FAS-IC cytotoxic cell activity.
Therefore, these results give reason to use different areas (i.e., active fragments or analogs) protein MORT-1 in various pharmaceutical applications. For example, the analogs or fragments or derivatives of protein MORT-1, which mostly contain only the C-terminal part of MORT-1, including its "domain of death", can be used for suppression mediated by the FAS-R cytotoxic effects in cells or tissues containing the FAS-R, and protection, in this way, these cells or tissues from the harmful effects of ligand FAS-R, for example, in such cases as acute hepatitis. On the contrary, those analogs or fragments or derivatives of protein MORT-1, which contain mostly only the N-terminal part of MORT-1, can be used to increase mediated by the FAS-R cytotoxic effects in cells or tissues, Sogno, for example, tumor cells or self-reactive T - and b-cells. The above application of different regions MORT-1 may be carried out, as described above, by using different recombinant viruses (for example, from cowpox) for embedding different sequences encoding parts MORT-1, in certain cells or tissue in need of treatment.
In addition, you can also obtain and use various other molecules, such as antibodies, peptides and organic molecules, sequences and molecular structures which meet the above areas MORT-1, to achieve the same desirable effects, what are mediated by these areas R-1.
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1. The DNA molecule encoding polypep the th sequence of SEQ ID No: 1 protein MORT-1;
(2) a nucleotide sequence that encodes an analogue of the specified protein MORT-1, where the specified analog differs from protein MORT-1 by a single amino acid residue and binds to FAS-IC;
(3) a nucleotide sequence consisting of nucleotide sequence that encodes a fragment of a specified protein MORT-1, where the specified fragment of the protein binds to FAS-IC; or
any obtained on the basis of the degeneracy of the genetic code variants of the nucleotide sequence specified in (1), (2) or (3).
2. The DNA molecule under item 1, characterized in that it has a DNA sequence encoding the amino acid sequence of SEQ ID No: 2.
3. The DNA molecule under item 2, characterized in that it has a DNA sequence SEQ ID No: 1.
4. The polypeptide able to bind with the intracellular domain of FAS-R encoded DNA under item 1.
5. The polypeptide under item 4, characterized in that it has a deduced amino acid sequence of SEQ ID No: 2.
6. Vector for expression in eukaryotic cells of polypeptide that binds to the intracellular domain of FAS-R, comprising the DNA sequence according to one of paragraphs.1-3, functionally linked to regulatory sequences.
7. The method of obtaining the floor of the new vector at p. 6, in conditions that ensure the expression of the specified polypeptide, an effective post-translational modification and its selection.
8. Mode of action of the ligand FAS-R cells carrying FAS-R, including treatment of these cells with the polypeptide under item 4 or 5, are able to bind with the intracellular domain of FAS-R and modulate the activity of FAS-R.
9. The modulation effect of ligand FAS-R cells, including application procedures using a ribozyme, in which the vector encoding the sequence of the ribozyme capable of interacting with a cellular mRNA sequence that encodes a polypeptide under item 4 or 5, is introduced into cells in a manner that provides the expression of the sequence of the ribozyme in these cells, while expression of the sequence of the ribozyme in said cells it interacts with the specified cellular mRNA sequence and cleaves the sequence of this mRNA, which leads to inhibition of expression of MORT-1 in these cells.
10. Pharmaceutical composition for modulation of the action of the ligand FAS-R cells, comprising as an active ingredient the polypeptide under item 4 or 5, or a mixture.
11. Pharmaceutical compositeactor virus animals carrying a sequence encoding a protein capable of contact with a receptor on the cell surface, and the sequence encoding the polypeptide under item 4 or 5.
12. Isolation and identification of a protein able to bind with the intracellular domain of FAS-R, including the application procedures of hybridization on Southern dipped in mild conditions with subsequent cloning by PCR, in which a sequence or part thereof according to any one of paragraphs.1-3 is used as a probe that binds to sequences from a cDNA library or genomic DNA having at least partial homology with the probe, with the above related sequences are then amplified and clone using PCR procedure to obtain clones encoding proteins having at least partial homology with these sequences on PP.1-3.