UA74529C2 - CASH (CASPASE HOMOLOGUE) WITH ôDEATH-EFFECTOR" DOMAIN, MODULATORS OF FAS-RECEPTOR FUNCTIONS - Google Patents

Abstract

The invention relates to the DNA sequence coding novel protein G1, able to contact or interact directly or mediately with MORT-1 and/or with any proteins which contact with MORT-1, and able to mediate an intracellular influence mediated by FAS-R or p55-TNF-R. Also disclosed is the preparation of such recombinant protein, methods for simulation of cells death or inflammatory processes in vitro methods for simulation of apoptotic processes or processes of the programmed cells death in vitro, and also pharmaceutical composition including the protein G1 as an active ingredient.

Description

Description of the invention

The present invention generally relates to the field of science that studies receptors that belong to the superfamily 2 TME/MOv-receptors and the control of their biological functions. The superfamily of TME/MOP receptors includes such receptors as the tumor necrosis factor receptor p5B5 and p75 (the so-called "TME-K", also designated as СО120a and СО120р, respectively: in this text they will be referred to as p55-K and p /7/5-K) and ERAz-ligand receptors (which are also designated as REAB/AROT or GAZ-K, or СОО5, and in this text they will be designated as РА5-К) and others. More specifically, this invention relates to novel proteins that bind to other proteins that themselves bind 70 to the pain MOCT-1 (or EAOI): they are also called MOCT-1-binding proteins, or to proteins that directly bind to the MOKT-1 bilayer, and more specifically, the invention relates to one such protein, designated herein as 1 (also designated as "SABN" for "caspase homolog," but hereinafter referred to as "(1")), which binds with the MOKT1-binding protein Mep4 (also designated/named SABR-10) and appears to bind to another MOKT1-binding protein called MASN (also designated/named CA5P-8) , and, 12 probably directly binds to MOKT-1 itself.

Accordingly, the present invention, in principle, relates to new proteins that are able to modulate or mediate the functions of MOKT-1 directly or indirectly or other proteins that bind to MOKT-1 directly or indirectly. In particular, this invention relates to C1 protein, its preparation and use, as well as certain novel C1 isoforms, their preparation and use.

Tumor necrosis factor (TME-o) and lymphotoxin (TME-r) (hereinafter the abbreviation TME will refer to both TMP-A and

TME-p) are multifunctional pro-inflammatory cytokines, which are produced mainly by mononuclear phagocytic cells, which exert various effects on cells (MMaiyasi, 1986, IP "Ipiepegop 7", ey.

Bou I.Sgezzeg, Asad. Rgezv, I opaop, pp. 83-122; and Vesheg 5 Segati, 1987). | TME-A and TME-D show their activity by binding to specific cell surface receptors. Some of their manifestations are probably positive for the body: for example, they can destroy tumor cells or cells infected with viruses (o) and cause the antibacterial activity of granulocytes. In this case, TME are the body's defense factors against tumors and infectious agents and participate in wound healing. Thus, TME can be used as an antitumor agent, when applied, it binds to its receptors on the surface of Fu 20 tumor cells and thus initiates events leading to the death of tumor cells. TMEs can also be used as anti-infective agents. with

However, both TME-A and TME-dD also have negative manifestations. There is evidence that overproduction of oyu

TME-o can play the main pathogenetic role in the development of some diseases. For example, the action of TME-o, primarily on the vascular system, is known as the main reason for the development of symptoms of septic shock |Ggaseu ei ai)., « 1986). In some diseases, TME can cause significant weight loss (cachexia) by suppressing the activity of adipocytes, leading to anorexia, which led to the designation of TME as cachetin. It has also been described that TME is a mediator of tissue damage in rheumatic diseases |Ivesheg 5 Segati, 1987) and the main mediator of damage observed in the syndrome of "graft against the host" |Ridzei ei « a!., 19871. In addition, TME known as a participant in the inflammatory process and many other diseases.

Two receptors that are clearly distinguished and independently expressed - pbo5 and p/5 TME-K - which specifically bind both TME-o and TME-D, initiate and (or) mediate the biological manifestations of TME described above. These two receptors include structurally different intracellular domains, which indicates the difference in their signaling activity | see Noptapp et al., 1989; Epdeitapp et al., 1990; Vhoskpaiz et al.,, 1990; I eoivspeg et al. , 1990; 5snaiY ei aiYi., 1990; Mornagh ei. , apparently, other factors - I are involved in the intracellular signaling to the receptors rb5b5 and p/5 TME-K. At the same time, there is an intracellular signaling pathway that is usually active after the binding of the receptors of its ligand, that is, TME (o or p), and which is associated with the initiation of a cascade of reactions that, ultimately, lead to the observed 1 cell response to TME. 7 50 From the point of view of the above-mentioned cytotoxic effect of TME, in most of the cell types studied to date, this manifestation is mediated mainly by receptor ro55 TME-K Antibodies to extracellular domains (i.e., iChe) domains of ligand binding) of the receptor po5 TME-K themselves can mediate the cytotoxic effect of Ivid. EP 412486), which correlates with the efficiency of cross-linking of the receptor with antibodies, which is considered as the first step in the initiation of the intracellular signaling mechanism. Next, mutational analysis | Vgakerizsi ei ai., 1992; Tapadia et al.,, 1993| showed that the biological functions of the TME-K gene depend on the assembly of its intracellular domain: accordingly, it was confirmed that the initiation of the intracellular signaling mechanism, which leads to the cytotoxic effect of TME, is a consequence of the binding of two or more intracellular domains of the TME gene - K. Moreover, TME (o; and B) is a homotrimer, and since it is so, it has been confirmed that the induction of the intracellular signaling mechanism through po5 TME-K is due to its ability to bind and form cross-links with receptor molecules, that is, to form aggregations receptors

Another member of the TME/MOE receptor superfamily is the RAb3 receptor (BAB-K), also called

EAZ3 antigen: This is a cell surface protein expressed in various tissues and shows similarity to a number of other cell surface receptors, including TME-K and MOB-K. The RHA5B-K receptor mediates the death of 65 cells by the type of apoptosis (MOP ei ai., 1991) and, apparently, works as a negative selector of auto-reactive

T-lymphocytes: that is, during the maturation of T-lymphocytes, EAB-K mediates the apoptotic death of T cells that recognize their own antigens. It was also established that a mutation in the RA5B-K gene (Irg mutations) causes a lymphoproliferative disorder in mice that resembles systemic lupus erythematosus, which is an autoimmune disease in humans | -K, as it is assumed, is

A molecule associated with the cell surface carried by, among other cell types, T-killers (or cytotoxic T-lymphocytes - STIs): accordingly, when such STIs come into contact with cells bearing GAB-K, they are able to induce apoptotic cell death , carrying GA5B-K. In addition, a monoclonal antibody specific for EA5-K was generated, this monoclonal antibody is capable of inducing apoptosis-like death of EA5-K bearing cells, including murine cells transformed with cDNA encoding human EA5-K 7/0 Shawn eia )., 19911.

While the cytotoxic action of lymphocytes is mediated by the interaction of a ligand produced by lymphocytes with the widespread cell surface receptor RBAB-K (СО95), which is capable of triggering the cell death mechanism, it has also been established that various other normal cells, in addition to

T-lymphocytes express HA5B-K on their surface and can be killed indirectly by the action of this receptor. 7/5 It is assumed that the uncontrolled induction of such death is a factor in tissue damage in certain diseases, for example, the destruction of liver cells in acute hepatitis. Accordingly, the discovery of ways to inhibit the cytotoxic activity of RAB-K may have therapeutic value.

On the other hand, since some malignant tumor cells and cells infected with HIV have also been found to carry EAB-K on their surface, antibodies against HA5B-K or the EA5B-K ligand can be used to control HAB-K-mediated cytotoxic effects on such cells and, thanks to this, serve as a way to fight against cells of malignant tumors or cells infected with HIV | see Moi ei ai., 1991). The discovery of some other ways of enhancing the cytotoxic activity of EAB-K may also have therapeutic value.

Obviously, there is an urgent need to develop a method of modulating cellular responses to TME (o, or B) and EA5B-K ligands. For example, in the pathologies described above, when there is an overexpression of TME or p

EA5-K ligand, it is desirable to suppress the cytotoxic effect induced by TME or RAZ5-K ligand, while in other situations, for example, during wound healing, it is desirable to enhance the effect of TME, or in i) the case of RGAB-K in in relation to tumor or HIV-infected cells, it is desirable to enhance the effect caused by EAZ-K.

Applicants have proposed a number of relevant approaches (see, for example, European patent applications Ge») 3o MoMo EP-186833, EP-308378, EP-398327 and EP-412486) aimed at controlling the adverse effects of TME by inhibiting the binding of TME on their receptors using antibodies to TME or using soluble TME receptors (that is, sufficient to dissolve the extracellular domains of such receptors) in order to create competition for TME binding on TME-K receptors located on cell surfaces. In addition, based on the fact that the binding of TMEs to their receptors is necessary for the manifestation of the respective induced effects of TMEs, in

335, the applicants proposed approaches (see, for example, ERO-568925) aimed at modulating the action of TME by changing the activity of TME-K receptors.

Briefly, the application EPO-568925 relates to a method of modulating signal transmission and (or) cleavage of TME-K in such a way that peptides or other molecules can interact either with the receptor itself or with effector proteins that interact with this receptor, as a result of which modulation of normal functions of TME-K. « 20 IV of the application ERO-568925)| the construction and characteristics of various mutant forms of po5 TME-K, which all have mutations in the extracellular, transmembrane, and intracellular domains of po5 TME-K, are described. In this case, sections of these domains were identified as part of TME-K proteins, which are key from the point of view of ensuring "" functions of the receptor, i.e. binding of its ligand (TME) and subsequent signal transmission and triggering of the intracellular signaling mechanism, which, ultimately -end, and lead to manifestations of TME action on the observed cells. In addition, a number of approaches to the isolation and identification of proteins, peptides, or other factors capable of binding to various regions of the TME-K domains mentioned above, proteins, peptides, and other factors that may be involved in the regulation or modulation of receptor activity are also described TME-K. Also (|in that application EPO-568925) a number of approaches to isolation and cloning of DNA sequences encoding such proteins and "sl peptides, for the construction of expression vectors intended for the production of such proteins and peptides, and obtaining antibodies or their fragments, which interact with TME-K or with the above-mentioned proteins and peptides that bind to different segments of TME-K. However, (application ЕРО-568925) does not specify the parameters of proteins and peptides,

Ge) acting to bind to the intracellular domain of TME-K receptors (eg, TME-K receptor), and does not describe a "yeast two-hybrid approach" to isolate and identify such proteins or peptides that bind to the intracellular domains of TME-K receptors K. Also | in the application EPO-568925) there is no description of proteins or peptides capable of binding to the intracellular domain of the HA5-K receptor.

Thus, if it is desired to inhibit the action of TME or the HA5B-K ligand, it should be desirable to reduce iF) the amount or level of TME-K or RAB-K activity on the cell surface, while increasing the amount or level of TME-K activity or RHA5B-K should be preferred when seeking to enhance the action of TME or ligand

EAB-K. In this regard, the promoters of the genes encoding both p55 TME-K and p75 bo TME-K were sequenced and analyzed: and some key sequence motifs were identified for which specificity was established relative to different transcription factors: this means, that the expression of these TME-K receptors can be controlled at the level of the corresponding promoters - that is, inhibition of transcription from the promoter leads to a decrease in the number of these receptors, and amplification of transcription from these promoters - to an increase in the number of these receptors (ER-606869 and

MO 95/31206). Also, there are still no reports on the study of EA5-K regulation at the level of the promoter of the gene coding for this receptor b5.

Although it is known that receptors for tumor necrosis factors (TME) and the structurally similar receptor EA5-K in response to stimulation by ligands produced by leukocytes mediate destructive activity within cells, leading to their own death, the mechanisms of such mediated effects are still not fully understood .

Mutation analysis indicates that BAB-K and the rb55 TME receptor (p55-K) involve different parts of their intracellular domains in signaling mechanisms of cytotoxicity |VhaKerizsi ei ai., 1992; Tapadiia ei ai., 1993; My 8 Madaiyya, 1993). These segments (so-called "death-domains" or "death-effector domains", or "CEOs") are characterized by similarity of amino acid sequences. "Death domains" in BAB-K and rb5B-K show a tendency to self-association. Such their self-association is believed to contribute to the formation of receptor aggregations, which are necessary for the initiation of signaling mechanisms | see bopo eyi ai., 1994; MUaMasy ei ai., 1994; 7/0 Voyaip et al., 1995), and a high level of receptor expression intensity can lead to the mediation of ligand-independent signaling mechanisms (Voyaip et al., 1995).

Some manifestations of cytotoxicity in lymphocytes are mediated by the interaction of a ligand produced by the lymphocyte with the RAB-K (CO95) receptor, a widespread cell surface receptor that is capable of mediating cell death (see also Madaya 48: Soivieip, 1995), and this cell death 7/5 The "ligand-receptor" complex in the composition of TME and its receptor p55-K (СО120), which is structurally similar to the complex of the HA5-K receptor and its ligand, is drawn in by mononuclear phagocytes (also see Mapdepabeiye ei ai., 1995). As in the case of other manifestations that are induced with the participation of receptors, the induction of cell death with the participation of TME and EA5-K receptors occurs through a series of protein interactions that begin with the binding of the ligand protein and the receptor with the possible activation of enzymatic effector functions, which in in the analyzed case, non-enzymatic interprotein interactions that initiate the cell death signal are determined by: binding of trimeric

TME or BAB-K receptor ligand molecules with their receptors, which occurs as a result of the interaction of their intracellular domains IVhaKerizsi ei a!.,1992; Tagadiia ei ai., 1993; Tsoi 5 Madaghia, 1993), determined by the property of "death-domains" (or "death-effector domains", OEO) to self-associate |Voidip et al., 1995a), and the induction of binding of two cytoplasmic proteins (which can also to connect with each other) with the intracellular domains of the receptors - protein MOKT-1 (or GAYU) with RA5-K |Voiiaip ei ai., 19956; Spippaiuap ei ai,, 1995; KizzyKe! ei and I., 1995)| and TKAYUO protein with p5B5-K |Nzy ei ai., 1995; Nzy ei ai., 1996). Three proteins (8) that bind to the intracellular domain of RA5B-K and p55-K through their "death domain" are involved in the induction of cell death by receptors through the formation of hetero-linked homologous segments and are also capable of independently mediating cell death independently of each other, were identified with Ge! using the procedure of dihybrid yeast screening. One of these proteins - MOKT-1 (Voiadip et al., 19950), also known as EAOO (Spippaiuap et al., 1995) specifically binds to RHA5B-K. Another such protein is p

TKAO see also Nzgy ei ai., 1995, 1996) - binds to p55-K, and the third such protein - KIR, see also yu eZiapdeg ei ai., 1995) - binds to both RAB-K and p55-K. In addition to the fact that they bind to EA5-K and p55B-K, these proteins are also able to bind to each other, which may determine the functional mechanism of "cross-talk" between receptors PA5B-K and p55-K. Such binding takes place with the participation of a conservative segment of the sequence - the so-called "module doom-domain" (also referred to as "SEU" by "Oevaii EPesiog

Botatype" - "death-effector domain") - common for receptors and proteins that bind to them. Moreover, although in the yeast two-hybrid test the MOKT-1 protein showed spontaneous binding to HA5-K, in mammalian cells such binding took place only after stimulation of the receptor: this confirms that MOKT-1 is involved in the "initiation process of the HA5-K signaling mechanism. The MOKT-1 protein does not contain any motif in its amino acid sequence that is characteristic for the manifestation of catalytic activity activity: that is, its ability to mediate cell death is believed not to be based on the activity of MOKT-1 itself, but, most likely, is caused by activation of some other proteins (protein) that bind MOKT-1 and act further down the signaling cascade.

Expression in cells of mutant variants of MOKT-1, which lost the M-terminal part of the molecule, was shown to block cytotoxicity induced by BAB/AROIT (BAB-K) or p55-K |Nzy ei aiYi., 1996; Spippaiup ei ai., -I 1996): this indicates that this M-terminal segment transmits the signal of cell death from both receptors through protein interactions. o Recent studies have revealed groups of cytoplasmic thiol proteases that show structural similarity to the protease SEOS of the free-living nematode Saepognabaizis eiedapz and to the ISE convertase of interleukin-1r5or of humans in connection with various physiological parameters of cell death processes | see reviews Kitag, 1995 and de Nepkaipi, 1996). There are also a number of indications that proteases (protease) of this family may be involved in

Ge) manifestation of cytotoxicity of cells, which is induced with the participation of EAB-K and TME-K receptors. For specific peptide inhibitors of proteases and two proteins-blockers of their functions encoded by viruses - the vaccinia virus protein sptA and the baculovirus protein rZb5 - protection of cells from the manifestation of cytotoxicity was shown (Epagi et al., 1995; oze et al. , 1995; Temagyi et al., 1995; Htse et al., 1995; Veidieg et al., 1995). Rapid cleavage of some specific cellular proteins, as it is assumed, by the action of proteases of the family iF) SEOZ/LCE can be demonstrated in cells shortly after stimulation of EAB-K or TME-K receptors. It should be noted that these proteases of the CEOZ/CE family, which are also called caspases, are produced as inactive precursors and are activated by proteolytic cleavage during the induction of cell death. These caspases are conservative cysteine proteases that cleave specific cellular proteins downstream of aspartic acid residues and, accordingly, play a key role in all currently known processes of programmed cell death. In addition to their homologous C-terminal regions, from the content of which mature forms of proteases are formed, the precursor proteins include unique M-terminal segments. The interaction of these "prodomains" with specific regulatory molecules ensures differential activation of various b5 caspases with the participation of various cell death induction signals Ivoiaip ei a!., 1996; Milio eyi a!., 1996; Yutsap 45. Rikhii, 1997, Map

Stiekipde eyi ai., 1996; Aptaasd ey! Ai., 1997).

Recently, one such protease and its various isoforms (including inhibitory forms) have been isolated, cloned and characterized, designated MASN (also designated SABR-8), which is a MOKT1-binding protein and serves as a modulator of MOKT-1 and, accordingly, EAZ- K and p55-K, and which can also act independently of MOKT-1; the possible use of this protease was also described, in particular, in the detailed list of cited materials, co-pending joint applications, Ina Israeli patent MoMo114 615, 114986, 115319, 116588 and 117932, as well as in the corresponding PCT - Mo PCT /LI596/105211), exactly as in the recent publication of the applicants

Ivoidip eyi ai., 1996). Other such protease, including its isoforms (including inhibitory forms), are indicated

Mena (also referred to as SABZR10) has also recently been isolated and characterized by the applicants of this invention (unpublished materials) and other researchers (Regpapdez-AIpetgi et al., 1996; Zgipimaztsa et al.,, 1996). This Msep4 protein is also a MOKT1-binding protein that serves to modulate the activity of MOKT-1 and, accordingly, appears to be a modulator of RHA5B-K and p55-K, and which may also act independently of MOKT-1. Thus, details regarding all aspects of the properties, characteristics, and uses of Mep are provided in the above-mentioned publications, which are fully incorporated in the citation list of this invention.

It should also be noted that caspases MASN (SAZBR-8) and Mep4 (SAZRI10), which include similar "prodomains", see

Voidip eyi ai., 1996; Milio eyi aiI., 1996; Regpapdez-AIpetgi ei aiI., 1996; Mipsepi "5 Oihii, 1997) interact with MOKT-1 through their "prodomains": this interaction is mediated by the motif "death-domain" or "death-effector domains" (OEbB), which are located in the M-terminal part of MOKT-1 and available in a duplicated form as part of the MASN (SABR-8) and Mena (SABR-10) | see Voyaip eyi a!., 1995; Spippaiuap eyi a!., 1995).

It is also worth mentioning that, from the point of view of the above, different proteins, enzymes and receptors involved in intracellular signaling processes leading to cell death received different names. To overcome possible inaccuracies, the following list of various names, including new names adopted in connection with the new standards, for each of the given proteins or their components, as well as the given names accepted for use in this text, taking into account the relevant text, is proposed below Amenities. o name with which the described name is used here for the first time

F zosch mov 00011 yu 00011111 yu 0011110 yu h with ENN MON po ОН in 0 ШЧЭШЧЕНОНННЫ "death-effector motif" (OEO), "death-domain motif", "MOKkT-modules" "MOKkT-modules" "prot. 00000000 00000010 protam jemovisye 77711111 1САВР 0 svo o s The object of the present invention is to present new proteins, including all their isoforms, analogs, fragments or derivatives, capable of binding to MOKT1-binding proteins, such as, for example, Mena proteins named above and

MASN and their isoforms are either capable of binding to MOKT-1 itself. Taking into account the fact that the MOKT-1 protein itself

Binds to the intracellular domain of the RA5-K receptor, new proteins according to the present invention due to -and binding to MOKT1-binding proteins and, accordingly, indirectly to MOKT-1, or due to direct binding to the protein of MOKT- 1, capable of inhibiting the intracellular signaling mechanisms initiated by the binding of RAb-ligand to its receptor: that is, in this way, the new proteins according to the present invention are modulators of EAB-K-mediated effects on cells. The MOKT-1 protein is also involved in the modulation of the TME effect on cells through its involvement in the modulation of p55-K: accordingly, the new proteins of the present invention are also considered as modulators of the effect of TME on cells through the p55-K receptor. Also, by

Ge) analogies with the above-described modulation of the effect on cells mediated by receptors

EAB-K and p55-K, proteins according to the present invention can also be mediators or modulators of other cytotoxic mediators or inducers by acting on general or combined intracellular signaling mechanisms in which T proteins according to the present invention participate - for example, 1 and its isoforms. These new proteins according to the present invention are designated as "C1" proteins and, as mentioned above, include the actual protein 1 (F, (discussed below), all its isoforms, analogs, fragments and derivatives. Another object of the invention is to provide antagonists ( for example, antibodies, peptides, organic compounds or even some isoforms of the above-mentioned new proteins 1, their isoforms, analogues, fragments and derivatives, which can be used to inhibit the signaling mechanism or, more specifically, the manifestation of cytotoxicity, if this is desired.

A further object of this invention is the use of the above-mentioned new proteins 1, their isoforms, analogues, fragments and derivatives in order to isolate and characterize additional proteins or factors that may be involved in the regulation of receptor activity, for example, other proteases that cleave new proteins with b5 converting them into biologically active ones, and (or) for the purpose of isolating and identifying other receptors located before the start of signaling processes that bind these new proteins, their analogues, fragments and derivatives

(for example, other EA5-K or related receptors), in the functioning of which, accordingly, they are also involved.

Another object of the present invention is to provide inhibitors that can be introduced into cells to bind or interact with protein 1 and possible isoforms of 1 having protease activity (protein 1

Includes a region homologous to the proteolytic segments of Mep4 and MASN), and inhibition of their intracellular activity, which, at least for some C1 isoforms, is proteolytic activity.

In addition, the object of this invention is the use of the above-mentioned new proteins 1, their isoforms, analogues, fragments and derivatives as antigens for the preparation of polyclonal and (or) monoclonal antibodies to them. In turn, these antibodies can be used, for example, to purify new proteins from various sources, such as cell extracts or transformed cell lines.

Further, these antibodies can be used for diagnostic purposes, for example, to identify disorders associated with abnormal functioning of cellular processes mediated by EBA5B-K or other related receptors.

Another object of the present invention is to provide pharmaceutical compositions that include the above-mentioned proteins 1, 7/5, their isoforms or analogs, fragments or derivatives, as well as pharmaceutical compositions that include the above-mentioned antibodies or other antagonists.

According to this invention, a new protein - 21 is isolated, which is able to bind or interact with Mep4, which itself binds to MOKT-1, which, in turn, binds to the intracellular domain of RA5B-K. 01 may also interact with another MOKT1-binding protein called MASN, and may also be able to bind or interact directly with MOKT-1. Protein 51 apparently functions as a modulatory factor of the cell death mechanism, which is initiated by the binding of the PAZ ligand to the PHA5-K receptor on the cell surface, which, apparently, is ensured by the presence of a proteolytic segment similar to the proteolytic segments of Men. and MASN: accordingly, protein (31 can also be an active intracellular protease. Further, depending on the processes of transcription and translation during the expression of 51, especially on its proteolytic segment, some isoforms of 51 can be expressed excluding the active proteolytic segment and such can thus serve as antagonists of proteolytic activity mediated, for example, by Meni and MASN.

EAB-K. The protein MOKT-1 (or GAYU) binds to the intracellular domain of PA5-K during the activation of this receptor, and the new protein 1 according to the present invention binds to MOKT1-binding proteins such as Ge! with MspA and, probably, also MASN, or directly with MOKT-1 protein. The C1 protein cloned and characterized in accordance with the present invention can exist as multiple isoforms, some of which include a segment of homology with c

SEOZ/LSE having proteolytic activity (proteolytic domain) similar to that of Mep and some isoforms

MASN, and which can cause cell death in case of expression in them. Thus, the activation of this new SEOZ/LSE homologue (i.e., different isoforms 1, which include the proteolytic domain) with the participation of ГА5Б-К (through "direct or indirect interaction with MOKT-1)" appears to form an effector component of mediated by

EA5-K mechanism of cell death.

Moreover, protein 51 also functions as an effector component of the cell death mechanism, which is initiated by the binding of TME to the p5-K receptor on the cell surface: in this case, by indirectly binding MOKT-1 to TKAOO protein, a protein that itself binds binds to the intracellular domain of ro5b5-K INzyme "81. 1995); after that, or at the same time, protein 51 binds to MOCT1-binding proteins, such as, for example, Msp4 or MASN, or binds to MOCT-1 directly, with the conversion of C1 into an active protease involved in mediating cell death. c It should also be noted that although (31) reveals at least some sequence parameters that are key to the functioning of the SEJOH/LCE proteases, it nevertheless has a number of distinctive sequence characteristics that may provide it with a unique and apparently tissue/cell-specific nature of activity.-I Thus, according to the present invention, there is provided a novel protein designated 1. This protein (31 was isolated and cloned using a dihybrid screen and was characterized as a Mena-binding molecule. pi As noted above, Mcep4 is a MOCTI-binding protein capable of triggering cell death, although, however, it should also be noted that some Mep4 isoforms have the opposite activity, namely, they inhibit the destruction of 5or cells. In addition, sequencing 1 showed that this protein includes in its M-terminal part two so-called "MOKT-MODULES" (MM), which are also found in the MOKT1-binding proteins Msp4 and MASN.

Ge) "MOKkT-modules" in compound 1 are believed to give it the ability to bind to Mep4, and may also be a factor in direct binding to MOKT-1 and binding to MASN or to specific MASN isoforms (specific variants MASN splicing). In the amino acid sequence ("31) following the M-terminal ov segment, which includes "MOKT-modules", there is also a long segment that shows similarity with the proteolytic segment of MASN and Mesp4. Moreover, based on the data of the initial analysis of the probable (F) localization of the sequence of gene 21 on human chromosomes, it became clear that gene 1 is located on the Mo2 chromosome in close proximity to the localizations of the Msy4 and MASN genes, which also indicates the presence of a connection between the oi proteins,

Mena and MASN. In more detail, at least three possible isoforms of protein 01 have been obtained according to the present invention (see example 1 below). Two of these isoforms have been isolated and cloned: they are believed to represent two alternative splicing variants of the new protein designated 1 (or SAZN). These two isoforms were called c19, (or

SAZNO) for the longer isoform and C1 p (or SAND) for the shorter isoform (since there is more than one short isoform, this short isoform 51p is designated as 1 Dt and the other short isoform as b5 5182: see Example 1 below) . Each of these 01 isoforms - A and B includes two M-terminal "death domains"/MOCT modules and can bind to each other indirectly through these "death domain motifs" and can also bind to MOCT- 1, Mesp4 and MASN are mediated through the same "death-domain motifs". The longer C19 isoform has a unique C-terminal segment (compared to the short C1p isoform), while this unique C-terminal segment is characterized by sequence homology with that in the protease activity segment of caspases. In terms of biological activity, the shorter C1p isoform inhibits cell death and cytotoxicity mediated by p55-K and EA5-K receptors, while, in contrast, the longer C19u isoform is cytotoxic against at least some cell types (eg, cells of line 293) despite the fact that this cytotoxicity involves their protease-homology segment. However, it should be noted that the long isoform of C19u can also inhibit cytotoxicity mediated by p5-K and PHA5B-K receptors in other 70 cell types (for example, Ne!a cells). These data determine that protein 1 (namely, its various isoforms) act as attenuators-inhibitors and (or) inducers-enhancers of signaling mechanisms of cell death mediated by p55-K and EA5B-K receptors.

It should also be noted that for the sake of simplicity in the designation of different isoforms of 1, for example C10 and 1D, the simple designation "51" will often be used in this text, but it should be clear that in these 75 cases the term "C1" will denote all isoforms (01 - that is, it will denote both inducers-enhancers of cytotoxicity and inhibitors-attenuators of cellular cytotoxicity. When specific isoforms of C1 are meant, they will be called in the appropriate way, for example, C1o or C1p. Taking into account the above, when the collective designation is used " C1", referring to different isoforms, it will often be referred to in this text as a "modulator": this means that it can exhibit both inhibitory and enhancing effects relative to the biological function under discussion.

In view of the foregoing, it is clear, as described above and discussed below, that 1 is a modulator of MOKT-1 activity and, accordingly, a modulator of receptor-mediated cellular effects.

EAZ-K and p55-K, as well as, apparently, other receptors from the TME/CHOE receptor family and others that may participate in similar components and mechanisms of intracellular signaling. with

Thus, since (31 has a protease-like segment (at least as part of the long isoform), it may He) be directly responsible for cytotoxicity and inflammation caused or induced by various stimuli, including those that enter through receptors of the receptor family TME/MOBE and probably other receptor families as well.

Also (31 can serve as an inhibitor of cytotoxicity and inflammatory processes due to the fact that it (22) is part of a complex of other proteins and, accordingly, can affect the cytotoxic and inflammatory activity of these other proteins (for example, proteins, Mesyp4 and MASN or even MY T-1), ultimately leading to inhibition of their cytotoxic or inflammatory activity.

Also, 01 can serve as an enhancer or determinant of cellular cytotoxicity and inflammatory processes, other proteins (for example, proteins, Mei4 and MASN or even MOKT-1), due to binding to them with

With subsequent binding to MOKT-1, or acting independently of MOKT-1, but in each case providing the cytotoxic activity of various proteins or their effect on the induction of inflammatory processes.

Also, in a similar way, protein 1 can serve as an inhibitor or facilitator of other intracellular mediators or modulators involved in mechanisms in which 1 itself actively participates.

Protein MOKT-1 (abbreviation "Mediayug oi Keseriog Tohisyu" - mediator of toxicity, receptors - (Voiadip ei a, 199551) is able to bind to the intracellular domain of EA5B-K. This EAb-binding protein, as it is assumed, acts as a mediator or modulator of the action of the RA5B-K receptor ligand on cells by "" mediating or modulating intracellular signaling mechanisms, which are usually triggered after "" binding of the EAZ-K ligand on the cell surface. In addition to the specificity of PA5-binding, the presence of other parameters was shown for MOKT-1 (see example-reference 1): for example, it contains a segment homologous to the segments of "death domains" (00) in the composition of ro5- TME-K and EA5-K (p55-00 and EA5Z-OO) - respectively, it is also capable of self-binding. Also, MOKT-1 itself is able to activate cellular cytotoxicity: this activity of theirs is apparently connected with the ability to self-associate. It was also found that co-expression of a segment of MOKT-1, which includes a sequence homologous to the "death-domain" (MOKT-OO - present in the C-terminal 1 part of MOKT-1), significantly disrupts RAB-induced cell death processes , which can be expected based on the ability to bind to the "death domain" in EA5-IS. In addition, under the same experimental conditions, it was found that co-expression of the part of MORT-1, which does not contain the MOKT-0O segment (M-terminal

Me; part of MOKT-1, amino acids 1-117: "the main part of MOKT-1"), results in the absence of interference with RA5Z-induced cell death, and even to some increase in HA5-induced cytotoxicity.

Accordingly, it is quite likely that MOZHKT-1 also binds to other proteins involved in intracellular signaling mechanisms. These MOKT1-binding proteins can also, accordingly, act as

GF! indirect mediators or modulators of the effect of EA5-K receptor ligands on cells by mediating or modulating MOKT-1 activity; or these MOCT1-binding proteins may act directly as mediators or o-modulators of the MOCT-1-related intracellular signaling mechanism by mediating or modulating the activity of MOCT-1, which, as described above, has an independent ability to activate 60 cell cytotoxicity. These MOKT1-binding proteins can also be used in any of the standard screening procedures to isolate, identify, and characterize additional proteins, peptides, factors, antibodies, etc., that may be involved in MOKT-1-related signaling mechanisms. or related to

EAB-K, or may be elements of other intracellular signaling processes. The following were selected

MOKT1-binding proteins and are described in the above-mentioned co-pending Israeli co-applications MoMo114615, 114986, 115319, 116588, 117932 and the corresponding PCT MePCT/O5-96/10521 (in the part relating to MASN and its isoform) and |in the articles of Regpapaev-AIpetgi ei a!., (1996) and Zgipimazshia ei ai., (1996),

(in the part that concerns Mep4 and other "Map" proteins). One such MOKT1-binding protein, previously referred to as MASN, was first cloned, sequenced, and partially characterized as having the following parameters: MASN cDNA encodes the open frame of OKE-B; MASN binds to MOKT-1 in a very rigid and highly specific manner; The MASN-binding site in MOKT-1 is located above the "death-domain motif" of MOKT-1; the OKE-B site for MASN is its site of interaction with MOKT-1; and MASN is characterized by the ability to self-associate and by itself can induce cellular cytotoxicity. In addition, later studies, also reflected in the above-mentioned pending joint applications, as well as in the publication of Voidip et al., (1996)), have shown that MASN does exist in the form of a series of isoforms. Moreover, the above-named OKR-B MASN is actually one of the isoforms of MASN, designated as

MASNDVI. In the aforementioned publications concerning the Mesp4 protein, it was shown that this protein also binds to MOKT-1 (or EAOO) and is directly involved in MOKT-1-mediated or independent cellular cytotoxicity by binding to its proteolytic activity.

Accordingly, the present invention provides a DNA sequence encoding protein 1, analogs or fragments thereof, capable of binding or interacting directly or indirectly with MOKT-1 and/or with MOKT1-binding proteins, such as, for example, Msp4 or MASN, while the specified protein (31, its analogs or fragments are capable of mediating the intracellular effect mediated by HA5B-K or p55-TME-K, or that the specified protein 1, its analogs or fragments are capable of also modulating or mediating intracellular effects the activity of other proteins with which they can bind directly or indirectly.

In particular, the present invention provides a DNA sequence selected from the group consisting of: a) a cDNA sequence derived from a segment encoding native protein 1;

B) DNA sequences capable of hybridizing with the sequence (a) under conditions of medium degree of rigidity and which encode biologically active protein 1; and c) DNA sequences that are degenerate due to changes in the genetic code in the DNA sequences identified by Ha

In paragraphs (a) and (B) and which encode biologically active protein 1.

Another special variant of the above-mentioned DNA sequence according to the present invention is the sequence o

DNA, including at least part of the sequence that encodes at least one of the isoforms of protein 1. Another variant of the above-mentioned DNA sequence is the sequence that encodes the C1 protein shown in Fig. 1 (isoform

C10). Another similar variant is the second isoform of protein C1 shown in Fig.2 (isoform C1p). Gee)

The present invention provides C1 proteins, their analogues, fragments or derivatives, which are encoded by any of the above-mentioned DNA sequences according to the present invention, with the fact that said proteins, their analogues, fragments or derivatives are capable of binding or interacting directly or indirectly with MOKT-1 and/or with any MOKT1-binding IF) proteins, such as, for example, Msp4 or MASN, and mediate an intracellular effect "mediated by HA5B-K or p55-TME-K, or the activity of any other mediators or inducers of cytotoxicity, with which the named proteins 1, their analogues, fragments or derivatives are able to directly or indirectly bind. -

A special variant of the present invention is related to C1 proteins, their analogues, fragments and derivatives. Another option provides any isoform of protein 1, their analogues, fragments and derivatives.

The present invention also provides vectors encoding the above-mentioned protein (31 and its analogs, fragments or "derivatives according to the present invention, which include the above-mentioned DNA sequences according to the present invention, so that these vectors are capable of expressing in suitable prokaryotic or eukaryotic - c host cells; transformed eukaryotic or prokaryotic host cells carrying such vectors; and the method of obtaining protein 51 or its analogs, fragments or derivatives according to the present invention is by growing such transformed host cells in conditions suitable for the expression of the specified protein, its analogs, fragments or derivatives and effective from the point of view of post-translational modifications of the specified protein in case it is necessary to obtain the specified protein and extract the specified expressed protein, its analogues, fragments or derivatives from the culture medium with the specified transformed cells or from their cellular extracts obtained from the specified tr unformed cells. All the given definitions include all isoforms of protein 1. 1 In another embodiment, this invention also provides antibodies or their active derivatives or fragments specific for protein 1, its analogs, fragments and derivatives according to the present invention.

In yet another embodiment, the present invention provides various ways of using the aforementioned DNA sequences or 3e) the proteins encoded by them, according to the present invention, wherein, among other things, such use involves the following: (A) A method of modulating cell death or inflammation processes, which includes the treatment of the specified cells with one or more proteins (31, their analogues, fragments or derivatives according to the present invention, as described above, while the specified treatment of the specified cells includes introducing into the specified cells one or more proteins, their analogues, fragments or derivatives in a form suitable for their i.e.) intracellular introduction, or introduction into the specified cells of a nucleotide sequence that encodes one or more of the specified proteins, their analogues, fragments or derivatives in the form of a suitable vector carrying the specified sequence so , so that the specified vector is able to ensure the inclusion of the named sequence in the specified cells in such a way that the named this sequence was expressed in the specified cells; and (B) A method of modulating cell death or inflammation, comprising treating said cells with one or more inhibitors of one or more proteins/enzymes that mediate said cell death or inflammation, wherein said inhibitors are selected from the group consisting of 65 includes: (1) one or more proteins (31, their analogs, fragments or derivatives according to the present invention, capable of inhibiting the death of the specified cells or inflammatory processes; and (2) inhibitors of one or more proteins 21 according to the present invention, with the fact that the specified one or more number of proteins 01 strengthen/enhance or mediate the death of the specified cells or inflammatory processes.

More specifically, the above-mentioned methods in accordance with the present invention include the following special options: 1) a method of modulating the effect of the EAB-K or TME ligand on cells bearing ГА5Б-К or р5Б5-К receptors, which includes the treatment of these cells with one or more proteins (31, their analogues, fragments or derivatives according to the present invention, capable of binding to MOKT1-binding proteins, such as Mei4 or

MASN, while, in turn, their associated MORT-1 directly binds to the intracellular domain

EAZ-K, or capable of binding directly or indirectly to MOKT-1 or to MOKT1-binding proteins as described above, but in this case MOKT-1 binds to TKAOO, which , in turn, binds to the intracellular domain of p5-K, which, thanks to this, provides the ability to modulate/mediate the activity of the specified EAB-K or p55-K receptors, while the said treatment of the specified cells includes introducing into the specified cells one or more the number of proteins, their analogs, fragments or derivatives in a form suitable for their intracellular introduction, or the introduction into the specified cells of the DNA sequence encoding /5 One or more specified proteins, their analogs, fragments or derivatives in the form of a suitable vector that carries the specified sequence, so that the specified vector is able to ensure the entry of the specified sequence into the specified cells in such a way that the specified sequence is expressed in the specified cells; 2) the method of modulating the effect of the HA5-K or TME ligand on cells according to item (1) described above, with the fact that the treatment of cells specified in item 2 includes the introduction of the specified protein (31 or its analogs, fragments or derivatives in the form , suitable for intracellular introduction or introduction into said cells of a DNA sequence encoding said protein 1 or its analogs, fragments or derivatives in the form of a suitable vector carrying said sequence, so that said vector ensures that said sequence enters said cells in such a way, so that the specified sequence is expressed in the specified c g cells;

C) the method according to item (2) described above, while the specified treatment of the said cells is carried out i) by transfection of the specified cells with a recombinant viral vector, which includes the following steps: (a) construction of a recombinant animal virus vector carrying the sequence, which encodes a viral surface protein (ligand), which is able to bind to a specific cell surface receptor on He! from the surface of cells that carry GA5-K or p55-K receptors, and a second sequence that encodes a protein selected from protein 1, its analogs, fragments and derivatives, so that when expressed in the specified cells, the ability to modulate mediating the activity of the specified EA5-K or p55-K, and (5) infecting the specified cells with the vector specified in point (a); 4) a method of modulating the effect of the EA5B-K or TME ligand on cells bearing EA5B-K or p55-K receptors, which includes the treatment of these cells with antibodies or their active fragments, or their derivatives according to this invention, so that said treatment is carried out by introducing a suitable composition, which includes said antibodies, their active fragments or their derivatives into said cells, with the fact that when at least part of the protein 01 appears on the cell surface, said composition is formulated for extracellular use, and when said proteins (31 are completely intracellular, then the specified composition is formed for "Intracellular introduction; in p. 5) a method of modulating the effect of the HA5B-K or TME ligand on cells bearing EAB-K or p55-K, which includes . treatment of these cells with an oligonucleotide sequence encoding an antisense sequence relative to, and? at least part of the protein sequence (31 according to the present invention, while the specified oligonucleotide sequence is able to block the expression of protein 1; 6) the method according to item (2) described above, for processing tumor cells, or cells infected with HIV, or other -I diseased cells, which includes: (a) construction of a recombinant animal virus vector carrying a sequence that encodes a viral surface antigen capable of binding to a specific receptor on the surface of tumor cells or to a receptor on the surface of cells infected with the HIV virus, or with the receptor of other diseased cells, as well as the sequence encoding the protein, which is selected from protein (31, its analogs, fragments and derivatives according to the present invention, in such a way that in the case of expression of the specified tumor, HIV-infected or other diseased cells

Ge) become capable of destroying the named cells; and (c) infection of the specified tumor cells or cells infected with the HIV virus, or other diseased cells with the vector specified in point (a); 7) a method of modulating the effect of the GAB-K or TME ligand on cells, which includes the use of a ribozyme procedure, in which a vector encoding a ribozyme sequence capable of interacting with the cellular sequence (F) of mRNA encoding protein 1 according to the present invention is introduced into said cells in such a form that allows said ribozyme sequence to be expressed in said cells, and when said ribozyme sequence is expressed in said cells so that it interacts with said cellular mRNA sequence and cleaves said mRNA sequence, resulting in inhibition of expression the specified protein 1 in the specified cells; 8) the method selected from the methods according to the present invention, with the fact that the sequence encoding the specified protein 1 includes at least one of the C1 isoforms, analogs, fragments and derivatives of any of the forms presented by the invention, which are capable of to bind directly or indirectly to MOKT-1 or to MOKT1-binding proteins, such as 65 As, for example, Mena or MASN, while MOKT-1 bound to them, in turn, specifically binds to

EAZ-IS, or which are capable of binding directly or indirectly to MOKT-1 or the above-mentioned MOKT1-binding proteins, while MOKT-1 bound to them, in turn, binds to TKAYUO or , in turn, binds to p5b5B-IS; 9) the method of isolation and identification of proteins according to the present invention capable of binding directly or indirectly to the MOKT-1 protein or to MOKT1-binding proteins, which includes the use of a yeast dihybrid test, in which the sequence encoding the specified MOKT- 1 or MOCTI1-binding proteins, is part of one hybrid vector, and the sequence from cDNA libraries or genomic DNA is part of the second hybrid vector, while the vectors are then used to transform yeast host cells and positive transformed cells are isolated with subsequent extraction of the indicated second hybrid /o vector in order to obtain a sequence that encodes a protein that binds to the indicated protein MOKT-1 or

MORET1-binding proteins; 10) the method according to any of items (1)-(9)U, described above, with the fact that the specified protein 51 is one of the C1 isoforms, an analog, a fragment or a derivative of any of them; 11) the method according to any of items (1)-(10) described above, with the fact that protein 51 or any of its 7/5 isoforms, analogues, fragments or derivatives are involved in the modulation of the cellular effect, mediated or modulated by any other cytotoxic mediator or inducer, with which the specified protein 1, its isoform, analogue, fragment or derivative can bind directly or indirectly; 12) a method of screening other compounds, such as, for example, peptides, organic compounds, antibodies, etc., in order to obtain specific drugs that would be capable of inhibiting the activity of 1, for example, inhibiting the protease activity of the Sto isoform, thus inhibiting cellular cytotoxicity or enhancing cytotoxicity due to inhibition of C1rD activity.

Variants of the above-mentioned method of screening according to clause (12) include: (1) a method of screening a ligand capable of binding to C1 protein according to the present invention as described above, which includes contacting the matrix in affinity chromatography, to which attached Ha specified protein, with cell extract so that as a result the ligand binds to the specified matrix with subsequent elution, selection and analysis of the specified ligand; o (2) a method of screening a DNA sequence encoding a ligand capable of binding to protein 1 according to the present invention, which includes the use of a yeast dihybrid test, in which the sequence encoding the specified protein is part of one hybrid vector, and the sequence from cDNA libraries or genomic FU)

DNAs are part of the second hybrid vector, with the transformation of the host yeast cells with the specified vectors, followed by the selection of positive transformed cells and the extraction of the specified second hybrid vector in order to obtain the sequence encoding the specified ligand; and (3) a method of identifying and producing a ligand capable of modulating cellular activity modulated/mediated by white MOKT-1 or MOKT1-binding protein, which comprises: C (a) screening a ligand capable of binding to a polypeptide comprising at least part protein MOKT-1 or h-

MOKT1-binding proteins selected from MASN proteins, Mei4 proteins and other MOKT1-binding proteins; (B) identification and characterization of a ligand, other than MOKT-1 or specified MOKT1-binding proteins or parts of a receptor that is part of the TME/MOR receptor family, found by said step of "screening for said binding ability; and (c) obtaining said ligand in essentially isolated and purified form; - c (4) a method of identifying and obtaining a ligand capable of modulating the cellular activity of a protein (31 according to the present invention, which is manifested in modulation or mediation, which includes: "" (a) screening of a ligand capable of binding to a polypeptide that includes at least part of the C10 sequence shown in Figure 1, or at least part of the C1p sequence shown in Figure 2; (B) identification and characterization of a ligand other than MOKT-1 or MOKT1-binding proteins or parts - and a receptor belonging to the TME/MOE receptor family found by said screening step for said binding ability; and (c) obtaining said ligand in a sufficiently isolated and purified form; (9) (5) method of identification and production of a ligand capable of modulating cellular activity modulated/mediated by C1 protein, comprising: (a) screening of a ligand capable of binding at least to part of the sequence (1 in shown in Fig. 1, or (Ce) with sequence with 1p, shown in Fig. 2; (B) identification and characterization of a ligand, other than MOKT-1 or MOKT1-binding proteins or parts of a receptor that is part of the TME/MOE receptor family, found by means of the specified step of screening for the specified binding capacity; and

Gee! (c) obtaining the specified ligand in essentially isolated and purified form; (6) a method of identification and preparation of a molecule capable of directly or indirectly modulating cellular co-activity modulated/mediated by C1, which includes: (a) screening of a molecule capable of modulating the activity of 1, which is detected in the modulation of mediation; 60 (c) identification and characterization of the specified molecule; and (c) obtaining said molecule in essentially isolated and purified form; (7) a method of identifying and obtaining a molecule capable of directly or indirectly modulating cellular activity modulated/mediated by protein 1 according to the present invention, which includes: (a) screening of a molecule capable of modulating the activity of the specified C1 protein, which is found in b5 modulation/mediation ; (c) identification and characterization of the specified molecule; and

(c) obtaining said molecule in essentially isolated and purified form.

The present invention also provides a pharmaceutical composition intended to modulate the effect of EA5-K ligand or TME on cells or the effect of any other cytotoxic mediators or inducers on cells as described above, comprising as an active ingredient one of the following components : (Y) C1 protein according to this invention and its biologically active fragments, analogues, derivatives of mixtures; (i) a recombinant animal virus vector encoding a protein capable of binding to a cell surface receptor and encoding protein 1 or its biologically active fragments or analogs according to the present invention; (ii) an oligonucleotide sequence encoding an anti-sense sequence relative to the sequence of protein 70 (31 according to the present invention, while the specified oligonucleotide may be the second sequence in the viral vector according to item (i) described above.

Also

I. A method of modulating the MOKT-1-induced effect or the MOKT1-binding protein-induced effect or the effect of any other cytotoxic mediator or inducer on cells, which includes the treatment of said 7/5 Cells according to the method according to any of paragraphs (1)-(10) described above using proteins 1, their analogues, fragments or derivatives or using sequences encoding proteins 1, their analogues or fragments, with the fact that the specified treatment leads to amplification or suppression of said influence caused

MOKT-1, and thus the effect mediated by RA5B-K and p55-K receptors, or the specified other cytotoxic mediators or inducers.

IP The method described above, with the fact that the specified protein 51, its analogue, fragment or derivative is the part of protein 1 that specifically participates in binding to MOKT-1 or to MOKT1-binding proteins, or to the specified by other cytotoxic mediators or inducers, or when the specified C1 protein sequence encodes that part of protein 01, which is specifically involved in binding to MOKT-1 or to MOKT1-binding proteins, or to other cytotoxic mediators or inducers. high school

Sh. The method described above, with the fact that the specified protein 51 is any of the isoforms c1, and the specified isoforms are capable of enhancing the effect associated with MOKT-1 or the effect associated with other cytotoxic i) mediators or inducers, thereby also enhancing the effect on the cells bound to the receptors

EAB-K or p55-K, or exposure to cells of another cytotoxic mediator or inducer.

IM. The method described above, with the fact that protein 51 is any of the C1 isoforms, and the specified isoforms are He! zo are capable of inhibiting effects associated with MOKT-1 or associated with another cytotoxic mediator or inducer on cells, thereby also inhibiting effects on cells associated with EA5B-K c or p55-K receptors, or effect on cells of another cytotoxic mediator or inducer. yu

As can be seen from all of the above, as well as from the detailed description below, 51 can also be used in conjunction with the MOZHKT-1 bilum for the purpose of processing (treating) cells or tissues. Isolation of proteins 1, their identification and characterization can be carried out using any of the standard search technologies used for the isolation and identification of proteins, for example, using the yeast two-hybrid test, affinity chromatography methods and any other well-known standard procedures that are used for similar purposes.

Moreover, some isoforms of the protein (31 may include only the protease-like segment (which shows homology with the above-described protease segments in other known proteases), but in the absence of She) with real protease activity, as a result of which such isoforms may serve primarily in quality of inhibitors as described above. ;" Further, since 51 or any of its isoforms may be involved in modulating intracellular mechanisms unrelated to MOKT-1, 51 or any of its isoforms may be used to modulate signaling in any other intracellular pathways and mechanisms. -I Other aspects and variants of the present invention are also provided based on the following detailed description of the present invention. It should be noted that, as used, the following terms - "modulation of the effect of RGAb-ligand or TME on cells" and "modulation of the effect of MOKT-1 or MOKT1-binding protein on cells" - are understood as those that overlap effects of both ip mygo and ip mimo, and, in addition, also involve suppression (inhibition), o and reinforcement/conditioning.

Ge) Fig1A is a schematic representation of the primary nucleotide sequence encoding one of the isoforms of protein 1, described in detail in example 1.

Fig1B8 is a schematic representation of the deciphered amino acid sequence of the isoform shown in Fig. 1A.

Fig. 2 is a schematic representation of the primary nucleotide sequence and the decoded amino acid (F) sequence of the second isoform of protein 1, described in detail in example 1. and Fig. 3 is a diagram comparing the amino acid sequences of splicing variants of protein 51 (or SAN) of humans (NSAZNo, PSAZNV) and mice (SAZNOo), as well as conservative motifs found in the composition of these proteins. because Fig. 3 shows a parallel comparison of amino acid sequences 1 o; mice (PGSAZNOo), C1o; human (PSABNoO) ota 518 (PSABNV), SABR-8 (MASN/RGISEIMsy5), SABR-10 (Mepa/RGISE2), SABR-Z (SRRZ2/apopain//ata) and SABR-1 (CE). SABR-1 and SABR-Z are shown without their prodomain segments. In each sequence, amino acid residues are numbered from the right. Dashed lines indicate sequence breaks made to optimize alignment. Shadowed modules of "death-domains" (OEB). 65 amino acids that are identical in more than three proteins are circled. Within the regions of "protease homology", amino acids that are mapped to residues in SABR-1 in such a way that their participation in providing catalytic activity was confirmed using crystallographic X-ray photography are marked as follows: residues that are assumed to be involved in catalysis and respond to histidine -237 and cysteine-285 in SABR-1 are intensely darkened and indicated by colored circles below the alignment column. Residues that form Kkarman binding for the carboxylate side chain of RiI-Agvr and correspond to arginine-179, glutamine-238, arginine-341 and serine-347 in SABR-1 are darkened less and marked by uncolored circles. The known and experimentally identified Agr-X cleavage sites and potential cleavage sites found in the same positions in 1 (SAN) are shaded. Horizontal arrows point to the M- and C-terminal ends of the small and large subunits of SABR-1. C-termini of proteins are marked with asterisks. 70 Fig. 4 (A and B) reproduce the autoradiograms obtained during the identification of transcripts 51 using the Northern blotting method in various human tissues (heart, brain, placenta, lungs, liver, skeletal muscles, kidneys, pancreas, spleen, thymus, prostate, testes, ovaries, small intestine, large intestine and peripheral blood lymphocytes-?RVI). Testing using the Northern blotting method was carried out as follows: an isotopically marked mRNA probe corresponding to the "death-domain" (ODD) in fold 1 (according to 7/5 Nucleotides 482-1070 in fold 18 (|see Fig. 2), i.e., the segment common to both cloned splicing variants 51 |SABNI) was obtained using T7 RNA polymerase (Rgotheoda) and applied to the analysis of multiple human tissue spots (Siopiesi) that include poly(A) "-RNA (2 μg per line ), originating from various human tissues.

Fig. 5 is a schematic representation of the results of the interaction analysis 10; (SAZNOo) and 1p (SAND) with other proteins that include "death domains" (OEO) in transfected yeast cells - for example, MOKT-1/PA0O,

MASNoi (SABR-8bVa1), MASNrI (SABR-8D1), MASNrV4 (SABR-8r4), Mesyp4 (SABR-10), Sto (SAZNO), 18 (SAND), rob5-K (ro5-IS), TKAOO, KIR and lamin (as a negative control). The binding parameters of 18 (SAZNrD), as well as 10, (SAZNOo), were evaluated in the reporter yeast strain ZEU526 (Siopiesni) using the DNA-binding domain in the vector pOovT9-cAI4 and the activator domains in the c vectors rOAOI1318 and rOAON-ZA14. Quantitative analysis of binding in yeast cells in the test for the expression of p-galactosidase was carried out as described in examples-references 1-3. The obtained results are expressed in the time required to obtain a clear color. In all cases tested, identical results were obtained when the tested nucleotide inserts were inserted in both combinations within

DNA-binding domain and constructs of the activator domain of plasmids. None of the tested non-F inserts interacted with a number of control proteins, including the intracellular domains of p5-K (СО120a), p75-K (СО1205),... DC EM

COa40, lamin, and empty SAGA vectors.

Fig. 6 (A-O0) presents the results of evaluating the impact of mutant variants 195; (SAZNo), 61rf8 (SAND) and Sto, (SAZNOo) on cell viability and induction of cell death. Quantitative analysis of cell death induced in And her cells! a-Raz (the data are schematically shown in the form of a diagram in Fig.bA) and in 293-T cells (the data are schematically presented in the form of a diagram in Fig.bB and 6C) by transfection of these cells with the defined constructs, was carried out as described in Example 1. Cells (5x109 cells of 293-T or 3x102 cells of Her a in the calculation of b-centimeter dishes) were transfected in an intermittent manner using the cDNA of certain proteins together with the pOMM-rbo1 plasmid using the method of precipitation in a calcium phosphate solution. Each cup was transfected with 5 μg of p(cDNA) construct; if the transfection was carried out with two differentiated shsh c constructs, then 2.5 μg of each of them plus 1.5 μg of the p-galactosidase-expressing vector. Cells were washed with ts for 6-10 hours after transfection and then incubated for another 14 hours without additional treatment. Monoclonal "» antibody to C0O5 (anti-RAZ-K) (SNI11 |Opsog, Saipegerigo, MO, O,bmcg/ml) and recombinant human TMEo (10µg/ml) were added to the cells simultaneously with cycloheximide (1µg/ml) followed by incubation for an additional 4 hours. The cells were then stained with 5-bromo-4-chloro-3-indoxyl-D-XO-galactopyranoside (X-boa1) and examined by phase-contrast microscopy. in the illustrated experiments, cell death was assessed 24 hours after transfection of Nei a-Raz cells and 20 hours after transfection of 293-T cells. The data presented (mean - 5.0.; n averaged 1 from at least three experiments) are the percentage of blue-stained cells, which are counted from g 20 taking into account their demonstration of blebbing ("bubbiness") of the membranes.

This invention in one of its aspects relates to novel proteins 21 capable of binding or interacting

Me; directly or indirectly with MOKT-1 or with MOKT1-binding proteins such as, for example, Mep4 or MASN, thereby binding to the intracellular domain of the RA5B-K receptor to which MOKT-1 binds, or , connecting to the intracellular domain of the receptor ro5TME-K, with which the TKAOO protein binds, which, in turn, binds the MOVT-1 protein. Accordingly, C1 proteins according to the present invention are considered as

GF! mediators or modulators of PHA5-K, thereby playing a role, for example, in the signaling mechanisms initiated by the binding of the EA5 ligand to the EAZ-K receptor, as well as playing a role in the signaling mechanisms initiated by the binding of TME to the p55 receptor K. The new protein 1 and its isoforms are included in the group of proteins 01 according to the present invention. 60 More specifically, the present invention provides a novel protein 51 (also called SAZN), which is apparently a homologue of the nematode SEHUOZ protease. This new C1 protein, although closely related, shows certain differences in structure and substrate specificity: accordingly, it can perform somewhat different functions in mammalian cells. Indeed, two different types of protease activity are known. The main function of ISE (which is also called CA5P-1) seems to be the processing of the precursor II -1p, while for SEOZ it was clearly shown to function as an effector of programmed cell death. This latter function is also believed to be a function of at least some homologous mammalian proteins, such as some isoforms of the MASN proteins (also called CA5BR-8) described in the above-mentioned co-pending patent applications, as well as those mentioned above related proteins Mei4 (also called SABR-10), with

HOW protein C1 according to the present invention binds. The amino acid sequence of MASN 91 shows a close relationship to SRRZ2 (also called SAZR-3), which is the closest known mammalian homolog

SEYUOZ. The substrate specificity of MASN is also similar to that of SRRZ2, except that MASN 491 appears to have a more limited substrate specificity compared to SRRZ32. Protease

CPPZ2 more appropriately cleaves peptides that are a substrate at the corresponding site in the composition of 70 poly-ADP-ribose)-polymerase (RACP), and also shows proteolytic activity relative to peptides corresponding to the cleavage site of the ISE protease in the composition of the precursor of interleukin-1 D. However, it seems

MASNYAO/1 is capable of cleaving exclusively the RAKR-matched sequence. This is the ratio of MASN 91 with SRRZ2 and

SEYUOZ and its lack of similarity with ISE corresponds to the assumption that MASNoOi! similar to SEYUOZ, it is a regulator of cell death. MASN 91 at the same time reveals a number of sequence parameters that distinguish 75 it from SEOZ and SRRZ2, as well as from other members of the SEOZ/LSE family. The C-terminal part of MASN 41, located above its section, homologous to SEYUOZLSE, shows the absence of any similarity with all similarly located segments of any of the corresponding homologues. In addition, there are a number of unique characteristics of the sequence and within the segment of homology with proteases of the CEOZ/LCE family. The presence of such differences confirms that МАСНе1 belongs to a separate evolutionary branch of this family and that its participation in the control of cell death differs to some extent from that previously described for homologues from the SEYUOZ/LSE family. Also, protein 51 according to the present invention and its probable isoforms show some clear differences within the segment of homology with the SEOZ/LSE proteases in sequence 1, and if so, then such differences may represent unique characteristics that reflect the specificity of activity 1 in mammalian cells. c 25 One important difference may relate to the mechanism by which the protease is regulated. Being involved in both the ontogenetically regulated processes of cell death and receptor-induced immune cytolysis, the cleavage of proteins by proteases of the SEOZ/LCE family must be regulated both by signals generated inside the cell and by signals coming from receptors located on the outside cell surface. In the ontogenetic processes of cell death, the activation of such proteases is believed to involve mechanisms affecting gene expression, resulting in an increase in the synthesis of these Gα proteases, as well as a decrease in the intensity of the synthesis of some proteins, such as ALL-2, which is antagonist of apoptotic induction. However, this does not exactly correspond to the case when cytotoxicity is mediated by RHAB-K or TME-K receptors. Cells can be destroyed with the participation of TME-K or RA5-K ligand even when the activity of protein synthesis inside them is completely blocked (in fact, they can be destroyed in this case with greater efficiency) and remain stimulus-dependent even in such conditions Activation of proteases in the family of SEOZLSE by TME and RA5B-K receptors can also occur by a mechanism that is independent of protein synthesis. Unique parameters of the sequence MASSOOI! may allow him to participate in the work of such a mechanism. In a similar way, the unique characteristics of the sequence of protein 1 according to the present invention also agree with its probable ability to participate in the work of such a mechanism. WITH

Thus, the novel protein 51 may be another member of a recently discovered group of proteases that includes the aforementioned MASN protease (and its isoforms) and Meni, for which connectivity has been established, either directly or through an adapter protein, with the intracellular domain of the cell surface receptor.According to the interaction of the mechanisms of action of receptor-bound proteins that have different catalytic properties, it seems likely that the binding of 1 to Me/4 or 1 to MASN (or the MASNα/1 isoform) and , in turn, the binding of Map4 or MASN to - 15 MOKT-1, or the direct binding of 1 to MOKT-1 allows to stimulate the protease activity of 01 and (or) May and (or) MASN in the course of HA5 binding -ligand on the EAB-K receptor. It can also allow the activation of the protease by the p55B-K receptor due to the binding of MOKT-1 to TKAOO, which, in turn, binds to rb5-K. sl For other family members SEHUOZ/LSE protease was found to be fully active only after proteolytic processing, which occurs either by self-cleavage, or under the influence of proteases of the same 50 family (review by Kitaz, 1995; NepkKagii, 1996). For example, as described in detail in the above co-pending MASC patent applications, the cytotoxic effect of co-expression of the two potentially self-cleavable main products of MACNOe/1, in contrast to the absence of cytotoxicity in cells in in the case of expression of the full-length section of SEYUOZLSE homology, agrees with the 5 probability that MASNo! also reveals its full activity only after processing. Enzyme activity observed in lysates of bacteria expressing the full-length segment appears to (F) reflect the presence of autoprocessing of this protein occurring under such conditions or processing carried out by

GI by some bacterial proteases. It is still not clear by what mechanism such processing takes place in mammalian cells and how it can be carried out through the mediation of receptors РА5Б-К and рб55-К, and it is still not clear what is the relative contribution of the protease activity of МАСНо/1 to cytotoxicity , which is induced by GAZ5-K and

TME. The assessment of such a contribution is complicated by the fact that there is also the expression of MASN rt, which does not contain a segment of homology with SEYUOZ/LSE, which leads to pronounced cytotoxicity. Apparently, this manifestation of cytotoxicity reflects the ability of МАСНРрИ1 to bind to МАСН "1. Due to this ability, the transfected MASN molecules can, due to aggregation, lead to conformational changes of the MASN molecules, which is dangerous for the transfected cell. Such a mechanism can also cause cytotoxicity, which is observed when molecules that act on MASN are overexpressed in cells (proteins MOKTI1, TKASO or "death domains" in the composition of either p55-K or RAB-K). At the same time, however, it cannot be ruled out that the cytotoxicity observed during the induction of the expression of MASN or molecules that act before its manifestation also reflects, in addition to the proteolytic activity of the homologous SEHUOZ/LSE segment in the composition of MASN, the activation of some other mechanisms, which are assumed to be involved in the cytotoxic manifestation of ГА5Б-К and рб55-К (for example, activation of neutral or acidic sphingomyelinases). Also, it cannot be excluded that the proteolytic activity of the homology segment

CEOSL/LCE serves functions other than induction of cytotoxicity. For a clearer understanding of the function of MASN o1, it would be necessary to identify the endogenous protein substrates that are cleaved as a result of the activation of MASN "1.

The discovery of ways to disrupt the activity of MASN 491, for example, by expressing inhibitory molecules, can also contribute to the understanding of the functions of this protein and thereby contribute to the elucidation of ways of regulating its activity, if this is desired.

Accordingly, the protein (31 according to the present invention and its probable isoforms can be active in a similar way compared to the above-mentioned MASN proteins in the presence or absence of direct interaction with other proteins, namely 1 can act directly by binding to MOKT-1 or act indirectly through 75 binding to Mep and/or MASN and, in turn, through binding of Mey and/or MASN to MOCT-1 or by some other, as yet undefined, protein-specific mechanism 1. In a similar way, regulation of the activity of 31 can be similar to that previously characterized for MASN proteins.

This may exist within cells that express natural inhibitors 51 relative to the protease activity exhibited by this protein. The existence of isoforms, which are formed as a result of alternative splicing, characteristic of other members of the CEOZLSE family, was determined as a factor of physiological limitation of the functions of these proteases. For some of the isoforms of such other proteases, their activity as natural inhibitors of full-length isoforms was reported, which is probably due to the formation of inactive heterodimers between them. This may also be true for 1 and some of its putative isoforms, such as those lacking the potential M-terminal cleavage site. The expression of such He inhibitory isoforms can determine the functioning of the cellular self-defense mechanism against cytotoxicity caused by EGAB-K and TE. 1 can also have other functions: for example, 51 or any of its isoforms can have an enhancing or strengthening effect on other proteins that have enzymatic activity, for example, on the proteolytic activity of various Mep4 and MASN isoforms; such enhancing or strengthening activity is carried out according to the mechanism in which 1 acts as an inducer of binding of other proteins to MOKT-1 (for example, Mei4 and MASN proteins). In addition, (31 or any of its isoforms may also play a role unrelated to cytotoxicity, but cm may act as a "shelter" for molecules (the so-called "depot site") that are involved in the effect of RA5B-K and TME is not associated with the manifestation of cytotoxicity.

Some of the specific isoforms of 01 according to the present invention are illustrated below in example 1. One of them C

C19, (SAZNO), - isolated in humans and mice (respectively, No1o/pSAZNo and tOLTo/pSAZNOo), apparently represents a splicing variant that includes a protease homologous segment and, at least in some cell types (for example, in 293T cells) has cytotoxic activity. Another isoform, designated 51p (SAZNV), isolated in humans appears to be a short splicing variant without a protease homologous segment, this isoform “which acts as an inhibitor of cell death signaling mechanisms.

Due to the unique ability of EA5B-K and TME-K receptors to cause cell death, as well as the ability of TME receptors to mediate various other types of activity associated with tissue damage, disruption of the functions of these receptors can have negative consequences for the body. Indeed, for both types of functions of these » receptors - excess and insufficient - participation in pathological manifestations of various diseases was demonstrated. The identification of molecules involved in the signaling activity of these receptors and the discovery of ways of modulating the functions of these molecules is a possible key step necessary for new treatment methodologies for such diseases. From the point of view of the supposed important role of 1 in the toxicity caused by the participation of BAB-K and TME, it is considered, in particular, important to create drugs that could block the proteolytic function of such molecules, which was previously done with respect to other members of the family of proteases SEHUOZ/ LSE The unique sequence parameters of the homologous CEOZLSE segment in the composition of 1 in 50 may allow the creation of drugs that would violate their protection against cytotoxicity caused by excess immune activity, without disrupting the physiological processes of cell death in which other

Me, members of the SEOZ/LSE family.

As noted above, 01 or any of its isoforms may also be involved in modulating other intracellular signaling mechanisms, such as, for example, the action of other cytotoxic mediators or

Inducers or other proteins acting independently of binding to MOKT-1 or to MOKT1-independently binding proteins. Further, 21, or at least its isoforms that include a protease-like segment but without actual protease activity, may be involved in a primary inhibitory function: i.e., to inhibit those mechanisms where (e.g., signaling mechanisms in general or cytotoxic mechanisms) in which 1 or its isoforms participate either by direct binding to the participants of these mechanisms, or by indirect binding 60 to other proteins, which, in turn, bind to the participants of these mechanisms.

Therefore, this invention also relates to the DNA sequence encoding the C1 protein and the C1 proteins encoded by such DNA sequences.

Moreover, this invention also relates to DNA sequences that encode biologically active analogues, fragments and derivatives of protein C1, as well as analogues, fragments and derivatives encoded by them. Preparation of such b5 analogues, fragments and derivatives is carried out using standard methods (see, for example, the methodical manual Zatgook ei a/!., 1989), by means of which in the DNA sequences that encode protein 1, one or more codons can be deleted, added or replaced by others, resulting in analogs having at least one replacement of an amino acid residue compared to the native protein.

A polypeptide or protein that "substantially corresponds" to protein (31) includes not only protein C1, but also polypeptides or proteins that are analogs of 01.

Analogues that substantially correspond to the C1 protein are polypeptides, in which one or more amino acids of the native amino acid sequence (31 are replaced by another amino acid, deleted and (or) inserted, as a result of which the resulting protein shows essentially the same or a greater level of biological activity compared to the C1 protein to which it corresponds. 70 In order to match the C1 protein, the changes in the amino acid sequence of proteins 1, such as its isoforms, must be relatively small. Although the number of substitutions can be greater than ten, it is more acceptable that it does not exceed ten substitutions, it is even more acceptable that the number of substitutions is no more than five, and most preferably no more than three such substitutions. While any techniques can be used to identify biologically active proteins that substantially correspond to proteins 1, One / 5 of such methods is the use of a standard mutagenesis procedure relative to the protein-coding DNA, with the result that the advantages of a small number of modifications. Proteins expressed by such clones can then be screened for their ability to bind to various MOCTI1-binding proteins such as

Mena and MASN, or even directly from MOKT-1, and (or) for their EA5-K - and po5-K-mediating activity and (or) the ability to mediate the activity of any other intracellular mechanisms in such a way as it was described above.

By "conservative" changes are meant those changes that are not expected to alter the activity of the protein and that are usually screened in the first place because they are not expected to result in a significant change in the size, charge, or configuration of the protein in question and, therefore, as expected, do not lead to a change in its biological properties. c 25 Conservative substitutions in proteins 51 include such an analogue in which at least one amino acid residue in the polypeptide is substituted in a conservative way by another amino acid. It is more acceptable i) to perform such substitutions according to the list given in Table 14A: such substitutions can be determined by routine experimentation with the aim of obtaining modified structural and functional characteristics of the synthesized polypeptide molecule while maintaining the biological activity characteristic of He! protein C1. 30 schu - zv tsi h 4 Z p. i» 6000 vm - in t, still sr

On the other hand, another group of substitutions in the composition of protein 51 is represented by such substitutions, when at least one amino acid residue in the polypeptide composition is removed and another residue is introduced in its place according to Table 18. The types of substitutions that can be made in the polypeptide composition can be based on the analysis about the frequency of amino acid substitutions between homologous proteins in different types of organisms, such as those presented in tables 1-2 Tu Zsptsi2 ei ai., 1978, Rgipsiriev oi Rgoiviiip Zigysitsge, Zrgipdeg-Megpad, Mem Mogk, MU, and in fig. 3-9 in Steidpiop T. Ye., 1983, Rgoieipv: Zigysige apa MoiIesshiag Rgoregpiev, GRgeetap 5 So., Zap Egapsizso, SA). for Based on similar analyses, alternative conservative substitutions are defined here as substitutions within one of the following five groups: vv

With |Polar, positively charged residues: Niv, Aga, Guv

The three amino acid residues, taken in the above table in parentheses, are characterized by a specific role in the spatial architecture of proteins. Glycine is the only amino acid devoid of any side chain and thus provides chain mobility. This, however, tends to form any secondary structure other than the o-helical. Due to its unusual geometry, proline ensures the density of chain packing and, in principle, provides a tendency to the formation of structures of the D-plane type, although in some cases /o the cysteine residue can participate in the formation of disulfide bonds, which are important for the spatial folding of the protein. It should be noted that the above cited Zsptsi7 ei a). combined the aforementioned groups 1 and 2.

It should also be noted that tyrosine, due to its potential to form a hydrogen bond, shows significant affinity with serine and threonine, etc.

Conservative amino acid substitutions according to the present invention, such as those described above, are well known in the art and are expected to maintain the biological and structural properties of the given polypeptide after the amino acid substitution. Most of the deletions and substitutions according to this invention are such that do not lead to radical changes in the characteristics of a given protein or polypeptide molecule. By "characteristics" are understood both changes in the secondary structure, for example, the presence of A-helices or p-planes, and changes in biological activity, for example, binding to MOKT1-binding proteins or to MOKT-1, or mediating the effect on the cells of the ligand GAB-K or TME.

Examples of amino acid substitutions in proteins that can be used to produce analogs of proteins 51 for use in the present invention include any known methods, such as those shown in US Patents KE-33653, 4959314, 4588585 and 4737462 to McEaly. ; 5116943 in Koiz eyi ai.; 4965195 in Matep ei aI.; 4879111 in Spopd eyi ai.; and 5017 691 in I ee ei a); and lysine-substituted proteins, provided by US patent 4,904,584, s.

In addition to the conservative substitutions discussed above, which should not significantly change the activity of protein 1, and conservative substitutions, and less conservative and "more random" changes that lead to an increase in the biological activity of protein analogs (31, are also included in the scope of this invention.

When the exact manifestation of a substitution or deletion is to be determined, it should be clear to one skilled in the art that such manifestation of substitution(s), deletion(s), etc. can be assessed using standard binding and cell death assays. Screening using such standard tests does not include any inappropriate experiments. yu

Acceptable analogs are those that retain at least the ability to bind to MOKT1-binding proteins as noted above, or to MOKT-1, or to other proteins, thereby as described above -

Зз5 mediating the activity of RAZ-K and p55-K receptors or other proteins that were mentioned above. Thus, analogs can be produced that show the so-called "dominant-negative effect", namely analogs that are defective either in binding to MOKT1-binding proteins (for example, Mep4 or MASN) or in tying with

MOKT-1, or with other proteins, or by subsequent signaling or protease activity after such binding.

Such analogues can be used, for example, to inhibit the effect of the GA5-K ligand or the effect of other proteins due to competition with natural MOKT1-binding proteins or other proteins. For example, it seems likely that the isoforms MASN-MASNeЯ2 and MASNе3 are "natural" analogues that serve as inhibitors of MASN activity due to competition for the binding of active (protease) isoforms of MASN with MOKT-1 , which appears to be essential for the activation of these MASN isoforms. As a result, active MASN isoforms cannot bind to MOKT-1, intracellular signaling mechanisms mediated by EAZ-K and p55-K receptors will also be inhibited. Similarly, 1 or any of its isoforms can also act as inhibitors of the RAZZ ligand by competing with various MOKT1-binding proteins for binding to MOKT-1 or by interacting with them in a way that diverts their binding binding to MOKT-1. Also, so-called "dominant-positive" analogs of C1 can be obtained, which will serve to enhance the effect of EAb3 ligand or TME. They may have the same or better ability to bind to MOKT1 -binding proteins or even the same or better properties with respect to binding to MOKT-1 and the same or better characteristics with respect to signaling mechanisms compared to natural proteins 1. (Che) At the genetic level, these analogues are in principle obtained by site-directed mutagenesis with respect to nucleotides in composition of DNA encoding protein 51, thus obtaining DNA encoding this analog, with subsequent synthesis of this DNA and expression of the recombinant polypeptide in cell culture. As a rule, analogs show the same or improved qualitative indicators of biological activity compared to the protein that occurs in nature: |(Aizibvbeii ei ai., 1987-1995, Systepi Rgoiosoiiv ip MoiIesciag Vioiodu, Sgeepe Ryri. 5 UMieu iF) Ip(egesi., Mem/ Mogk, MU; ZatrgbooK ei aiYi., 1989, MoiIesshag Siopipd: A Iarogaigu Mapiai, Soiy 5rgipd Nagrog ko I ab., Soia 5rgipd Nagbrog, MU).

Preparation of protein 1 as described herein or an alternative nucleotide sequence encoding the same polypeptide, which differs from the natural sequence due to changes that take into account the known degeneracy of the genetic code, can be carried out by site-specific mutagenesis of DNA encoding a previously prepared analog or native protein variant 51. Site-specific mutagenesis allows obtaining analogues by using specific oligonucleotide sequences encoding the DNA sequence with the desired mutation, as well as a sufficient number of neighboring nucleotides to obtain a seed sequence 65 of sufficient size and complexity, necessary for the formation of a stable duplex on both sides of "intersecting" deletion connection. A primer consisting of approximately 20-25 nucleotides, with approximately 5-10 complementary nucleotides on each side of the sequence being replaced, is usually more acceptable. In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by the publication of Adey!tap e! a!., 1983 (OMA, 2, 183), data included in this text by citation.

It should be taken into account that the technique of site-specific mutagenesis usually uses a phage vector that exists in both single-stranded and double-stranded forms. Common vectors used in the method of directed mutagenesis include vectors such as phage M13, for example, as described by Messing et al. | Mezvipud ei ai., 1981, according to Siemeyap butr. op MasgotoYescez 5 Kesotbripapi OMA, ey. A. UmUaiyuop,

EIvemieg, Atziegdati, whose data are included here by way of citation. These phages are readily available on a commercial 7/0 basis and their use is generally well known to those skilled in the art. On the other hand, plasmid vectors carrying a single-stranded phage replication initiation site can also be used to obtain single-stranded DNA (Meiga et al., 1987, Meijoaz Epgutoi)., 153, 31.

In general, site-specific mutagenesis as described here is first performed by obtaining a single-stranded vector that includes within its sequence a DNA sequence that encodes the required 7/5 polypeptide. An oligonucleotide primer carrying a sequence that is mutated in the desired way is obtained synthetically by means of automatic DNA-oligonucleotide synthesis. This primer is then annealed with a single-stranded vector containing the protein-coding sequence and exposed to

DNA-polymerizing enzymes, such as a fragment of Klonov polymerase | E. coli, in order to complete the synthesis of the chain that carries this mutation. Thus, like the mutated sequence, the second strand carries the desired mutation. Such a heteroduplex vector is then used to transform suitable cells, such as UM101 cells of E. soii, and clones are selected by including recombinant vectors carrying the mutant sequence.

After such a clone is selected, the mutant C1 protein can be isolated and inserted into a suitable vector, usually a transformation or expression vector of the type that can be used to transfect a suitable host organism.

Accordingly, the gene or nucleic acid encoding protein 51 can also be detected, obtained and (8) (or) modified by ip miigo, ip zi and/or ip mimo by using known DNA amplification techniques or

RNA, such as polymerase chain reaction (PCR) and chemical oligonucleotide synthesis. PCR allows you to amplify (quantitatively increase) specific DNA sequences by repeating reactions with the participation of Ge!

DNA polymerases. This reaction can be used instead of cloning: all that is required in this case is to know the composition of the nucleotide sequence. In order to perform PCR, primers are formed that are complementary to the sequence of interest. These primers are then generated by automated DNA synthesis. Because the primers can be designed to hybridize to a part of a specific gene, conditions can be created that allow errors in base complementarity to be skipped.

Amplification of such sites that carry errors can lead to the synthesis of a mutant product, resulting in the formation of a peptide with new properties (ie, in essence, this is site-directed mutagenesis). See also A!zyBbei's manual ei aiI.), quoted above, chapter 16. Also by combining the synthesis of complementary

DNA (ccDNA) using reverse transcriptase and the RNA PCR method can be used as a starting material for the synthesis of the extracellular domain of the prolactin receptor without the cloning step. "

Moreover, PCR primers can be designed to include new restriction sites or other elements such as termination codons at the ends of the gene segment to be amplified. That's it. the placement of restriction sites at the 5- and 3-ends of the amplified gene sequence enables aimed at obtaining gene segments that encode protein 51 or its fragments, formed so as to be suitable for lysing to other sequences and (or) cloning sites in the composition of vectors.

PCR and other methods of RNA and/or DNA amplification are well known in the art and can be used in accordance with the present invention without undue experimentation, based on the advice given in this text. Known methods for amplifying DNA or RNA include, but are not limited to, polymerase chain reaction (PCR) and similar amplification processes |see, for example, US Patents 4,683,195, 4,683,202, 4,800,159, 4,965,188 (Wash et al.), 4,795,699, and 4,921,794 (Tarog ei a), 5142033 (Ippiv), 5122464 (UMivop ei ai.), 2091310 (Ippiv), 5066584 (SuPepviep ei a), 4889818 (SeMapa ei a), 4994370 (Zimeg ei ai.), 4766067 yu (Vizvm 'az), 4656134 (Kipdoi4) and the manual Ippiz ey aiY. Iedv3, RSK Rgoiosoiiv: A Scide yuyu Meiyod4 apa

Ge) Atriyisayop|, as well as RNA-mediated amplification, which is based on the use of RNA that is antisense to the target sequence and is used as a matrix for the synthesis of double-stranded DNA (US patent 5130238 - Maiek et al.: trademark MAZVA) and immune PCR, which is a combination

DNA amplification with antibody labeling (Kygiska et al., 1993, Zsiepse, 260, 487; Zapo et al., 1992, Zsiepse, 258, 120; Zapo et al., 1991, Vioyesppidoez, 9, 1378), at because the full content of the patents and articles that (F, cited) is fully incorporated by reference. In a similar way, biologically active fragments of proteins 1 (for example, such as any proteins (31 or its isoforms) can be obtained as as it was described above in relation to the preparation of analogues of proteins 51. Suitable fragments of proteins (31) are those that retain the inherent protein (31) ability to mediate the biological activity of RA5B-K and p55-K receptors or other proteins as described above. Accordingly , the following protein fragments (31) can be obtained, which have a "dominant-negative" or "dominant-positive" effect as described above for the characterization of analogs. It should be noted that these fragments represent a special type of analogs according to the present invention, namely they define areas of proteins 51, which is deriv and from the complete 65 sequence of protein 1 (for example, from the sequence of any 1 or their isoforms), while such a part or fragment had any of the above-mentioned desired characteristics. Such a fragment can be, for example,

peptide

In a similar way, derivatives can be obtained by standard modifications of the side groups of one or more amino acid residues in the composition of protein 1, its analogues or fragments, or by connecting protein 1, its analogues or fragments with another molecule, for example, an antibody, enzyme, receptor etc., which is well known in the art. Accordingly, the term "derivatives" in this text means derivatives that can be obtained from functional groups located on the side chains of amino acid residues or M- or C-terminal groups using known techniques, and they are included in this invention. Derivatives can be chemical components, such as hydrocarbon or phosphate residues, as a result of which the resulting 7/0 fraction will have the same or higher biological activity compared to proteins 1.

For example, derivatives may include aliphatic esters of carboxyl groups, amides of carboxyl groups obtained as a result of reactions with ammonium or with primary or secondary amines, M-acyl derivatives or free amino groups of amino acid residues formed by acyl components (for example, alkanoyl groups or aroyl groups with carbon ring) or 0-acyl derivatives of free hydroxyl groups (for example, in residues 7/5 of serine or threonine), formed by adyl components.

The term "derivative" should include only those derivatives that do not change one amino acid to another of the 20 naturally occurring amino acids.

Although protein 51 is a protein or polypeptide, it is a sequence of amino acid residues. A polypeptide including a longer sequence that contains the complete sequence of protein 1, according to the definitions adopted here, should also be included in the parameters of such a polypeptide, since the additions do not affect the main and novel characteristics of the invention: that is, if it preserves or enhances the biological activity protein 1 or if the elongated polypeptide can be subsequently cleaved to yield a protein or polypeptide having the biological activity of protein 1. Thus, for example, this invention also includes fusion proteins that consist of protein 1 and other amino acids or peptides. high school

The novel C1 protein, its analogues, fragments and derivatives are suitable for use in a number of directions, for example: (1) The C1 protein, its analogues, fragments and derivatives can be used to mimic or enhance i) the functions of MOKT1-binding proteins, such as, for example, Msp4 or MASN (including their various isoforms) or even MOKT-1 itself and, therefore, the EA5-K ligand or TME, or other proteins in cases where it is desired to enhance the effect of the RAB-K ligand or TME, or another protein, as, for example, when used with antitumor, anti-inflammatory, Ge! zo anti-HIV, etc. goal, the achievement of which is desirable to induce cytotoxicity with the participation of the EAB-K ligand or

TME or other proteins. In this case, protein 1, its analogues, fragments or derivatives, which enhance the effect of the HA5B-K ligand, or TME, or another protein, i.e. cytotoxic effect, can be introduced into cells using standard procedures known up to now. For example, when the protein 51 is completely intracellular (as it is supposed to be) and must be introduced only into cells in which the manifestation of the effect of the ligand EA5-K, " or TME, or another protein is desired, a special system for intracellular introduction must be used. squirrel. One of the ways to solve this problem is to create a recombinant animal virus, for example, derived from Massipia, into the DNA of which the following two genes must be embedded: a gene that codes for a ligand that binds to cell surface receptors that are specifically expressed by cells, for example, such a ligand as the antigen dr120 of AIDS (HIV), which specifically binds on some types of cells (on « 04 lymphocytes and related leukemic cells), or any other ligand that specifically binds to cells carrying EA5B-K or p55-K receptors, so that such a recombinant viral vector would be able to bind such cells carrying GA5B-K or p55-K; and the gene that codes for C1 protein. So, ;" the expression on the surface of the virus of an antigen that binds on the surface of the target cell will allow to precisely direct this virus to a tumor cell or another cell carrying PHA5B-K or p55-K so that after

This sequence, which encodes the C1 protein (for example, the sequence 51 y) was introduced into cells by this virus and, accordingly, expressed in these cells, which as a result will lead to an increase in the effect of the EAB-K or TME ligand and further lead to death of tumor cells or other cells carrying EAB-K or p55-K, if it is desired to destroy them. The construction of such a recombinant animal virus is carried out using standard procedures (see, for example, Zatgook et al., 1989). Another possible way to introduce the 5p sequence of protein (51) (for example, or protein (1, or any of its isoforms)) is to introduce oligonucleotides in the form of o, which can be absorbed by the cell with their subsequent expression inside it.

Ge) Another way of strengthening such cellular cytotoxicity can be suppression of the activity of isoforms 1 (for example, C1p), which are inhibitors of cellular cytotoxicity by themselves. Ways of inhibition of such isoforms 1 are numerous and include those considered in the following clause (2) in connection with their use for specific inhibition of inhibitory isoforms of protein C1, such as, for example, isoform C1p. (2) They can be used to inhibit the effect on cells of EAZ-K ligand, or TME, or other proteins, for example, in such cases as tissue damage in septic shock, rejection in graft-versus-host syndrome, or acute hepatitis , that is, when it is desirable to block the intracellular signals induced by the PA5B-K or TME ligand from EAB-K or from p55-K, or signals induced by other proteins. In this case, it is possible, for example, to introduce into the cells using standard procedures oligonucleotides having antisense coding sequences relative to the sequence that encodes protein 1, which provides effective blocking of the translation of mRNAs that encode protein 1, and thus blocking its expression. which leads to the suppression of the effect of the HA5B-K ligand, or TME, or another protein. Such oligonucleotides can be introduced into cells using the above-described method of constructing 65 recombinant animal viruses, the second built-in sequence of which is this oligonucleotide sequence.

Further, as already noted above and will be considered below as an example, at least one isoform of protein 21 was isolated, which is considered as a "natural inhibitor" of cellular cytotoxicity - it is 1 p1.

Accordingly, such an isoform of protein 31 can be introduced into cells directly or using a suitable vector that

It carries the nucleotide sequence that encodes this isoform: therefore, when expressed in these cells, this 01 isoform will act as an inhibitor of cytotoxicity.

Another possibility is provided by the use of antibodies specific for protein 01 to inhibit its intracellular signaling activity.

Another way to inhibit the effect of EAB-K ligand or TME is the recently developed "ribozyme 70 approach". Ribozymes are RNA molecules that have catalytic activity aimed at splitting molecules

RNA. Ribozymes can be artificially engineered in such a way as to selectively cleave an RNA target, for example, an mRNA that encodes protein 01 according to the present invention. Such ribozymes must be characterized by a sequence specific to protein 51 mRNA and must be able to interact with it (by complementary binding) with subsequent cleavage of the mRNA, resulting in a decrease (or 7/5 complete cessation) of C1 protein expression, and the degree of decrease expression depends on the intensity of ribozyme expression in the target cell. Any suitable vector, such as a plasmid, animal virus-based vectors (retroviruses), which are commonly used for similar purposes, can be used to introduce ribozymes into a selected cell (e.g., cells bearing HA5B-K or p55-K receptors). (see also clause (1) described above, where viral vectors are described, which include as a second integrated cDNA sequence, the coding sequence of the ribozyme to be selected). As reviews of the methods of using ribozymes | see Snap ei a)., 1992; 2pao 85. Risk, 1993; Zpoge ei ai., 1993; dozerp " VygKe, 1993; Zpitauata eyi ai., 1993; Sapiog eii ai., 1993;

Vahipada, 1993; Stevie! ei a!., 1993 and Koigiti ei a!., 1993). (3) Protein 1, its analogues, fragments or derivatives can also be used for the isolation, identification and cloning of other proteins of the same type, i.e. those that bind to the intracellular domain c of the RHAB-K receptor or to functionally related receptors, or those , which are related to those described above

MOCTI-binding proteins, or those that bind to MOCT-1 protein and thus bind to functionally related receptors, such as BRAB-K and p5bB-K, and, accordingly, are involved in intracellular signaling processes. In this case, the above-mentioned system of dihybrid yeast screening can be used, or the recently developed Southern blot hybridization system with subsequent PCR cloning under non-stringent conditions can be used (Gumiike, 1989). This publication described the identification and cloning of two potential protein tyrosine kinases using p

Southern blot hybridization in non-stringent conditions followed by PCR cloning based on the known sequence of the main kinase motif. This approach can be applied in accordance with the present invention using the sequence of protein (31) in the identification and cloning of those proteins related to the MOKTI-binding proteins. (4) Another approach to the use of protein 51 or its analogs, fragments or derivatives according to the present invention is their use in affinity chromatography for the purpose of isolation and identification of other proteins or factors with which they can bind, for example, MOKT-1 protein, MOKT1-binding proteins or other proteins or factors, involved in intracellular signaling processes. In the case of such application, protein 1, its analogs, fragments or derivatives according to the present invention can be separately attached to matrices for affinity chromatography and then exposed to cell extracts or isolated proteins or factors that , are believed to be involved in intracellular signaling processes. At the end of the "" affinity chromatography procedure, other proteins or factors that found to be bound to protein 51 or to its analogues, fragments or derivatives according to the present invention, can be eluted, isolated and characterized. -and (5) As mentioned above, protein (31) or its analogs, fragments or derivatives according to the present invention can also be used as antigens (immunogens) for the purpose of producing antibodies specific to them. These antibodies can also be used for the purpose of purifying this of protein 51 (eg, 1c itself or any of its isoforms) from both cell extracts and transformed cell lines producing protein 51 or its analogs or fragments.In addition, these antibodies can be used for diagnostic purposes to identification of disorders associated with abnormal functioning of EAB-K ligand systems or TME,

Ge) for example, excessive or insufficient cellular influence induced by the ligand РГА5Б-К or TME. Thus, despite the fact that such diseases are associated with a malfunction of the system of intracellular signaling mechanisms, which involves the MOKT-1 protein or any others, for example, the above-mentioned MOKT1-binding proteins or the C1 protein itself, such antibodies will serve as an important diagnostic tool.

It should also be noted that isolation, identification and characterization of protein 1 according to this iF) of the invention can be carried out using any of the known standard screening procedures. For example, as one of these screening procedures, the yeast dihybrid test described in this text was used to identify the protein MOKT-1 and subsequently various MOKT1-binding proteins and protein 1 according to the present invention. Also, as described above and will be described below, other approaches such as affinity chromatography, DNA hybridization techniques, etc., which are well known in the art in connection with the isolation, identification and characterization of protein 51 according to the present invention, may be used or with the selection, identification and characterization of other proteins, factors, receptors and the like that are able to bind to protein 1 according to the present invention. 65 As indicated above, protein 51 can be used to produce antibodies specific for protein 51, for example, for (31 and its isoforms). These antibodies or fragments thereof can be used as described in more detail below, and it should be understood , that in such cases these antibodies or their fragments are those that are specific for proteins 1.

Based on the data of the present invention that at least some of the proteins 51 or their possible isoforms are proteases similar to the proteases of the SEHUOZ/LCE family, the following special medical applications are proposed for these proteins 1 and their isoforms: it has been found that specific inhibitors already exist other SEHUOZ/CE proteases, which are able to enter the cell and can effectively block programmed cell death. Therefore, according to the present invention, it is possible to form such inhibitors that can prevent ligand-induced cell death

EAB-K or TME - that is, the mechanism in which protease C1 is involved. Further, from the point of view of the unique parameters of the 70 sequences of these new proteases 251, it seems possible to design such inhibitors that would be highly specific for the effect of TME and the GAB-K ligand on cells. The data of the present invention also provides a way to study the mechanism in which the "cell-killing protease" is activated in response to the ligand EAB-K and TME: accordingly, it allows the development of drugs that would be able to control the extent of such activation. There are quite a few diseases in the treatment of which such drugs could provide significant help. Among such /5 diseases - acute hepatitis, in which acute lesions of the liver tissue, apparently, reflect the death of liver cells, mediated by the ligand of the BAZB-K receptor; autoimmune response-induced cell death, such as β-cell death of pancreatic islets of Langerhans, leading to diabetes; cell death during rejection of transplanted tissue (for example, kidneys, heart and liver); death of brain oligodendrocytes in multiple sclerosis; and self-destruction of T-lymphocytes is suppressed in AIDS, as a result of which the reproduction of the AIDS virus and, therefore, the development of AIDS itself is ensured.

As noted above, it is possible that 1 or one or more of its possible isoforms (e.g., the C1p isoform) may serve as a "natural" inhibitor of C1 protease or C1 protease isoforms, and this may also be used as noted above for specific inhibitors of such proteases 1. Also, other compounds, such as peptides, organic compounds, antibodies, etc., can be screened in order to obtain Ha-specific drugs capable of inhibiting proteases 1.

A non-limiting example of how peptide protease inhibitors 51 could be prepared and i) screened is based on previously conducted studies of peptide inhibitors of ISE or ISE-like proteases, substrate specificity of ISE, and epitope analysis strategies using peptide synthesis.

The minimum condition for effective cleavage of the peptide by ISE protease, as it was established, is the presence of He")

Four amino acids to the left of the cleavage site with strict greater acceptability of the presence of an aspartic acid residue in the Ру position and the sufficiency of methylamine presence to the right of the Р residue. Nomagya ei aiYi.,, 1991; Trogpreggu ei aiYi., 1992). Moreover, it was established that the fluorogenic peptide substrate (which is a tetrapeptide) - acetyl-Azr-cSISH-MaI-Azr-a-(4-methylcoumaryl-7-amide) - abbreviated

As-OEMO-AMS, which corresponds to the sequence of ADP-ribose polymerase (RAKR), is cleaved in cells through C

A short time after stimulation of the GAZB-K receptor, as well as other apoptotic processes (Kashtapp, 1989; h-

Kashtapp ei ai., 1993; And agerpik ei aI., 1994), and is efficiently cleaved by protease CPPZ2 (which is a member of the family of proteases SEHUOZ/CE) and proteases MASN (and also, probably, proteases c1).

Taking into account the importance of the presence of the Azr residue in the P position - in the composition of the substrate, tetrapeptides that "have aspartic acid in the fourth position and a different combination of amino acids in the first three positions can be quickly screened for binding to the active site of protease 01 using - with, for example, the method developed by Heisen | Seusep, 1985; Seuzepei AI., 1987), in which a large number of peptides attached to a solid support are screened for specific interaction with antibodies. The binding of MASN proteases to "" specific peptides can be detected using various methods well known to those skilled in the art, such as isotopic labeling of proteases 31, etc. It has been shown that using this method according to

Geisen can test at least 4,000 peptides in one working day. -I Since the construction of peptide-based inhibitors that selectively inhibit proteases 1 without any interference with the physiological processes associated with cell death, in which other members of the family of proteases SEHUOZ/LCE participate, the pool of peptides that bind to proteases C1, for analysis in such a test 1, which was described above, can be further synthesized in the form of a fluorogenic peptide substrate intended for testing selective cleavage by proteases 1 in the absence of cleavage by other proteases of the SEHUOZ/LCE family. Peptides for which selective cleavage by C1 proteases has been determined can (Che) then be modified to enhance intracellular permeability and inhibitory activity against (C1-mediated cell death, both in a reversible and an irreversible mode of action. Thornbury et al. PPPogpbeit et al., 1994) reported that tetrapeptide-(acyloxy)-methylketone

As-Tug-MaI-AIa-Azr-SNYOS(O)-(2,6-(СЕ3)2)| RA is a strong inactivator of ISE protease. In a similar way, Miligen et al. (Miyidap ei ai., 1995) reported that tetrapeptide inhibitors, which include chloromethylketone o or aldehyde groups, respectively, irreversibly and reversibly inhibit ISE protease. In addition, inhibition of ISE by benzyloxycarboxyl-Azr-CH 50C(0)-2,6-dichlorobenzene (0OSB) was shown |Mazpita et al., 1995).

Accordingly, tetrapeptides that selectively bind to proteases (31) can be modified by using, for example, an aldehyde group, chloromethylketone, (acyloxy)-methylketone or cSnHOC(O)-OSB groups when solving the problem of creating a peptide inhibitor of protease activity 1.

Since some specific inhibitors of other proteases of the SEHUOZ/LCE family are characterized by intracellular permeability, the intracellular permeability of peptide inhibitors needs to be strengthened.

For example, peptides can be modified or converted into their derivatives chemically in order to increase their permeability through the cell membrane and facilitate the transport of such peptides through the membrane into the cytoplasm. Muranishi et al. (Migapispi et al., 1991| reported on the preparation of a thyrotropin-releasing hormone derivative using lauric acid with the formation of a lipophilic lauroyl derivative that has good characteristics of passing through cell membranes. Zakaria et al. I(/asnagia et al., 1991) also reported that oxidation of methionine to sulfoxide and replacement of the peptide bond with its ketomethylene isoester (COCH") leads to the facilitation of transport of the peptide across the cell membrane. There are a number of modifications of the derivatives that are well known to those skilled in the art.

In addition, drugs or peptide inhibitors capable of inhibiting the activity of 01 relative to cell death, or their possible isoforms, can be combined or included in complexes with molecules that will provide easier penetration into the cell. 70 (US Pat. No. 5,149,782) discloses the association of a molecule intended for transport across a cell membrane with a membrane-binding agent such as slitogenic polypeptides, polypeptides that form ion channels, other membrane polypeptides, and long-chain fatty acids, such as myristic acid and palmitic acid. These agents, which connect to the membrane, embed the molecular conjugate inside the lipid bilayer of the plasmalemma and facilitate its penetration into the cytoplasm.

Low et al. (om ey ai-) in US patent 5108921) consider acceptable methods of transmembrane delivery of molecules, such as (but not limited to) proteins and nucleic acids, based on the mechanism of receptor-mediated endocytosis activity. These receptor systems are systems that recognize galactose, mannose, mannose-b6-phosphate, transferrin, asialog-lycoprotein, transcobalamin (vitamin B"),

A-2-macroglobulins, insulin and other peptide growth factors such as epidermal growth factor (EGF). Lowe with 2o co-author. indicates that food receptors, such as biotin and folate receptors, can mostly be used to intensify transport through the cell membrane, due to the peculiarities of the localization and abundance of biotin and folate receptors on the surface of most cells and the processes of transmembrane transport associated with these receptors. Thus, a complex formed by a compound intended for transport into the cytoplasm and a ligand such as biotin or folate contacts the cell membrane that carries biotin or folate receptors in order to initiate the mechanism of transmembrane transport mediated by these receptors, thanks to this, ensuring the entry of the desired compound into the cell. and)

ISE protease is known to be tolerant of free substitution at the P "5" position, and this tolerance of free substitution was used in the development of an efficient, highly selective biotin-containing affinity label (Ppogpreggu et al., 1994). Accordingly, the position P 5, as well as, apparently, Ge!

The M-end of the tetrapeptide inhibitor can be modified or used to obtain derivative compounds, for example, by attaching a biotin molecule, in order to increase the permeability of these peptide inhibitors through the cell membrane. yu

In addition, it is known in the art that the fusion of a desired peptide sequence with a signal (leader) peptide sequence to produce a "chimeric polypeptide" can be used to transport -

From such a "chimeric polypeptide" through the cell membrane into the cytoplasm of this cell. uh-

As should be well understood by those skilled in the art of peptides, it is believed that peptide inhibitors of proteolytic activity according to the present invention should include peptidomimetic drugs or inhibitors that can be rapidly screened for protease binding (31 in order to obtain the most stable possible inhibitors."

It should also be understood that the same methods of facilitating or enhancing the transport of peptide c inhibitors across cell membranes discussed above are also applicable to the protein itself.

O1 or its isoforms, as well as other proteins and peptides that exert their influence inside cells. ;" As used herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies (tAbs), chimeric antibodies, anti-idiopathic (anti-Ia4) antibodies to antibodies that

Can be seen in soluble or bound form, as well as their fragments, obtained in any of the known ways, such as (but not limited to) enzymatic cleavage, peptide synthesis or recombinant technologies. Polyclonal antibodies are heterogeneous populations of antibody molecules isolated from the serum of animals immunized with some antigen. A monoclonal antibody includes an essentially homogeneous population of antibodies specific for antigens, while these populations include essentially similar epitopes of binding sites. Monoclonal antibodies can be prepared using methods known to those skilled in the art

Ge) technical fields: |see, for example, Copy 5 Miiviviip, 1975, Maishge, 256, 495-497; US patent 4376110; A!yzyvei ei aiI., edv. Nagom/ 5 Iape 1988, Apiirodiez: A I aroghaigu Mapiai, So Zrgipd Nagrog ar. and Soiydap ei ai.,

Sitepi Rgoiosoiiv ip Ittypoiodu, edv. Stgeepe Riri. Avvos. 5 UMPeu Ipiegesiepse, MU (1992-96)), the full content of which is given in this text as references. Such antibodies can belong to any class of immunoglobulins - Id, DM, I9ZE, IZA, SI - and any of their subclasses. Hybridomas producing monoclonal (F) antibodies in accordance with the present invention can be cultured ip miigo, ip zyi or ip mimo. Obtaining high titers of monoclonal antibodies ip migo or ip zya makes this approach even more acceptable for their production.

Chimeric antibodies are molecules in which different parts come from different animal species, for example, antibodies that include variable domains isolated from mouse monoclonal antibodies and constant domains of human immunoglobulins. Chimeric antibodies are primarily used to reduce immunogenicity during their application and to increase product yield, for example, when mouse monoclonal antibodies are characterized by a higher yield with a hybrid, but higher immunogenicity in human cells, therefore chimeric "human-mouse" monoclonal antibodies can to be used Chimeric antibodies and methods of their 65 production are well known in science: I(Sabiyu ei ai., 1984, Rgos. Mai. Asad. Zei. OBA, 81, 3273-3277; Moggtizop ei ai., 1984, Rgos. Mai. Asai. Zei. OBA, 81, 6851-6855; Voshiappe et al., 1984, Maishge, 312, 643-646; Sabriyu et al., European patent application Mo125023 (published on November 14, 1984); Metzbregdeg et al., 1985 ,

Maiige, 314, 268-270; Tapidispi ei aI. European patent application No. 171496 (published on February 19, 1985); Mogtizop ei AI., application for European patent Mo173494 (published on March 5, 1986); Meibregdeg ei ai., PCT UUO application 8601533 (published on March 13, 1986); Kido ei aII., application for a European patent

Mo184187 (published on June 11, 1986); Zapadap ei ai., 1986, 9. Ittipoi, 137, 1066-1074; Kobipzop ei AI., application for international patent MO 8702671 (published on May 7, 1987); Pi eyi AI., 1987, Rhos. May). Asavd.

Zeim. OBA, 84, 3439-3443; Zyup eyi aiYi.,, 1987. Rhos. May Asai. bey BOTH, 84, 214-218; VeTseg eyi ai., 1988,

Zsiepse, 240: 1041-1043; and Napomu/ 8. I ape, Apiibodiez: A I arogaiogu Mapiai|, cited above. These works are fully 7/0 Incorporated herein by reference.

An anti-idiotypic (anti-I4) antibody is an antibody that recognizes unique determinants that are essentially bound to the antigen-binding site of the antibody. An anti-idiotypic antibody can be prepared by immunizing an animal of the same species and genetic type (for example, an inbred line of mice) as a source of monoclonal antibody against which an anti-idiotypic antibody is obtained. The immunized animal must recognize and respond to the idiotypic determinants of the immunizing antibody by producing antibodies against these idiotypic determinants (anti-Ia antibody). |(See, for example, US Patent 4,699,880), which is incorporated by reference.

The anti-idiotypic antibody can also be obtained in the form of an "immunogen" for the purpose of inducing an immune response in another animal that produces the so-called anti-antiidiotypic antibody. Anti-anti-idiotypic antibody can be identical in terms of epitope parameters to the original monoclonal antibody that induced anti-id. Thus, by using antibodies to the idiotypic determinants of a monoclonal antibody, it is possible to identify other clones expressing antibodies that have identical specificity.

Accordingly, monoclonal antibodies that have been generated to proteins 1, their analogs, fragments or derivatives according to the present invention, can be used to induce anti-idiotypic antibodies in suitable animals, such as mice of the BA line! V/s. The cells of the spleen of such immunized mice are used to produce anti-I4 hybrids, which secrete anti-idiotypic monoclonal antibodies. Next, anti-idiotypic monoclonal antibodies can be combined with a carrier, such as mucosal hemocyanin (KI H), and used to i) immunize new mice of the VAII V/s line. The serum of these mice will contain anti-anti-idiotypic antibodies that have the ability to bind to the original monoclonal antibody specific for the epitope of the protein 31 described above or its analogs, fragments or derivatives. b z o Anti-idiotypic monoclonal antibodies thus have their own idiotypic epitopes, i.e. "Idiotopes", similar in structure to the epitope being evaluated, such as KV protein-a. with

The term "antibody" also refers to both intact molecules and fragments thereof, such as, for example, Rab and

K(iav)2, which are able to bind to the antigen. Fragments of Rar and E(abB)2 are devoid of the E-domain of an intact antibody, are more easily released from the circulation and may have a lower level of non-specific tissue binding compared to an intact antibody (Umani et al., 1983, 9. Mysi. Mead., 24 , 316-325). uh-

It should be understood that Rab and E(ar)2 and other antibody fragments that are applicable in accordance with the present invention can be used to detect and quantify protein 1 according to the methods described herein for intact antibody molecules. Such fragments are usually obtained by proteolytic cleavage using enzymes such as papain (for obtaining Bar fragments) or pepsin (for obtaining P(ab)2 fragments). z c When an antibody is said to be "capable of binding", it means the ability to specifically react with a molecule in such a way that binding of that molecule to the antibody occurs. The term "epitope" means that;» part of any molecule that can be bound by an antibody and that can also be recognized by that antibody.

Epitopes or "antigenic determinants" usually consist of chemically active surface groups of molecules, such as side chains of amino acids or sugars, and have specific three-dimensional structural -I characteristics, as well as specific charge parameters. An "antigen" is a molecule or part of a molecule capable of binding to an antibody and, moreover, capable of inducing the production of an antibody capable of binding to the epitope of this antigen in an animal. An antigen can have either one or more epitopes. The specific reaction mentioned above means that the antigen will react with a high level of recognition with the corresponding antibody and with one of the many other antibodies that can bind to other antigens.

Ge) Antibodies, including fragments of antibodies that are applicable according to the present invention, can be used for qualitative and quantitative other detection of protein 1 in a sample or to detect the presence of cells expressing protein (31 according to the present invention. This can be accompanied by two immunofluorescence techniques , which use a fluorescently labeled antibody (see below), combined with the use of light microscopy, flow cytometry or fluorometry for such (F, detection. ka Antibodies (or their fragments), applicable according to the present invention, can be used at the histological level with the use of immunofluorescence or immuno-electron microscopes for detection of the protein in the protein (31 according to the present invention. The detection of the protein can be accompanied by the selection of a histological sample from the patient and the transfer of the fluorescently marked antibody to such a sample.

It is more acceptable to transfer the antibody (or fragment) by introducing the observed antibody (or fragment) into a biological sample. With the use of such a procedure, it becomes possible to determine not only the presence of protein 1, but also to assess its distribution in the examined tissue. With the use of the present invention, those skilled in the art can easily understand that any of a large group of histological methods (such as tissue staining techniques) can be modified to perform similar IP detection.

Such tests for the detection of C1 protein according to the present invention usually involve incubating a biological sample, such as a biological fluid, a tissue extract, freshly obtained cells such as lymphocytes or leukocytes, or cells that have been incubated in tissue culture, in the presence of a labeled labeled antibody, capable of identifying the protein (31 and detecting the antibody by any of a number of methods well known in the art.

The biological sample can be treated with a solid support or carrier such as nitrocellulose or another solid support or carrier capable of immobilizing cells, cell particles, or soluble proteins. The substrate or support can then be washed with suitable buffers, followed by treatment with the labeled/o detected antibody of the present invention as described above. The solid-phase substrate or support can then be washed with buffer a second time to remove unbound antibody. The amount of bound label on said solid substrate or carrier can then be determined using standard techniques. "Solid-phase support", "solid-phase support", "solid support", "solid carrier", "substrate" or "carrier" are any supports or carriers capable of binding antigen or antibodies. Well-known substrates or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and synthetic celluloses, polyacrylamides, gabbro, and magnetite. The nature of the carrier can be either partially soluble or it can be insoluble based on the objectives of the present invention. The substrate material can visually have any possible structural configuration as long as the attached molecule is able to bind the antigen or antibody.

Thus, the configuration of the substrate or carrier can be spherical, spherical, cylindrical, like the inner surface of a test tube, or the outer surface of a rod. On the other hand, the surface can be flat, such as a record, test tape, etc. More acceptable substrates or carriers are polystyrene granules (balls). Those skilled in the art can select other suitable carriers for antigen or antibody binding, or can determine this through routine experimentation. with

The binding activity of this set of antibodies, as set forth above in this invention, can be determined using well-known methods. Those skilled in the art can determine the conditions for i) rapid and optimal tests for each definition based on routine experiments.

Other steps such as washing, mixing, shaking, filtering, and the like can be added to the He! zo tests as recommended by manufacturers or required by a specific situation.

One of the ways in which the antibodies according to the present invention can be detected is the connection of the antibody with an enzyme using enzyme immunoassay (EIA). This enzyme, in turn, when it is subsequently exposed to the corresponding substrate, will react with this substrate in such a way as to form a chemical component that can be detected, for example, spectrophotometrically, fluorometrically - or visually. Enzymes that can be used to detect labeled antibodies include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, d5-steroid isomerase, yeast alcohol dehydrogenase, A-glycerophosphate dehydrogenase, triose phosphatase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Detection can be accompanied by a colorimetric method based on the use of a substrate that is chromogenic for this enzyme. - c Detection can also be accompanied by a visual comparison of the degree of enzymatic reaction of the substrate compared to standards prepared in a similar way. "» Detection can be accompanied by the use of any of a number of other immunoassays. For example, by using isotopic labeling of antibodies or antibody fragments, it becomes possible to detect K-PTRase using KIiIA | (radioimmunoassay). A qualitative description of the KII method can be found in the manual "I arogag - I Tesppidcez 5 Viospeptivigu ip Moiesciag Viiodu" (MuUogk, T. 5. ei aiYi.,, 1978, Mopy NoMyapa Rybri. So., MM) with specific attention to the section entitled "Introduction to radioimmunoassay and similar techniques", by T. Chard (T. Saga), which is incorporated herein by reference. Radioactive isotopes can be detected in 1 ways such as using a Geiger counter or scintillation counter, or by autoradiographic analysis. o It is also possible to detect antibody according to of this invention by means of a fluorescent compound.When the (Che) fluorescently labeled antibody is exposed to light of a suitable wavelength, its presence can be detected by fluorescence. Fluorescein isothiocyanate, rhodamine, phycoerythrin, picocyanin, allocycocyanin, o-phthalaldehyde and fluorescamine are among the most frequently used fluorescent compounds for labeling.

The antibody can also be detected by labeling using fluorescent emitting metals such as 122E or others from the lanthanide group. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (ETRA). ime) The antibody can also be obtained by combining it with a chemiluminescent substance. Then, the presence of the chemiluminescent type of antibody is determined by detecting the presence of 60 luminescence that occurs during the chemical reaction. Examples of chemiluminescent compounds specifically used for labeling are luminol, isoluminol, thermal acridine ether, imidazole, acridine salt, and oxalic ether.

Bioluminescent compounds can also be used for labeling antibodies according to the present invention. Bioluminescence is a variant of chemiluminescence found in biological systems in which a protein 65 having catalytic activity increases the efficiency of the chemiluminescence reaction. The presence of bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for labeling purposes are luciferin, luciferase, and aequorin.

The antibody molecule according to the present invention can be adapted for use in immunometric testing, also known as "two-site" or "sandwich" testing. In a typical immunometric test, an amount of unlabeled antibody (or antibody fragment) is bound to a solid support or carrier and an amount of labeled soluble antibody is added in such a way that it is possible to detect and/or quantify the ternary complex between the solid-phase antibody and the antigen. and labeled antibody.

Usually, and more appropriately, immunometric tests include "forward" tests, in which the antibody bound to the solid phase first contacts the tested sample in order to isolate the antigen from the sample and form a binary complex "antigen-antibody" in the solid phase After a suitable incubation period, the solid support or support is washed to remove any remaining liquid sample, including unreacted antigen, if any, and then contacted with a solution containing an unknown amount of labeled antibody (which functions as a "reporter molecule"). After the second incubation period required for the formation of a complex of the labeled antibody and the antigen bound to the solid substrate or carrier through the unlabeled antibody, the solid substrate or carrier is then washed a second time to remove the unreacted labeled antibody.

Another type of "sandwich" method that can also be used with antigens according to the present invention is the use of so-called "simultaneous" and "reverse" tests. A "simultaneous" test involves a single incubation step in which both the antibody bound to a solid substrate or carrier and the labeled antibody are added to the test sample at the same time. At the end of the incubation, the solid substrate or carrier is washed in order to remove the remaining liquid sample and the observed antibody that was not included in the complex. The presence of labeled antibody bound to a solid substrate or carrier is then determined as in a standard "forward sandwich" test. high school

In the "reverse" test, the gradual addition of a solution of the labeled antibody to the liquid sample is used, followed by the addition of the unlabeled antibody bound to a solid support or carrier after i) a suitable incubation period. After the second incubation, the solid substrate is washed in a standard way to free it from the remains of the tested sample and the solution of the observed antibody that did not react. Determination of the observed antibody bound to a solid substrate or carrier is then carried out in the same way as in Ge! with "simultaneous" and "forward" tests.

Proteins 51 according to the present invention can be obtained using any of the existing standard recombinant DNA procedures (see, for example, ZatbgookK ei ai!., 1989 and A!izibei ei ai., 1987-95, and cited above) , in which suitable eukaryotic and prokaryotic host cells, well known in the art, are transformed using suitable eukaryotic and prokaryotic vectors that include sequences encoding these proteins. Accordingly, this invention also relates to such expression vectors and its transformed host organisms designed to produce these proteins in accordance with the present invention. As noted above, these proteins also include active analogs, fragments, and derivatives thereof, and thus vectors encoding them also include vectors that express analogs and fragments of these proteins, and transformed host organisms include those that produce these analogues and fragments. Derivatives of these proteins produced by such transformed host organisms are derivatives obtained by standard modification of these proteins or their analogs or fragments.

This invention also relates to pharmaceutical compositions comprising recombinant vectors based on animal viruses that encode C1 proteins, so that such a vector also encodes a viral surface antigen capable of binding to specific target cells (for example, tumor cells) with surface proteins in order to direct the introduction of protein sequences 51 into cells. In addition, pharmaceutical compositions -I according to the present invention include as an active component (a) an oligonucleotide sequence encoding an antisense sequence relative to the sequence encoding protein 1, or (B) drugs that block the proteolytic activity of protein 1 or its isoform Pharmaceutical compositions according to the present invention include an amount of active ingredient sufficient to achieve its intended purpose. Additionally, the pharmaceutical compositions may include suitable, or pharmaceutically acceptable carriers, including excipients and suitable components that facilitate processing

Ge) active components in the composition of preparations that can be used in pharmaceutical methods and that can stabilize such preparations, intended for administration to patients who need them, according to methods well known to specialists in this field of technology.

Protein 1 and its isoforms or isotypes are supposed to be expressed in different tissues at distinctly different levels and, apparently, at different parameters of isotypes, similar to the expression of bileu MASN and its

F) of different isotypes, as described in the above-mentioned joint applications under consideration. These differences may, apparently, determine the tissue-specific parameters of the response to the ligand RAB/APO1 and

TME. As in the case of other homologues of SEYUOZLSE |M/apo ei ai., 1994; AIpetgi ei a!., 1995), the applicants have previously shown (according to the above-mentioned patent applications) that MASN isoforms, which include incomplete segments of homology with SEOZ/LSE (for example, MASNAZ), have an effect of inhibiting the activity of MASN molecules and MASNAZ, which are simultaneously expressed; for them, too, the blocking of the induction of death by participation was established

EAB/AROY and p55-K. The expression of such inhibitory isoforms in cells can determine the mechanism of cellular self-protection against cytotoxicity mediated by RHAB/APO1 and TME. A similar inhibitory effect is also assumed in at least one of the C1 protein isoforms. The high level of heterogeneity of MASN isoforms and also the expected similar heterogeneity of C1 isoforms, which significantly exceeds the heterogeneity of isoforms of any other proteases of the CEOZ/CE family, should allow fine-tuning of the functions of active MASN isoforms, and, by analogy, also in active isoforms C1, according to the present invention.

It is also possible that some of the putative C1 isoforms have other functions. For example, the ability of MASN p1 to bind to both MOKT-1 and MASN was previously discovered by the applicants (which was noted above). confirms that this isoform can indeed enhance the activity of catalytically active isoforms. The average cytotoxicity observed in the cultures of the 293-EVMA and MCE7 lines transfected with this isoform, and the more significant cytotoxic effect found in the cells of Ne! and apparently reflects the activation of endogenously expressed MASN molecules from binding to transfected MASN p1 molecules. It is possible that some of the 70 MASN isoforms can also act as "depot sites" for molecules involved in other, unrelated associated with cytotoxicity, EAB/AROT1 and TME receptor manifestations. Therefore, in a similar way, protein-C1 and (or) its isoforms can also have the same enhancing activity or serve as "depot sites" for other such molecules.

Due to the unique ability of RHAB/AROA and TME receptors to cause cell death, as well as the ability of TME receptors to mediate other types of activity associated with tissue damage, disruption of 75 functions of these receptors can be negative for the body. However, participation in the pathogenesis of some diseases was demonstrated for both excessive and insufficient functions of these receptors | Magzzaiyi, 1992;

Madaya 8: (Szoiviviip, 1995). The identification of molecules involved in the signaling function of receptors and the discovery of ways to modulate the activity of such molecules can lead to new therapeutic approaches. In view of the presumed key role of protein 51 in toxicity mediated by RAB/AROA and TME, it seems important to develop drugs that would block the possible proteolytic function of protein 51, which has previously been done for other proteins of the CEZLSE family. !., 1994; MiiPeg ei ai., 1995;

Mazpita eyi ai., 1995; Miydap eyi aiYi.,, 1995; ЕЕпаги ей айЙ.,, 1995; 105 of the Russian Federation, 1995). The unique sequence parameters of the SEOZ/LCE homologue, apparently present in 01 molecules, may make it possible to prepare drugs that would specifically inhibit their activity. Such drugs can provide protection against Ha

From excessive, immunity-related cytotoxicity with the participation of 1 without any interference with the physiological processes of cell death, in which other proteases of the CEOZLSE family are involved. and)

Other aspects of the present invention will be apparent from the following examples.

The invention will now be described in more detail in the following non-limiting examples and accompanying drawings. Gee!

It should also be noted that the following procedures: (I) dihybrid screening and dihybrid test for p-galactosidase expression; c (i) induced expression, metabolic labeling and immunoprecipitation of proteins; yuyu (ii) binding ip migo; (im) assessment of cytotoxicity; and C (M) Northern blotting and sequencing, referred to below in reference examples 1 |see. also Voiyaip ei ai., h- 199561, 2 ii Z Idiv. also Voiliap et al., 1996), relative to MOKT-1 and MOKT1-binding proteins (eg, MASN), respectively, are equally applicable (with some modifications) to the corresponding isolation, cloning, and characterization of 1 and its putative isoforms according to the present invention. These procedures, therefore, "should be taken as a complete representation of such procedures used to isolate, clone and characterize 1 according to the present invention, which is discussed in detail below in Example 1. (The following examples-references 1-3 also submitted in a form similar to or equivalent to the one typical for joint, pending Israeli patent applications MoMo114615, 114986, 115319, 116588 and 1179321, "" as well as for the corresponding |PCT application MORST/О5О96/ 105211).Moreover, some details of the experimental procedures carried out in accordance with the present invention have already been included in the above "Brief Description of the Drawings" section, and they form a part of the following example 1 in terms of a complete presentation of the present invention, respectively, and should be considered together with what is stated in example 1.

Reference example 1.

Cloning and isolation of the MOKT-1 protein that binds to the intracellular domain of the EA5-K receptor (9) (1) Dihybrid screening and dihybrid test for p-galactosidase expression 7 50 In order to isolate proteins that interact with the intracellular domain of the EA5-K receptor K, a dihybrid yeast system was used (Rieidz 5 opo, 19891). Briefly, this two-hybrid system is a (Che) yeast-based genetic test designed to detect specific interprotein interactions and mimo by reconstitution of a eukaryotic transcriptional activator, such as ZA/4, which includes two distinct domains, a DNA-binding and an activator domain: these domains, when expressed, form a reconstituted OA 4 protein and are able to bind to an upstream activator sequence that, in turn, activates a promoter that controls the expression of a reporter gene, such as Ias/ or NI53, whose expression can be readily detected in cultured cells. In this system, the genes that code for the expected interacting proteins are cloned into separate expression vectors. In the composition of one expression vector, the gene sequence of one of the expected proteins is cloned together with the sequence encoding the 60/DNA-binding domain of AG 4 in order to form a hybrid protein that has a DNA-binding domain in its composition

CA14; another vector includes a sequence that encodes a second putative protein along with a sequence that encodes the CA14 activator domain in order to obtain a hybrid protein that includes the CA14 activator domain. The two hybrid vectors are then simultaneously transformed into host cells of a yeast strain carrying a reporter gene - Ias7 or NIZZ, which is under the control of a promoter segment that has 65 CA 4 binding sites. Only those transformed host cells (co-transformants ), in which two hybrid proteins are expressed and able to interact with each other, are found to be able to express a reporter gene. In the case of using the Ias reporter gene, the host cells expressing this gene turn blue when X-daiY is added to the culture medium. Therefore, blue colonies are indicators of the fact that two cloned proteins--"candidates" are able to interact with each other.

Using a two-hybrid system, the EAZ-IIS intracellular domain was separately cloned into the rOOVT9 vector (carrying the sequence encoding the DNA-binding domain of CA 4, provided by Siopiesp, USA: see below) to obtain a fusion protein comprising the DNA - binding domain CA 4. To clone HA5B-K into the composition of rOVTO, a clone was used that encodes the full-length cDNA sequence of BAB-K |M/O 95/31544), from which the intracellular domain (1C) was excised using standard procedures using 7/0 different restriction enzymes, then isolated using standard procedures and integrated into the pOOVT9 vector opened at its multiple cloning sites (MC5) using the appropriate restriction enzymes. It should be noted that the ERA5-IS domain occupies amino acid residues 175-319 of intact EAB-K: this segment, which includes amino acids 175-319, which are the EAZ-IS domain, and were integrated into the rovt9 vector.

The hybrid (chimeric) vector described above was then co-transfected together with the elements of the cDNA library of human Nea cells, cloned into the pOAYU-OH vector carrying the SAI14 activator domain, into host cells of the yeast strain HE?7c (all the above-mentioned vectors - roVTt9 and roAb-CH, which includes parts of the cDNA library of cells

Nega, as well as the yeast strain, were purchased from Siopiesp I ar. Ips., USA. in the form of components of the MaisptakKeg dihybrid system MoR'T1265-1). Co-transfected yeasts were selected for their ability to grow in a medium without histidyl (NO medium), the grown colonies were found to be positive transformants. The selected yeast clones were then tested for their ability to express the as gene, that is, for their GAS activity, which was determined by adding X-yes! into the culture medium, which was catabolized by p-galactosidase (an enzyme encoded by the Ias/7 gene) into a blue colored product. Thus, blue colonies are an indicator of an active gene as.

In order to ensure the activity of the Ias7 gene, it is necessary that the transcription activator (ZA! 4) be present in an active form in the transformed clones, namely, that the DNA-binding domain of AG 4, which is encoded by the above-mentioned hybrid vector, precisely connects with by the activator domain of CAG4 encoded by another hybrid vector.This combination is possible only when two proteins fused to each of the OA 4 domains are able to stably interact (bind) with each other. Thus, the histidine-positive and blue (GAS") colonies that were isolated were colonies that had been co-transfected with a vector encoding the ERAZ-IS domain and a vector encoding a protein product associated with cell origin It is a human, able to stably connect with RA5-IS. Ge»)

Plasmid DNA from the above-mentioned yeast colonies from Niv, GAS" was isolated and electroporated into the cells of E. soy strain NVI101 using standard procedures, followed by the selection of Ge" and ampicillin-resistant transformants: these transformants are those carrying the ROAO hybrid vector -OH, which includes coding MU sequences and Atr" and Hec2. Such transformants, thus, are clones carrying sequences that encode newly identified proteins capable of binding to RA5B-IS. Plasmid DNA was then isolated from these

From the transformed cells of E. soya and re-tested by: c. (a) their retransformation using the original hybrid plasmid including the intracellular domain

EAZB-K (hybrid plasmid roVTt9 carrying EGAZ-IS) yeast strain NO? as described above. As a control, vectors carrying a foreign protein-coding sequence, for example, pOOVT9 or pAST-lamin, were used for co-transformation together with a plasmid encoding RA5-IS-binding protein (ie, MOKT-1).

Then, co-transformed yeast cells were tested for growth in a histidine-free environment without or with different contents of 3-aminotriazole; and from" (B) retransformation of plasmid DNA and the original hybrid plasmid from RAZ-IS and the control plasmids described in pa) in the host yeast cells of strain 5ZEM526 and analysis of GAS activity "efficiency of p-galactosidase formation, i.e., blue coloration) -1 15 The results of the tests described above showed that the parameters of the growth of colonies on the medium without histidine were identical to the parameters of GAS activity, which were evaluated by the appearance of the coloration of the colonies: that is, "Niv" colonies also showed GAS ". Next, the activity of AS in liquid culture (as more acceptable conditions for cultivation) was evaluated after transfection with hybrids encoding DNA-binding and activator domains

SAI4, host cells of yeast strain ZEU526, which are characterized by better inducibility of GAS with the participation of the transcriptional activator SAI 4 compared to this process in host cells of yeast strain HE7. c Using the procedure described above, a protein originally named NET and now designated MOKT-1 was identified, isolated, and characterized.

Moreover, it should also be noted that in some of the dihybrid tests for p-galactosidase expression described above, this expression was also evaluated in a filtration test, which is more acceptable. Screening showed (F) that five out of 3 million cDNAs contained the MOKT-1 insert. The co-isolated cloned MOKT-1 cDNA inserts were then co-sequenced using standard DNA sequencing procedures. The amino acid sequence of MOKT-1 was deciphered from the DNA sequence (for an overview of the nucleotide and amino acid sequence of MOKT-1, see joint pending Israeli patent applications

MoMo 112022, 112692 and 114615 and the corresponding application PCT Mo M/O 96/186411). The numbering of amino acid residues in proteins encoded by cDNA inserts is provided according to the Zm/izz-Rgoi database. Deletion mutants were obtained using PCR, and point mutants were obtained using the technique of oligonucleotide-directed mutagenesis (Sitepi Rgoiosoiv and Moyissiag Vioidou, 1994). c5 (ii) Induced expression, metabolic labeling and immunoprecipitation of proteins

Linked at the M-end with RI As-octopeptide MOKT-1 IRGAS-MOKTI, Eazitap Kodak, Mem Namep, ST, USAI,

EAZ-IS, RAB-K p5bB-K, a chimera that includes the extracellular domain of rB-K (amino acids 1-168), fused with the transmembrane and intracellular domains of RAZ-K (amino acids 153-319), and KkKDNA luciferase, which is a control , were expressed in Ne a cells. Expression was carried out using a tetracycline-controlled expression vector in a clone of Neia cells (NETA-1), which expresses a tetracycline-controlled transactivator | (Sozzep 8 Vciaga, 1992; see also Wojaip ei a!., 1995). Metabolic labeling using (3551-methionine and (3981-cysteine) (Oiropi, Umiitipdyup, OE, USA and Ategzpat, Viskipopatvpige,

UK) was performed 18 hours after transfection by an additional 4-hour incubation at 372 in Dulbecco's modified Eagle's medium, without methionine and cysteine, but with the addition of 295 dialyzed 70 fetal calf serum. Then the cells were lysed with KIRA buffer (10 mM Tris-HCl - pH-7.5, 150 mM Mass, 195

MP-40, 195 deoxycholate, 0.195 ZO and 1 mM EOTA), and the lysate was pre-clarified by incubation with a third-party rabbit antiserum (µm/ml) and Sepharose beads with protein-c (RNagtasia, Orrzaia, Sweden; bOm/ml). Immunoprecipitation was carried out by one-hour incubation at 42C of 0.3 ml aliquots of lysate with mouse monoclonal antibodies (μl per aliquot) to the octopeptide GG As (M2; Eavitap Kodak), to ro5-K (|Mo18 and 75. Mo20; EpdeItapp ei a !., 19901) or to GAB-K (284; Katiua Zotsiyapa OSakz, CA, USA) or radioactive mouse antibodies used as a control, followed by incubation for another hour with sepharose beads with protein-O (ZOmcl per aliquot). (iiii) Binding of ip mygo

Glutathione-Z-transferase (351) fusions with wild-type BAZB-IS or mutant ERA5-IS were obtained and adsorbed by glutathione-agarose beads |see Voidip eyi ai., 1995; Sispepi Rgoiosoiiv ip Moiessciag Vioiodu, 1994; Rhapdiopi 8: Meey, 1993). The binding of the metabolically labeled P AS-MOCTI1 fusion protein to Z5T-EA5-IS was assessed by incubating beads for 2 hours at 42C with extracts of metabolically labeled NegH a cells

with I3581|-methionine (bOmCy/ml) expressing EI AS-MORVT. These extracts were prepared in a buffer containing 5 mM Tris-HCl - pH-7.5, 150 mM Masi, 0.195 mM MP-40, mM dithiothreitol, 1 mM EOTA, 1 mM s-phenylmethylsulfonyl fluoride, 2 µg/ml aprotonin, 20 µg/ml leupeptin, 10 mM of sodium fluoride and 01 mM of sodium vanadate (1 ml per 5x109 cells). (m) Evaluation of cytotoxicity mediated by induced expression of MOKT-1

The cDNA of MOKT-1, RAZ-IS p55-IS and luciferase were integrated into a tetracycline-controlled expression vector and transfected into NITA-1 cells (a cell subline) (Bozzep 5 Vciaga, 1992)| together with the cDNA secreted by placental alkaline phosphatase placed under the control of the 5M40 promoter, the vector reVo-2; with

IOitke ei ai!., 1993). Cell death was assessed 40 hours after transfection either using the neutral red absorbance test (UmMaijasi, 1984) or, in the case of specific death assessment of cells expressing the transfected cDNA, by determining the amount of placental alkaline phosphatase. !I., 1988), which is secreted into the culture medium in the last 5 hours of incubation. 3o In another series of experiments, which should analyze the area of the MOKT-1 protein, which is involved in binding with

EAZ-IS, the following proteins were expressed in an intermediate type from the tetracycline-controlled expression vector РОНО10-3 in Nega cells, which carry the tetracycline-controlled transactivator (NITA-1): only human HA5B-K; Human EA5B-K together with the M-terminal part of MOKT-1 (amino acids 1-117 - the so-called "head of MOKT-1"); BAB-K of a person together with the C-terminal part of MOKT-1, which includes a region of homology with the C "death domain" (amino acids 130-245 - "MOKT-1 00"); РГАС-55.11 (amino acids 309-900 of the 55.11 protein, c are connected to the M-end of the RAS octopeptide, while the 55.11 protein is a protein that specifically binds to

From" work-IS). 12 hours after transfection, the cells were treated with trypsin and replated at a concentration of 30,000 cells per well. After a 24-hour incubation, the cells were treated for 6 hours with a monoclonal antibody to the extracellular domain of GAB-K (monoclonal antibody CH-11: Opsog, (Zaypegzryga, MO, USA) at various concentrations (0.001-100 μg/ml of monoclonal antibody) in the presence of 1 μg /ml cycloheximide 7 Cell viability was then determined using the neutral red absorbance test, and the results of "" were reported as the percentage of surviving cells compared to the total number of cells incubated with cycloheximide alone (ie, in the absence of CH-11 monoclonal antibody to EAZ5-K). i-th (M) Northern blotting and sequencing ka 20 Polyadenylated RNA was isolated from the total RNA of Nei a cells (reagent set Oiidoyekh-4i tKkMA Kiii.

Oiadep, Niyidep, Germany). Northern blotting using MOKT-1 cDNA as a probe was carried out using standard procedures | see Voyaip eyi a!., 1995). The nucleotide sequence of MOKT-1 was determined in both directions by dideoxy chain termination.

The sequencing data of MOKT-1 cDNA cloned in the dihybrid test showed that this cDNA encodes 22 new proteins. Further application of the dihybrid test in order to assess the binding specificity of this protein

GF) (MOKT-1 - "Meadiayug oi Keseriog-ipdised Tohisyu") with EA5-IS and the identification of a specific segment in the composition of EAZ-IS, with which it binds, led to the following results: (a) MOKT-1 protein binds to EA5-IS in both human and mouse, but not to a number of other proteins tested, including three receptors of the receptor family

TME/MOE (p55-TME and p75-TME and CO40 receptors); (5) mutations for the substitution of amino acid-225 (isoleucine) in the 60th member of the "death domain" of BABZ-K led to the disappearance of the signal of both ip omygo and ip mimo (mutation

Irg-9 Mu/agapare-Rykypada eyi ai!., 1992; Moi 5 Madaiia, 19931), which also prevents binding of MOKT-1 to EAZ-IS; (c) the site of binding to the MOKT-1 bilium in EAB-K falls on the "death domain" of this receptor; and (4)

MOKT-1 is capable of binding to itself. Such self-binding and binding of MOKT-1 to RHA5B-K involves different regions of this protein: a fragment of MOKT-1 corresponding to amino acid residues 1-117 binds to full-length MOKT-1, but does not bind to either with itself, nor with RA5B-IS. On the other hand, the fragment corresponding to amino acid residues 130-245 binds to EA5B-K, but does not bind to MOKT-1.

Moreover, it is clear from the obtained results that the "death-domain" section in the composition of REAB-K is critical for the self-binding of EAZ-IS, which is also characteristic of the "death-domain" in the composition of ro5-K for self-binding in p5b5 - Iss.

Deletions flanking these "death domains" do not impair their ability to self-associate in any way, whereas, however, deletion within the "death domains" suppresses self-association. In the case of MOKT-1, the binding of MOKT-1 to EPA5-IS also appears to be dependent on the completeness of the GAZ5-K death domain, while it is also independent of segments outside the GAZ5-K death domain. on connection with GA5-IS.

The interaction of proteins encoded by constructs carrying the DNA-binding domain and the activator domain of 7/0 ZAGA4 (roVTZ and roAO-OH) in transfected cells of yeast strain ZEU526 was evaluated in a filtration test with p-galactosidase expression. Constructs with the DNA-binding domain included 4 constructs from human BAZ5-IIS, four constructs from mouse RA5B-IS, including two full-length constructs carrying the substitution mutations "isoleucine to leucine" and "isoleucine -2 alanine" in the 225th position (respectively, 1225M and 1225A) and three designs with MOKT-1. Constructs carrying the activator domain included three MOKT-1 constructs, with 7/5 because the MOKT-1 portion was the same as the construct carrying the sequence encoding the DNA binding domain and the full-length PA5B construct -IS human, part of the EPA5-IS was the same as in the previous construct, carrying the DNA-binding domain. Intracellular domains of human p55-TME receptor (p5B5-IC: residues 206-426), human Cp40 (CO40-IC: residues 216-277) and human p7/5-TME receptor (p75-IC: residues 287-461), yes as well as lamin, cyclin-O and "empty" ZAG 4 (roVt9) vectors were taken as a negative control in the form of constructs carrying a DNA-binding domain. ZME1 and ZME4, taken in the form of a construct with a DNA-binding domain (in the case of ZMET1) and with an activator domain (ZME4) served as positive controls. "Empty" CA 4 vectors (ROAYU-OH) also served as a negative control in the form of constructs carrying the activator domain. The symbols "in" and "zh", which were used in the presentation of the obtained results of the analysis described above, mean the appearance of a clear color within 30 and 90 minutes of the test, respectively; the sign "-" means the absence of p

Coloring within 24 hours.

The expression of MOKT-1 molecules, connected at their M-end with the octopeptide BIG AS (RIG AS-MOKT-1), leads to the release of four proteins of a certain size - approximately 27, 28, 32 and 34kD in i) Her cells. The interaction of MOKT-1 with ERA5-IS of migo was detected by performing immunoprecipitation of proteins from extracts of Nea cells transfected with the RGAS-MOKT-1 fusion protein or luciferase cDNA as a control: immunoprecipitation was performed with He» using an antibody against RGAS (ag As ). The interaction of ip mygo has also been demonstrated between MOKT-1 and

EAZ-IS, despite the fact that MOBT-1 was in the form of metabolically labeled 393-methionine fusion proteins

EGAS-MOKT-1 isolated from extracts of transfected cells is not a, but HA5-IS is in the form of fusion proteins.

Human and mouse O5T-EA5-IS, including one of the variants carrying a mutation at position 225 of the EAZ-IIIS polypeptide, and all the fusion proteins З5T-РА5B-IS were obtained in E. soy cells. The 5 fusion proteins were attached to t glutathione beads before interaction with extracts containing the P AS-MOKT-1 fusion protein, and after such interaction, 5O5 electrophoresis was performed in PAAG. Thus, the interaction of ip and mygo was evaluated by the author-diographically with further ZOB-electrophoresis in PAAG, as binding of metabolically marked Z55-methionine. MOVT-1, produced in the cells of Not and in the form of a fusion with the octopeptide RGAS " (RGAO-MOKT-1) with O5T, which is included in the fusion in a row with EAZ-IS of a human or mouse (Z5T-PEAZ-IS or z5T-tEAZ- IS), or with 51 in fusion with EA5-IS, which includes the substitution of isoleucine for alanine in the 225th position of the polypeptide. It was shown that all four proteins of RIH AC-MOKT-1 showed the ability to bind to RA5-IS under the conditions of incubation with the fused protein Z5T-RAB-IS. As in the yeast dihybrid test, MOKT-1 did not bind to the fused protein "O5T-EAZ-IS, characterized by substitution at the mutant site of Irg-9 (1225A).

The protein encoded by the RGAS-MOKT-1 cDNA also demonstrated the ability to bind to the intracellular domain of the EA5-E receptor, as well as to the intracellular domain of the EAZ-B chimera, in which the extracellular domain is replaced by the p55-K receptor (rbo55 -PA5), in the case of co-expression with these receptors in their cells. and. In this case, the interaction of MOKT-1 with EAZ-IS in transfected cells No! and, that is, ip mimo, was demonstrated using immunoprecipitation of various variants of transfected cells of Her! a, as the interaction of mimo and the specificity of the interaction between MOKT-1 and RAZ-IS in cells co-transfected with constructs encoding these proteins. Thus, the RGAS-MORVT-1 fusion protein was expressed and metabolically labeled with C355-cysteine (20 μCy/ml) and 358-methionine (40 μCy/ml) in cells of Ne! and separately or together with the human EAZ-B, the EAZ-K chimera, in which the extracellular domain of the EA5B-K was replaced by the corresponding part of the human p55-K receptor (p5o5-PA5), or the human rob-K, taken as a negative control. Cross-immunoprecipitation of MOKT-1 with the co-expressed receptor was carried out using different specific antibodies. The obtained results 59 showed that RGAS-MOKT-1 is able to bind to the intracellular domain of EAZ5B-K, as well as to

GF) by the intracellular domain of the EBAB-K-p55-K chimera, which includes the extracellular domain of the p5bbB-K receptor and the 7th intracellular domain of BABZ-K, in the case of co-expression with these receptors in Her cells. Further, immunoprecipitation of RI AO-MOKT-1 from extracts of transfected cells also leads to precipitation of the co-expressed HA5B-K receptor or the co-expressed p5o5-EA5 chimera. On the other hand, immunoprecipitation of these receptors leads 60 to simultaneous precipitation of P AS-MOKT-1.

Northern blotting using MOKT-1 cDNA as a probe indicates the presence of a single hybridizing transcript in Nei cells. In a Northern blot in which polyadenylated RNA (0.µg) from transfected cells was hybridized with MOCT-1 cDNA, the size of the RNA transcript (approximately 1800 nucleotides) was found to be very close to that of MOCT-1 cDNA (approximately 1702 nucleotides). Because during sequencing, it was found that the cDNA includes an open reading frame that codes for approximately 250 amino acids. Nucleotide and amino acid sequences of MOKT-1 were shown in the joint, pending application | see MUO 96/18641|. In these sequences, the "death domain" motif is underlined, as are the probable methionine start codon (position 49: bold, underlined letter M) and translation stop code (asterisk under the codon at position 769-771). This "death-domain" motif is similar to known "death-domain" motifs in ro5-K and REA5B-K (ro55OrO and EABZ-0O). In order to determine the exact C-terminus in the composition of MOKT-1 and to obtain convincing evidence of the accuracy of the determination of the M-terminus (the original methionine residue) in

MOKT-1 additional experiments were performed as follows:

Using the methods described above, a number of constructs encoding MOKT-1 molecules were merged in their 7/0. M-end with RIAO octopeptide (RI AS-MOKT-1), were formed and expressed in Her cells! and with the use of metabolic labeling of expressed proteins using Z585-cysteine and Z55-methionine. Molecules

EGAS-MOKT-1 was encoded by the following cDNAs, which include different parts of the sequence that encodes MOKT-1: (Y) cDNA of the octopeptide RH AS, connected to the 5-end of the cDNA of MOKT-1, from which nucleotides 1-145 were deleted ; (ii) cDNA of the octopeptide RI AS, connected to the 5-end of the full-length cDNA of MOKT-1; (ii) EGAS octopeptide cDNA, connected to the 5-end of the MOKT-1 cDNA, from which nucleotides 1-145, as well as nucleotides 832-1701 were deleted, and the ZSS codon at position 142-144 was mutated into a TSS codon in order to preventing the start of broadcasting on this site.

After the expression of the fusion products P AS-MOKT-1 described above, immunoprecipitation was carried out as described above, using either monoclonal antibodies to RI AS or, as a control, antibodies to rtT5-TME-K, followed by ZUOS-electrophoresis in 1090th PAAG and autoradiography. The results of the analysis of the RIG AS-MY T-1 fusion products described above confirmed the position of the C-terminus of MOKT-1 and provided evidence that

The M-end of the MOKT-1 polypeptide can be located at position 49 of this sequence.

In addition, through additional experiments on the expression of the MOKT-1 protein without the addition of the octopeptide BIG As by

Finally, it was shown that the remainder of May" serves as an effective site of initiation of transmission. p

A search in the databases of SepeVapkK and Rgoieipy Vapk showed that, until now, they do not have any sequences that would correspond to the MOKT-1 sequence described above. Thus, MOKT-1 is a new protein specific for binding to HA5-IS.

Intense expression of ro5-IS leads to the mediation of a harmful effect on cells (Voidip et al., 1995).

EAZ-1IS expression in Her cells! and also has such an effect, although expressed to a lesser extent, which can be due to 0) it is detected only when using a sensitive test. Thus, ligand-independent mediation of the deleterious effect upon transfection of MOKT-1 cells, as well as p55-IS and RA5-IS, was analyzed. The effect of intermittent cm expression of MOKT-1, human RA5-IS, human ro55-IS or luciferase taken as a control on the viability of IS o) Neia cells was evaluated using a tetracycline-controlled expression vector.

Cell viability was assessed 40 minutes after transfection with these cDNAs either in the presence or absence of tetracycline (1 µg/ml, to block expression), together with a cDNA encoding secreted placental alkaline phosphatase. Cell viability was determined either using the neutral red absorbance test, or, when specifically determining the viability of those specific cells expressing the transfected DNA, by measuring the amount of placental alkaline phosphatase secreted into the culture medium.

The analysis described above showed that the expression of MOKT-1 in Ne cells results in significant cell death, more significant than that caused by EAZ-IS expression. These cytotoxic manifestations of all a factors - p55-IS, EAZ-IS and MOKT-1 - are apparently associated with segments of "death domains" present in the composition of all these proteins, while "death -domains" have the property of self-binding and thus probably cause the manifestation of cytotoxic effects.

From the point of view of the characteristics of MOKT-1 described above, namely the specific binding of MOKT-1 to a specific segment of RAZ-K, which is involved in the induction of cell death, and the fact that even a weak change in the structure of this segment, which stops signal (Irg 99 mutation) and also averts binding to MOKT-1, determines that this protein plays a role in signaling or directly mediating cell death. This reasoning is also supported by the revealed ability of MOKT-1 itself to mediate a deleterious effect on 50 cells. Thus, the MOKT-1 protein may function as (1) a modulator of EA5-K self-binding through its own ability to bind to RHA5B-K in the same way that it can bind to itself, or (2) serve

Me) a "depot site" for additional proteins involved in the signaling mechanism involving RA5-K, i.e. MOKT-1 can be a "depot protein" and thus can bind receptors other than EAZ-K, or (3) to form part of a separate signaling system that interacts with the EA5-K signaling system. 22 In order to further analyze areas of MOKT-1 involved in binding to RA5-IS and modulation of cellular

Gee! manifestations mediated by RA5B-K (cytotoxicity), the experiments described above were carried out using vectors that encode parts of MOKT-1 ("head of MOKT-1" - amino acids 1-117, and "ZhMOKT-1 Jur" - where amino acids 130 -245) (separately), and a vector encoding human EAB-K intended for co-transfection of cells

Nega In these experiments, various proteins and combinations of proteins were expressed in an intermediate manner in Nega cells, 60 which included a tetracycline-controlled transactivator (NETα line) by inserting the protein-coding sequences into a tetracycline-controlled expression vector. rYinoO1o-3.

Control transfection was carried out using vectors encoding only GAZ-K and vectors encoding the RI As-55.11 fusion protein (protein 55.11 is specific for binding to the ro55-IS protein, the part of which including amino acids 309-900 was fused at its M-end with the RI Ac octopeptide). 65 After the stages of transfection and incubation, the transfected cells were treated with different concentrations of the monoclonal antibody CH-11 to RHA5B-K, which specifically binds to the extracellular domain of the receptor

EAB-K expressed by these cells. Such binding of the antibody to RAB-K induces the aggregation of BA5B-K on the cell surface (with a higher probability than with the ligand RA5B-K) and induces the activation of an intracellular signaling mechanism mediated by EAZ-IS, which ultimately leads to death. cells (i.e., EA5-K-mediated cellular cytotoxicity). The applied concentrations of the CH-11 monoclonal antibody to EGAB-K ranged from 0.01 to 1 Ωkg/ml: as a rule, the concentrations were as follows - 0.005, 0.05, 0.5 and

Bbmkg/ml. Cells were treated with an antibody to EA5-K in the presence of 1 µg/ml cycloheximide.

The results of the analysis described above show that the expression of the RAB-K receptor in transfected cells leads to increased sensitivity to the cytotoxic manifestation of antibodies to RAB-K. Next, co-expression of segment 7/0. MOKT-1, which includes a region of homology with the "death domain", and REA5-K enter into strong contradictions with the induced

EABZ (i.e. EAB-K mediated) cell death, which can be expected based on the ability of the "death domain" (00) of MOKT-1 to bind to the "death domain" (GAB-OU) of GAZ-K. Moreover, co-expression

The M-terminal part of MOZHKT-1 and RGAB-K does not interfere with BAB-K-mediated cell death and, if it does occur, to some extent enhances cytotoxicity (that is, a weak enhancement of cell /5 Death is observed).

Thus, the above results clearly indicate that the MOKT-1 protein has two different segments that function to bind to GAZ-1IS and mediate the cytotoxic activity of EAZ-IS.

These results thus also provide a basis for the use of different parts (ie, active fragments or analogs) of MOKT-1 bileu for different pharmaceutical applications. For example, analogues or fragments or derivatives of the MOKT-1 protein comprising essentially only the C-terminal part of MOKT-1 containing its "death domain" can be used to inhibit EA5-K-mediated cytotoxicity in cells or tissues. , carrying the РГА5Б-К receptor, and thereby protecting these cells or tissues from the action of the damaging EA5Б-К ligand in cases such as acute hepatitis. On the other hand, analogs or fragments or derivatives of the MOKT-1 protein, which essentially include only the M-terminal part of MOKT-1, can be used to enhance the ГА5-K receptor-mediated cytotoxic effect in cells or tissues that carry EA5- K, which thus leads to increased destruction of such cells or tissues when it is desired, for example, in such i) cases as tumor cells and autoreactive T- and B-cells. As was described in detail above, such application of different segments of MOKT-1 can be carried out using different recombinant viruses (for example, Massipia) in order to introduce sequences that encode segments of MOKT-1 into specific cells or He! from fabric that it is desirable to subject them to such processing.

In addition, it is also possible to obtain and use various molecules, such as antibodies, peptides and organic compounds, which include sequences or molecular structures that correspond to the above-described MOKT-1 segments in order to achieve the same desired effect mediated by these segments.

MOV-1. in

Moreover, the MOZKT-1 protein can be used for the specific identification, isolation and characterization of other proteins that are able to bind to MOKT-1 (ie, MOKT1-binding proteins): see reference examples 2 and 3.

Reference example 2.

Isolation of MOKT1-binding protein " (I) Dihybrid screening and dihybrid test with p-galactosidase expression in c Analogous to the procedures described in example-reference 1, using the intracellular domain and receptor roB5-TME-K (p55-IS) and MOKT-1 as a "bait" and screening the B-lymphocyte library and? human, two cDNA clones encoding a protein product capable of binding to both MOKT-1 and Rob-IS were isolated. For both clones, the presence of identical nucleotide sequences from the 5-end was shown (see common ones at the application stage (МО 96/18641 and PCT/О596/105211). - and (i) Binding parameters of new cloned cDNAs during dihybrid screening

Using the yeast two-hybrid assay described above, a construct that includes the cDNA of a novel MOKT1-binding protein was used as a "bait" to which several "bait" constructs were added in different reactions to determine MOKT1 binding specificity - binding protein encoded by this

KDNK. These "baits" included constructs that encode the MOCTI protein, parts of the MOCT-1 protein ("head of MOCT-1" - where amino acids 1-117; "tail of MOCT-1" - amino acids 130-245), p55-IS (amino acids 206 -426 p55-K) or his

Ge) part of amino acids 326-426 - the "death domain" in p55-K; or other domains located above the "death domain" - amino acids 206-326). The obtained results are presented below in Table 2. vagovzzhr vagovyh 0 spell: where vvvizimo

EAV-IS -

zagaves 001 vmoe 010 tuo 01003 spish nin chno 1 demno 111 70 The above data of a dihybrid test with p-galactosidase expression from binding of a clone with a larger set of "bait" confirmed that the protein encoded by this clone specifically binds to "death-domains" of both the receptor po5-TME-K and the MOKT-1 protein.

In general, a MOKT1-binding protein can be used directly to modulate or mediate a MOKT-1-related effect on cells, or indirectly to modulate or mediate a 7/5 HA5-K receptor ligand effect on cells, when such an effect is modulated or mediated by MOKT-1 protein. The same holds true for other intracellular proteins or intracellular domains of transmembrane proteins, as specifically demonstrated here for the p55-TME-K receptor.

MOKT1-binding proteins include those proteins that specifically bind to the complete MOKT-1 protein or those proteins that bind to different segments of the MOKT-1 protein, such as the M- and C-terminal segments described above protein M.OKT-1. MOKT1-binding proteins that specifically bind to such segments can be used to modulate the activity of these segments and, therefore, the specific activity of MOKT-1 determined by these segments.

Reference example 3.

Isolation and characterization of MASN protein, another MOKT1-binding protein c (Y) Dihybrid screening, dihybrid test with p-galactosidase expression, sequencing and sequence analysis

Using the procedure described above in Examples References 1 and 2, the full-length construct encoding the human MOKT-1 protein was used as a "bait" in a yeast two-hybrid system to isolate a cDNA clone encoding an additional novel MOKT1-zv' protein This new protein was first designated MOKT-2, and then renamed and designated as MASN (abbreviation "MOKT1-associaiei SEYUOZ He") potood" -- a homologue of SEYUOZ that binds to MOKT-1"), based on its characteristics, which are discussed in detail below. p

This cDNA clone was sequenced using the standard procedures described above in Reference Examples 1 and 2. Analysis of the sequencing data using standard techniques and computer simulations (see Reference Examples 1 and 2) indicated that this cDNA includes a new sequence and encodes a new C protein (neither DNA nor amino acid sequences are found in the SepVapk or PgoieipvVapk databases). Next, cha

The cDNA encoding MASN includes an OKE-B open reading frame that shows a significant degree of homology to the segment preceding (i.e., flanking on the 5-side) the "death domain" of the MOKT-1 protein (see example reference 1 ).In joint pending Israeli patent applications MoMo114615, 114986, 115319, 116588 and 117932, as well as (in the PCT application MoPCT/LIBO6/10521)| corresponding to them, the structure of that part of the clone is shown cDNA MASN, which includes OKE-B (encodes 235 amino acid residues), and the deciphered amino acid sequence of MASN OKRE-B and the nucleotide sequence of the cDNA molecule MACN. y The region corresponding to OKE-B shows a high level of homology with the MOKT-1 segment, located above and" "death-domain" motif MOKT-1.

In addition, a yeast dihybrid test was performed to assess the specificity of MASN binding to MOKT-1, in particular, to determine the segment in MOKT-1 to which MASN binds, as well as to determine which of the open frames MASN interacts with MOCT-1: the relevant procedures were described above in Reference Examples 1 and 2. Briefly, various constructs of MOCT-1 and MASN were prepared to test the t-interaction of proteins encoded by constructs that include sequences that encode DNA- binding and sl activator domains of the transcriptional activator ZAI4, within the transfected cells of the yeast strain

ZEU52 6 with an evaluation in the filtration test for p-galactosidase expression. The constructs with the DNA-binding o domain were obtained on the basis of the pROVTO9 vectors, and the constructs with the activator domain were obtained on the basis of the pROAYU-OM vectors. Full-length cDNA was used for the construct with the activator domain

MASN, which became a construction that encodes only the segment corresponding to OKE-B (MASN-B). Control constructs with an activator domain were those constructs that included the full-length coding sequence of MOKT-1 (MOKT-1 - positive control), as well as those that did not have inserts, that is, were "empty" vectors (pRSAO-OM). For the constructs with the DNA-binding domain, full-length cDNA (MOCT-1) was used, i.e., they were constructs encoding only the upper segment of MOCT-1 (ie, MOCT-1 BO: amino acids co 130-245). Control constructs with a DNA-binding domain that were generated to determine the specificity of MASN binding included constructs encoding lamin, amino acid residues 287-461 60 of the intracellular domain of the human p75-TME-K receptor (human p75-IS ), cyclic-O (susb), ZME1, amino acid residues 206-42 6 of the intracellular domain of the human p55-TME-K receptor (human p55-IS), the "death-domain" segment in the human EAB-K intracellular domain (RGAB-OYU human), amino acid residues 216-277 of the intracellular domain of human CO40 (human CO40 IS), vectors without an insert or "empty" vectors rOvT9 (rOVTO - negative control) and a construct encoding the OKE-B segment of MASN 65 ("MASN-B) . In this test, the appearance of the color was determined: the more intense the color, the stronger the interaction between the structures encoding the DNA-binding domain and the activator domain.

Manifestation of color was indicated by symbols, among which "no" indicates the appearance of a clear color after 30 and 90 minutes of the test, respectively, and the sign "---" indicates the absence of color during 24 hours of the test.

In cases where the interaction was not tested, the symbols were not inserted. The obtained results of the evaluation of the various interactions of the tests described above are shown below in Table 3, and the results of the various interactions of the MASN isoforms are shown in the above-mentioned joint pending application |PCT/I596/10521) and the corresponding applications filed in Israel. and dnkvyakhuvaliydomen | | 11 11101011 MAsNIMASnvIMovty RoAosN pu NN PO MON NNYA NANNYA my 018 nn

The communication statement is 0001 and movtet 00000011

Movtiboiatute 5030

Test of persistence 301 lm 0111-11 vtsludny 0011

Fe

ЕХо2 НЕ ПИННИ НОЯ NANNYA PA

BI of a person 11111

VAV Orlyudim 1-11 sradielyudym 01-11 sch

Rovya 11-11 o'clock Masnya 11111

Thus, as follows from the results of Table 3, it is clear that: (a) MASN binds to MOKT-1 very tightly and in a specific manner; Gee! (B) The MASN-binding site in MOKT-1 is located before (i.e. higher in sequence) the "death-domain" motif of MOKT-1, i.e. it falls into the MOKT-1 segment corresponding to amino acids c 1-117 in the composition of MOKT -1; (c) OKE-B segment in MASN is the site of interaction with MOKT-1 of the MASN protein; (4) the OKE-B segment in MASN is capable of self-binding. C (ii) Cytotoxic action mediated by the ability of the MASN protein to self-associate

The finding that the MASN protein is capable of self-binding, in particular, that the segment that corresponds to OKE-B self-binds, and given that the previously revealed correlations between self-binding and cytotoxicity, established for the intracellular domains of the p5-TME-K receptors and EA5-K, as well as the one that was discovered for

MOKT-1 (see example-reference 1) - all this confirms that self-binding in MASN can also be involved "in the manifestation of cytotoxicity." - c In order to test this possibility, constructs encoding MASN were obtained on the basis of the tetracycline-controlled vector (for details, see example-reference 1) M. These constructs "" were used for transfection of Neg/a cells, in which these vectors were expressed by of the intermediate type

МАСН-carrying constructs, other control constructs were used to assess the effect of intermittent expression on the viability of Na cells, on which the effect of МАСН-carrying constructs can be determined for the purpose of -I comparison. These other constructs include MOZHKT-1, human RAB-1C, and His (luciferase). Additionally, co-transfection of Nei cells was also tested using constructs carrying MOKT-1 and MASN, in order to determine that the effect may be due to an interaction between these proteins. After transfection, Sl Neg cells were incubated and cell viability was assessed 4-8 hours after transfection either in the presence or

Absence of tetracycline (μg/ml), designed to block expression. Cell viability was determined using the neutral red absorption test.

Ge) Based on the results of the analysis described above, it became clear that MASN protein induces a very powerful cytotoxic effect against Neg/a cells, i.e. induced overexpression of MASN cDNA in Nei cells! and as a result causes a powerful cytotoxic manifestation. Apparently, this manifestation of cytotoxicity is due to the ability of MASN to self-associate. (ii) Northern blotting iF) Using well-known procedures (see example-ref. 1), the analysis of a number of cell lines by the Northern blotting method was carried out using the cDNA of MASN as a probe. The results of this analysis show that a large number of cell lines, in particular, the cell lines of SEM, Kaiyi, BOatsai, Nega, AIhepaeg, Odigtkai and Ab7Z, because they detect two hybridizing transcripts approximately 3200 nucleotides long.

In light of the above, the MASN protein, particularly MASNrI (ie, OKE-B as part of MASN) can be used directly to modulate or mediate MOKT-1-related effects on cells, or indirectly to modulate or mediate the effects of a HA5 receptor ligand K on cells when this effect is modulated or mediated by the MOKT-1 bilem. The fact that MASN specifically binds to the upstream 65 segment of MOCT-1 and shows homology to MOCT-1 defines a specific pathway by which MASN or MASN

OKE-B can be used to modulate this specific segment in MOKT-1 and, therefore,

specific activity of MOKT-1 determined by this upper segment. Further, MASN or MASN OKE-B can be used as a modulator or mediator of intracellular manifestations, similar to the pathway that is characteristic of MOKT-1 itself (see above), based on the ability of the MASN protein to self-associate and induce

It is the manifestation of cytotoxicity.

Further analysis of the MASN protein and the DNA sequences encoding it was carried out as described below.

In addition, it was shown that OKE-B from MASN is not only one of the series of MASN isoforms. Therefore, the MASN protein and the DNA sequences that encode it have now been renamed, the reasons for which will become clear from what follows. 70 (a) Dihybrid screening of proteins that bind to MOCT-1 identifies a novel protein with a sequence motif similar to MOCT-1:

As mentioned above, to identify the proteins involved in the induction of cell death by MOKT-1, the dihybrid test was used to screen cDNA libraries for proteins that bind to

MOKT-1. Dihybrid screening of the human B-lymphocyte library (Oipei, AI., 1993)| using kKDdNK

MOKT-1 as a "bait" led to the isolation of cDNA clones corresponding to the MOKT-1 protein itself, which reflects the self-binding ability of this protein, as well as clones corresponding to the TKAOO protein, with which MOKT-1 effectively binds (see example-reference 2). The screening also led to the selection of cDNA clones with a new sequence that encodes a product that specifically binds to white MOKT-1. This protein, originally designated MASN and then, after the discovery that it occurs in multiple isoforms (see below), renamed MASNVI1, also demonstrated the ability to bind to itself in a dihybrid assay, but was unable to bind to the EA5-K receptor (see the above-mentioned co-pending application PCT/О596/10521, which also includes all further analyzes and the results obtained therein).

MOKT-1 and MASNVI and their deletion constructs, as well as the MASNAT isoform (MASNAT is a mutant variant in which the catalytic cysteine-360 is replaced by serine - MASNAT1 (C3605)), and the intracellular Ha domain of human EAB-K (HA5-IS) were expressed in transfected yeast cells of the ZEU526 strain as part of constructs that include sequences that encode the DNA-binding and activator domains of i) transactivator of transcription AG 4 (respectively, pOoVTtTe9 and rdAO-OH). their interaction was evaluated in a filtration test with B-galactosidase expression as described by Boldin et al. (Voidip eyi ai., 19950). The results are presented in the form of periods of time necessary for the formation of a permanent color. None of the inserts tested. FU did not interact with a series of negative control variants, including vectors carrying the intracellular domains of human p5-TME-K receptor, p75-TME and CO40 receptors, as well as lamin, cyclin-O, and an "empty" vector with

SAI4. MASNVI was cloned using a digihybrid screening of a CA 4-AU-labeled library

of human B-lymphocytes (Oipei et al., 1993) in order to detect proteins that bind to MOKT-1, using the reporter yeast strain HE7c. With the exception of specially indicated cases, all experimental procedures for such a search have been described above (see also Voidip et al., 1995). Deletion analysis showed that MASNVI1 binds to the M-terminal part of MOKT-1, which is involved in the induction of cell death (Spippaiuap ei ai., 1995). MASNVI is also capable of self-binding in transfected yeast cells. However, it does not bind to some control proteins and, unlike the MOKT-1 protein, it is unable to bind to the EAZ-K receptor. "

The expression of MASNVI molecules in mammalian cells leads to the production of a protein with a molecular weight of З4kD, which binds to MOKT-1 molecules, which are simultaneously expressed with it. It was also able to communicate with - with the fusion protein 55T-MOKT-1 ip miigo. A comparison of the amino acid sequences of MASNVI and MOKT-1 showed that these two proteins have a "" sequence motif common to these two proteins (designated as the "MOKT-module"), which differs from the "death-domain" motif, through which the MOKT-1 protein binds to by the RAZ-K receptor. This motif is present in a single copy in MOKT-1 and in two copies in the MASNVI sequence. The same motif is also found in the REA-15 - -I phosphoprotein of astrocytes, the functions of which are still unknown. Preliminary data confirm that the "MOKT-module" motif is involved in the binding of MASNVII (and other isoforms of MASN proteins) to the protein MOKT-1. e The deciphered amino acid sequence of MASNVI is submitted in the above-mentioned application PCT/O596/10521 and (9! corresponding to it) Israeli patent applications , in particular, application II 117932. Two "SHOCT-modules" are shown, as well as the C-termini of the two deletion mutants of MASNVI used are determined. The sequence homology of the modules comprising MASNV1, MOKT-1 and the REA-15 gene (depository MoX86809) was also submitted in of the above-mentioned common, (Che) pending applications, in which identical and similar amino acid residues were marked by space in closed lines and shaded, respectively.

Also, in the above-mentioned joint applications, "death-domain" and "MOKT-modules" and sections of EAB/AROT, MASNVI and MASNAT, which show homology with the proteases of SEOZLSE, are presented in the form of diagrams.

For the MOKT-1 segment, which includes such a "MOKT-module", it was shown to be involved in the induction of cell death i) with the participation of this protein (see the above example-reference 1). It was also shown to be involved in self-binding of the MOKT-1 protein, although it is not sufficient for this (see example-reference 1). Analysis of binding parameters in deletion constructs of MASNVI in transfected yeast cells showed similar participation of 60 "MOKT-modules" in self-binding of MASNVI, as well as in its binding to MOKT-1 protein: deletion constructs in which the segment located after (i.e. downstream of) the "MOKT module" was missing, were unable to bind to each other, but retained the ability to bind to the full-length protein

MOKT-1 and with full-size MASNVI. Further shortening, in which a part of the "MOKT-module" sequence was also delegated, resulted in the loss of the ability to bind to these proteins. To 65 further evaluate the involvement of "MOKT modules" in these interactions, deletion mutant MASNUI, fused with an octopeptide

EGAS (RGAS-MOKT-1) were expressed in Her cells! and with further evaluation of their binding of miigo to a fusion protein produced by bacteria, which consists of glutathione-Z-transferase and the protein MOKT-1 (55T-MOKT-1).

In a similar manner to the binding found in the yeast dihybrid assay, it was found that such IP binding appeared to be dependent on segment interactions within the MASNVI1 modules. Metabolically marked using 355 MASNVI, MASNVI fused at its M-end with the octopeptide RIH As (RI AO-MASNVI), mutant for shortening the C-terminus of RGAS-MASNVI and luciferase as a control were obtained in transfected cells of Ne! and. Expression was carried out using a tetracycline-controlled expression vector in the NETA-1 cell clone of NegH a cells, which expresses a tetracycline-controlled transactivator. 70 Evaluation of protein expression and determination of their molecular weights were carried out by means of immunoprecipitation of cell lysates using antibodies to RGAS. The antibodies used were as follows: rabbit antiserum to MASNVI and to MOKTI was formed against the fusion proteins ZZT-MASNVI and z5T-MOKT-1.

Mouse monoclonal antibodies to the octopeptide RIH As (M2) and to EABZ/AROT1 (SNI11) (Mopepaga ei a/!., 1989) were purchased from the companies Eazitap Kodak and Opsog | Sainpegzrygo, MO, USA), respectively. Mouse monoclonal antibodies to epitope 75 NA MP12SAB: Rivia et al., 1988) and antibodies to TME were obtained in the applicant's laboratory according to standard techniques well known in the art. Results showing the affinity binding of proteins to o5T-MOCT-1 adsorbed on glutathione agarose beads (or, as a control, to 51 or fusions of O51 and the RA5/-AROT intracellular domain) and immunoprecipitation data of various fusion constructs involving MOCT- 1

Its MASN using different specific antibodies are submitted in the above-mentioned joint pending applications, in particular, in applications PCT/О5З96/10521 and I 117932. (B) Existence of MASN in the form of multiple isoforms:

Application of the Northern blotting method using cDNA MASNVII as a probe showed a small amount of transcript(s) approximately 3000 nucleotides long in a number of different cell lines.

Briefly, the method of Northern blotting of the total pool of RNA (14 μg per lane) or polyadenylated RNA (2 μg), with

Isolated from some cell lines were used using cDNA MASNVII as a probe. All the tested cell lines - TA7O, SEM, Kaii, Oatsai, Nei a, AIhepaeg, digkai and Ab7Z - originate from human tissues and were formed, respectively, on the material of breast carcinoma, acute lymphoblastoid T-cell leukemia, Burkitt's lymphoma, epithelioid carcinoma, human hepatoma, acute T-cell leukemia and rhabdomyosarcoma. The sufficiently blurred shape of the hybridization bands on Northern blots indicates that these "F) transcripts are characterized by size heterogeneity ranging from 2.85 to 3.5 thousand pairs of nucleotides. Signs of both the number and size of transcripts vary when comparing different human tissues and, moreover, do not correlate with the levels of expression of MOKT-1 or RAB/APO1 proteins (Muaiapare eyi a, 1992). In testes and skeletal muscles, for example, MASN transcripts were weakly detected, even though these tissues express significant amounts of MOKT-1 protein. On the other hand, in resting peripheral blood mononuclear leukocytes, in which the expression level of M MOKT-1 is extremely low, a very high level of MASN expression was detected. The activation of leukocytes by cha lectin results in a significant change in the dimensional parameters of MASN transcripts together with the induction

ILO-1.

Given the nature of such size heterogeneity, cDNA libraries were screened for transcripts that would hybridize with the MASNVI1 cDNA probe. FAT! and FAT? were cloned from a 70 Spagop-B5 cDNA library derived from mRNA isolated from the human thymus. The library was screened under stringent conditions with an 8s cDNA probe. MASNVI spotted using the Capdot-Rgitip Kii random priming reagent kit (Voepgipdeg Mappeit). Other MASN isoforms were cloned using PCR with reversion, which was performed on the basis of total RNA isolated from cells of the Ka line (MASNA, A2, AZ, BZ and B4) and cells of the Oatsai line (MASNA?2, 82, BZ, B4 and B5), which are human lymphoblastoid cells.Reverse transcriptase reaction

It was carried out using oligo-di adapter primer - 5-SASTOOASTSTASASTSOACIT) 47-3 - and -I reverse transcriptase of Ziregzosgiri-! (Sbibso-VKI) based on the recommendations set out in the manufacturer's instructions. The first round of PCR was carried out using the reagents of Ekhrap Gopd Tetriaie RSK Zuviet e (Voepgipdeg Mapppeit) and the following sense and antisense primers: 5-ААСТОАССАСАСААААТТОАО-5 ос (corresponds to nucleotides 530-551 in the composition of the MASNVI cDNA) and 5-САСТООАСТСТАСАССАТСОАС-3, respectively .

The second round was carried out using Mapi-polymerase (MEV) and the following sense and antisense primers: - o 5-SVAOSATSSSSSAAADATOSAAAASTOSATOATOAS-3 and 5-VSSASSASSTAAAADASATTSTSAA-3, - derivatives of (Che) MASNVI cDNA sequence, respectively. To confirm the presence of translation-initiating codons in MASNVZ and MASNVA, longer 5' regions of these isoforms were cloned from RNA pools of cells

Kaya. A reverse PCR reaction using the adapter oligo-dT primer described above was completed with two rounds of PCR using Mapi polymerase (MEV) and the following sense and non-sense primers: 5-TTHIOSATSSASATOSASTTSSASSAAAAATSTT-3 and U-ATTISTSAAAASSSTOSATSSAASTO-3 , - derivatives and F) from the MASNVI sequence. The last of the oligonucleotides is specific for B isoforms. Among the clones obtained in this way, those that include nucleotides that encode the amino acids of the "second block" (the presence or absence of which MASNV3 and MASNV differ from MASNVI and MASNV2) were completely sequenced. Nucleotide sequences of all cloned isoforms were determined in both directions using the Sanger method. Only partial cDNA clones were obtained that correspond to MASNAZ and

MASNVZ In general, the performed screening confirmed the existence of multiple isoforms of the MASN protein. The amino acid sequences of seven of these isoforms have been studied in detail. The results of this analysis in the form of diagrams and illustrative examples are given in the aforementioned joint pending applications 65 PCT/ОБО96/10521 and I 117932, where the amino acid sequences of these three isoforms are compared with known homologues.

The loss of 65 nucleotides, which encode the "second block" in MASNAT, causes a change in the nucleotides that encode the "third block" in MASNVI and MASNV2. Thus, in these isoforms, these nucleotides encode other amino acids that together make up their unique C-terminal region. On the other hand, in the composition of open frames

MASNVZ and MASNVA "third block" is preserved, but at the same time, the absence of nucleotides encoding the segment of homology with SEHUOZ/LSE, and part of the 3Z'i non-coding segment, as a result, lead to a change in the composition of the open frame of nucleotides located below. As a result of this change, the larger 5'-part of this non-coding downstream segment encodes 10 amino acids, which make the C-terminal part of these two isoforms unique. 70 isoforms were cloned from the human B-lymphocyte cDNA library (MASNVI), from the human thymus cDNA library (MASNAT and A2) from the mRNA of lymphoblastoid cells Kai and (MASNAT, A2, AZ, VZ, B4 and VB5) and cell lines

Batsai of a person (MASNAZ2, 82, B3, B4 and B5). Cloning of mRNA from Kaia and Satsai cells was carried out using

PCR with reversion using oligonucleotides corresponding to the C-noncoding segment and the sequence within the second "MOKT-module" as part of MASNVI. Thus, the start codon in clones isolated in this way is localized within the second "MOKT module".

The sequences of the different isoforms are related to each other as follows: (a) all MASN isoforms are characterized by a common 182-amino-acid M-terminal segment, which includes "MOKT-modules", while being located closer to the C-terminus (i.e., from the Z'end below") to these modules, as well as non-coding segments vary; (B) according to the parameters of their C-end sequences, these isoforms are divided into two subgroups - four isoforms, which are defined as subgroup B, have different C-ends due to a change in the coding frame; two isoforms (MASNVI and

MASNVZ2) share a common C-terminus found in the isoform initially cloned during dihybrid screening, and two more isoforms (MASNVZ and MASNVA) share a different C-terminus; the three isoforms identified as subgroup A have a longer C-terminal segment that largely resembles the parameters of the CEOSYLSE family of proteases (see below); (c) segments located between the "MOKT-module" site and the C-terminal segment, by which subgroups are determined, varies from one isoform to another. However, similar testing revealed that these intermediate regions include different combinations of the same three amino acid blocks (blocks 1, 2, and 3). and)

Amino acid sequence variability among different clones reflects two types of nucleotide sequence variability that most likely occur as a result of alternative splicing: (a) insertion or absence of either of two nucleotide sequences—one 45 nucleotides long and the other He! from 65 nucleotides, - or both of them, located below the nucleotides that encode the residue of lysine-184; (B) the presence of an additional insert within the site, which in MASNVI1 represents a 3'-non-coding segment. This variability affects both the composition of the open frame and the length of the encoded polypeptide. yu

Part of the MASN isoforms includes a region homologous to SEYUOZ/LSE. The analysis of the data banks showed that the C-terminal segment of the isoforms of the MASNA subgroup, including the "third block" and the sequence that goes down from it, to a large extent "resembles the proteases of the SEYUOZ/LSE family." A comparison was made of the sequences of this segment in the composition of MASN and various known members of this family, which represent the human body, as well as the seiZz protein of the nematode Saepogparaiiv eiedapv |EPYv8 5 Nogmiyi7, 1986; Mitsap et al.,, 1993), as well as well-known human proteases from the protease family of SEZLSE: SRRZ2 (Gretapadez-AIpetgi et al., 1994), which is also called apopain (Mispoyivop et al., 1995 and Mata Temagi et al. .,, 19955), Mep2A (Regpapdez-AIpetgi ei ai., 1995), « 40. 1sp-1 (Mapo ei ai., 19941), a human protein homologous to the mouse Med?2 protein (Kitag ei ai!., 19941 ), ISE 611 (Mypdau ei shu) s ai!., 1995), which is also called TX and Isn2 |Raispei ei a|Yi., 1995; Katepz ei ai., 19951), ii ISE | Progpregtu ei ai!., 1992; Seitetsi ei ai., 19921). ;" The above-mentioned C-terminal segment of MASN is most similar to SRRZ2 (with 4195 identities and 6290 homologies) and SEOZ (with 34905 identities and 5695 homologies). A significantly lower similarity with ISE (with 2890 identities and 50905 homology) was shown with its most closely related homologues ISE,e(11 (also called -I TX and sn) and ISE e(11. Similarity was found over almost the entire segment starting from tyrosine-226 within the "third block" all the way to the C-terminus of the MASNA isoform.ve Two specific features of this similarity attract attention: c (a) All known proteases of the SEOZ/LSE family cleave proteins at sites determined by their presence 5o Avr residue in the PI position and the presence of a small hydrophobic amino acid residue in the P position". However, their specificity differs due to other structural features of the substrate, including

Ge) the nature of the residues located in the P2-P4 positions. Accordingly, active site residues involved in catalysis (corresponding to histidine-237, glycine-238 and cysteine-285 residues in ISE) and residues forming the "binding pocket for the carboxylate side chain of Ri-Agr (arginine-179 , glutamine-283, arginine-341, and, apparently, serine-347), are conserved in these proteases. These residues are also conserved in MASNAT. There is a single exception, although it is related to the conservative substitution of serine for threonine in the site , which corresponds to serine-nu-347 in the (F) ISE fold. Another small but potentially important sequence difference between MASNA isoforms and other members of the protease family is the replacement of an arginine with a glutamine in the site corresponding to arginine-286 in the ISE fold . This residue, which is adjacent to the potential catalytic cysteine residue, because it is characterized by complete invariance in all other members of the SEOZ/LSE family. Also, part of the residues in the sites located near substrate residues P2-P4 differ in isoforms MASNA from those found in other members of the SEOZ/CLE family. (B) SEYUOZLSE family proteases include auto-cleavage sites. For some proteases, the passage of the self-processing stage is indeed known, and for others - the dependence of this processing on the manifestation of maximum catalytic activity. their fully biologically active form consists of two non-covalently bound cleavage products that differ in size (p2O0 and p17 in ISE; p17 and p12 in

SRRZ2). The presence of potential autocleavage sites in other members of this family confirms that they are subjects for similar processing, and in a similar way there is a dependence of the manifestation of maximum catalytic activity on such processing. Such potential autocleavage sites are present in MASNAT in almost the same positions as in SRRZ2. The site corresponding to the M-terminus of the p1!7 subunit of CPPZ2 is located in the second conserved block of amino acids, only a few amino acid residues upstream of the M-terminus of the region of homology with CEOZL/LCE (downstream of aspartic acid residue-216). The site corresponding to the cleavage site between the two subunits of CPP2 is located, as in all other members of the CEOZ/CE family, in which cleavage is known, several amino acid residues downstream of the catalytic cysteine residue (below aspartic acid 7/0 Acid-374). This conservatism confirms that the SEOZLSE homology region in MASNAT1 is subject to proteolytic processing. The size of the two putative products of such cleavage is very close to that of the two subunits of the processed CPPZ2 molecule. (c) The presence of proteolytic activity in regions of homology with SEOZ/LSE in the composition of MASN

In order to find out whether the segment of homology with SEYUOZ/LSE in the composition of MASNA exhibits proteolytic activity, /5 applicants conducted experiments on the expression of the segment that goes from the site of potential cleavage up to this site, between residues Azvr-216 and bZeg-217 , up to the C-end of this protein, in bacterial cells in the form of a fusion protein, ZT. Bacterial lysates were tested for the ability to cleave fluorogenic peptide substrates that had previously been shown to be cleaved by other SEHUOZ/LCE homologues.

Two peptide substrates were used. First of all, it is acetyl-Azr-siSh-MaI-Azr-a--4-methylcoumaryl-7-amide) (AC-OEMO-AMS), which corresponds to the sequence of ADP-ribose polymerase (RACP), a nuclear protein for which the its cleavage in cells almost immediately after RA5-K stimulation |Temagi et al., 199551, as well as in other apoptotic processes (Kashitapp, 1989; Kashitapp et al., 1993; I agerpik et al., 1994). This fluorogenic substrate is efficiently cleaved by CPPZ2 protease. The second fluorogenic substrate - acetyl-Tug-MaI-AIa-Azr-AMS (As-ZhMAYUB-AMS) - which corresponds to the substrate site for the ISE protease in the chain sequence of the precursor 1-18. This fluorogenic substrate is cleaved by ISE protease. Bacterial lysates expressing a segment of homology with SEYUOZLSE from the composition of MASNAT effectively cleaves the RAKR-derived i) fluorogenic substrate. However, they did not have visible proteolytic activity relative to the sequence derived from the precursor of interleukin-1B as a fluorogenic substrate (control) As-UMAO-AMS, which is the cleavage site for ISE protease in the composition of PRO-II.-18 |Pogpregtgu ei ai. , 1992). Proteolytic activity of Ge! zo was blocked by iodoacetic acid (5 mM): this confirms that it is mediated by a thiol protease. No cleavage was observed when testing lysates containing a 5T-fused region of MASN, homologous to SEOZ/LSE proteases, in which the catalytic residue of cysteine-360 was replaced by serine. Also, bacterial lysates that expressed the full-length MASNAT!T protein in the form of a 5T fusion protein do not cleave the substrate

As-OEMO-AMS, apparently, due to the absence of bacterial enzymes capable of processing the full-sized "z5 Molecule. No cleavage occurred when testing lysates containing either of the two potential cleavage products of the segment of homology with SEYUZ/-ISE proteases.

Kinetics of cleavage of the fluorogenic substrate Ac-OEMO-AMS (50 mM) derived from the RAKR sequence by the extract of E. soybeans expressing the 35T-fusion protein of the SEOZLSE MASNAT homology segment (from serine-217 to

C-end of this polypeptide), was detected compared to the absence of such cleavage by the extract of bacteria "that express OST-fusion proteins, full-length MASNAT molecule or any one of the two products from c proteolytic cleavage of the segment of homology with SEHUOZ/LCE (from serine- 217 to aspartic acid-374 and from aspartic acid-374 to the C-terminus of this polypeptide). ;" Next, the dependence of the cleavage of the As-YOEMO-AMS substrate on its concentration was demonstrated, incubated for 180 minutes with an extract of bacteria that express a segment of homology with SEHUOZ/LSE from the composition of MASNA1, fused with O5T. No cleavage was detected in the presence of iodoacetic acid (5MM). The non-I extracts showed activity relative to As-UKHMAYUB-AMS - a fluorogenic substrate that corresponds to the site in the composition of the precursor of I! -Ib, which is a substrate for ISE protease. ve Briefly, 55T-fusion proteins were produced in Xi and blue bacteria using the expression vector pPOEXZ. Bacteria were lysed by ultrasound in a buffer containing 25 mM NEREB (pH-7.5), 0.195 mL of 3-IZ-(cholamido-propyldimethylamino|-I-propanesulfonate, 5 mM EOTA and 2 mM O0O0T) followed by centrifugation at 160,009 for 10 minutes. Analysis using ZYUOBZ-electrophoresis in PAAG

Ge) confirmed the presence of a similar content of various fusion proteins in the lysates. Aliquots of extracts of 5 µm (4 mg/ml of total protein) were incubated at room temperature for a certain period with a total volume of the reaction mixture of 50 µm with a fluorogenic substrate at certain concentrations. The AMC output was determined spectrofluorometrically at wavelengths of 30nm (excitation) and 46nm (emission). The concentration of AMC was determined according to the calibration curve. Both peptides used as fluorogenic substrates were

F) received from Reriide Ipziyshe Ips. (OzaKa, Japan). For other proteases of SEHUOZ/LSE, the manifestation of full ka activity was shown only after proteolytic processing, which occurs either by the mechanism of self-cleavage or by cleavage by other proteases | see review Kitag, 1995; Nepkagi, 1996). The data obtained by the applicants that the lysates of bacteria expressing 5T-MASNAT molecules do not show enzymatic activity, in contrast to the activity shown by the lysates of bacteria expressing a segment of homology with SEHUOZ/LCE, confirm that processing is also necessary for the activity of MASNAT. The pathway by which MASNA processing occurs in mammalian cells and how this processing may be mediated by HA5B-K or p55-K receptors is still unclear. MOKT-1 was shown to bind in cells with activated ERAZ-K, as well as with some other 65 proteins of the CKisnkei ei!., 1995). These proteins appear to include FAT! and other isoforms of MASN. It seems quite likely that the binding of MOKT-1 to EA5B-K in the complex with MASNA holds together some molecules of MASNA or induces their conformational changes and that these changes either mediate the autolytic processing of MASNA or make MASNA susceptible to cleavage by other proteases. Stimulation of the p55-K receptor may mediate the self-cleavage of MASNA in a similar, albeit less directed, manner by holding together several TKAYO molecules or inducing conformational changes in them, which in turn induce a change in the formation or status or aggregation of the MOKT-1 protein and the molecule bound to it.

The substrate specificity of MASNA seems very "death oriented". Although it could cleave a peptide substrate that corresponds to the cleavage site in the "death-substrate" composition of RAKR (Ac-YUEMO-AMS),

MASNA does not detect proteolytic activity against the peptide that corresponds to the processing site of the 7/0 precursor of interleukin-18 by the ISE protease (As-UMA0-АМС). The identification of cellular proteins that would serve as substrates for the cleavage of bilum MASNA will allow to determine more distant events in the process of induction of cell death by this protease. Probable substrates for cleavage by the MASNA protein are other members of the SEOZ/CE family, such as SRRZ2 and ISE. Some of these proteases are actually processed after being mediated by EAB-K or TME-K receptors

IIMishga ei ai.,, 1995; ZspIede! ей айЙ.,, 1996; Snippaiiuap ei aiYi, 1996Й. It is not excluded that proteases that are not 7/5 INCLUDED in the CEOZ/LSE family are also activated by MASNA protein either directly or indirectly through the activity of other CEOZ/LSE proteases. The involvement of multiple proteases in cell death processes is consistent with the known ability of various protease inhibitors, including serine protease inhibitors and cleavage inhibitor

ISE, as well as antisense cDNA ISE, protect cells from toxicity induced by EAB-K and TME-K receptors |Meygep 5 Sgapdeg, 1980; Kiddiego eyi a!., 1987; Epagi ei ai., 1995; I ov ei ai., 1995).

Various groups of other enzymes, including phospholipases, sphingomyelases, and protein kinases, may participate in the induction of cell death mediated by TME and RA5B-K receptors, see Eizspep eyi ai., 1994; Mapdepabreie eyi ai.,, 1995; Spope ei ai!., 1995 and bibliography in these articles). Some of these enzymes can be activated by proteolytic cleavage initiated by MASNA protein. It also seems possible, however, that at least some of these other "death-related" activities are stimulated by separate SC signaling mechanisms that are independent of stimulation by MASNA. The involvement of more than one signaling cascade in the induction of cell death—some common to p55-K and RAZ/AROT1 and some inducible only (8) by one of them—should be consistent with the message that there are both common and distinct characteristics of death processes. of cells induced by these two types of receptors (Ogeiyi, 1994;

Zsptsige-Oviyoii ei ai., 1994; M/opd "Soedavei, 1994; Sietepi 5 (atepkomis, 1994). Gee! from (4) MASNA! connects to both MOKT-1 and MASNVI1:

To find out whether MASNAT1 can bind to MOKT-1 in the same way as it binds to MASNVI, we first studied the interaction of these proteins in transfected yeast cells. This effect was manifested in a significant decrease in the yield of yeast colonies that would express the protein encoded by the vector, which includes the activator domain (AU) (the expression level of which is higher than the expression level of the vector, which includes "

DNA-binding domain - OVO). On the other hand, MASNVI, in the composition of which the catalytic residue of cysteine-360 was replaced by serine (MASNAT |СЗ605)), did not show cytotoxicity either against mammalian cells (see below) or against yeast. Like MASN, MASNAT |SZ605) bound in transfected yeast cells with

MOKT-1 and himself. He also contacted MASNVI1. Also, yeast cells expressing wild-type MASNAT1 together with MOKT-1 or MASNVI showed interaction of the transfected proteins. However, the intensity of the color caused by the Ias2 product varied between yeast colonies: in yeast transfected with MASNAT in the composition of She) from both vectors (AS and OBO), the colored product was not detected at all, probably due to the cytotoxic manifestation of MASN1. wild type Furthermore, despite this variability, yeast that expressed FAT! or in combination and? with MOKT-1, or in combination with MASNVI, showed a clear positive reaction from the interaction of transfected proteins.

Unlike MASNVI, MASNAT did not reveal self-to-self interaction in the dihybrid test.

Y MASNAT1 |SZ3605), and MASNVI were immunoprecipitated simultaneously with MOKT-1 from lysates of human embryonic kidney-I cells 293-EVMA, which indicates their binding to MOKT-1 also in mammalian cells. Further testing of MASNAAAT for the possibility of binding to MOKT-1 was also carried out in mammalian cells, and expression of MASNA!T or MASNVTI, fused with the octopeptide BIG AS, was carried out along with molecules of MOKT-1 labeled with the HA epitope. os Metabolically tagged with 355 FAT! and MASNVYAI, fused at their M-ends with the BAS octopeptide "BAS-MASNA! and VI), and MOKT-1, fused at its M-end with the NA epitope Tsrieia ei ai., 1988), were also expressed in Neg cells. Immunoprecipitation of proteins from the lysates of these cells was carried out using mouse monoclonal (Che) antibodies to GAS octopeptide (M2: Eavitap KoaakK), HA epitope (12C5: Ryvid ei ai., 1988) or p7B5-TME-K receptor |Mo9: Vidda ei ai ., 1994), taken as a control. Proteins were analyzed using zO5-electrophoresis in a 1296-polyacrylamide gel followed by autoradiography. | MASN!T and MASNV1 were immunoprecipitated together with MOKT-1 from cell lysates, which confirms their binding to MOKT-1 bilium.

The efficiency of the interaction of MASNAT with MOKT-1 protein seems to be lower compared to that of MASNVI1.

F, (e) Ability to mediate cell death by molecules of MASN containing a segment of homology with the name) СЕОЗЛСЕ

In order to evaluate the participation of MASN in the induction of cell death, the effect of overexpression of different isoforms of MASN on cell viability was studied. The analysis was carried out by transfection of MASN-expressing vectors together with a vector expressing B-galactosidase, taken as a transfection marker, into human embryonic kidney cells 293-EVMA and human breast carcinoma MCE7 cells.

Briefly, 293-EVMA cells, human MCE7 breast carcinoma cells, and Nega NITA-1 cells were grown in Dulbecco's modified Eagle's minimal medium supplemented with 1095 fetal calf 65 serum, replacement amino acids, 100 bod./ml penicillin, and 100 μg/mg streptomycin. Dishes with cell cultures (5x109 293-EVMA cells, 3x109 MCE7 cells, or 3x109 Ne!a cells in 6-cm dishes) were transfected according to the intermediate type using the calcium phosphate precipitation method, using the cDNA of the specified protein together with a vector expressing B- galactosidase. In one group of experiments, each dish was transfected with 3.5 μg of the MASN construct and 1.5 μg of pAM-B-CaII, and in the other group of experiments, each dish was transfected with 2.5 μg of the specified MASN variant and the MOKT-1 construct (or, as a control, "empty " vector) and 1.5 μg of rem-V-Cai. Cells were washed 6-10 hours after transfection. 293-EVMA and MSE7 cells were then incubated for another 18 hours without additional treatment. Her cells! and incubated after transfection for 26 hours and then for another 5 hours in the presence of either antibodies to EAB/APO1 (SHI1: 0.5 µg/ml) or to TME (10 µg/ml), in both cases with the addition of cycloheximide (1 µg/ml) . The intensity of cell death at the end of the incubation period was assessed by the level of B-galactosidase expression as described in (Kiptag ei ai!., 19941.

Cultures transfected with vectors expressing MASNAT1T or MASNA2 exhibited massive cell death, manifested by rounding, desiccation of cells, roughening of their surface, and eventual detachment of cells from the cup surface. 20 hours after transfection, the majority of transfected cells, which were identified by B-galactosidase staining (X-CaY), showed a clear morphological condensation, /5 typical for the process of apoptosis. On the other hand, cells expressing the "empty" vector survived.

In order to evaluate the participation of the segment of homology with SEOZ/LSE in the composition of MASN isoforms in the manifestation of such an apoptotic effect, cells were transfected with a vector expressing the MASNVI isoform devoid of the segment of homology with

SEOZLSE, as well as the vector expressing the MASNAZ isoform lacking the M-terminal part of this segment, and the vector expressing MASNAT1 |СЗ6О051), and the vector expressing the C-terminally shortened mutant form of MASNAT |MASNAT (1-415 )), which are devoid of one of the amino acid residues, which is considered critical for ensuring SEYUZ/LSE-protease function (Corresponds to serine-347 in the composition of ISE). No death (with the exception of a very low level detected upon transfection with an "empty" expression vector) was not detected in 293-EVMA or MCE7 cells transfected with vectors expressing MASNAZ, MASNA1 (1-415) or MASNAT1 |СЗ360О5). Moreover, cells transfected with MASNAT at the same time as these vectors showed very weak cell death: this indicates the manifestation of MASN molecules, which include an incomplete segment of homology with SEHUOZ/LSE, a negative dominant effect relative to the activity of wild-type molecules. (8)

Cultures expressing MASNVI, which are completely devoid of the segment of homology with CEOS/LSE, showed a weak degree of cell death. This manifestation of MASNVI, which is most likely the result of the activation of endogenous MASNAT molecules, was for a number of reasons more pronounced in transfected cells of No! and. Moreover, Ge! In NeHa cells, MASNAZ, MASNAT (1-415) or MASNAT |C36051 variants were also cytotoxic to some extent. with

The activity of MASNA appears to be the earliest catalytic stage in the signal cascade of the cytotoxic manifestation of HAB/AROT and p55-K. The ability of MASNVI to bind to MOKT-1 and MASNAT confirms that this isoform enhances the activity of catalytically active isoforms. It is likely that some isoforms of MASN "

Zv have additional functions. The ability of MASNVI to bind to both MOKT-1 and MASNAT confirms that this isoform should enhance the activity of catalytically active isoforms. The average cytotoxicity observed in the cultures of 293-EVMA and MCE7 cells transfected with this isoform, and the rather more pronounced cytotoxic effect that it exhibits in Her cells! and probably reflects the activation of endogenously expressed MASNA molecules by their binding to transfected MASNVI molecules. It is suggested that "some of the MASN isoforms can also act as "depot sites" for molecules involved in other, unrelated to the cytotoxicity of expression of RAB/AROT1 and TME receptors. . (9 Blocking the function of MASNA prevents the induction of cell death by the EAZ/AROT1 and p55-K receptors and?" To assess the contribution of MASNA to cytotoxicity mediated by RHAB/AROY and p55-K, MASNAS, as well as non-functional mutants MASNAT (1-415) or MASNA IC36051 were expressed in cells in which cytotoxicity was induced. Cytotoxicity induced by p55-K was included in 293-EVMA cells as a result of -I intermittent overexpression of this receptor (|Voidip et al., 19958), and cytotoxicity due to EGAZ/APO1 , by overexpression of chimeric molecules that include the extracellular domain of po5b-K and the transmembrane and intracellular domains of RAB/APO1. This chimera had a significantly stronger cytotoxic effect compared to the normal RAB/APO1 receptor. Cytotoxic activity in Her cells was also induced by their treatment 50 ME or an antibody to RAZ/AROT1 in the presence of cycloheximide, which is a blocker of protein synthesis. Her cells became susceptible to RGAB/AROT1 as a result of intermittent express of this receptor. In all tested

Ge) MASNAZ systems and non-functional MASNAT1 mutants provided effective protection against cytotoxicity induced by REAB/APO1 or p55-K. Such protection was also manifested, as previously described |Nzy ei ai., 1996; SVippaiuap ei aiYi.,, 1996), in cells transfected with a mutant version of MOKT-1 with a deletion in the M-terminal part, devoid of the MASN-binding segment - MOKT-1 (92-208). These protective manifestations indicate that MASNA is a necessary component for signaling cascades to induce cell death involving and

F) EAZV/ARO!, and rB5B-V. The MASN protein is expressed in different tissues at significantly different levels and, apparently, with a significantly differentiated isotype composition. Probably, these differences determine the tissue-specific parameters of reactions to the RAZ/AROT1 ligand and to TME. As is the case with other ACE/ACE homologues (Mapa et al., 1994; Alpettgi et al., 19951), MASN isoforms that contain an incomplete segment of homology with CEOSLCE (eg, MASNASE) have been found to inhibit the activity of the molecules МАСНА1 or МАСНА?2, expressed at the same time: blocking of the induction of cell death by the factors RAZ/АРОТ1 and p55-K was also found for them. The expression of such inhibitory isoforms in cells may represent a mechanism of cellular self-protection against cytotoxicity mediated by 65 EAZ/АРОТ1 and TME The wide spectrum of heterogeneity of MASN isoforms, which is significantly greater compared to any other proteases of the SEHUOZ/LSE family, should lead to the presence of narrower functional specializations in specific MASN isoforms.

Example 1.

Cloning and isolation of a protein (1 that binds to the MOKT1-binding protein Mep4 (Y) Dihybrid screening and dihybrid test with expression of B-galactosidase

Using the procedures described herein above in examples-references 1-3, a new protein, designated C1, was isolated, which appears to be homologous to, and therefore likely to be, a member of the CEOSLCE family of proteases.

Protein 51 includes two modules showing homology to modules in MOKT, namely homology to

M-terminal part of MOKT-1, modules of MOKT of MASN proteins (see above-described examples-references 1-3 and Voidip 70 ei aI.,, 1996) and with modules of MOKT of another related MOKT1-binding protein Mep | see Regpapaev-AIpetgi ei aI,, 1996; Zgipimaztssha ei aiYi., 1996). In addition, the SI protein includes a catalytic (protease-like) segment, which appears to be homologous to the catalytic (protease) segments of proteases of the SEOZ/LSE family (for example, the protease segments of MASN and Mena proteins and others).

Briefly, a clone of protein C ("clone (31")) was obtained after dihybrid screening of a human digkai/5 T-cell cDNA library using May protein as a "bait". A human digkai T-cell cDNA library tagged with the activator domain of SAG4 was used ; screening was carried out using the reporter yeast strain HE7c (Siopiesi, Rayu A, SA, USA) in the absence of 3-aminotriazole according to the MaisptakKegtm protocol

Tmjo-Nubgiy Zuziet Rgoioso! (Siopiesp). The Msp4 sequence was taken from the EMVI database. Using this extracted sequence, primers for PCR were generated based on the OG IbOm program, and the DNA fragment that

Corresponding to the coding part of Mepy4, it was isolated using PCR with reversion (CT-PCR) on the material of total RNA obtained by standard methods from the line of primary cells of the human umbilical vein endothelium (HOME).

The coding part of Mep4 was then cloned into the pOAYUSnH vector (Siopiesi) and used as a "bait" as described above for the dihybrid screening procedure. In the course of this dihybrid screening, 11 clones were obtained: all of them encoded a protein that appears to be a splice variant of a protein that includes two motifs of homology to MOKT-1. Analysis of the preliminary partial sequence of 31 sequences of the aBevzi database and the human genome database (level 1) revealed several EzT sequences (unidentified by transcribed tags) that include fragments of the sequence of "clone (31). Sequencing of these E5Ts showed that that there may be a series of splicing variants of protein 51 that include a sequence encoding a polypeptide motif homologous to ISE-like proteases, and that this sequence is located 3' to the He» of the C1 sequence identified in the dihybrid screening.

To isolate the complete sequence of isoform 1, which includes the protease-like catalytic segment, the reverse transcription reaction was carried out on the basis of total RNA isolated from various cell lines, using as a primer an oligonucleotide that includes the dTt 15 motif and an adapter sequence in order to form cDNA molecules. These cDNA molecules were then used as templates for PCR, in which 3-5 primers were selected and synthesized in the form of oligonucleotides comprising the sequence derived from the 5-coding part 1 and an adapter sequence: these primers were used in the first round reactions

PCR. After that, in the second round of PCR, additional oligonucleotide primers were used, which include the sequence of the 5-coding part of 51, including the ATO start codon, as well as the adapter sequence. In this case, it was possible to obtain the complete sequence of the predicted splicing variant of the protein 40. 51, which includes the catalytic (protease-like) segment. It represents all but one of the putative /-- c isoforms 1. The predetermined sequence of one such isoform 1 - splicing variant 1 - is shown in "» Fig. 1: in it, the upper sequence is a nucleotide sequence, and the lower one is deciphered amino acid sequence according to OKE, starting with the ATO codon (482nd nucleotide) and ending with TAA (nucleotide 1921) - these initial and final nucleotides in the given nucleotide sequence are marked with asterisks (""). Splicing -I variant 1, shown in Fig.1, is also pre-labeled as "ia".

Fig. 2 schematically shows the predetermined sequence of another isoform (31 - short e splicing variant 1, which includes two "MOKT-modules", previously designated as "z18B", - in which the upper (9! sequence is a nucleotide sequence, and the lower - the decoded amino acid sequence according to OKE, starting with the 482nd nucleotide (ATO start codon) and ending with the 1145th nucleotide (TOA stop codon): o start and stop codons in the nucleotide sequence are marked with asterisks (7). Che) In addition, it should be noted that the originally isolated "C1 clone" obtained with the Mei4 sequence taken as a "bait" was at least part of the short variant (31, named 518 (see Fig. 2). This originally isolated "Clone 1" is a part of the clone of the new KkKDNA, which, like MASN (SAB5R-8) and Mep4 (СА5BR-10), includes two death-domain motifs - MOKT-modules (or "death-effector domains" OEB), are located immediately below its M-terminus (see also Fig. 3).Using the original clone of It was then possible to isolate and characterize cDNA clones corresponding to the larger splice variant - CA, and the shorter splice variant 518. The larger splice variant includes a C-terminal segment homologous to the protease segment of caspases and, therefore, is a likely new a member of the caspase family. Therefore, 01 bo was also designated by the abbreviation SAZN, which is used to denote "caspase homologues". A comparison of the sequences of CTA (SAZNA) and 518 (CA5HB) with the sequences of other caspases was carried out (for more details, especially regarding the isolation of the mouse 31 sequence, see below, as well as the drawing description section above in the comments section of Fig.3). The results of such a comparison are schematically presented in Fig. 3: all details, in particular, the key for determining the sequence belonging to this figure, are included in section 65 "Brief description of the drawings".

After the initial cloning and sequencing of C1 described above, in particular its OA and 518 isoforms, further analysis of these proteins was carried out. (ii) Northern blot analysis and additional sequencing

Analysis by Northern blotting showed that protein (31) exists in at least three size variants of transcripts - 2000, 2400 and 4400 nucleotides, the ratio of which varies significantly in different tissues (Fig. 4). To obtain the full-length cDNA of protein 1 (SAZN), a cDNA library of human skin fibroblasts (Siopiesi) was used for screening with a cDNA probe corresponding to the sequence (31. Two types of cDNA corresponding to the two splicing variants 1 (see also above) were detected. The proteins encoded by these

cDNAs share a common M-terminal region that includes the "death-effector domain", but their C-terminal 7/0 regions differed. One of them (BS18-SAZNV) has a short C-terminus, which corresponds to that contained in the original cloned cDNA. Another variant (ZIA-SAZNA) had a long C-end.

The amino acid sequence of this long C-terminal segment of STA showed a sufficiently high level of homology with that of the segments in the composition of MASN (SABR-8) and Mesp4 (SABR-10), which are precursors to the protease domains (Fig. 3). However, STA has lost some amino acids believed to be critical for protease activity: /5 Che confirms that this protein may be devoid of cysteine protease activity. It is considered interesting that STA includes a caspase-substrate sequence in a site that corresponds to a proteolytic processing site in the protease segment of MASN (SABR-8). | Mesp4 (SABR-10) (shaded in Fig. 3). Preliminary data confirm that C1A can indeed be cleaved at this site by MASN (data not shown).

Based on the nucleotide sequence of the E5T clone, for which a match was found to the mouse homologue in the th part of the "death-domain" (0EO) region in composition 21, the cDNA of both splicing variants of the mouse - SAZNA and

SAZNV - were cloned from the mRNA library of mouse liver cells using reverse transcription PCR. Clone EZT (depository MoAA198928 of the SepVapkK database) was identified as a mouse homolog relative to part of the OEO segment from composition 51. Based on this sequence, the splicing variants of mouse STA (SAZNA) and 18 (SAZNV) were cloned from an mRNA library of liver cells using Reverse PCR. The reverse transcriptase reaction was carried out using the oligodeoxythymidine adapter primer 5-САСТСОАСТСАСАСТСОАС-(Т) 17-3 and AMM reverse transcriptase (Rgoteda), using i) manufacturers' recommendations. The first round of PCR was carried out using Ehrap Gopd Tetriaie RSK buviet (Voepgipdeg Mappypeiiit) and the following sense and anti-sense primers: 5-SOSTSTOOTOOTTSSSASSASS-3 and 5-SASTOOASTSTASASTOSOAS-3 (adapter), respectively. The second round was carried out using polymerase Ge! zo Mep (MEV) and the semantic "concentrated" seed 5-TOSTSTTOSTOTOTASATO-Z and the adapter.

Comparison of the sequences showed a high level of conservatism in the length of the STA molecule (SAZNA (7190 s of identity by the OBO segment and 5995 by the protease homology segment): this confirms that both the OBO domain and the I segment of the protease homology of this protein are essential for determining its functions (see Fig. 3). (ii) Dihybrid analysis of binding specificity of isoforms 1 "

Dihybrid analysis of interaction parameters of CTA (SAZNA) and C1B8 (SAZNV) variants (Fig. 5) showed that both of these variants interact with MOKT-1/RABO and MASN (СА5Р-8), most likely indirectly through their shared OEO domain . Although initially cloned by dihybrid screening, Mep4 binding proteins (SABR-10), 18 (СА5РВ), it was found that in this test the binding to Mep4 (SABR-10) was weak, as well as STA (SAZNA ), also weakly bound to Mep4. However, it should be noted that the original dihybrid screening resulted in "

Cloning of proteins 1, which differ in the specificity of their binding during dihybrid screening: therefore, from c we can actually assume that both CTA and 51B bind to MASN and Mep in the same way as the original showed. cloning Two variants of 01 also show self-association and connect with each other, but not at the same time? bind to KIR or TKAUO (adaptor proteins that, like MOKT-1/EAOO, include "death domains" but lack OEUO) and do not bind to a number of irrelevant proteins (eg, lamin) , which were used as a specific control. -I For further analysis of functions (31, two of its variants were expressed in an intermediate type in Nei a and 293-T cells, and in them the effect of transfected proteins on the signaling mechanism induced by p55-K ve (СО120а) and involved in the manifestation of cytotoxicity was evaluated , which is activated by the participation of TME or overexpression of this receptor, as well as the signaling mechanism induced by GEAB-K (СО95) and involved in the manifestation of cytotoxicity, which is activated by an antibody that cross-links with HA5-K or by overexpression of a chimeric receptor, which includes the extracellular domain of p55-K (СО120a) and the intracellular domain of BAB-K (СО95) (see Fig. b).

Ge) cell lines, the expression of 518 (SAZNV) by itself does not affect the viability of cells, but significantly inhibits the induction of cell death with the participation of rbo5-K (СО120а), as well as the GAB-K receptor (СО95). The expression of the STA variant (SABNA) affects the two tested cell lines to a large extent differently. In Neg cells, it inhibits the cytotoxic effect of p55-K (СО120а) and EAB-K (СОО95), similar to that of С1В (САЗНВ). However, in cells 293-- it causes significant cytotoxicity. Similar cytotoxicity was found when the STA protein

F) expressed in 293-EVMA cells (not shown). This cytotoxic effect could be completely blocked by co-expression of p35 - a caspase inhibitor derived from baculoviruses, see p35 | also Siet ei ai., 1991;

Htse 5 Nogmiig, 19951). In order to assess the contribution of the protease homology region in STA (SABNA) to the cell-damaging effect exerted by it, the functions of two mutant variants of this protein were studied. These mutants are as follows: (zZYSAZNA(1-385) and ZYSAZNA(1-408) - that is, they have C-terminal deletions in the segment corresponding to the part of the protease domain from which the small subunit of the mature protease is derived. Both mutants did not produce any cytotoxic Moreover, like 518 (SAZNV), they protected cells of line 293 from the induction of death by receptors in rZO-K (СО120a) and RAB-K (СО95) (Fig. 6C and 60).

It should be noted that the following methods and materials were used to carry out the procedures described above:

(1) Deletion mutants of STA (SAZNA) and chimera p5o55-K/RAB-K (СО120а/С095) were obtained using the PCR method and (or) standard cloning. Splicing variants 1 (SAZN), signaling cascade proteins

EAB-K (CO095) and rb55-K (CO120a) (all derived from human tissues) and the baculovirus protein pZ35 were expressed in mammalian cells using the expression vector rsOMAZ (Ipigodep). B-galactosidase was expressed using the RSMMU-B-CA1 vector (Rgoteda). (2) Human embryonic kidney cells 293-T and 293-EVMA and human cervical carcinoma cells Nei a (Ne a-RAB: clone NINA-1) stably expressing transfected human RA5B-K (CO95) (developed in the applicant's laboratory ), were grown in Dulbecco's modified Igla's minimal medium with the addition of 70 1090 fetal calf serum, replacement amino acids, 10 bod./ml penicillin and 100 mg/ml streptomycin.

The results described above show that protein (31) can interact with components of the rob-K and RGAB-K signaling complexes and that it inhibits the induction of death through a pathway that may be different depending on the type of splice variant 1 and the type of cells in which it expressed

Suppression of the induction of cytotoxicity by the C18 variant, and in the case of Nega cells - also by the CTA variant, 7/5 is apparently mediated by the "death domain" (DDM) segment of this protein. This, perhaps, reflects the presence of OEO competition in the C1 composition with the corresponding segments in the MASN (SABR-8) and Mena (SABR-10) for binding to MOKT-1/eА0О.

In terms of the pathway by which SABNA causes cell death 293, the ability of the p35 protein to block the cytotoxic effect indicates that cytotoxicity is mediated by caspase activity. However, CA, even with significant homology with caspase sequences, can really lose cysteine-protease activity due to the fact that it lacks several amino acids conservative for caspase in the active site.

Thus, STA may act by activating other molecules that have caspase activity.

Another possibility is related to the fact that C1A, although it is not able to act as a protease itself, can nevertheless be part of an active protease molecule. Crystallographic analysis of the structure of SABR-1 and SABR-3 indicates that the small and large subunits of the protease in the composition of each processed enzyme are derived from different pro-enzyme molecules-precursors (MUaiYKeg ei ai., 1994; MUyivop ei aiYi., 1994; MIN ei aiYi., 1997; Kofopda ei (8) ai!., 1996). In connection with the revealed dependence of the cytotoxic activity of STA on the integrity of the segment corresponding to the small subunit of the protease (Fig. 6C), it may be that this segment in the composition of STA (SAZNA) is able to bind to the large subunit of some caspases (caspases) along such a path, which as a result leads to the rearrangement of the catalytically active molecule. The resulting active hetero-tetramer should subsequently be able to activate other caspases, thus including the cell death mechanism. with

Further, it was also found (results not shown) that protein 51 has at least some level of homology with another protein, named MUYUO88, which is involved in the signaling mechanism mediated by interleukin-1. Thus, protein (31) can also be involved in other mechanisms that are "initiated/mediated by other cytokines.

From the point of view of the above regarding the cloning and isolation of protein (31 (at least two of its isoforms), the following characteristics and possibilities of use of 1 are noted: (1) Protein 21 was cloned using dihybrid screening in the form of a molecule that binds to

MOKT-binding protein Mena and, therefore, its participation in modulating the activity of Mesp4 and MOKT-1 proteins is possible: "accordingly, according to the mechanisms defined above, protein (31 is obviously involved in the modulation of cellular processes, with those initiated by EA5 receptors K and p55-K Since Mep is able to bind to MOKT-1 and is directly involved in the manifestation of cytotoxicity and, ultimately, the induction of cell death, and since some Mep4 isoforms are known as inhibitors of cell death, it is noted that protein C1, including its various isoforms, can be directly or indirectly involved in the manifestation of cytotoxicity and cell death, and, equally, in the inhibition of death

CELL -And (2) It is obvious that protein 51 is characterized by an M-terminus, which includes two "MOKT-modules", homologous to two "MOKT-modules" in MASN (see the above-described examples-references 1-3) and Mep4. The presence of these pi "MOKkT-modules" in composition 21 thus testifies to its ability to bind to May4, and also, possibly, to the binding of C1 (or at least some of its isoforms) to MASN (or some of its isoforms), as well as 5o Z MOKT-1 directly. Accordingly, 51 may be able to modulate the activity of MOKT-1 (and, in turn, the activity of ГА5Б-К and p55-К receptors) directly, by direct binding to MOKT-1, or indirectly by binding to

Ge) Mena and (or) from MASN, which, in turn, are known for their ability to bind to MOKT-1. (3) Analysis of the sequence of 01 according to the databases described above and their screening showed that the gene encoding the protein

O1 is apparently localized in the immediate vicinity of the Msp4 and MASN loci on human chromosome 2: this two determines the close relationship between the genes encoding these proteins also at the chromosomal level. (4) At least some of the isoforms (31 (for example, the isoform of Sioya - shown in Fig. 1) include a segment, (F) located downstream of the "MOKT-modules", which shows similarity with the catalytic, i.e. protease region of MASN and Mena, and since this is the case, protein 51 may be a member of the ACE/LCE family of proteases.(5) The presence of a protease-like segment in at least some isoforms of protein 31 (e.g., CTA) suggests that 51 or such isoforms may be directly involved in the manifestation of cytotoxicity and inflammatory processes caused by various stimuli, including receptors from the TME/MOE receptor family (for example,

EAZ-K and p55-K) and other factors acting directly or indirectly through the activity of cellular proteases to induce cytotoxicity and inflammation. (6) C1 protein can act as an enhancer or determinant of the activity of other proteins, such as, for example, 65 MASN and Mena proteins (including their various isoforms), in the volume of intracellular mechanisms that ensure the manifestation of cytotoxicity, the development of the inflammatory process, and other related effects mediated by receptors from the TME/MOE receptor family and other factors that have common intracellular effectors.

The enhancing or conditioning effect of the protein (31 (or at least some of its isoforms)) can determine its binding to such other proteins (according to the type of binding mentioned above, 01 with May. and also possible binding to proteins MASN and MOCT-1), thereby enabling its binding to MOCT-1 (including self-binding of MOCT-1) or acting independently of MOCT-1. (7) Protein 51 can also act as an inhibitor of the activity of other proteins, and this can happen by entering the 51 part into a complex of other proteins with which it binds (for example, Mepy4 and possibly also

MASN and MOKT-1), thereby disrupting their cytotoxic effect to the extent characteristic of inhibition of such activity. Further, in a similar way, as was described above for some isoforms of MASN, as well as some isoforms of Mesp4, there may also be isoforms of the C1 protein that specifically exhibit |inhibitory activity. One such isoform (01) may be the C18 isoform shown in Figure 2, which includes two "MOKkT modules" but lacks a clear protease-like segment similar to those of other known proteases.

Having completed the full description of the present invention, it should be clear to those skilled in the art that the same /5 can be carried out for a wide range of equivalent parameters, concentrations and conditions without any departure from the spirit and scope of the present invention and without unplanned experiments.

Since this invention has been described in connection with its specific variants, it should be understood that it assumes its further modifications. This application shall cover any variations, uses or embodiments of the present invention, substantially adhering to the principles of the present invention and including such deviations from the present disclosure as are within the known or customary practice of the art to which the invention belongs and which may to be applied essentially according to the scope of the following claims.

All materials cited herein, including journal articles and abstracts, published or corresponding US or foreign applications, issued US or foreign patents, are incorporated herein by reference, including all data, tables, figures and text contained in the publications , which are cited. In addition, the full content of the references cited within the cited works is also fully included in the bibliographic list. and)

References to known methods, standard methods, known methods, and standard methods in no way imply that any aspect, description, or variant of the present invention has already been claimed, filed, or supported in the art. The foregoing description of specific embodiments was intended to set forth the gist of the present invention so fully that anyone skilled in the art (including the information contained in the references) may easily modify and/or adapt it to different types of application of such specific options without unplanned experiments and without deviation from the basic concept of the present invention. Thus, such adaptations and modifications are considered to be within the spirit and range of equivalents of the claimed variants based on the guidelines and descriptions herein. It should be understood that the phraseology or terminology herein is intended to be descriptive, but not restrictive, that is, such terminology or phraseology in this specification should be interpreted by those skilled in the art in light of the explanatory and guiding descriptions provided herein, in conjunction with the knowledge of the skilled artisan. in this field of technology.

Link "

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LIST OF SEQUENCES

(1) GENERAL INFORMATION () APPLICANT: b zo (A) NAME: Heda Kezeagsi 5 Oemeiortepi So. | (a. (B) STREET: UUeigtapp Ipziyshe oi 5siepse s (C) CITY: R.O. Voh 95 y (0) STATE: Kepomoi (E) COUNTRY: Israel « (R) ZIP CODE: 76100 cha (b) TELEPHONE : (972-8) 934-4093 (H) FAX: (972-8) 947-0739 (A) NAME: UMaiiasp, Yuamia (B) STREET: 24 Vohospom s. (E) COUNTRY: Israel

І (Р) POSTAL CODE: 7 6406 и?" (A) NAME: Soiivem, Miha (B) STREET: U/ei2tapp Ipviishe oi Zsiepse, Veii Sioghe (C) CITY: Kepomoi -I (E) COUNTRY: Israel (RE) POSTAL CODE: 76100 ve (A) IM I: Komaiepko, Apagei s (B) STREET: U/ei2tapp Ipviishe oi Zsiepse, Veii Sioghe (S) CITY: Kepomoi yu (E) COUNTRY: Israel

Ge (R) POSTAL CODE: 76100 (A) NAME: MapoYoteem, Eidepe (B) STREET: U/ei2tapp Ipviyshe oi Zsiepse, Veii Sioghe (C) CITY: Kepomoi (E) COUNTRY: Israel (F, (R) POSTAL CODE: 76100 ka (A) NAME: Vgodiapeki, Madit (B) STREET: Kespom Nama I tsizKu 10/12 vo (C) CITY: Kepomoi (E) COUNTRY: Israel (R) POSTAL CODE: 76251 (its NAME THE INVENTION: SAZN (homolog of caspase) with a "death-effector" domain, modulators of functions

EAZ receptors 65 (NUMBER OF SEQUENCES: 4 (m) FORM SUITABLE FOR COMPUTER

(A) COMMUNICATION TYPE: Floppy disk (B) COMPUTER: PC compatible (C) OPERATING SYSTEM: RO-BO5/M5-005 (0) PROGRAM: Raiepiip Keyeaze 241.0, Megwiop 21.30 (ERO) (m) CURRENT APPLICATION PARAMETERS

APPLICATION NUMBER: I PCT/LI 98/00098 (my) APPLICATION PRIORITY DATA (A) APPLICATION NUMBER: I. 120367 70 (B) SUBMISSION DATE: From March 1997 (my) APPLICATION PRIORITY DATA (A) APPLICATION NUMBER: I. 120759 (B) SUBMISSION DATE: May 1, 1997 (2) INFORMATION FOR 5EO IO MO 1 () SEQUENCE CHARACTERISTICS (A) LENGTH: 2243 nucleotide pairs (B) TYPE: nucleic acid (C) STRAND NUMBER: single-stranded (B) TOPOLOGY: linear (s) MOLECULAR TYPE: cDNA (hi ) DESCRIPTION OF THE SEQUENCE: ES IU MO 1 s (8) (22) sc

IS) « and - « shi s z -I sh» 1 of 50 3e)

F) name) 60 b5

SSASOTSSAS OSATTASAAT SOSbAAASSA ABSSATABSA TSAdDASZOoSo AbSTToSASS in

STSASSSASO AOTSTSAAST AAAASbOASTI SSSOBAOSTA SoSoTObbbA STSSOSSTSSA 120 and САССТОАСТ oССoОsStTATT СОАСТТТИТОТ ССАСТОАСАС СТСАСАСААС ААОБАССАСо 180

СОАССАССТо ТАССАСАСАА ОСБССбСоАА САССОАТСОС ССАбСАССАА СбТоСоСТТеС 240 70 дОобСТТТСоС ТТТСстТтТоСс ТОСАТСТТоО ОТОСОССТТС ССбОСОТСТА СООБАСССАд 300 бОСТСАССТо ССАССОбСАС САСАСТССОС ССОССАСАбС АССААСТОСС ССАСТОСААА 360 /5 ОСАТТСТОАА АОДААТОААС ТСАОСССТСА САААТОААСТ ТоАСТОоССТОо СстТоОСТТТос 420

TOTTOASTOB SSSObAbSTO TASTOSAASA SOSTIoTolLo STTSSSSTAST STAASASTAo 480

SATOTSTOST OAASTSATOS ATSASSTTTOA ASAASSASTT SATASASATO ASAASSAbAT 540 7 bSToSToTTtT TToTOoSSobo ATSTTTOSTAT ASATSTOSTTT SSASSSTAATO TSASOSASST 690

TSTObBATATTI TTASOOBALA SAOSTAASST OTSTOoTSoob BASTTOOSTO DASTOSTSTA 6 29 SABASTOAOS SOATTTBASS TOSTSAAASO TATSTTOAAS ATOBASASAA AdDOoSstTOoTovA 129 o

BASSSASSTO STSASSAASS STSASSSTIST TTSOOASTAT ABASTOSTSA TOSSAbASAT tvo zo TOBTBABOAT TTOSATAALAT STOATOTOTS STSATTAAATT TTSSTSATOA AbSATTASAT 849 iy soosssAsos AASATAASSA ABBASADSAS TTTSTTObBAS STTOTOSTTo ASTTOBABAA 300 yu

ASTADATTTO STTSSSSSSA ATSAASTOSA TTTATTASAAA AAATOSSSTAA ASLASATOSSA 960 Z 7 SAOAATASAS STODASASAA ADATSSASAAA OTASAASSAS TSTOTTSAAb SAbSASSOPdS 1020 M

AALOTTASABOS AATSSTISTSSS AASSASSAAT SSAAAASAOT STSAASOATS STTSAAATAd 1080 « s 40 StTtSASOSTO SATAATOSOSA SAASTALASA ASADASASTTI AASOSASASASS TTbOoSOSTSA 1140 not with» ASAVSAASSA STOAASALAT SSATTSASSA: ATSASASAST TTASTSB0SATASS STO 1 А2

TOAASASASA TASAASATOA ASVOSAAbSS SSTBSAATS TOSSTOATAA TOSATTOSAT 1260

В ТООСААТОАб АСАСАССТТС ТТОСАБАСАС СТТСАСТТСС СТООССТАТС ААСТССАСЛА 1320 с АТТСТТОСАТ СТСАСТАТОС АТОСТАТАТС ССАСАТТСТІ БОССААТТТОо ССТОТАТОСС 1380 зо ССАССАСССА САСТАССОАСА ОСТІТОоТОоТОо ТОТОСТОбТо АоСССАбодо бСТОССАСВо 1440 т ТСТОТАТОСТ ОТОбАТСАСА СТСАСТСАбО бСТОССССТО САТСАСАТСА СОАОбАТОТІ 1509

F) ko 60 bo -AB-

SATOSOASAT TSATOSOSTT ATSTASSAOO SAAOSSADAS ATOTTITTTTA TTSASAASTA 1550

TOTOOTOTSA BASBOSSASS TOSASAASAAb SAOSSSTOSTTO SASOTOobATO OOSSASSBSSAT 1520

OAASAATOTO SAATTSAAOS STSABAAOSO AbOOSTOoTOoS ASAOSTTSASS bAbAASSTOd 1580

STTSTTStTOb AOSSSTOTOTA STOSOBASAT STOSSTOSTo SABSASTSTS ASAOSTSASS 1740 70 OtTSSStTOTAS STOSASTOSS TSTSSSABAA ASTSASASAA SAAABAADAS OSSSASTOST 1500

SSATSTTSAS ATTOAASTSA ATOSSTASAT STATOATTOS AASAAbSASAS TTTStTOSSAA 195860 75 BBASAAATAT TATOoTSToOS TSSASSASAS TSTOASADAS AAASTTATSS TSTOSTASAS 1920

АТАААААСС ААААСОоСТоОС ССоТАСТООС TSASASSTOT AATSSSASSA STTTOOBASo 1980

ССААОСАбо САСАТСАСТ САбСТСАССА PTTSBABASS AOSSTOOSSA DSATOSTAAAd 2040

SOSTOTSSST ASTAAAAATO SAADAAATTAS SstosboToToo oTtoToOoStTAS STOoTOoTTSSSS 2100

AOTTIASTTSO BAObSToAba TOOSAbOATS TTTTOAASSS ASSASTTSAb OSBTSATAvbSA 2160 s TOSTOTOATT OTOSSTASOA ATABSSASTO SATASSAASS TOBOSAATAT ABSAAOATSS 2720 o

SATSTSTITA ADAAAADAADAAA ALLA 2243 (2) INFORMATION FOR 5EO IYU MO 2 () SEQUENCE CHARACTERISTICS b" (A) LENGTH: 480 amino acids (B) TYPE: amino acid (C) NUMBER OF THREADS: single-stranded o (B) TOPOLOGY: linear "E ( i) MOLECULAR TYPE: protein 3o (hi) SEQUENCE DESCRIPTION: 5EO I MO 2 - "shi s"; -I sh» 1 of 50 3e)

F) name) 60 b5

Mes beg Aia bij Ma1i i1e Ni bij Mai bij bi Aiz Bez Abzo TpPtg Ar 1 . 5 19 15? bij uz bi Meh Bec re Rpe Pec Suz Ago Azr Mai A1ia ii Azr Uai 20 25 30

Ma1 Rgo Rgo Azp o Mai Ag AzroPeia Gay Azr o Yi1e Seb Ako 613 At9 b1Uu to 35 19 45

There was Bep back Mai b01u Azr Pec dia bij Bei o Sets Tug Aga Uvi Agoya Ago 50 55 bo

Rpe Ar Gets Gen o Buz Agu Te Pec Buv Mes Ar Agu Tuz A1a MUai 01 65 70 . 75 8о 70 TVE No Bez yeech Azp Rgo No Tez Uzi bek Azr Tukh Agu Mai Tez Meh 85 - 90 95

Aza biv tze siu bij d5o cez Azr Guz Bek Avr Ua! back back This Iie s what 100 ' 105 110 o

Rpe pbets Mebe B0uz A5r Tukh Meb b1u Aga Sbiu BUuz I1e bek Bbuz bij Guz 115 120 125 o s

Zek Rpe iets Azr o Sech Uai Mai bi cen bii Dub Bez Azp Bez Mai Aa oyu « v. « t s with -I their go with 50 s

F) o) because b5

130 135 140

TRIO Azr obip Pets Azrobesh without fight Puz Sub this Buz Azp I1e Niz Agd 145 150 155 160

Iiie A5r Tsep puz Tig o buz T1e b)ia Buz Tuk Puz bip Beh Uai bij siu 185 170 . 175 70 Aia biu TnI bek Tur Ag Avp Uai Pem bil Aia Aza Y1e siy Guz beg 180 , 185 190 rech Guz Azr o Rko Zak Azp Ap Rpe Akgu Ge Ni Azp biu Agud bek Buv 195 200 205 15 bi biv Ag Gey tsuz bii bip ceo biu Aia beep beep beep Rko Mai Buz 210 215 220

Buz beh Ii biv bii bek biz Aza Rpee Pec Rko bip bBei YIT1e Rgo bir go 225 230 235 240 siz Aka Tut Puz Mes Puz bek Puz Rko be biu I1iv Suye Pe) i1e Iie 245 250 255 s ov VA5r o Suz I1e b1iu Azp bii tt bin be pey Agu AzroTVpg Re Tv beg 260 255 270 (8)

Tem biu Tuk bish Uai Sip tsPubye RBe Te Ni Pes Bei Mei Niz Siu 112

I 215 280 285 o 30 century beep I1e Bei biu beep Rpe Aza Sub Mek Rgo bitz Niz Agch Ar Tug s 290 295 09 oyu

Ar Beh Re Ua1i Suz Mai be Uai Becht Akad Siu b1iu Bek bip bek MUa1 "I 305 310 315 329 35 ich-

Tukh sSiu Uai Azr bip TBg Ni bet biu Bey Rgo Bets Ni5 Ni Y1e Ago9 , 325 330 335 «

Ag o Mes Rpe Mee biu Ar 5eg Su Rkgo Tut Bez A1a biu MDuv Rko Gu Z 340 345 350 s h» Mes Rie Re I1iie bip D5p Tuhk Mai1! Mai bek biz biu bip Gay 019 Azp " 355 360 365 bek bek yets Pec biy Uai1i Avr biu Rgo Aza Mee uz A5p Ua! bi Rpe i "370 375 zvo i I , tsTsuz A1t1a Sip buye Ag biu yets Suz Tyh Viz Aku biy Aza Azr Re o z85 390 395 400 m)

Rpe Ter bZei Gay Suv TpK Aia dD5r Mei 5Zei Pe) ces bi b1ip Beh No

I3e) 405 419 415 bZeikh Bei Rgo bei Pyem Tug tsets bip Suz Bec bek bip tsuz Be Aku biy 420 125 430 o Si Agu yuz Ag Rego Hey Be Azr o Bech Ni T1e bi Pe Abp b1u Tug ime) 435 "440 145 bo Mek Tuk Ar TEr Azp bek Akd Uai bek Aia Buz bish Guz Tuk Tukh Mai 450 455 460

Ter Pe bip Niz Tig Bez Ag Buz Oak5 Ppeh I1e Pemz bei Tug Tpg bih 465 470 475 150 65 Y (2) INFORMATION FOR 5EO IO MO Z

() SEQUENCE CHARACTERISTICS (A) LENGTH: 1373 nucleotide pairs (B) TYPE: nucleic acid (C) NUMBER OF THREADS: single-stranded (B) TOPOLOGY: linear (i) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: 5EO AND MO Z

SSASOTSSAS OSATTASADT SOSSALASSA ASSSATASSA TOAAASASSO DOoSTTOSAoS BO

STSASSBASO AOTSTSAAST AAAASObBAST SSSOBASSTA SOSOTOBOSA STSSbSSTSA 120

САСАСТОАООТ ОССООСТАТ ООАСТТТТОТ ССАСТОАСАС СТСАСААС ДАОСАССАСО 1850 72 ObBAbSASSTOo TAbbABASAd bSOoSSOSOAA SAOSOATSOS SSAbSASSVA STOSOSTTOS 240

AoOSTTTtSbo TTISTTTTOSS TSSATSTtTbo SIbSSSSTTTS SSOOSOTSTA BObOAbSSAd 300 uh vostTbAoostTo OSABSObSAb sAbAoToSo0 SSOSBASASO ASOAASTOSS SSASTOVAAA 360

BOATISTOAA ABADATOAAS TOVOSSSTSA SAAATSADOT TOASTTOoSStTo stTobSstItToS 429

TOTTOASTOS SSSbSASSTo TASTOSAASA SOSTTOTOAS STTOSSSTAST STAASASTAS 480 s

I bATOTSTOST SAASTSATSS ATSABOTISA ABADOSASIT BATASASATO ABAASOAssAT 540 o bStTOoStTSTttT tTTotToSSsobo ATOTTTOSTAT ABATOTOSTI SSASSTAATO TSAboSASStT in (22) z TtTStTOSatTATT TTASSObBADA SASSTADOST otSToTSobo SASTTOOSTO AASTOSTSTA bo s

SABASTOASO SSATTTOAASS TOSTSAAASO TATSTTODAS ATOSASASAA ADOSTOTOoSA 729 o « zv OASSSASSTO STSABSAdSS STSASSTTOT TISSSASTAT ABASTOSTOA TOOSAbBAbAT 780 M

ТОСОТОСАбСАТ ТТООАТАААТ СТОАТОТОТС СТСАТТААТТ ТТОСТСАТСА АБОАТТАСАТ 9840 бОбСССАСОС ААСАТААССА АССАСААСАС ТІТСТТССАС СТІСТОСТТО АСТТОСАСАА за « ші с АСТАААТТТО СТТСССССАС АТСААСТОСА ТТТАТТАСАА АААТОССТАА АСААСАТССА 360 » САСААТАБАС СТОААСАСАА АААТССАСАА СТАСААОСАо ТСТОТІСАдДо САБСАбСоАС 1020 -1 45 ААСТТАСАСО ДАТОТТСТОС ААБСАбСАДТ ССАДААСАСТ СТСААбСАТС СТТСАЛАТАА 1080 їз СТІСАССАТО АТААСАСССТ АТОСССАТТО ТССТСАТСТО ААААТІСТТо БАААТТОТІС 1150 and 50 SATOTOATTA ASATOSAAST OSSTSTASTT AATSATISTO ADATOATTALA TSOTTTSATT 1200 ime) with TTSTAAATST STTATAATOT SITTAbSSST TISTTOTTOS TOTATOTTTA SATOSTTTISS 1250

AATSTITTOT TASTASTAAT DATOSTATAA AATAAATATS STIOTASTTS TTAAADADAd 1320

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA ADA 1373

F)

GI (2) INFORMATION FOR 5EO AND MO 4 () SEQUENCE CHARACTERISTICS in (A) LENGTH: 222 amino acids (B) TYPE: amino acid (C) THREAD NUMBER: single-stranded (B) TOPOLOGY: linear (s) MOLECULAR TYPE: protein 65 ( xi) DESCRIPTION OF THE SEQUENCE: ES IU MO 4

Claims (1)

The formula of the invention 1. A DNA sequence that encodes a polypeptide having C1 protein activity, while said polypeptide: 2 (i) is capable of binding or interacting directly or indirectly with MOKT-1 and/or with any proteins that bind to MOKT-1, or to other intracellular mediator/modulator proteins; and (i) able to mediate intracellular effects mediated by EA5-K or p55-TME-K or other cytotoxic mediators or inducers; while the indicated DNA sequence is selected from the group including: 70 (a) DNA sequence encoding isoform 1 having the amino acid sequence shown in Fig.1; (c) DNA sequence encoding isoform 1 having the amino acid sequence shown in Fig.2; (c) a DNA sequence encoding at least part of a polypeptide having the amino acid sequence shown in Figure 1 or Figure 2; (4) a DNA sequence that includes at least part of the sequence shown in Fig.1 and encodes at least one isoform of protein 1 - isoform 1 5; (e) a DNA sequence that includes at least part of the sequence shown in Fig.2 and encodes at least one isoform of protein 1 - isoform O1 B; (9) a DNA sequence derived from a segment encoding a native isoform of protein 1; (9) a DNA sequence encoding a fragment, analog, or derivative of a polypeptide encoded by the DNA sequence 720 of any of (a)-(i); ( n) a DNA sequence capable of hybridizing with a sequence according to any of (a)-(e) under conditions of medium stringency and which encodes a biologically active isoform of protein 1; and (І) a DNA sequence which is degenerate according to the genetic code with respect to DNA sequences, according to any of (a)-(H), and which encodes the biologically active isoform of protein 1. p 29 2. The DNA sequence according to claim 1, which is used for the preparation of a pharmaceutical composition for Ge) modulation of the effect of the EAB ligand on cells -K or TME or other proteins for the treatment of septic shock, transplant rejection, acute hepatitis, diabetes, multiple sclerosis, or AIDS. C. A vector comprising the DNA sequence of claim 1. 4. The vector according to claim 3, capable of being expressed in a eukaryotic or prokaryotic host cell. F 5. Vector according to para. C or 4, which is a recombinant vector based on an animal virus, containing a Ge DNA sequence that encodes a viral surface protein (ligand) capable of binding to a specific cell surface receptor bearing PHA5B-K or p55-K and the second sequence, including the DNA sequence according to claim 1. "I 6. The vector according to claim 5, which is characterized by the fact that the cell surface receptor is specific for tumor cells, HIV-infected cells or other diseased cells. - 7. The vector according to claim 5, which is used for the preparation of a pharmaceutical composition for modulating the effect on cells of the ligand EAB-K or TME or other proteins for the treatment of septic shock, transplant rejection, acute hepatitis, diabetes, multiple sclerosis or AIDS. 8. The vector according to claim 6, used for the preparation of a pharmaceutical composition for the treatment of tumors, AIDS or other diseases manifested in the production of specific cell surface receptors. c 9. The host cell carrying the vector according to any one of claims 3-6, which is eukaryotic or prokaryotic. :z» 10. The method of obtaining a polypeptide that has the activity of the C1 protein, which includes growing the transformed host cell according to claim 9 in conditions suitable for the expression of the specified polypeptide, ensuring post-translational modifications, if necessary, to obtain the specified polypeptide and isolating -1 75 designated polypeptide. 11. A polypeptide having the activity of a C1 protein, while the said polypeptide: CHK" (i) is capable of binding or interacting directly or indirectly with MOKT-1 and/or with any proteins that bind to MOKT-1, or with other intracellular mediator/modulator proteins; and (i) able to mediate intracellular effects mediated by EA5-K or p55-TME-K, or other cytotoxic mediators or inducers; with the fact that the specified DNA sequence is selected from the group including: (a) a DNA sequence encoding isoform 1 having the amino acid sequence shown in Fig.1; (c) DNA sequence encoding isoform 1 having the amino acid sequence shown in Fig.2; (c) a DNA sequence encoding at least part of a polypeptide having the amino acid sequence shown in Figure 1 or Figure 2; GF) (4) a DNA sequence that includes at least part of the sequence shown in Fig. 1 and which encodes 7 at least one isoform of protein 1 - isoform 01 Zh; (e) a DNA sequence that includes at least part of the sequence shown in Fig.2 and which encodes at least one isoform of protein 1 - the C1 p isoform; 60 (9) a DNA sequence derived from a segment encoding a native isoform of protein 1; (9) a DNA sequence encoding a fragment, analog or derivative of a polypeptide encoded by the DNA sequence of any of (a)-(i); ( n) a DNA sequence capable of hybridizing with a sequence according to any of (a)-(e) under medium stringency conditions and encoding a biologically active isoform of protein 1; and (Y) a DNA sequence that is degenerate according to the genetic code relative to to DNA sequences, according to any of (a)-(H), and which encodes a biologically active isoform of protein 1, or obtained by the method according to claim 10. 12. The polypeptide according to claim 11, which is used for the preparation of a pharmaceutical composition for modulating the effect on cells of the ligand RGAB-K or TME or other proteins for the treatment of septic shock, transplant rejection, acute hepatitis, diabetes, multiple sclerosis or AIDS. 13. An antibody specific for the polypeptide according to claim 11. 14. The antibody according to claim 13, which is used for the preparation of a pharmaceutical composition for modulating the effect on cells of the ligand RHA5B-K or TME or other proteins for the treatment of septic shock, transplant rejection, acute hepatitis, diabetes, multiple sclerosis or AIDS. 15. The method of modulating cell death or inflammatory processes of the myo, which includes the treatment of the specified cells with one or more polypeptides according to claim 11, while the specified treatment of the specified cells includes the introduction into the specified cells of the specified one or more polypeptides in a form suitable for their intracellular introduction or introduction into the specified cells of the DNA sequence /5 according to claim 1 in the form of a suitable vector containing the specified sequence, while the specified vector is able to ensure the introduction of the specified sequence into the specified cells in such a way that the specified sequence is expressed in the specified cells. 16. A method of modulating the effect of the EAB-K or TME ligand on cells containing EAB-K or p55B-K, i.e., which includes the treatment of said cells with one or more polypeptides according to claim 11, provided that said treatment of said cells includes the introduction into the specified cells of the specified one or more polypeptides in a form suitable for their intracellular introduction or the introduction into the specified cells of the DNA sequence according to claim 1, which encodes the specified one or more polypeptides in the form of a suitable vector containing the specified sequence, while , that the specified vector is able to ensure the introduction of the specified sequence into the specified cells in such a way that the specified sequence is expressed in the specified cells. 17. The method of modulating the effect of the HA5B-K or TME ligand on cells containing EAZ-K or p55-K, i.p. claim 16, which is characterized by the fact that the specified treatment of cells includes the introduction into the specified cells of one of the specified polypeptides, a DNA sequence encoding one of the specified polypeptides in the form of a suitable vector containing the specified sequence, while the specified vector is capable of providing introducing the specified sequence into the specified cells in such a way that the specified sequence is expressed in the specified cells. with 18. The method of modulating the effect of the HA5B-K or TME ligand on cells containing EAZ-K or p55-K, according to claims 16 or 17, which is characterized by the fact that the specified treatment of the specified cells is carried out by transfection of the specified cells with a recombinant vector on based on an animal virus according to claim 5. 19. A method of modulating cell death or inflammatory processes in the myo, which includes the treatment of these cells with one or more inhibitors of one or more proteins/enzymes that mediate the specified cell death or inflammatory processes, while the indicated inhibitors are selected from the group , which includes: () one or more polypeptides according to claim 11, capable of inhibiting the indicated cell death or inflammatory processes; and c (her) inhibitors of one or more polypeptides according to claim 11, when said one or more polypeptides enhance/cause or mediate said cell death or inflammation;" processes. 20. The method of modulating the effect of the HA5B-K or TME ligand on cells containing RA5B-K or p55B-K, i.p. myo, which includes the treatment of the specified cells with an antibody according to claim 13, while the specified treatment is carried out with the use of a suitable composition , which includes the specified antibody relative to the specified cells, with the fact that when the protein 51 or its parts are exposed on the outer surface of the specified cells, the specified composition is created for extracellular use, and when the specified C1 proteins are intracellular, then the specified composition is created for intracellular application. 21. A method of modulating the effect of EA5-K or TME ligand on cells containing EA5B-K or p55-K, i.e., including treatment of these cells with an oligonucleotide sequence that encodes an antisense sequence of Ge) relative to at least part of the DNA sequence, according to claim 1, while the specified oligonucleotide sequence is able to block the expression of protein 1. 22. The method of modulating the effect of the EAB-K or TME ligand on cells containing EAB-K or p55B-K, according to L. 21, which is characterized by the fact that the specified oligonucleotide sequence is introduced into the specified cells using the vector according to claim 5 , while the specified second sequence of the specified virus encodes the specified (F) oligonucleotide sequence. ka 23. A method of modulating the effect of the EAB-K or TME ligand on human cells, which includes the use of a ribozyme procedure, in which a vector encoding a ribozyme sequence capable of interacting with a sequence of cellular mRNA encoding a polypeptide according to claim 11 is introduced into the specified cells in a form that allows expression of said ribozyme sequence in said cells, and when said ribozyme sequence is expressed in said cells, it interacts with said cellular mRNA sequence and cleaves said mRNA sequence, resulting in inhibition of expression of said polypeptide in the specified cells. 65 24. The method of modulating the effect of EAZ-K or TME ligand on ip migo cells according to any of claims 16-18, 20-23, which is characterized by the fact that the indicated polypeptide is able to bind directly or indirectly to MOKT-1-B4 - mAbo with any proteins that bind to MOCT-1, where MOCT-1, in turn, specifically binds to PA5-IS, or which are able to bind directly or indirectly to MOCT-1 , and/or with any proteins that bind to MOKT-1, while MOKT-1, in turn, binds TKABO, which, in turn, binds to ro5-16. 25. The method of modulating cell death or inflammatory processes according to any one of claims 15 or 19, which is characterized in that said polypeptide is able to bind directly or indirectly to MOKT-1 mAb or to any binding proteins with MOKT-1, while MOKT-1, in turn, specifically binds to EAZ-IS, or which are able to bind directly or indirectly to MOKT-1, and/or to any of the 70 proteins , which bind to MOKT-1, while MOKT-1, in turn, binds TKABO, which, in turn, binds to ro5-16. 26. The method of isolation and identification of the polypeptide according to claim 11, which includes the use of the yeast dihybrid method, in which the sequence encoding the specified MOKT-1 protein and/or the protein binding to MOKT-1 is contained in one hybrid vector, and the sequence from the cDNA or from the genomic DNA library /5 is in the composition of the second hybrid vector, with the fact that the vectors are then used to transform host yeast cells, and the positively transformed cells are isolated by subsequent extraction of the specified second hybrid vector in order to obtain a sequence that encodes a protein that binds to the indicated MOKT-1 protein and/or to MOKT-1 binding proteins. 27. The method of modulating the effect of the EAZ-K or TME ligand on human cells according to any of claims 16-18, 20-23, which is characterized by the fact that the indicated polypeptide is at least one of the C1 isoforms, one of its analogues, a fragment or derivative 28. The method of modulating cell death or inflammatory processes according to any one of claims 15 or 19, which is characterized in that the specified polypeptide is at least one of the C1 isoforms, one of its analogues, a fragment or a derivative. high school 29. The method of isolating and identifying a polypeptide according to claim 26, which is characterized by the fact that the specified polypeptide is at least one of isoform 1, one of its analogues, a fragment or a derivative. (8) 30. A method of modulating the MOKT-1-induced effect or the effect of a protein that binds to MOKT-1, or the effect induced by other proteins on ip migo cells, which includes processing said cells in a manner according to any of claims 15-29 with a DNA sequence according to claim 1 or the polypeptide according to claim 11, with the fact that the indicated processing of Ge! leads to the enhancement or suppression of the specified effect mediated by MOKT-1 or a protein binding to MOKT-1, or another effect mediated by proteins and thereby also the effect of GAB-K or srob-K or other cytotoxic mediators or inducers mediating this influence. yu 31. The method of modulating the MOKT-1-induced effect or the effect of a protein that binds to MOKT-1, or the effect induced by other proteins on ip mygo cells according to clause ZO, which is characterized by the fact that the specified "polypeptide is that part of the polypeptide that is specifically involved in binding to MOKT-1 or to a protein that binds to MOKT-1 or to another protein. 32. The method of modulating the MOKT-1-induced effect or the effect of a protein that binds to MOKT-1, or the effect induced by other proteins on the cells of the ip miigo according to pp. 30 or 31, which is characterized by the fact that said polypeptide is any of the C1 isoforms capable of enhancing the effect caused by MOKT-1, or a protein that binds to MOKT-1, or another effect on cells associated with with proteins, and thus the effect of GAZ-K or po5-K or pl») with other cytotoxic mediators or inducers affecting cells. 33. The method of modulating apoptotic processes or processes of programmed death of cells of the brain, which;" includes the treatment of the specified cells with one or more polypeptides according to claim 11, while the specified treatment of the specified cells includes the introduction into the specified cells of the specified one or more Quantity of polypeptides in a form suitable for their intracellular introduction or introduction into the specified cells of the DNA sequence according to claim 1 in the form of a suitable vector containing the specified sequence, while the specified vector is able to ensure the introduction of the specified sequence into the specified cells in such a way that the specified sequence is expressed in the specified cells. c 34. A method of screening a ligand capable of binding to a polypeptide according to claim 11, which includes contacting a 5p affinity chromatographic matrix, to which the specified polypeptide is attached, with a cell extract so that the ligand binds to the specified matrix, followed by elution, isolation and analysis of the indicated He) ligand. 35. A method for screening a DNA sequence encoding a ligand capable of binding to a polypeptide according to claim 11, which includes the use of a dihybrid yeast method, in which the DNA sequence encoding the specified polypeptide is part of one hybrid vector, and the sequence from cDNA or from the DNA genomic library are included in the composition of the second hybrid vector, the transformation of the indicated yeast F) host cells with the indicated vectors, the selection of positively transformed cells and the extraction of the indicated second hybrid vector ka in order to obtain the DNA sequence encoding the indicated ligand. 36. A method of identifying and obtaining a ligand capable of modulating cellular activity modulated/mediated by MOKT-1 or proteins binding to MOKT-1, which includes: (a) screening for a ligand capable of binding to a polypeptide that includes at least part of MOKT-1 or MOKT-1-binding proteins selected from MASN proteins, Mena proteins and other proteins binding to MOT-1; (5) identification and characterization of a ligand, other than MOKT-1, or specified proteins that bind 65 to MOKT-1, or parts of a receptor from the TME/ChMOR receptor family, identified during said screening for the ability to bind yawning; and (c) obtaining said ligand in essentially isolated and purified form. 37. A method of identifying and obtaining a ligand capable of modulating cellular activity modulated or mediated by a polypeptide according to claim 11, which includes: (a) screening a ligand capable of binding to a polypeptide, including a polypeptide encoded by a DNA sequence according to any with (a)-(c) for claim 1; (5) identification and characterization of a ligand, other than MOKT-1, or proteins that bind to MOKT-1, or parts of a receptor from the TME/MOR family of receptors identified in said screening for said binding ability; and 70 (c) obtaining said ligand in essentially isolated and purified form. 38. A method of identifying and obtaining a ligand capable of modulating cellular activity modulated/mediated by protein 1, comprising: (a) screening a ligand capable of binding to a polypeptide comprising a polypeptide encoded by a DNA sequence of any of ( a)-(c) for claim 1; p) identification and characterization of a ligand, other than MOKT-1 or proteins that bind to MOKT-1, or parts of the receptor from the TME/MOR receptor family, detected during the specified screening for the specified binding ability ; and (c) obtaining said ligand in substantially isolated and purified form. 39. A method of identifying and obtaining a molecule capable of directly or indirectly modulating cellular activity modulated/mediated by protein 1, which includes: (a) screening of a molecule capable of modulating activity modulated/mediated by protein 1; (5) identification and characterization of the specified molecule; and (c) obtaining said molecule in essentially isolated and purified form. 40. The method of identification and production of a molecule capable of directly or indirectly modulating cellular tissue activity modulated/mediated by a polypeptide according to claim 11, which includes: (a) screening of a molecule capable of modulating activity modulated/mediated by a polypeptide according to i) claim 11; (5) identification and characterization of the indicated molecule; and (c) obtaining said molecule in essentially isolated and purified form. Gee! zo 41. A pharmaceutical composition that includes as an active ingredient a substance selected from the group consisting of a DNA sequence according to claim 1, a vector according to claims 5 or 6, a polypeptide according to claim 11, or an antibody according to claim 13 and, optionally, a pharmaceutically acceptable carrier. yu 42. Diagnostic composition, including the antibody according to claim 13. " and - - and? -i sh» 1 name) iChe) name) 60 b5 -58v-