The dna sequence encoding a polypeptide that binds to mort-1 (options), polypeptide (variants) and a method thereof, a vector, a method of modulating the action of the ligand fas-r or tnf on cells (options), a method of processing cells, isolation and identification of proteins, the method of modulation induced mort-1 action or induced mort-1 binding protein activity in cells
The invention relates to biotechnology, in particular genetic engineering, and can be used to modulate the action of ligand FAS-R or TNF on cells. The polypeptide has the ability to communicate with MORT-1 and affect intracellular process signal initiated by binding of FAS ligand to its receptor or the binding of TNF to P55 TNF-R, is obtained by culturing cells transformed by a vector containing a DNA encoding the polypeptide. Modulation of action of the ligand FAS-R or TNF on cells or modulation induced MORT-1 action is performed with the use of the polypeptide, binding to MORT-1, or a nucleotide sequence that encodes it. Pharmaceutical composition for modulation of the action of the ligand FAS-R or TNF on cells contains a polypeptide, binding to MORT-1. The invention allows to develop products for treating tumor or HIV-infected cells. 13 N. and 42 C.p. f-crystals, 37 ill., table 4.
The scope of the invention
This invention relates in General to the field of receptors belonging to the superfamily of receptors of the TNF/NGF, and regulation of their biological functions. TNF/NGF-receptor superfamily includes receptors, as P55 and P75 receptors fact called FAS-R) and others. More specifically, this invention relates to new proteins that are associated with protein MORT-1 (or FADD), and more specifically, it refers to one such MORT-1-binding protein, referred to here as the IAIS.
Thus, the present invention relates in General to new proteins that can modulate or mediashout function MORT-1 or other proteins that are directly or indirectly associated with MORT-1. In particular, this invention relates to IAIS, its reception and its applications, as well as to various new isoforms IAIS, their preparation and applications.
Prerequisites related field knowledge
The tumor necrosis factor (TNF-) and Lymphotoxin (TNF-) (hereinafter TNF will be treated as a TNF-and TNF-) is a multifunctional Pro-inflammatory cytokine, produced mainly by mononuclear phagocytes, which have many effects on cells (Wallach, D. (1986) In: Interferon 7 (Ion Gresser, ed.), pp.83-122, Academic Press, London; and Beutler and Cerami (1987)). As TNF-and TNF-initiate their effects by binding to specific cell-surface receptors. Some of these effects JW is rowanne virus cells and to increase the antibacterial activity of granulocytes. The way TNF helps to protect the body against tumors and infectious agents and contributes to the recovery from damage. Thus, TNF can be used as an antitumor agent, where it binds to its receptors on the surface of tumor cells and thereby initiates the events leading to tumor cell death. TNF can also be used as anti-infective agent.
However, as TNF-and TNF-also have adverse effects. There is evidence that overproduction TNF-can play a major pathogenic role in some diseases. For example, it is known that the actions of TNF-primarily on vascular network, are the main cause of symptoms of septic shock (Tracey et al., 1986). In some diseases, TNF can cause weight loss (cachexia) by suppressing the activity of adipocytes and induce anorexia, and therefore, TNF-was named cajetina. He was also described as a mediator of tissue damage in rheumatic diseases (Beutler and Cerami, 1987) and as the main mediator of the damage observed is m the process and many other diseases.
Two different, independently expressed, receptor, TNF-R P55 and P75, which bind specifically as TNF-and TNF-initiate and/or mediashout described above biological effects of TNF. These two receptors are structurally different intracellular domains, suggesting different transmit their signal (See. Hohmann et al., 1989; Engelmann et al., 1990; Brockhaus et al., 1990; Leotscher et al., 1990; Schall et al., 1990; Nophar et al., 1990; Smith et al., 1990; Heller et al., 1990. However, cellular mechanisms, for example, various proteins, and possibly other factors that are involved in intracellular signaling of TNF-R P55 and P75, is yet unknown. This intracellular signal transmission, which usually occurs after binding of the ligand, i.e., TNF (or), with the receptor responsible for the beginning of a cascade of reactions that eventually lead to the observed cell response to TNF.
With regards to the above causes cell death actions of TNF, in most studied so far cells this effect is triggered mainly TNF-R P55. Antibodies against the extracellular domain (landscapebased domain) TNF-R P55 can start causing cell death dececco consider the first stage in generating intracellular process of signal transmission. Further, mutational studies (Brakebusch et al., 1992; Tartaglia et al., 1993) showed that the biological function of TNF-R P55 depends on the integrity of its intracellular domain, and therefore an assumption was made that the initiation of intracellular signal transduction, leading to the calling cell death action of TNF, occurs as a consequence of the Association of two or more intracellular domain of TNF-R P55. In addition, TNF (and) occurs in the form of homotrimer and it was assumed that, as such, it induces intracellular signaling through the TNF-R P55 through its ability to bind and cross-react with receptor molecules, i.e., to cause aggregation of the receptor.
Another member of the superfamily of TNF/NGF receptor is FAS-receptor (FAS-R), which was also called FAS-antigen, protein cell surface expressed in various tissues and has homology with a number of cell surface receptors, including TNF-R, NGF-R and FAS-R mediium the death of cells in the form of apoptosis (Itoh et al., 1991) and, apparently, serves as a negative selectivity agent self-reactive T cells, i.e., during maturation of T cells FAS-R Mediaset poptopics the death of T-cells recognizing autology, is such autoimmune disease of human systemic lupus erythematosus (SLE) (Watanabe-Fukunaga et al., 1992). The ligand for FAS-R is likely to be associated with cell surface molecule, which are, among others, killer T cells (or cytotoxic T-lymphocytes CTLs), and therefore, when such CTL come into contact with cells bearing FAS-R, they are able to induce apoptopic death bearing FAS-R cells. Next, were obtained antibodies that are specific for FAS-R, and these antibodies are able to induce apoptopic death bearing FAS-R cells, including murine cells transformed with cDNA that encodes FAS-R man (Itoh et al., 1991).
Although some of the cytotoxic effects of lymphocytes mediasource interaction produced by lymphocytes ligand with widely occurring cell surface receptor FAS-R (CD95), which has the ability to run cell death, it was also found that various other normal cells are T lymphocytes that Express FAS-R on their surface and can be killed by running this receptor. There is a suspicion that uncontrolled induction of this process of killing cells contributes to the destruction of tissue in some diseases, nadikai activity of the FAS-R may have therapeutic potential.
In contrast, since it was also found that some cancer cells and HIV-infected cells carry on their surface FAS-R, antibodies against FAS-R or FAS ligand-R can be used to start Mediaroom FAS-R cytotoxic effects in these cells and thereby provide a means of dealing with such malignant cells or HIV-infected cells (see Itoh et al., 1991). Finding other ways to increase the cytotoxic activity of FAS-R may, therefore, also to have a therapeutic perspective.
Have long felt need to provide a way of modulating the cellular response to TNF (or) and FAS ligand-R. for Example, in the above-mentioned pathological situations, where the overexpression of TNF or FAS ligand-R, preferably the inhibition induced by TNF or FAS ligand-R causing cell death effects, although in other situations, for example, when used in the healing of wounds, preferably enhancing effect of TNF, or in the case of FAS-R, tumor or HIV-infected cells, it is desirable amplification mediated FAS-R effect.
A number of approaches have been taken by the laboratory of the applicants (see, for example, European Application Nos. EP 186833, Yeri using antibodies against TNF or by using soluble TNF receptors (which are essentially soluble extracellular domains of these receptors) to compete for binding of TNF related with cell surface TNF-R. Next, on the basis that the binding of TNF to its receptors is required for induced TNF cellular effects, laboratory applicants (see, for example, EPO 568925) were taken approaches to modulate this effect of TNF by modulating TNF-R.
Briefly, EPO 568925 describes a method of modulating signal transduction and/or cleavage of TNF-R, resulting in peptides or other molecules can interact either with the receptor or effector proteins that interact with the receptor, thus modulating the normal function of TNF-R. EPO 568925 described the construction and characterization of various mutants of TNF receptors with mutations in the extracellular, transmembrane and intracellular domains of TNF-R P55. In this way, the areas in the above domains of TNF-R P55 were identified as essential for the functioning of this receptor, i.e., binding of ligand (TNF) and subsequent signal transduction and intracellular signal transduction, which in the end leads to the observed effect of TNF on these cells. Further, there is also described a number of approaches for separation and identification of proteins, peptides or other factors that can communicate with various areas in the above domains of TNF-R approaches for isolation and cloning of DNA sequences encoding such proteins and peptides; to construct expressing vectors to obtain these proteins and peptides; and to generate antibodies or their fragments that interact with the TNF-R or with the above proteins and peptides that bind to different regions of TNF-R, also described in EPO 568925. However, EPO 568265 does not feature the actual proteins and peptides that bind to the intracellular domain of TNF-R (e.g., TNF-R P55) and does not describe a yeast dvuhserijnyj approach for isolation and identification of such proteins and peptides that bind to the intracellular domain of TNF-R. similarly, up to the present time have not been described proteins or peptides capable of binding to the intracellular domain of FAS-R.
Thus, when it is desirable inhibition effect of TNF or FAS ligand-R, it would be desirable to decrease the amount or activity of receptors of the TNF or FAS-R on the cell surface, whereas the increase in the number or activity of receptors of the TNF or FAS-R, it would be desirable, when the reinforced effect of TNF or FAS ligand-R. For this purpose, the promoters as TNF-R P55 and TNF-R P75 were sequenced, analyzed, and was found several key sequence motifs that are SPE is to wirematic at the level of their promoters, i.e., inhibition of transcription from these promoters to reduce the number of receptors and increase transcription from these promoters to increase the number of these receptors (ER 606869 and WO 9531206). Should be communicated to relevant studies concerning the regulation of FAS-R at the level of the promoter of the gene FAS-R.
Although it is known that receptors of the tumor necrosis factor (TNF) and structurally related receptor FAS-R run in the cells, the stimulation produced by leukocytes ligands, destructive activity, which lead to their own death, the mechanisms of this launch is still little understood. Mutational studies show that in the signal transmission FAS-R and TNF-R p55 (p55-R) for cytotoxicity involves various areas in their intracellular domains (Brakebusch et al., 1992; Tartaglia et al., 1993; Itoh and Nagata, 1993). These areas (the"domain of death") are similar sequence. "The death domains of both receptors FAS-R and p55-R tend to self. Their self-Association is likely to enhance the aggregation of the receptors, which is required for the initiation of signal transmission (see Song et al., 1994; Wallach et al., 1994; Boldin et al., 1995) and at high levels of expression of receptors can trigger ligand-independent signaling (Bolding et al., 1995).
Thus, it is improper to the superfamily of TNF/NGF, such as the action of TNF or FAS ligand-R cells by meditirovaniya intracellular process of signal transmission, which is reputed to be controlled to a large extent the intracellular domain (IC) receptors belonging to the superfamily of receptors of the TNF/NGF, such as the intracellular domain of TNF receptors, i.e., TNF-R P55 and P75 (RS and RS, respectively), as well as FAS-R.
Some of the cytotoxic effects of lymphocytes is mediated by the interaction produced by the lymphocytes of the ligand to FAS-R (CD95), a widely occurring cell surface receptor, which is able to run cell death (see Nagata and Goldstein, 1995). In killing of cells by mononuclear phagocytes involved a pair of ligand-receptor, TNF and its receptor p55-R (CD120), which is structurally related FAS-R and its ligand (see also Vandenabeele et al., 1995). Like other induced receptor effects, induction of cell death receptors TNF and FAS-R is carried out through a series of protein-protein interactions, leading from binding of the ligand to the receptor to the final activation functions enzymatic effector that in the case of these particular receptors leads to cell death. Previous studies have found the interaction of non-enzymatic protein-protein, initiating peritumorally their intracellular domains (Brakebusch et al., 1992; Tartaglia et al., 1993; Itoh and Nagata, 1993), increased predisposition motives domains of death to self (Boldin et al., 1995a) and induced the binding of two cytoplasmic proteins (which can also communicate with each other) with the intracellular domains of the receptor - MORT-1 (or FADD with FAS-R (Boldin et al., 1995b; Chinnaiyan et al., 1995; Kischkel et al., 1995) and TRADD with the p55-R (Hsu et al., 1995; Hsu et al., 1996).
Three proteins that bind to the intracellular domain of FAS-R and p55-R when the area of the "domain of death", which participate in the induction of cell death receptors through heteroassociation homologous areas and are independently able to start cell death were identified using the method yeast twohybrid screening. One of them is protein MORT-1 (Boldin et al., 1995b), also known as FADD (Chinnaiyan et al., 1995), which binds specifically with FAS-R. Second, TRADD (see also Hsu et al., 1996), is associated with the p55-R and the third, RIP (see also Stranger et al., 1995), is associated with FAS-R and p55-R in Addition to their binding to FAS-R and p55-R, these proteins are also able to communicate with each other, which provides a functional cross-talk" between the FAS-R and p55-R. These bindings are made through conservative motif sequence, the module domain of death", the total for these prescriptions is is spontaneously with FAS-R, in mammalian cells, this binding takes place only after stimulation of the receptor, which suggests that the MORT-1 is involved in initiating the communication signal FAS-R. MORT-1 does not contain any motif sequence characteristic of enzymatic activity, and, consequently, its ability to run cell death, apparently, is not associated with the inherent MORT-1 own activity, but rather the activation of some other proteins (protein) that bind MORT-1 and act later in the cascade signal. It has been shown that cellular expression of mutant MORT-1 without N-terminal part of the molecule, inhibits the induction of cytotoxicity of FAS/APO1 (FAS-R or P55-R (Hsu et al., 1996; Chinnaiyan et al., 1996), suggesting that this N-terminal region is involved in the signal transmission of the calling cell death activity of both receptors via protein-protein interactions.
Recent studies have shown the participation of a group of cytoplasmic thiol proteases, which are structurally similar to the CED3 protease Caenorhabditis elegans and turns interleukin-1mammalian enzyme (ICE), the occurrence of various physiological processes of cell death (review in Kumar, 1995, and Henkart, 1996). There were also some who and induced FAS-R and TNF-R. it Was found that specific inhibitors of these proteases and coded by two virus proteins that block their function, protein cowpox virus crmA and protein R35 baculovirus protect cells against cellular cytotoxicity (Enari et al., 1995; Los et al., 1995; Tewari et al., 1995; Xue et al., 1995; Beidler et al., 1995). Rapid cleavage of specific cellular proteins, apparently mediated by a protease (protease) family CED3/ICE observed in cells shortly after stimulation of the FAS-R or TNF receptors. To date no reports of identity specific D3/ICE-related proteases (proteases) and on the mechanisms of activation of these proteases receptors.
The purpose of this invention is the provision of novel proteins, including all their isoforms, analogs, fragments or derivatives, which is able to connect with MORT-1, which itself binds to the intracellular domain of FAS-R, and these new proteins influence the process of intracellular signal transduction by binding of FAS ligand to its receptor.
Another purpose of this invention is to provide antagonists (e.g. antibodies, peptides, organic compounds, or even some isoforms) Viseu the I process of signal transmission or more specifically, cellular cytotoxicity, when this is desirable.
A further purpose of this invention is the use of these new proteins, their analogs, fragments and derivatives for isolation and characterization of additional proteins or factors that may be involved in the regulation of receptor activity, for example, other proteases, which break down new proteins, making them biologically active, and/or to highlight and identify other receptors above during signal transmission, with which these new proteins, their analogs, fragments and derivatives of contact (for example, other receptors FAS or related receptors) and, consequently, the functioning of which they also participate.
Another purpose of this invention is the provision of inhibitors, which can be introduced into cells to bind or interact with the protease IAIS and inhibition of their proteolytic activity.
In addition, the purpose of this invention is the use of vysheupomyanutyh new proteins and their analogs, fragments and derivatives as antigens to obtain them polyclonal and/or monoclonal antibodies. These antibodies, in turn, can be used, nab, the e cell line.
Further, these antibodies can be used for diagnostic purposes, for example, for identifying disorders related to abnormal functioning of the cellular effects mediated FAS-R or other related receptors.
A further purpose of this invention is to provide pharmaceutical compositions containing the above-mentioned new proteins or their analogs, fragments or derivatives, and pharmaceutical compositions containing the above-mentioned antibodies or other antagonists.
In accordance with this invention, was discovered a new protein, IAIS, which is able to communicate or interact with MORT-1, which itself binds to the intracellular domain of FAS-R IAIS may function as effector path component of cell death initiated by the binding of FAS ligand to FAS-R on the cell surface, and this is due to the fact that at least some isoforms IAIS, apparently, are active intracellular proteases. It was suggested that protease family CED3/ICE involved in apoptosis triggered by FAS-R. MORT-1 (or FADD) binds to the intracellular domain of FAS-R when you activate this receptor, and new proteins IAIS managemenet, really exists in the form of multiple isoforms, some of the isoforms have a region of homology CED3/ICE that has proteolytic activity (domain proteolytic activity), and causes cell death upon expression in these cells. Thus, activation of this new homolog of the CED3/ICE (i.e., different isoforms IAIS having proteolytic domain) FAS-R (through interaction with MORT-1), apparently, is the effector component mediated FAS-R path (cascade reactions) cell death.
In addition, the IAIS, apparently, it also functions as the effector component of the path cell death initiated by the binding of TNF to p55-R on the cell surface, which occurs through an indirect mechanism linking MORT-1 with TRADD, a protein that binds to the intracellular domain of the p55-R (Hsu et al., 1995), with subsequent (or simultaneous) binding IAIS with MORT-1 activation IAIS to an active protease that is involved in the implementation of cell death.
It should also be noted that, although the IAIS, in particular, isoforms MACH1, shows all the signs of a sequence, which are crucial for the function of proteases CED3/ICE, but she really has some different their own signs succession is I.
MORT-1 ("the Mediator of the Toxicity of the Receptor", Boldin et al., 1995b), previously called the HF1, are able to bind with the intracellular domain of FAS-R. This FAS-IC-binding protein, apparently, acts as a mediator or modulator of the action of the ligand FAS-R cells by meditirovaniya or modulation process, intracellular signal transduction, which usually occurs after binding of the ligand FAS-R at the cell surface. It was shown that, in addition to its FAS-IC-binding specificity, MORT-1 has other characteristics (see Example 1), for example, it has a region homologous to regions of "death domain" (DD) TNF-R P55 and FAS-R (p55-DD and FAS-DD), and therefore also capable of self. MORT-1 itself can also activate cellular cytotoxicity, and this ability may also be related to its ability to self. It was also found that coexpression this area in MORT-1 (HF1), which contains sequence homology "domain of death" (MORT-1-DD, present in the C-terminal part of MORT-1), strongly counteracts the induced FAS cell death, as would be expected on the basis of its ability to communicate with the "domain of death" FAS-IC. Further, in some experimental conditions, it was found that coexpressed part of MORT-1, the address induced FAS cell death and to a slightly increased induced FAS cellular cytotoxicity (if it ever was).
Thus, it is likely that the MORT-1 binds with other proteins involved in intracellular signal transduction. Therefore, MORT-1-binding proteins can also act as indirect mediators or modulators of the action of the ligand FAS-R cells by meditirovaniya or modulate the activity of MORT-1; or these MORT-1-binding proteins can act as mediators or modulators associated with MORT-1 intracellular process of signal transmission through meditirovaniya or modulate the activity of MORT-1, which, as noted above, has apparently independent ability to activate cellular cytotoxicity. These MORT-1-binding proteins can also be used in any of the standard ways of screening to select, identify and characterize additional proteins, peptides, factors, antibodies, etc. that can participate in the associated with MORT-1 or associated with FAS-R the process of signal transmission, or may be elements of other intracellular processes the transmission signal. Such MORT-1-binding proteins have been isolated and described herein (see Example 2 and Example 3). Some of these MORT-1-binding proteins, referred to here as the IAIS, was originally cloned, Sequeira ORF-B; IAIS binds to MORT-1 is strongly and specifically; the binding site IAIS in MORT-1 is against the course of transcription from the motive of "domain of death" MORT-1; district ORF-B MACH is interacting with MORT-1 part; and MACH able to self and to the self-induction of cellular cytotoxicity.
In accordance with this invention, at the present time it has been shown (as mentioned above) that MACH really exists in several isoforms. In addition, ORF-B MACH, mentioned above, is in fact one of the MACH isoforms, named here IAIS1 (see below).
Thus, this invention provides a DNA sequence encoding a protein, its analogs or fragments, the ability to communicate or interact with MORT-1, and this protein, its analogs or fragments capable of mediashout intracellular effect, mediarray FAS-R or p55-TNF-R.
In particular, this invention provides a DNA sequence selected from the group consisting of:
(a) cDNA sequence derived from the coding region of the native MORT-1-binding protein;
(b) DNA sequences capable of gibridizatsiya sequence (a) under conditions of moderate stringency and encoding biologically is ordinati of the genetic code, relative to the DNA sequences defined in (a) and (b) and which encode biologically active MORT-1-binding protein.
Another characteristic variant of the above-mentioned DNA sequences of the present invention is a DNA sequence that contains at least part of the sequence that encodes one protein isoforms IAIS selected from isoforms IAIS, named here MACH1, MACH2, MACH3, MACH1, IAIS2, IAIS3, IAIS4 and IAIS5.
Other typical variants of the DNA sequence of this invention, as noted above, are DNA sequences coding:
(a) isoforms IAIS selected from MACH1, MACH1 and IAIS3, having the amino acid sequence represented in SEQ ID No. 7, 5 and 8, respectively, and analogs and fragments of each of them;
(b) MACH1, having the amino acid sequence represented in SEQ ID No. 1 and its analogues and fragments;
(c) MACH1, having the amino acid polinomialnoi sequence, represented in SEQ ID No. 8, and its analogs and fragments.
This invention provides MORT-1-binding proteins and their analogs, fragments or derivatives encoded by any of the above sequences of the invention, and these proteins, analogs, fragments and derivatives are able to bind or interact with MORT-1 and mediashout intracellular effect, mediarray FAS-R or P55 TNF-R.
Characteristic variant of this invention is the MORT-1-binding protein, its analogs, fragments and derivatives are selected from at least one isoform IAIS group containing MACH1, MACH2, MACH3, MACH1, MACH2, IAIS3, IAIS4 and MACH5, which have at least part of its amino acid sequence.
This invention also provides vectors encoding the above-mentioned MORT-1-binding protein and its analogs, fragments or derivatives of this invention which contain the above DNA sequence of the present invention, and these vectors can be expressed in suitable eukaryotic or PR is operasie such vector; and the method for the MORT-1-binding protein, or analogs, fragments or derivatives of the present invention by growing the transformed host cells under conditions suitable for expression of the indicated protein, analogs, fragments or derivatives, perform post-translational modifications of the protein, as is necessary to obtain this protein, and extraction of the expressed protein, analogs, fragments or derivatives from the culture medium of transformed cells or cellular extracts of transformed cells. The above definition is intended to include all isoforms of the protein IAIS.
In another aspect, the invention also provides antibodies or their active derivatives or fragments specific to MORT-1-binding protein and its analogs, fragments and derivatives of the present invention.
Another aspect of the invention achieved through a variety of applications described above DNA sequences or proteins that they encode, in accordance with this invention, and these applications include (among others):
(i) Method of modulating the action of the ligand FAS-R or TNF on cells carrying FAS-R or p55-R capable of handling these cells one or more MORT is with MORT-1, which binds to the intracellular domain of FAS-R, or the ability to communicate with MORT-1, which binds to TRADD, which binds to the intracellular domain of p55-R, and consequently capable of modulating/mediaremote activity of the FAS-R or P55 TNF-R, and treatment of these cells involves the introduction into the cells of one or more proteins, analogs, fragments or derivatives in a form suitable for intracellular introduction, or introducing into the cells a DNA sequence that encodes one or more proteins, analogs, fragments or derivatives in the form of a suitable vector, bearing this sequence, and this vector is able to carry out the introduction of this sequence into these cells in such a way that this sequence is expressed in the cells.
(ii) a Method of modulating the action of the ligand FAS-R or TNF to cells at (i) above, in which the cell treatment involves the introduction into the cells of MORT-1-binding protein, or its analogs, fragments or derivatives in a form suitable for intracellular introduction, or introducing into the cells a DNA sequence that encodes a MORT-1-binding protein, or analogs, fragments or derivatives in the form of a suitable vector carrying this posledovatel, this sequence is expressed in the cells.
(iii) the Method according to (ii) above, in which the processing cells performed by transfection of cells with recombinant viral vector of an animal, comprising the stage of:
(a) constructing a recombinant viral vector of the animal carrying a sequence encoding a viral surface protein (ligand) that is capable of contact with a specific cell surface receptor on the surface of the carrier FAS-R or p55-R cells, and a second sequence encoding a protein selected from the MORT-1-binding protein and its analogs, fragments and derivatives, which upon expression in these cells capable of modulating/mediaremote activity of the FAS-R or p55-R; and
(b) infection of these cells by the vector (a).
(iv) a Method of modulating the action of the ligand FAS-R or TNF on cells carrying FAS-R or p55-R capable of handling these cells with antibodies or their active fragments or derivatives, in accordance with this invention, and this processing is performed by the provision of suitable compositions containing these antibodies, active fragments or derivatives of these cells, while exhibiting MORT-1-binding proteins or parts of these nazyvausia proteins are intracellular, the above composition is prepared for intracellular applications.
(v) a Method of modulating the action of the ligand FAS-R or TNF on cells carrying FAS-R or p55-R capable of handling these cells oligonucleotide sequence that encodes the antisense sequence of at least part of the protein sequence MORT-1-binding protein of the present invention, and this oligonucleotide sequence capable of inhibiting expression of MORT-1-binding protein.
(vi) the Method according to (ii) above for the treatment of tumor cells or HIV-infected cells or other with pathology of cells, including:
(a) constructing a recombinant viral vector of the animal carrying a sequence encoding a viral surface protein that can bind to specific receptor surface of tumor cells or other receptor with pathology of cells, and a sequence encoding a protein selected from the MORT-1-binding protein, analogs, fragments and derivatives of the present invention, when expression in the tumor, HIV-infected or another with the pathology of the cell can kill the cell;
(b) tumor or infection HIV infection is yrs, involving ribozymes the way in which a vector encoding the sequence of the ribozyme capable of interacting with a cellular mRNA sequence that encodes a MORT-1-binding protein according to this invention, is introduced into cells in a manner which allows expression of the ribozyme sequence in these cells, and, when the sequence of the ribozyme is expressed in cells, it interacts with the cellular mRNA sequence and cleaves the sequence that leads to inhibition of expression of MORT-1-binding protein in these cells.
(viii) a Method selected from a method in accordance with this invention, in which the MORT-1-binding protein that encodes the sequence contains at least one of the isoforms of IAIS, its analogs, fragments or derivatives according to this invention, which can specifically bind with MORT-1, which, in turn, specifically binds to FAS-IC, or who are able binds to MORT-1, which, in turn, binds to TRADD, which, in turn, binds to the P55-IC.
(ix) the Method of separating and identifying proteins, in accordance with this invention, capable of binding the th encoding a protein MORT-1, is located on one hybrid vector and sequence from a cDNA library or genomic DNA is on the second hybrid vector, and these vectors are used to transform yeast host cells and the positive transformed cells release with subsequent extraction of the second hybrid vector to obtain a sequence that encodes a protein that binds to the protein MORT-1 and represents the MORT-1-binding proteins.
(x) the Method according to any of items (i) to(ix), in which the MORT-1-binding protein is an isoform IAIS, here called MACH1, its analogs, fragments and derivatives.
(xi) the Method according to any of items (i) to(ix), in which the MORT-1-binding protein is an isoform IAIS, here called MACH1, its analogs, fragments and derivatives.
(xii) the Method according to any of items (i) to(ix), in which the MORT-1-binding protein is an isoform IAIS, here called IAIS3, its analogs, fragments and derivatives.
This invention also provides a pharmaceutical composition for modulating actions of ligand FAS-R or TNF on cells containing as an active ingredient any of sletty, analogs, derivatives, or mixtures;
(ii) a recombinant viral vector of the animal that encodes a protein capable of binding a cell surface receptor and encoding MORT-1-binding protein, or biologically active fragments or analogs, in accordance with the invention; and
(iii) oligonucleotide sequence encoding the antisense sequence of the sequence MORT-1-binding protein of the present invention, and this oligonucleotide can be second sequence of the recombinant viral vector of the animal (ii) above.
This invention also provides:
I. a Method of modulating induced MORT-1 action or induced MORT-1-binding protein activity in cells, capable of handling these cells, in accordance with the method of any of paragraphs (i) to(ix) above, MORT-1-binding proteins, their analogs or fragments, and this processing leads to the enhancement or inhibition mediamoo MORT-1 action and consequently also mediamoo FAS-R or p55-R steps.
II. The method corresponding to that described above in which the MORT-1-binding protein, its analogue, fragment or derivative is the part of MORT-1-binding protein, which specifically involved in associates that part of MORT-1-binding protein, which is specifically involved in binding to MORT-1 or by MORT-1-binding protein.
III. The method corresponding to that described above in which the MORT-1-binding protein is one of the isoforms IAIS selected from MACH1, MACH1 and MACH3, can enhance associated with MORT-1 action on cells and thereby increase associated with FAS-R or p55-R action on the cells.
As can be seen from the foregoing and from the detailed description below, IAIS can be used independently of the MORT-1-binding proteins, their identification and characterization can be performed in any of the standard ways of screening used for separation and identification of proteins, for example, yeast twohybrid way, ways affinity chromatography and any of the well known standard methods used for this purpose.
Provided other aspects and variations of the present invention arising from the following further detailed description of the invention.
It should be noted that throughout the description the following terms: "Modulation of the actions of the FAS ligand or TNF on cells; Modulating the actions of the MORT-1 or MORT-1-binding protein in cells" should be understood as vkljucenosti MORT-1 in the transformed yeast according to estimates twohybrid test expression-galactosidase.
Fig.2 depicts schematically a preliminary nucleotide (SEQ ID No. 1) and the decoded amino acid sequence (SEQ ID No. 2) MORT-1 (HF1), in which the "domain of death" is underlined, as well as the possible start site of translation, i.e., the underlined residue methionine at position 49 (bold, underline, M). The asterisk indicates the stop codon of translation (nucleotides 769-771). At the beginning and in the middle of each line given two numbers indicating the relative position of a nucleotide and / or amino acid sequence relative to the beginning of the sequence (5`-end), where the first digit indicates the nucleotide, and the second number refers to the amino acid.
Fig.3 - preliminary partial nucleotide sequence encoding a MORT-1-binding protein derived from the cDNA clone.
Fig.4A-C schematically depicts cDNA IAIS and coded it protein, where Fig.4A shows the structure of a cDNA IAIS, which encodes two open reading frames IAIS (ORF A and ORF-B), and the shaded area the ORF-B shows an area that has homology with the protein MORT-1; Fig.4B shows the decoded amino acid sequence (SEQ ID No. 5) for region ORF-A IAIS, and the underlined amino acid residues are, th is activity (SEQ ID No. 4) of the whole molecule cDNA IAIS (indicated MACH1).
Fig.5 depicts the results illustrate the interaction of MORT-1 and IAIS in transfected yeast cells.
Fig.6 depicts a chart ligand-independent start causing cell death effects in HeLa cells, transfected with a tetracycline-controlled expressing vectors, encoding the IAIS, in comparison with these effects in the same cells transfected with such vectors encoding other proteins, such as luciferase (luc, negative control), FA5-IC, MORT-1, and cells, cotransfection vectors, encoding the MORT-1 and IAIS.
Fig.7A and 7B show the amino acid sequence (SEQ ID No. 5) MACH1 (figs.7A). Fig.7B shows the sequence homology module MORT in MACH1, MORT-1 and PEA-15 (SEQ ID No. 6).
Fig.8 is a view diagram of interactions between a receptor and targets involved in the induction of cell death by FAS/APO1 and the P55 and the module domain of death is listed stripes, module MORT indicated in gray and the area of the CED3/ICE is indicated in black.
Fig.9A-C depicts the results illustrate the interaction in vitro MACH1 and its deletion mutants with MORT-1. Fig.9A shows the expression of these proteins and their molecular size and these proteins with GST-MORT-1, adsorbed on glutathione-agarose pellets (or, as a control, GST or GST fused to the intracellular domain of FAS-APO1). Fig.9C shows the results of immunoprecipitate merged various designs MORT-1 and IAIS using a variety of specific antibodies.
Fig.10 is a view diagrams of different isoforms IAIS.
Fig.11 is a schematic kulinarne building (analysis of primary structure) of the amino acid sequences of isoforms IAIS, MACH1 (SEQ ID No. 7), MACH1 (SEQ ID No. 5) and MACH3 (SEQ ID No. 8), and various well-known members of the family of protease CED/ICE, CED3 (SEQ ID No. 9), Ich-11/Nedd2 (SEQ ID No. 10), ICErelIII (SEQ ID No. 11), Tx/Ich/ICErelII (SEQ ID No. 13), CPP-32 (SEQ ID No. 30), Mcn2(SEQ ID NO. 31). Amino acid residues are numbered on both the left and right of each sequence. The dashed line; the gaps in the sequence allow optimal alignment. Amino acids that are identical in at least three members of a family of proteases CED3/ICE, enclosed in blocks. Modules MORT progress against transcription relative to the region of homology CED3/ICE enclosed in blocks. The sites C-terminal to the divisions used in this study is indicated by a broken line isoforms IAIS (blocks 1-4), indicates the upper lines. In the region of homology CED3/ICE amino acid residues that are aligned with the remains inside the ICE, which may participate in catalytic activity according to x-ray diffraction analysis indicated the following: Residues presumably involved in catalysis, matching His237/ Gly238and Cys285in ICE, marked by solid circles below the line of the building (n). Residues that constitute the binding pocket for the carboxylate side chain of the P1 Asp corresponding to Arg179, Gln283, AGD341and Sr347in ICE, marked with white circles below the line of building. The remains l progress against transcription from the residues corresponding to Cys285and ICE and residues Arg and Gly in the course of transcription relative to s are conservative in all previously described protease family CED3/ICE. The remains, relatively proximal residues of the P1-P4substrate, marked by triangles below the line of building). Known previously proposed sites of cleavage of the Asp-X and the potential cleavage sites found in similar positions in the IAIS, enclosed in blocks. Arrows indicate the N - and C-terminal ends of subjudice is illustrating protease activity MACH1 at 15 min (Fig.12A), 30 min (Fig.12V), 60 min (Fig.12C), 90 min (Fig.12D), 180 min (Fig.12E). Fig.12F shows proteolytic activity in time when one characteristic of the substrate concentration.Fig.13A and 13B show protease active region of homology CED3/ICE in IAIS. And, the Kinetics of cleavage derived from PARP-sequence fluorogenic substrate, AC-DEVD-AMC (50 μm), extracts of E. Coil, expressing the GST-fused protein region of homology CED3/ICE MACH1 (Ser217across From the end of this protein (), in comparison with the absence of cleavage extracts of bacteria expressing GST-fused protein full-MACH1or region of homology CED3/ICE, which s360replaced by Ser (), or extracts of bacteria expressing GST-fused products of any of the two potential proteolytic products of the region of homology CED3/ICE (Ser217- s373(and Sr375- s478With the end of this protein (). In the Substrate concentration dependence of the cleavage of Ac-DEVD-AMC. The substrate was incubated for 180 min with extracts /82/820857.gif">). Cleavage of this substrate extracts inhibited in the presence of iodixanol acid. Ac-YVAD-AMC, fluorogenic substrate corresponding to the cleavage site in the precursor of IL-1that was not digested ().
Fig.14A-D show cell death, mediated MACH1 and IAIS2.
Fig.15 graphically depicts cell death, mediated MACH1 and IAIS2.
Fig.16A-D show the morphology of cells in which cell death was induced or blocked.
Fig.17 shows a chart that molecules IAISthat contain a non-functional area of the CED3/ICE, inhibit the induction of cell death receptor p55-R.
Fig.18 shows that the molecules IAISthat contain a non-functional area of the CED3/ICE, inhibit the induction of cell death by FAS/APO1.
Fig.19 shows the death of HeLa cells, which temporarily Express FAS/APO1.
Detailed description of the invention
This invention relates, in a first aspect, to a new MORT-1-binding proteins, which are able to communicate or interact with MORT-1 and thereby to bind the nom P55 TNF-R, associated protein TRADD (see Example 2 and described above) and which protein TRADD binds to MORT-1. Thus, the MORT-1-binding proteins of the present invention are considered as mediators or modulators of FAS-R, participating, for example, in the transmission signal, which is initiated by the binding of FAS ligand to FAS-R, and in this way participate in the process of signal transmission, which is initiated by the binding of TNF to p55-R. Of the MORT-1-binding proteins of the present invention includes newly discovered isoforms IAIS, amino acid and DNA sequences which are new sequences, not found in the data banks of DNA sequences or amino acid sequences "GENBANK" or "PROTEIN BANK.
More specifically, in accordance with this invention, have been described several homologues of mammalian protease nematodes CED3. They were identified as isoforms IAIS (isoforms IAISand IAIS), which, although they are closely related, discover indeed some differences in the structure and substrate specificity, and therefore may have slightly different functions in mammalian cells. Indeed, izvestnikh IL-1while CED3, as clearly shown, is an effector of programmed cell death. This last role is, apparently, the role of at least some of the homologues from mammals (some isoforms IAIS). Amino acid sequence MACH1 shows a close similarity with SRR, the closest known homologue of mammalian CED3. The substrate specificity of the IAIS also similar substrate specificity SRR, except that MACH1, apparently, has a more limited substrate specificity than SRR. SRR predominantly cleaves substrate peptides corresponding to the site of cleavage at the poly(ADP-ribose)-polymerase (PARP), although it also has some proteolytic activity against peptide corresponding to the site of cleavage ICE in the precursor of IL-1. However, MACH1 able, apparently, to cleave only produced from PARP sequence. This similarity MACl SRR and CED3 and no similarity with ICE are consistent with the idea that MACH1 operates like CED3 as a regulator of cell death. MACHall other family members CED3/ICE. C-terminal part of the MACH1, against the course of transcription relative to the region of homology CED3/ICE, never finds affinity with this area of progress against the transcription of any of the other homologues. There are also some unique characteristics in relation to the region of homology CED3/ICE of this protein. These differences suggest that MACH1 belong to different evolutionary branches of this family and its involvement in cell death differs from participation previously described homologues of the CED3/ICE.
One important difference may relate to the manner in which regulated the function of this protease. Because they are involved in development-related processes of cell death and induced receptor immune cytolysis, the splitting of proteins by proteases family CED3/ICE must comply with the regulation as signals, which are formed in the cell, and the signals emanating from receptors on the cell surface. In a related development processes of cell death activation of these proteases include, apparently, the mechanisms that affect gene expression, leading to increased synthesis of these proteases, as well as to reduced synthesis of proteins, like BCL-2, which are antagonists and can be killed or TNF ligand FAS-R, even when their activity protein synthesis completely ingibirovany (actually in this case they are killed more efficiently), and remain stimulus-dependent under these conditions. Thus, the activation of proteases family CED3/ICE receptors TNF and FAS-R is carried out through a mechanism that does not depend on protein synthesis. The unique properties of the sequence MACH1 can allow her to take part in this mechanism.
As far as known to applicants, to date has not been found another protease, communicates directly or through adaptery protein intracellular domain of the receptor to the cell surface. Although on the basis of the method steps associated with the receptor proteins that have other enzymatic activity, it seems likely that binding of MACH1 and MORT-1 makes it possible to stimulation by activity MACH1 when you run FAS-R. This may also allow the activation of this protease receptor p55-R through binding to MORT-1 with TRADD, which is associated with the p55-R.
It was found that other members of the family CED3/ICE show full activity only after proteolytic processing, which is either the toxic effect, resulting from the co-expression of two major potentially self-cleaving products MACH1, in contrast to the absence of cytotoxicity in cells that Express the full-sized region of homology CED3/ICE, consistent with the possibility that MACH1 acquires full activity only after processing. The enzymatic activity observed in lysates of bacteria expressing the full-sized region of homology CED3/ICE, apparently, reflects the self-processing protein produced under these conditions, or processing some bacterial proteases. How is this processing within mammalian cells and how this can be caused by running FAS-R and p55-R, unknown and it is also unclear what the relative contribution gives proteasa activity MACH1 in induced FAS-R and TNF-R cytotoxicity. Evaluation of this contribution is complicated by the fact that also the expression of MACH1 deprived region of homology CED3/ICE, causing significant cytotoxicity. Presumably, this cytotoxicity reflects the ability MACH1 contact MACH1. Due to this the molecules MACH1, which are endogenous to transtitional cells. Such a mechanism may well explain the cytotoxicity observed when molecules that act early in the process regarding IAIS (MORT-1, TRADD, or domains of cell death p55-R or FAS-R) sverkhekspressiya in the cells. However, at present it cannot be excluded that the cytotoxicity observed in induced expression IAIS or molecules acting before IAIS, reflects, in addition to the proteolytic activity of the region of homology CED3/ICE in the IAIS, the activation of some other mechanisms that are perceived to be involved in the cytotoxic action of FAS-R and p55-R (for example, the activation of neutral or acidic sphingomyelinase). Also, it is possible that the proteolytic activity of the region of homology CED3/ICE has other functions besides the induction of cytotoxicity. A clearer understanding of the functioning of MACH1 should be achieved by the identification of endogenous substrate proteins that are broken down when you activate MACH1. The means of the destruction of optional activity MACH1, for example, the expression of inhibitory molecules, will also contribute to the understanding of funkcijas1, it can be natural inhibitors of this protease. It was shown that the existence of alternative splaisiruemym isoforms for some other family members CED3/ICE is a way of physiological restriction of the function of these proteases. It was reported that some isoforms of these other proteases act as natural inhibitors full isoforms, apparently, through the formation of inactive heterodimers with them. It may be for some isoforms IAIS, for example, IAIS3, in which there is no potential N-terminal cleavage site, and mutants MACH1, the region of homology CED3/ICE which is defective. The expression of these inhibitory isoforms may be the mechanism of cellular defense against the cytotoxicity of FAS-R and TNF. A wide heterogeneity isoforms IAIS, which significantly exceeds the heterogeneity observed for any of the other protease family CED3/ICE, may allow a particularly refined the configuration of the active form of this protein. It seems also possible that some isoforms IAIS perform other functions. The ability MACH1 to contact with MORT-1 and IAISDue to the unique ability of FAS-R and TNF-R to cause cell death, as well as the ability of TNF receptors to run the various other damaging tissue activity, deviation from the norm of the function of these receptors may be particularly harmful for the body. Indeed, it was shown that both excessive and insufficient function of these receptors leads to pathological manifestations of various diseases. Identification of molecules involved in the signal transmission for the activity of these receptors, and finding ways to modulate the functions of these molecules is a potential key for new therapeutic approaches to these diseases. In connection with the proposed Central role MACH1 in toxicity FAS-R and TNF appears to be particularly important preparation of medicines that can inhibit the proteolytic function of this molecule, as it was made is in molecules MACH1 can allow the creation of medicines, which may affect the protection against excessive immunomediated cytotoxicity without resistance to the processes of physiological cell death, involving other members of the family CED3/ICE.
Thus, the invention also relates to DNA sequences coding MORT-1-binding protein, and MORT-1-binding proteins encoded by this DNA sequence.
In addition, this invention relates to DNA sequences encoding biologically active analogs, fragments and derivatives of MORT-1-binding protein, and coded their analogs, fragments and derivatives. The receipt of such analogs, fragments and derivatives perform a standard way (see, for example, Sambrook et al., 1989), in which the DNA sequences encoding the MORT-1-binding protein, can be deleterows attached or replaced by other one or more codons with the formation of analogues having at least one substitution of amino acid residue compared to the native protein.
The polypeptide or protein, "mostly appropriate" MORT-1-binding protein, includes not only the MORT-1-binding protein, but also polypeptides or proteins that are th polypeptides, in which one or more amino acids of amino acid sequence of MORT-1-binding protein has been replaced with another amino acid, deleterow or inserted, provided that the resulting protein exhibits basically the same or higher biological activity, which has a corresponding MORT-1-binding protein.
In order to "mostly correspond to the" MORT-1-binding protein changes in the sequence of MORT-1-binding proteins, such as isoforms IAIS are relatively small. Although the number of changes may be more than 10, preferably has no more than 10 changes, more preferably not more than 5 and, most preferably, not more than three of such changes. Although you can use any method for finding potentially biologically active proteins, which mainly correspond to MORT-1-binding proteins, one of these ways is the use of traditional methods of mutagenesis of DNA that encodes this protein, leading to a small number of modifications. The proteins expressed by these clones can be subjected to screening for MORT-1-binding and/or Medeiros FAS-R and p55-R activity.
"Conservative substitutions are substitutions that do not have a preference for the men, which presumably should not basically change the size, charge or configuration of this protein, and therefore presumably should not alter its biological properties.
Conservative substitutions MORT-1-binding proteins include analog, in which at least one amino acid residue in the polypeptide has been conservatively replaced by a different amino acid. Such substitutions preferably are carried out in compliance with the list presented in Table IA, these replacements can be determined by routine experimentation to provide modified structural and functional properties of a synthesized polypeptide molecules with biological activity characteristic of the MORT-1-binding protein.
Alternatively, another group of substitutions of MORT-1-binding protein can be replaced, in which at least one amino acid residue in the polypeptide has been removed and a different residue was inserted in its place in accordance with Table IB. The types of substitutions which may be made in the polypeptide, can be based on analysis of the frequencies of amino acid substitutions between homologous proteins of different species, such as those presented in Table 1-2 of Schulz et al., G. E. Principle of this analysis, alternative conservative substitutions are defined as exchanges within one of the following five groups:
1. Small aliphatic nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly).
2. Polar negatively charged residues and their amides: Asp, Asn, Glu, Gln.
3. Polar, positively charged residues: His, Arg, Lys.
4. Large aliphatic nonpolar residues: Met, Leu, Ile, Val (Cys).
5. Large aromatic residues: Phe, Tight, Thr.
Three amino acid residue in parentheses have a specific role in the architecture of the protein. Gly is the only residue that does not have a side chain and, therefore, gives the flexibility of the chain. However, this tends to enhance the formation of secondary structures differing from-spiral. Pro, because of its unusual geometry, hard pulls on the chain and usually seeks to strengthen local structures, such as-round, although in some cases Cys may be able to participate in the formation of disulfide bonds, which are important for protein folding. It should be noted that Schulz et al., supra, brings together Groups 1 and 2 (above). Note also that Tight, due to its ability to form hydrogen bonds has significant similarity to Ser and Thr, and so on
Conservative amino acid substitutions according to this invention, for example, presented above, is known in that the amino acid substitutions. Most deletions and substitutions in accordance with this invention are those which do not produce radical changes in the characteristics of the protein molecule or polypeptide. "Specifications" are related to both changes in secondary structure, for example,-spiral or-plate, as well as changes in biological activity, such as binding MORT-1 or meditirovaniya actions ligand FAS-R or TNF on cells.
Examples of making amino acid substitutions in proteins which can be used for obtaining analogs of the MORT-1-binding proteins for use in this invention include any stage known methods, such as that shown in U. S. patent RE 33653, 4959314, 4588585 and 4737462 Mark et al.; 5116943 Koths et al., 4965195 Namen et al. 487911 Chong et al.; and 5017691 Lee et al., presents for lysine-substituted proteins in U. S. patent No.4904584 (Shaw et al.).
In addition to conservative substitutions discussed above, should not significantly alter the activity of the MORT-1-binding protein, any conservative substitutions, or less conservative and more random replacement, which lead to increased biological activity of analogues of the MORT-1-binding proteins, are within the scope of this invention.
Specialist in this field is elechi (deletions) and so on you should evaluate the routine tests of binding and cell death. Screening using this standard test does not require undue experimentation.
Acceptable are analogs that retain at least the capacity of binding to MORT-1 and as a result, as noted above, mediashout activity of the FAS-R and p55-R (for example, through by activity in at least some isoforms IAIS). Thus can be obtained analogues, which have a so-called dominant-negative effect, namely, similar to that of the defective or binding to MORT-1, or in the subsequent signal transmission or by activity after such binding. Such analogs can be used, for example, for inhibiting the action of FAS-ligand by competing with the natural MORT-1-binding proteins. For example, it seems likely that the isoforms IAIS, IAIS2 and IAIS3 are "natural" counterparts, which are used for inhibiting the activity IAIS by competing for binding to MORT-1 active (ProcessName) isoforms IAIS that, apparently, is essential for activation of these isoforms IAIS. If the active isoforms IAIS not the s. Similarly, can be obtained from the so-called dominant-positive counterparts, which will serve to enhance the effects of FAS ligand or TNF. They will have the same or better MORT-1-binding properties and the same or better properties of signal transmission, in comparison with properties, which have natural MORT-1-binding proteins.
At the genetic level, these analogues are usually site-directed mutagenesis of nucleotides in the DNA coding MORT-1-binding protein, which results in DNA coding similar, with subsequent synthesis of DNA and expression of the polypeptide in the culture of recombinant cells. Typically, the analogs exhibit the same or increased qualitative biological activity, which is a natural occurring protein, Ausubel et al., Current Protocols in Molecular Biology, Greene Publications and Wiley Interscience. New York, NY, 1987-1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Col Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.
Getting the MORT-1-binding protein, in accordance with this or another nucleotide sequence that encodes the same polypeptide, but differing from the natural sequence due to changes permitted by the known degeneracy of the genetic code, can be achieved site-Belka. Site-directed mutagenesis allows to obtain analogues through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a significant number of adjacent nucleotides, to provide primerno sequence of sufficient size and of sufficient complexity sequence for the formation of a stable duplex on both sides cross the deletion junction. Generally, the preferred primer length of approximately 20-25 nucleotides with approximately 5-10 complementary nucleotides on each side of the variable sequence. In General, the method of site-directed mutagenesis well known in this field, for example, described in the publications Adelman et al., DNA 2: 183 (1983), incorporated herein by reference.
As will be clear, the way the site-directed mutagenesis typically uses a phage vector that exists in single-stranded form, and in double-stranded form. Typical vectors applicable to site-directed mutagenesis include vectors such as the M13 phage, for example, described by Messing et al., Third Cleveland Symposium on Macromolecules and Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), the description is included here as A area. Alternatively, to obtain single-stranded DNA can be used plasmid vectors containing single-stranded beginning of replication of phage (Veira et al., Meth. Emymol. 153:3, 1987).
In General, site-directed mutagenesis, in accordance with this invention, perform receiving first single-stranded vector that includes within its sequence a DNA sequence encoding the corresponding one or another polypeptide. Oligonucleotide primer bearing the desired mutated sequence, are synthetically using an automated DNA/oligonucleotide synthesis. Then this primer is annealed to single-stranded containing the sequence of the protein vector and subjected to the action of a DNA polymerase enzyme, such as a piece maple DNA polymerase I of E. coli, to complete the synthesis of the mutation carrier chain. Thus, the mutated sequence and the second circuit carrying the desired mutation. This heteroduplex vector will then use to transform appropriate cells, such as E. coli JM101, and select clones that include recombinant vectors bearing the arrangement of the mutated sequence.
After selection of this clone sequence mutated MORT-1-binding of the PR type, which can be used for transfection of a suitable host.
Accordingly, a gene or nucleic acid encoding a MORT-1 binding protein can also be detected, obtained and/or modified in vitro, in situ or in vivo using well-known methods of amplification of DNA or RNA, such as PCR and chemical synthesis of oligonucleotides. PCR allows amplification (increase in number) of specific DNA sequences through a repeating DNA polymerase reactions. This reaction can be used instead of cloning; all that is required for this, this knowledge nucleic acid sequence. For PCR design primers, complementary to the target sequence. Then these primers receive automated DNA synthesis. Because they can be designed primers for hybridization with any part of the gene can be created such conditions that may be possible (portable) incorrect pairing to complementary base pairing. Amplification of these is incorrectly paired areas can lead to the synthesis of mathenesserlaan product, leading to the formation of the peptide with new properties (i.e., may be site-nab is ementary DNA (cDNA) using reverse transcriptase PCR with the obtained RNA can be used as a starting material for the synthesis of the extracellular domain of the prolactin receptor without cloning.
In addition, can be designed PCR primers to enable the new restriction sites or other signs, such as termination codons at the ends of the gene segment that should be amplified. This is the location of the restriction sites on 5`- and 3`-ends of the amplified gene sequence allows to obtain the gene segments encoding the MORT-1-binding protein or fragment specifically designed for ligating the other sequences and/or cloning sites in the vector.
PCR and other methods of amplification of RNA and/or DNA are well known in this field and can be used in accordance with this invention without undue experimentation, based on the descriptions and guidance presented here. Known methods of amplification of DNA or RNA include, but are not limited to) polymerase chain reaction (PCR) and related methods of amplification (see, for example, U. S. patent Nos. 4683195, 4683202, 4800159, 4065188 Mullis et al.; 4795699 and 4921794 Tabor et al.; 5142033 Innis; 5122464 Wilson et al.; 5091310 Innis; 5066584 Gyllensten et al.; 4889818 Gelfand et al.; 4994370 Silver et al.; 4766067 Biswas; 4656134 Ringgold; and Innis et al., eds. Pcr Protocols: A Guide to Method and Applications) and Mediaroom RNA amplification using antisense RNA to the target sequence as a matrix for sin is the amplification of DNA tagging of antibodies (Ruzicka et al., Science 260:487 (1993); Sano et al., Science 258:120 (1992); Sano et al., Biotechniques 9:1378 (1991)), the entire contents of these patents and publications are incorporated herein as references.
Similarly, biologically active fragments of the MORT-1-binding proteins (e.g., active fragments of any of the isoforms IAIS) can be obtained, as described above with respect analogues MORT-1-binding proteins. Suitable fragments of the MORT-1-binding proteins are fragments that retain the MORT-1-binding capacity and which can mediashout biological activity of FAS-R and p55-R, as noted above. In line with this, can be obtained fragments of the MORT-1-binding proteins, which have a dominant negative or dominant-positive effect, as noted above with respect analogues. It should be noted that these fragments represent a special class of analogues of the present invention, namely, they are certain parts of the MORT-1-binding proteins, originating from the full sequence MORT-1-binding protein (for example, from any sequence of one of iform IAIS), and each of these parts (or fragments) has any of the above activities. Such a fragment can be, for example, a peptide.
Podobnyesayty residues MORT-1-binding protein, its analogs or fragments, or pairing R-1-binding protein, its analogs or fragments with another molecule, e.g., antibody, enzyme, receptor, etc., as is well known in this field. Thus, the term "derivative", in the application here, covers derivatives which may be obtained from the functional groups which are present as side chains on the residues or the N - or C-terminal groups, by means known in this field, and derivatives are also included in this invention. Derivatives may have chemical side, such as carbohydrate or phosphate residues, provided that such fraction has the same or higher biological activity than the activity of MORT-1-binding proteins.
For example, derivatives may include aliphatic esters of the carboxyl groups, amides of the carboxyl groups obtained by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of amino acid residues formed with acyl part (for example, alkanoyloxy or carbocyclic arolina group), or O-acyl derivatives of free hydroxyl group (for example, hydroxyl group ceilinga or traviling OS is, not substitute one amino acid for another of the twenty commonly occurring natural amino acids.
Although MORT-1-binding protein is a protein or polypeptide, it is a sequence of amino acid residues. Polypeptide consisting of a larger sequence, which includes the entire sequence MORT-1-binding protein, in accordance with these definitions here, also intended for inclusion in the list of such polypeptides, provided that the additions do not affect the basic and novel characteristics of the present invention, i.e., if they either retain or increase the biological activity of MORT-1-binding protein, or may be split with the formation of a protein or polypeptide having the biological activity of MORT-1-binding protein. Thus, for example, the invention includes a fused proteins MORT-1-binding protein with other amino acids or peptides.
New MORT-1-binding protein, its analogs, fragments and derivatives have a number of applications, for example:
(i) MORT-1-binding protein, its analogs, fragments and derivatives can be used to simulate or enhance the function of MORT-1 and, hence, ligand FAS-R or TNF, in situations where genene, and so on, where the desired induced ligand FAS-R or TNF cytotoxicity. In this case, the MORT-1-binding protein, its analogs, fragments or derivatives, amplifying the effect of the ligand FAS-R or TNF, i.e., cytotoxic effect, can be introduced into cells by standard methods known per se. For example, as MORT-1-binding proteins are intracellular and should be entered only in cells in which it is desirable effect of ligand FAS-R or TNF, a system is needed for the specific introduction of this protein in cells. One way to do this is to create a recombinant animal virus, for example, made of Vaccinia DNA which must be entered the following two gene: a gene that encodes a ligand that binds to cell surface proteins that are specifically expressed in these cells, such as Dr protein of AIDS virus (HIV), which specifically binds to certain cells (CD4 lymphocytes and related leukemia), or any other ligand that binds specifically to cells bearing FAS-R or p55-R, so, the recombinant viral vector is capable of binding such bearing FAS-R or p55-R cells; and the gene encoding MORT-1-binding protein. So brazaletes to a tumor cell or other carrier FAS-R or p55-R cell, then the coding sequence of the MORT-1-binding protein is introduced into cells by the virus and expression in these cells will lead to enhanced activity of ligand FAS-R or TNF, leading to tumor cell death or other carrier FAS-R or p55-R cells that you want to kill. The design of such a recombinant animal virus is a standard procedure (see, for example, Sambrook et al., 1989). Another possibility is the introduction of sequences MORT-1-binding protein (for example, any of the isoforms IAIS) in the form of oligonucleotides that can be adsorbed by cells and expressed in them.
(ii) They can be used for inhibiting the action of the ligand FAS-R or TNF, for example, in cases such as tissue damage in septic shock, graft rejection according to the reaction of graft-versus-host or acute hepatitis, in which it is desirable to block the intracellular response induced by ligand FAS-R or TNF signaling FAS-R or p55-R. In this situation, for example, to enter the cells by standard methods, oligonucleotides having antisense coding sequence for the MORT-1-binding protein, which will be effective blokirovki to the inhibition of ligand FAS-R or TNF. Such oligonucleotides can be introduced into cells by using the above described approach with the recombinant virus, and the second sequence transferred by this virus, is the oligonucleotide sequence.
Another possibility is the use of antibodies specific to MORT-1-binding protein for the inhibition of the activity of intracellular signal transduction.
Another way of inhibiting the action of the ligand FAS-R or TNF is a recently developed approach using a ribozyme. Ribozymes are catalytic RNA molecules that specifically break down RNA. Ribozymes can be designed for cleavage of the target RNA by choice, for example, mRNA encoding the MORT-1-binding protein of the present invention. Such ribozymes can have sequence-specific mRNA MORT-1-binding protein, and be able to interact with it (through complementary binding) with subsequent cleavage of this mRNA, which leads to the reduction (complete loss) of the expression of the MORT-1-binding protein, and a low level of expression depends on the level of expression of the ribozyme to the target cell. For the introduction of ribozymes in cells (for example, Kladruby) vectors (virus animal), which is usually used for this purpose (see also (i) above, and this virus has the second sequence of cDNA encoding a selected sequence of the ribozyme). (To explore review, methods, etc. concerning ribozymes, see Chen et al., 1992; Zhao and Pick, 1993; Shore et al., 1993; Joseph and Burke, 1993; Shimayama et al., 1993; Cantor et al., 1993; Barinaga, 1993; Crisel et al., 1993 and Koizumi et al., 1993).
(iii) MORT-1-binding protein, its analogs, fragments or derivatives can also be used for isolation, identification and cloning of other proteins of the same class, i.e., proteins that bind to the intracellular domain of FAS-R or functionally related receptors, or proteins, binding to MORT-1 and thereby functionally related receptors, such as FAS-R and p55-R, and are involved in intracellular signal transmission. In this case, the application can be used dvuhserijnaya system yeast or can be used a recently developed system using hybridization on Southern in mild conditions with subsequent PCR cloning (Wilks et al., 1989). In the publication Wilks et al. describes the identification and cloning of two alleged proteincontaining by applying fuzzy hybridization conditions for Southern with the POS sequence of the kinase. This approach can be used in this invention using sequence MORT-1-binding protein (for example, any of the isoforms IAIS) to identify and clone isoforms IAIS, related MORT-1-binding proteins.
(iv) Another approach is to use the MORT-1-binding protein, or its analogs, fragments or derivatives of the present invention is their use in methods of affinity chromatography for isolation and identification of other proteins or factors with which they are able to communicate, for example, MORT-1 or other proteins or factors involved in the intracellular signal transmission. In this application MORT-1-binding protein, its analogs, fragments or derivatives of this invention can be separately attached to the matrices affinity chromatography and then brought into contact with cell extracts or selected proteins or factors, the alleged participants in the process of intracellular signal transduction. After the procedure, affinity chromatography, other proteins or factors that are associated with MORT-1-binding protein, or its analogs, fragments or derivatives of this invention can be suirvey, isolated and characterized.
(v) As OTL also used as immunogens (antigens) to obtain specific antibodies them. These antibodies can also be used for the treatment of MORT-1-binding protein (e.g., isoforms IAIS) either from cell extracts or from lines transformed cells that produce the MORT-1-binding protein, or its analogs or fragments. Further, these antibodies can be used for diagnostic purposes for identifying disorders related to abnormal functioning of the system of the ligand FAS-R or TNF, for example, an overactive or inactive induced by ligand FAS-R or TNF cellular effects. So, if these disorders were associated with violations functions of intracellular signal transduction involving protein MORT-1 or MORT-1-binding protein, such antibodies could serve as an important diagnostic tool.
It should be noted that the selection and characterization of MORT-1-binding protein (e.g., isoforms IAIS) of this invention can be performed using well-known standard methods of screening. For example, one of these methods of screening, yeast dvuhserijnyj the method described herein (Example 1) was used to identify protein MORT-1 and then MORT-1-binding proteins (Examples 2-3) this invention. Also, as noted above and below, can be ispolnie in this area how the selection, identification and characteristics of the MORT-1-binding protein of this invention or selection, identify and characterize additional proteins, factors, receptors, etc. that are able to bind with protein MORT-1 or MORT-1-binding protein of the present invention.
As described above, MORT-1-binding protein can be used to generate antibodies specific to MORT-1-binding protein, for example, isoforms of IAIS. These antibodies or fragments thereof can be used, as described in detail below, and it is clear that in these applications, the antibodies (or fragments thereof) are antibodies that are specific to MORT-1-binding proteins.
Based on the discoveries described in this invention that at least some of the isoforms IAIS (see above and Example 3 below) are proteases, related protease family of proteases CED3/ICE, the following specific applications in medicine are assumed for these isoforms IAIS: it was discovered that there are already specific inhibitors of other proteases CED3/ICE, some of which can penetrate into cells, which can effectively block the processes of programmed cell death. Thus, in accordance with this invention can skonstruirovali isoforms protease IAIS. Further, due to the unique characteristics of the sequence of these new protease IAIS, it is possible to design inhibitors that are highly specific in relation induced by TNF and FAS ligand-R effects. The opening of the present invention also provide a method of study of mechanism in which "killing (killing) protease" is activated in response to ligand FAS-R and TNF, which makes possible the subsequent development of drugs that can control the degree of activation. There are diseases in which these drugs can be of great help. Among others, acute hepatitis, in which acute liver damage, apparently, reflects Mediaroom ligand FAS-R the death of the liver cells; autoimmune-induced cell death, such as death-cells of the islets of Langerhans of the pancreas, leading to diabetes; death of cells in graft rejection (e.g., kidney, heart and liver); the death of oligodendrocytes in the brain in multiple sclerosis; and inhibiting AIDS suicide T-cells, which causes the proliferation of the AIDS virus and, consequently, the disease AIDS.
As mentioned herein above and below, who work together as "natural inhibitors protease isoforms IAIS, and therefore, they can be used as the above-mentioned specific inhibitors of these proteases IAIS. Other substances, such as peptides, organic compounds, antibodies, etc. may be subjected to screening to obtain specific medicines that are able to inhibit protease IAIS.
A non-limiting example of how can be constructed and subjected to a screening of peptide inhibitors of proteases IAIS, based on previous studies of peptide inhibitors of proteases ICE and ICE-like protease substrate specificity ICE and strategies analysis of epitopes using peptide synthesis. It was found that the minimum required for efficient cleavage of the peptide ICE is the presence of four amino acids to the left of the cleavage site with a strong preference for aspartic acid at position1and with methylamine to the right from the position of the P1(Sleath et al., 1990; Howard et al., 1991; Thornberry et al., 1992). In addition, fluorogenic substrate peptide (tetrapeptide), acetyl-Asp-Glu-Val-Asp-a-(4-methylcoumarin-7-amide), denoted by the abbreviation Ac-DEVD-AMC, corresponds to the sequence in poly(ADP-ribose)-polymerase (PARP), which, as it was found, broken down in the cells in depleats effectively SRR (a member of a family of proteases CED3/ICE) and protease IAIS.
Because Asp at position P1substrate, apparently, is important, tetrapeptide with Asp as the fourth amino acid residue and various combinations of amino acids in the first three positions, can be quickly skanirovaniya binding with the active site of proteases IAIS, for example, using the method developed by Gasana (Geysen et al., 1987), in which a large number of peptides on solid substrates were skanirovali specific interaction with antibody. The binding of proteases IAIS with specific peptides can be detected by various well-known methods of detection within the training of specialists in this field, such as radioactive labelling protease IAIS, etc., it Was shown that the method of Hasina allows you to test at least 4000 peptides for each working day.
As can be advantageous design of peptide inhibitors that selectively inhibit protease IAIS without intervention in the processes of physiological cell death, involving other members of the family proteases CED3/ICE, it is possible to synthesize, then, the pool of peptides that bind to proteases IAIS in the test, such as described above, in the form of fluorogenic substrate CED3/ICE. Then the peptides that were identified as selectively cleaved by proteases IAIS, can be reversibly or irreversibly modified to facilitate the permeability of cells and inhibiting the activity of cell death IAIS. Thornberry et al. (1994) reported that tetrapeptide (acyloxy)-ketone Ac-Tyr-Val-Ala-Asp-CH2OC(O)-[2,6-(CF)2]Ph is a strong inactivation ICE. Similarly, Milligan et al. (1995) reported that tetrapeptide inhibitors with chloromethylketone (irreversibly) or aldehyde (reversible) group, inhibited ICE. In addition, it was shown that benzyloxycarbonyl-s-CH2OS(O)-2,6-dichlorobenzene (DCB) inhibits ICE (Mashima et al., 1995). Thus, tetrapeptide that selectively bind to proteases IAIS, can be modified, for example, aldehyde group, chloromethylketone (acyloxy)-methylketone or CH2OS(O)-DCB-group to obtain a peptide inhibitor of the activity of proteases IAIS.
Although some specific inhibitors of other proteases CED3/ICE can penetrate into the cells, it may be necessary to increase the permeability of the cells of peptide inhibitors. For example, the peptides can be chemically modified or to be replaced by their derivatives to increase their pron is shi et al. (1991) reported receiving a derived thyrotropin-releasing hormone with lauric acid, lipophilic laurelbrooke, with good characteristics of penetration through the cell membrane. Zacharia et al. (1991) also reported the oxidation of methionine to sulfoxide and replacement of the peptide bond her betamethasonum itapira (PINES2to facilitate the transport of peptides across the cell membrane. These are just some of the known variants and derivatives, which are well known to specialists in this field.
In addition, drug or peptide inhibitors that can inhibit the activity of cell death IAIS1 and IAIS2, can be conjugated or kompleksirovanii with molecules that facilitate entry into the cell.
In U. S. Patent 5149782 described the conjugation of the molecule, which must be transported across the cell membrane, with membranophones agent such as capable of fusion polypeptides that form ion channels polypeptides, other membrane polypeptides, and long-chain fatty acids such as myristic acid, palmitic acid. These membranophones agents embed molecular conjugates in Linden is accessible ways transmembrane delivery of molecules, such as (but not limited to, proteins and nucleic acids, using the mechanism mediaremote receptor endocytotic activity. These receptor systems include systems that recognizes galactose, mannose, mannose-6-phosphate, transferrin, asialoglycoprotein, transcobalamin (vitamin B12),-2-macroglobulin, insulin and other peptide growth factors such as epidermal growth factor (EGF). Low et al. reports that the receptors nutrients, such as receptors, Biotin and folate, can be advantageously used to enhance transport across the cell membrane due to the location and multiplicity of receptors, Biotin and folate on the surfaces of the membranes of most cells and associated with them (Mediaroom receptors) processes transmembrane transport. Thus, the complex formed between the compound to be delivered into the cytoplasm, and a ligand such as Biotin or folate, is brought into contact with the cellular membrane, bearing receptors Biotin or folate, to initiate receptor-mediated mechanism transmembrane transport and thereby create the possibility of occurrence of the desired compounds in the cell.
It is known that ICE has spoodle the development of a strong and highly selective affinity label, containing bitenova tag (tag) (Thornberry et al., 1994). Therefore, the position of the P2and , possibly, N-end tetrapeptide inhibitor can be modified or used to create derivative, for example, by attaching molecules of Biotin, to enhance the permeability of these peptide inhibitors through the cell membrane.
In addition, in this field of knowledge is known that the fusion of the desired peptide sequence with the sequence of the leader/signal peptide to create a "chimeric peptide" will allow such "chimeric peptide" be transported through the cell membrane into the cytoplasm.
As will be clear to experts in this field, the peptide inhibitors of the proteolytic activity of IAIS this invention also include coworkers peptide drugs or inhibitors that can be subjected to rapid screening for binding to the protease IAIS, to create, perhaps, a more stable inhibitors.
Also it should be clear that some of the ways to facilitate or enhance the transport of peptide inhibitors through cell membranes discussed above, is also applicable to themselves isoforms IAIS, as well as other peptides and proteins, praclical polyclonal antibody monoclonal antibodies (mAb), chimeric antibodies, antiidiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments thereof, provided by any known method, such as, but not limited to enzymatic cleavage, peptide synthesis or recombinant methods.
Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with the antigen. Monoclonal antibodies contain essentially homogeneous population of antibodies specific against the antigen population, which contain essentially the same binding sites epitope. Mab can be obtained by methods known to experts in this field. See, for example, Kohler and Milstein, Nature, 256:495-497 (1975); U. S. Patent No. 4376110; Ausubel et al., eds., Harlow and Lane ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory (1988); and Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N. Y., (1992-1996), the contents of which are fully incorporated by reference. Such antibodies may belong to any class of immunoglobulins, including IgG, IgM, IgE, IgA, GILD and any of their division. Hybridoma producing mAb of the present invention, it is possible to cultivate in vitro, in situ or in vivo. Getting high titers ate are molecules the various parts which come from different animal species, such as molecules having variable region derived from a murine mb, and a constant region from a human immunoglobulin. Chimeric antibodies are used primarily to reduce immunogenicity in their application and to increase yields, for example, when the mouse mb have higher outputs when using a hybrid, but most immunogenicity in man, so that the use of chimeric mAb man/mouse. Chimeric antibodies and methods for their production are known in this area (Cabilly et al., Proc. Ntl. ad. Sci. USA 81:3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne et al., Nature 312:643-646 (1984); Cabilly et al., European Patent Application 125023 (published November 14, 1984); Neuberger et al., Nature 314:268-270 (1985); Tanigushi et al., European Patent Application 171496 (published February 19, 1985); Morrison et al., European Patent Application 173494 (published March 5, 1986); Neuberger et al., PCT Application WO 8601533 (published March 13, 1986); Kudo et al., European Patent Application 184187 (published June 11, 1986); Sahagan et al., J. Immunol. 137:1066-1074 (1986); Robinson et al., International patent Application No. WO 8702671 (published May 7, 1987); Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Better et al., Science 240:1041-1043 (1988); and Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, supra, (fully included as references).
Antiidiotypic (anti-Id) antibody is an antibody which recognizes unique determinants, mainly St. the species and genetic type (e.g., strain mice), as the source of the mAb, which receive anti-Id. Immunized animal will recognize idiotypical the determinants of the immunizing antibody and to respond by producing antibodies to these idiotypical determinants (anti-Id antibodies). (See, for example, U. S. Patent No. 4699880, incl. fully herein by reference).
Anti-Id can also be used as an immunogen for the induction of an immune response in another animal with obtaining the so-called anti-anti-idiotypic antibodies. Anti-anti-Id can be epitope-identical to the original mAb, which induced anti-Id. Thus, by using antibodies to idiotypical determinants mAb is possible to identify other clones expressing antibodies of identical specificity.
Accordingly, mAb generated against the MORT-1-sveeiuose proteins, their analogs, fragments or derivatives of this invention can be used for the induction of anti-Id antibodies in suitable animals, such as mice BALB/c mice. Spleen cells taken from such immunized mice used for antiidiotypic hybrids secreting antiidiotypic antibodies (anti-Id mAb). Further, these antiidiotypic antibodies may be associated with a carrier, such as emotionengine anti-anti-idiotypic antibodies, which may have binding capacity of the original mAb specific against epitope MORT-1-binding protein, or its analogs, fragments and derivatives.
Thus, the anti-Id mAb have their own idiotypical epitopes, or "idiotopes" structurally identical with estimated epitope, such as GRB-protein-A.
The term "antibody" includes both intact molecules and fragments thereof, such as, for example. Fab and F(ab`)2, which are capable of binding antigen, fragments Fab and F(ab`)2 are deprived of the Fc fragment of intact antibody, are excreted more rapidly from the bloodstream and may have less non-specific binding with cloth than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).
It should be clear that the fragments Fab and F(ab`)2 and other fragments of the antibodies used in this study can be used to detect and quantify MORT-1-binding protein as described here, the methods for intact antibody molecules. Such fragments are formed, as a rule, proteolytic cleavage with the use of enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab`)2 fragments).
Antibody "capable of binding" a molecule if it is capable of specifically re is olekuly, able to be bound by the antibody, which can also be recognized by this antibody. Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules, such as amino acids or sugar side chains and have specific three dimensional structural characteristics, as well as specific charge characteristics.
"Antigen" is a molecule or portion of a molecule capable of contacting an antibody that is additionally capable of inducing an animal to produce antibodies capable of contact with the epitope of this antigen. The antigen may have one or more epitopes. Specific reaction means that the antigen will react, with high selectivity, with its corresponding antibody, but not with the multitude of other antibodies which may be induced by other antigens.
Antibodies, including antibody fragments, are applicable in this invention can be used for quantitative or qualitative detection of MORT-1-binding protein in a sample or to detect presence of cells expressing the MORT-1-binding protein of the present invention. This can be accomplished by immunofluorescence methods using fluorescently metricheskie or fluorometrically methods of detection.
The antibodies (or fragments thereof) applicable in this invention, it is possible to use histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection MORT-1-binding protein of the present invention. The in situ detection can be performed by taking a tissue sample from the patient and ensure the labeled antibodies of the present invention to such a specimen. The antibody (or fragment) is preferably provided by applying or by layering labeled antibody (or fragment) onto a biological sample. By applying this procedure, you can determine not only the presence of the MORT-1-binding protein, but also its distribution on the examined tissue. When using this invention, the specialists of ordinary skill in this field will readily understand that a great variety of histological methods (such as staining procedures) can be modified to achieve such in situ detection.
Such tests on the MORT-1-binding protein of the present invention typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leukocytes, or in order to identify this MORT-1-binding protein, and detecting the antibody by any of the well-known in the field of methods.
The biological sample can be processed by solid-phase support or carrier such as nitrocellulose, or other support or carrier which is capable of mobilitat cells, cell particles or soluble proteins. Then the support or carrier may be washed with suitable buffers followed by treatment detektirano labeled antibody in accordance with this invention, as described above. Solid-phase support or carrier can then be washed a second time with buffer to remove unbound antibodies. The amount of bound label on the solid support or carrier can then be determined by standard methods.
By "solid phase support", "solid carrier", "solid support", "solid carrier", "support" or "carrier" means any carriage or carrier, capable of binding an antigen or antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified cellulose, polyacrylamides, gabbros, and magnetite. For the purposes of this invention, by its nature, the media can be strukturnoi configuration, provided that he is able to contact the antigen or antibody. Thus, the configuration of the support or carrier may be spherical, in the form of pellets, cylindrical, as in the case of the inner surface of the test tube or the outer surface of the rod. Alternative this surface may be flat, such as vinyl, test strip, etc. Preferred supports or carriers include granules of polystyrene. Professionals in this field can be known other suitable carriers for binding antibodies or antigen, or they can pick them up yourself through routine experimentation.
Binding activity of a specific batch of antibodies of the present invention, described above, may be determined by well known methods. Specialists in this field will be able to determine the working and optimum test conditions for each determination by routine experimentation.
Other such stages as washing, stirring, shaking, filtering, etc. can be added to these tests is usually necessary for the specific situation.
One of the ways in which the antibody of the present invention can be detektirano marked, is linking it with parentstate will react with the substrate with the formation of chemical molecules, which can be detected, for example, by spectrophotometric, fluorometric ways or visually. Enzymes that can be used for informative labelling of antibodies, include (but not only) a malate dehydrogenase, staphylococcal nuclease, Delta-5-steroisomers, alcoholdehydrogenase yeast, alpha glitserofosfatdegidrogenazy, triosephosphate, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphatedehydrogenase, glucoamylase and acetylcholinesterase. Detection can be performed by colorimetric methods using a chromogenic substrate for the enzyme. Detection may also be performed by visual comparison of the extent of enzymatic reaction of the substrate in comparison with similarly prepared standards.
Detection can be performed using any of a variety of other immunoassays. For example, using radioactive labelling of antibodies or fragments of antibodies can be used to detect R-PTP-ABC using radioimmunoassay (RIA). A good description of the RIA can be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T. S. et al., North Holland Publishing Company, NY (1978) concreteidentity isotope can be detected by means of as using a g-counter or a scintillation counter or autoradiography.
According to this invention, it is also possible to label the antibody with a fluorescent compound. When imaging a fluorescently labeled antibody is exposed to light of a suitable wavelength, its presence can be detected due to fluorescence. Among the most common are used for labeling compounds are isothiocyanate fluorescein, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, Italiy aldehyde and fluorescamine.
The antibody can also detektirano mark using emit fluorescence metals such as152E or other metals of the series of lanthanides. These metals can be attached to the antibody using such chelating the metals of groups as diethylenetriaminepentaacetic acid (ATPA).
The antibody can also be detektirano marked by his connection with the chemiluminescent compound. The presence chemiluminescence-labeled antibodies define then by detecting the presence of luminescence that arises during a chemical reaction. Examples are especially applicable chemiluminescence-aiming compounds are luminal, theromatic ester acridine, imidazole, salt acridine bretania. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and acorin.
The antibody molecule of the present invention may be adapted for use in immunometric analysis, also known as "two-sity" or "sandwich"assay. In a typical immunometric analysis of some amount of unlabeled antibody (or fragment of antibody) is associated with a solid support or carrier and add some amount detektirano labeled soluble antibody for detection and/or quantitative determination of ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
Typical or preferred immunodeficiencies tests include "direct" tests, in which the antibody associated with the solid phase, is first brought into contact with the test sample extraction of antigen from the sample by education dvukhkomponentnogo to remove any remaining liquid sample, containing unreacted antigen, if he was present, and then brought into contact with a solution containing a quantity of labeled antibody (which functions as a "reporter molecule"). After the second incubation period for the formation of a complex of the labeled antibody with the antigen bound to a solid support or a carrier through the unlabeled antibody, the solid support or carrier is washed again to remove the unreacted labeled antibody.
In the "sandwich"-the analysis of another type, which may also be applicable to the antigens of the present invention, use of the so-called "simultaneous" or "reverse" tests. "Simultaneous" test includes a single stage incubation, when the antibody associated with the solid support or a carrier and a labeled antibody together and simultaneously added to the test sample. After incubation, the solid support or carrier is washed to remove the residue of the liquid sample and complexional labeled antibodies. The presence of labeled antibody associated with the solid support or carrier, define then, as in the standard direct sandwich assay.
In "reverse" test use stepwise addition of the first solution and the carrier after a suitable incubation period. After the second incubation, the solid phase is washed with a standard way to release her from the rest of the test sample and the solution of unreacted labeled antibody. Then labeled antibody associated with the solid support or carrier, determine, as in "simultaneous" and "direct" tests.
MORT-1-binding proteins of the present invention can be obtained by any standard method of recombinant DNA (see, for example, Sambrook, et al., 1989 and Ansabel et al., 1987-1995, supra), in which a suitable eukaryotic or prokaryotic cells are the masters, well-known in this area, transforming suitable eukaryotic or prokaryotic vectors containing sequences encoding these proteins. Therefore, this invention relates to such expressing vectors and transformed hosts, used for protein of the present invention. As mentioned above, these proteins also include biologically active analogs, fragments and derivatives, and therefore, the vectors encoding them, include vectors encoding analogs and fragments of these proteins, and transformed hosts include owners, producing such analogs and fragments. Derivatives of these proteins, producer lcov or their analogues or fragments.
This invention relates also to pharmaceutical compositions containing recombinant virus (derived from animal viruses) vectors encoding the MORT-1-binding proteins, and that this vector encodes a protein surface of the virus, capable of binding surface proteins specific target cells (e.g. cancer cells) to the inserted sequences MORT-1-binding protein in these cells. Other pharmaceutical compositions of this invention contain as the active ingredient (a) oligonucleotide sequence encoding the antisense sequence of the sequence MORT-1-binding protein, or (b) drugs that block the proteolytic activity of isoforms of IAIS.
The pharmaceutical compositions according to this invention include a sufficient amount of the active ingredient to achieve the envisioned goal. In addition, the pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and additives, which facilitate processing of the active compounds into preparations which can be used pharmaceutically, and which can stabilize the same, the>ORT-1-binding protein IAIS is expressed in various tissues with different levels and, obviously, with different distributions of isotopes. These differences may contribute to tissue-specific features of the response to the ligand, FAS/APO1, and TNF. As in the case of other homologues of the CED3/ICE (Wang et al., 1994; Alnemri et al., 1995), found that the isoforms IAIS, which contain incomplete areas of the CED3/ICE (e.g., IAIS3) have inhibitory effect on the activity coexpression molecules IAIS1 and IAIS2; also found that they inhibit the induction of cell death by FAS/APO1 and p55-R. Expression of these inhibitory isoforms in cells may be a mechanism of cellular defense against mediated by FAS/APO1 and p55-R cytotoxicity. A wide heterogeneity isoforms IAIS, which greatly exceeds the heterogeneity observed for any of the other protease family CED3/ICE, should make possible a particularly fine-tuning function is active isoforms of IAIS.
It is also possible that some of the isoforms IAIS perform other functions. The ability MACH1 to contact with MORT-1 and MACH1, suggests that this isoform could be the in the cultures of 293-EBNA and MCF7, transfected this isoform significant cytotoxic effect expressed in HeLa cells, reflect, apparently, activation of endogenous expressed molecules IAISwhen linking with transfitsirovannykh molecules MACH1. It is clear that some isoforms IAIS could also function as docking sites for molecules that are involved in other, recitations the effects of FAS/APO1 and TNF receptors.
Due to the unique ability of FAS/APO1 and TNF receptors to cause cell death, as well as the ability of TNF receptors to run other damaging tissue activity, disturbances in the function of these receptors could be particularly harmful for the body. Indeed, it was shown that both excessive and insufficient functioning of these receptors contributes to the pathological manifestations of various diseases (Vassalli, 1992; Nagata and Golstein, 1995). Identification of molecules involved in the activity of signal transmission of these receptors, and finding ways of modulating the activity of these molecules could form the basis of new therapeutic approaches. In connection with the proposed Central role of the IAISas an intermediate step proteolytic function IAISas was done for some other protein family CED3/ICE (Thornberry et al., 1995; Enari et al., 1995; Los et al., 1995). Unique features of the homolog of the CED3/ICE inside molecules IAISallow to design drugs that could specifically affect its activity. Such drugs could protect against excessive immunopositive cytotoxicity involving IAISwithout intervention in the processes of physiological cell death, involving other members of the family CED3/ICE.
Other aspects of the present invention will be clear from the following examples.
The invention will be described hereinafter in more detail in the following non-limiting examples and accompanying drawings.
It should also be noted that the procedures (i) twohybrid screening and twohybrid test-galactosidase; (ii) induced expression, metabolic labeling and thus proteins; (iii) binding in vitro; (iv) evaluation of cytotoxicity; and (v) Northern blotting and sequence analysis set forth in Example 1 (see Boldin et al., 1995b) and 2 below, in respect of MORT-1 and MORT-1-binding protein, are equally applicable (with nekotoryye, therefore, built as a complete description of those procedures used for the isolation, cloning and characteristics of the IAIS in accordance with this invention, as described in detail in Example 3 below.
Example 1: Cloning and isolation of protein MORT-1, which binds to the intracellular domain of FAS-R
(i) Dvuhserijnyj screening and dvuhserijnyj test expression-galactosidase
Separation of proteins interacting with the intracellular domain of FAS-R, used yeast twohybrid system (Fields and Song, 1989). In short, this dvuhserijnaya system is a genetic analysis of the yeast-based detection of specific protein-protein interactions in vivo by restoring eukaryotic activator of transcription, such as GAL4, which has two separate domain, DNA binding domain and activation domain, and these domains in the expression and connection together form the recovered protein GAL4, which is able to communicate with lying against the progress of the transcription activating sequence, which, in turn, activates the promoter that controls the expression of reporter gene such as LacZ or HIS3 whose expression is easily seen in expressing vectors. In one expressing the vector sequence of the intended protein clone in phase with the sequence of the DNA binding domain of GAL4 for the formation of a hybrid protein with the DNA-binding domain of GAL4, and the other vector sequence of the second alleged interacting protein clone in phase with the sequence domain of the GAL4 activation for the formation of a hybrid protein with the activation domain GAL4. Then two hybrid vector cotransporter in yeast strain-host with reporter gene LacZ or HIS3 under the control of GAL4 binding sites against the course of transcription. Only transformed cells (cotransformation), which expressed these two hybrid protein, which can interact with each other, will be capable of expression of the reporter gene. In the presence of the reporter gene LacZ, the cells of the host expressing the gene will become blue when adding X-da to the cultures. Therefore, blue colonies indicate that two cloned candidate protein is able to interact with each other.
Using this twohybrid system has cloned the intracellular domain of FAS-IC, separately in vector pGBT9 (bearing DNA-binding sequence of the FAS-R in pGBT9 used the clone encoding the full-size cDNA sequence of FAS-R (WO 9531544), from which the intracellular domain (IC) cut out by standard procedures using various restricts and then embedded in the vector pGBT9, opened in his area of multiple cloning sites (MCS), the relevant restrictase. It should be noted that FAS-IC extends from amino acid residue 175 to balance 319 intact FAS-R, and this part, containing the remains of 175-319 is FAS-IC, built-in vector pGBT9.
The above-described hybrid (chimeric) vector was cotranslationally then, along with a library of cDNA from HeLa cells humans, cloned in the vector pGAD GH, bearing the activating domain of GAL4, a yeast strain is the master Nes (all of the above vectors pGBT9 and pGAD GH carrying cDNA library of HeLa cells, and the strain of yeast were purchased from Clontech Laboratories, Inc., USA, as part of twohybrid system, MATCHMAKER, NO. RT-1). Cotransfection yeast were selected on their ability to grow in medium without histidine (is environment), and the growing colonies testified about the positive transformants. Then selected yeast clones were tested for their ability to Express the LacZ gene, i.e., the activity of LACZ, and this was done by adding X-gl to the culture medium, to which, what armenta encoded lacZ gene. Thus, the blue colonies indicated the presence of active gene lacZ. For the activity of the lacZ gene is necessary that the activator GAL4 transcription was present in active form in the transformed clones, namely, that the DNA-binding domain of GAL4, encoded by the above-described hybrid vector, properly connected with the activating domain of GAL4 encoded by other hybrid vector. Such a connection is possible only if two proteins are fused with each of the GAL4 domains, can stably interact (communicate) with each other. Thus. His+and blue (LACZ+) colonies that were selected are the colonies that were cotranslationally vector coding for FAS-IC, and a vector coding for the protein product derived from HeLa cells humans, which can stably contact the FAS-IC.
Plasmid DNA from His+, LACZ+yeast colonies were isolated and introduced by electroporation into the strain E. coli HB 101 standard procedures with subsequent selection Leu+and ampicillin-resistant transformants, and these transformants are transformants carrying the hybrid vector pGAD GH, which had coding sequences as AMRRand L proteins, the ability to communicate with FAS-IC. Then from these transformed cells of E. coli were isolated plasmid DNA and re-tested with:
(a) re-transformation of the source containing the intracellular domain of FAS-R hybrid plasmid (hybrid pGBT9 bearing FAS-IC) in yeast strain HF7, as described above. As controls were used only vectors carrying sequences encoding the foreign protein, for example, age-Lamin or pGBT9, for cotransformation with a plasmid that encodes FAS-IC-binding protein (i.e., MORT-1). Then cotransformation yeast were tested for growth on one of His-environment or with different levels of 3-aminotriazole; and
(b) re-transformation of plasmid DNA and the original FAS-IC-hybrid plasmid and a control plasmid, described in (A), in a yeast cell host strain SEY526 and definitions LZ+activity (efficiency-gl, i.e. the formation of blue color).
The results of the above tests revealed that the distribution of colony growth in is-environment was identical to the distribution of LACZ activity, as assessed by the color of the colonies, i.e., His+colonies were also LACZ+. Next, LACZ activity in liquid culture (predpochtitelnye domain of GAL4 in yeast host SFY526, which are the best indutsiruemoi LACZ activator GAL4 transcription than cell-hosts HF7.
Using the procedure described above was identified, isolated and characterized a protein as indicated earlier MORT-1, now called MORT-1 ("the Mediator-induced receptor toxicity").
In addition, it should also be noted that in the range described above twohybrid-galactosidase test expression-galactosidase activity was evaluated also preferred test on the filter. In this screening, it was found that five of the approximately 3106cDNA containing an insert of the MORT-1. Then selected so the cloned cDNA insert MORT-1 sequenced using standard procedures for DNA sequencing. Amino acid sequence of MORT-1 (SEQ ID No. 2) transcribed from this DNA sequence. The numbering of residues in the proteins encoded by these cDNA inserts corresponds to the numbering of the data Bank, Swiss-Prot. Deletion mutants were obtained using PCR and point mutant oligonucleotide-directed mutagenesis (Current Protocols in Molec. Biol., 1994).
(ii) Induced the expression, metabolic labeling and immunoprecipitation proteins
In HeLa cells E. the extracellular domain of the p55-R (amino acids 1-168), fused with the transmembrane and intracellular domain of FAS-R (amino acids 153-319), and luciferase cDNA serving as control. Expression was performed using a controlled tetracycline expressing vector clone of HeLa cells (HtTA-1), which expresses controlled by the tetracycline transactivator (Gossen and Bujard, 1992); see also Boldin et al., 1995). Metabolic labelling with [35S]-methionine and [35S]-cysteine (DUPONT, Wilmington, DE, USA and Amershama, Buckinghamshire, England) was performed 18 h after transfection further 4-hour incubation at 37In the modified Dulbecco environment the Needle, not containing methionine and cysteine, but containing an additional 2% cialisbuynow calf serum. Then the cells were literally in RIPA buffer (10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% NP-40, 1% deoxycholate, 0.1% of LTOs and 1 mm EDTA) and the lysate was pre-osvetleni by incubation with irrelevant rabbit anticorodal (3 μl/ml) and pellet Protein G-Sepharose (Pharmacia, Uppsala, Sweden; 60 μl/ml). Immunoprecipitation was performed by incubation for 1 h at 4With an aliquot of 0.3 ml of lysate with mouse monoclonal antibodies (5 μl/aliquot) against oktapeptid FLAG (M2; Eastman Kodak), p55-R (No. 18 and No. 20); Engelmann et al., 1990) or FAS-R (ZB4; Kamiya Southand Oaks, CA., what Finance for 1 h to pellet Protein G-Sepharose (30 μl/aliquot).
(iii) Binding of in vitro
Slit proteins glutathione-S-transferase (GST) with FAS-IC wild-type or mutated FAS-IC received and absorbed onto glutathione-agarose pellet; see Boldin et al., 1995; Current Protocols in Molecular Biology, 1995; Frangioni and Neel, 1993). The binding of metabolically labeled fused protein FLAG-MORT-1 with GST-FAS-IC was assessed by incubation of these granules for 2 h at 4With extracts of HeLa cells, metabolically labeled with [35S]-methionine (60 µci/ml) that Express FLAG-MORT-1. The extracts were prepared in buffer containing 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% NP-40, 1 mm dithiothreitol, 1 mm EDTA, 1 mm phenylmethylsulfonyl 20 μg/ml Aprotinin, 20 μg/ml Leupeptin, 10 mm sodium fluoride, and 0.1 mm sodium Vanadate (1 ml per 5105cells).
(iv) evaluation of the cytotoxicity of running-induced expression of MORT-1
cDNA MORT-1, FAS-IC, p55-R and luciferase was built in a controlled tetracycline expressing vector and transfusional cells HtTA-1 (cell line HeLa) (Gossen and Bujard, 1992) together with cDNA Sekretareva placental alkaline phosphatase, were placed under the control of the SV40 promoter (vector pSBC-2, Dirks et al., 1993). Cell death was assessed after 40 h after transfection, or using test absorption of the dye neutral shall determine the quantity of placental alkaline phosphatase (Berger et al., 1988), Sekretareva in the environment for cultivation in the last 5 h of incubation.
In another series of experiments for the analysis region of the protein MORT-1, is involved in binding to the FAS-IC, the following proteins are temporarily expressed in HeLa cells containing controlled by the tetracycline transactivator (HtTA-1), using controlled tetracycline expressing vector (pUHD10-3): one FAS-R person; FAS-R person, as well as N-terminal part of MORT-1 (amino acids 1-117, "the head of MORT-1"); FAS-R person, as well as C-terminal part of MORT-1, contains a region of homology its "domain of death" (amino acids 130-245, "MORT-1 DD"); Flag-55.11 (amino acids 309-900 protein 55.11, fused at N-end with oktapeptidom FLAG, and protein 55.11 is specifically the P55-IC-binding protein. After 20 h after transfection the cells were trypsinization and re-sown at a concentration of 30,000 cells per well. After 24 h of additional incubation, the cells were treated for 6 h with monoclonal antibody against the extracellular domain of FAS-R (monoclonal antibody CH-11, Oncor, Gaithersburg, MD, USA) at various concentrations (0.001 to 10 μg/ml monoclonal antibody) in the presence of 10 μg/ml cycloheximide. Then determined the cell viability test the absorption of neutral red, and these resulta (in the absence of monoclonal antibody CH-11 against FAS-R).
(v) Northern analysis and sequence analysis
Poly And+-RNA was isolated from total RNA of HeLa cells (Oligotex-dT-mRNA kit. QIAGEN, Hilden, Germany). Northern analysis using cDNA MORT-1 as a probe visonary conventional methods (see Bolding et al., 1995). The nucleotide sequence of MORT-1 was determined in both directions using the method of dideoxyadenosine chain.
Sequence analysis of the cDNA MORT-1, cloned twohybrid way, showed that it encodes a novel protein. Further application twohybrid test to assess the binding specificity of this protein (MORT-1 denotes the Mediator-induced receptor toxicity") with FAS-IC, with whom he is associated, has led to the following results (Fig.1): (a) Protein MORT-1 binds with both human and mouse FAS-IC, but not associated with several other proteins tested, including three receptor family, receptors of the TNF/NGF (TNF receptors P55 and P75, and CD40); (b) it has Been shown that substitution mutation at position 255 (Il) in the "domain of death" FAS-R destroyed signaling both in vitro and in vivo (lpr mutationDM(Watanabe-Fukunga et al., 1992; Itoh and Nagata, 1993), also prevents binding to MORT-1 FAS-IC; (C) the binding Site MORT-1 FAS-R is inside the "domain of death" various sections of this protein: a Fragment of MORT-1, the corresponding residues 1-117, associated with a full MORT-1, but not associated with itself and with the FAS-IC. In contrast, the fragment corresponding to residues 130-245, binds to FAS-R, but is not associated with MORT-1 (Fig.1). In addition, from the results in Fig.1 shows that the district "domain of death" FAS-R is crucial for the self-FAS-IC, as well as the area of "domain of death" p55-R for self-p55-R=IC. Deletions on both sides of this "domain of death" does not affect the ability of their self, however, a deletion within these "domain of death" really affect the self-Association. In the case of MORT-1 binding to MORT-1 FAS-IC also depends on the full "domain of death" FAS-R, but it also depends on areas outside the district "domain of death" to bind FAS-IC.
In Fig.1 shows the interaction of the proteins encoded by the structures of the DNA-binding domain and an activating domain of GAL4 (pGBT9 and pGAD-GH) in transfected SFY526 yeast, activaloe test expression-galactosidase filter. Design DNA-binding domain comprised of four designs FAS-IC man, four design murine FAS-IC, including two full-sized design with substitution mutations Il to Leu or Il on l in position 225 (I225N and I225A, appropriate domain included three designs MORT-1, and part of MORT-1 corresponds to this part in the construction of DNA-binding domain; and construction of a full-sized FAS-IC one, and part of the FAS-IC corresponds to this part in the design of the DNA-binding domain. The intracellular domain of TNF-R P55 person (residues 206-426 p55-R-IC), human CD40 (CD40, remains 216-277) and TNF-R P75 person (P75-IC, remains 287-461), and Lamin, cyclin D and "empty" vectors GAL4 (pGBT9) served as negative controls in the form of structures of the DNA-binding domain. SNF-1 and SNF-4 served as positive controls in the form of structures of the DNA-binding domain (SNF1) and an activating domain (SNF4). "Empty" vectors GAL4 (pGAD-GH) were also used as negative controls in the form of designs activating domain. The symbols "++" and "+" indicate the development of a strong staining for 30 and 90 min of the test, respectively; and "-" means no development of color in the span of 24 hours Combinations that are not assessed not been tested.
The expression of molecules MORT-1, fused to the N-end with oktapeptidom FLAG (FLAG-MORT-1), resulted in the formation of proteins in HeLa cells of four different sizes - approximately 27, 28, 32 and 34 KD. The interaction of the MORT-1 FAS-IC in vitro was observed by conducting thus proteins from extracts of letiiu performed with antibody against FLAG (FLAG). It was also illustrates the interaction in vitro between MORT-1 and FAS-IC, where MORT-1 is in the form of fused proteins metabolically labeled with [32S]-methionine FLAG-MORT-1, obtained from extracts of transfected HeLa cells, a FAS-IC is in the form of a fused protein between human and mouse GST-FAS-IC, including a fused protein having a substitution mutation at position 225 in FAS-IC, all fused protein GST-FAS-IC was producirovanie in E. cli. GST-fused proteins were added to the granules of glutathione before interaction with extracts containing protein MORT-1-FLAG, after this interaction, and electrophoresis was performed in LTO-PAG. Thus, the interaction in vitro was determined by assessment, autoradiography after electrophoresis in LTO-page, binding35S]-labeled metabolic MORT-1, produced in transfected HeLa cells in the form of a fused protein with oktapeptidom FLAG (FLAG-MORT-1), with GST fused protein GST with human or murine FAS-IC (GST-hu FAS-IC, GST-mFAS-IC) or a fused protein with GST FAS-IC, containing substitution mutation Il on l in position 225. It was shown that all four protein FLAG-MORT-1 showed the ability to bind to FAS-IC by incubation with fused protein GST-FAS-IC. As in yeast twohybrid test (Fig.1), MORT-1 was not associated with the merged Pelkosenniemi contact with the intracellular domain of FAS-R, and with the intracellular domain of FAS Chimera-R extracellular domain which was replaced by the domain of the p55-R (p55-FAS), with the co-expression of these receptors in HeLa cells. In this case, the interaction of the MORT-1 FAS-IC in transfected HeLa cells, i.e., in vivo observed in immunoprecipitates different transfected HeLa cells showed an interaction in vivo and specificity of the interaction between MORT-1 and FAS-IC cells, cotransfection constructs encoding these proteins. So, protein FLAG-MORT-1 expressed and were metabolically labeled with [32S]-cysteine (20 µci/ml) and [32S]-methionine (40 µci/ml) in HeLa cells, separately or together with the FAS-R human, chimeric FAS-R, in which the extracellular domain of FAS-R was replaced by the corresponding region of the human p55-R (p55-FAS), or p55-R as a negative control. Cross-immunoprecipitation MORT-1 coexpression receptor were performed using specic antibodies. The results showed that FLAG-MORT-1 are able to bind with the intracellular domain of FAS Chimera-R-p55R with the extracellular domain of the p55-R and the intracellular domain of FAS-R, when the co-expression of these receptors in HeLa cells. Further, immunoprecipitate FLAG-MORT-1 from extracts of transfected cells also Pretoria of these receptors has led to coprecipitation FLAG-MORT-1.
Northern analysis using cDNA MORT-1 as a probe detected a single transcript hybridization in HeLa cells. In Northern-blotting, in which poly And+-RNA (0.3 ág) from transfected cells hybridized with cDNA MORT-1, it was found that the size of the RNA transcript (~1,8, etc., ad) was close to the size of the cDNA MORT-1 (~1702 nucleotide).
In sequeiros the analysis, it was found that this cDNA contains an open reading frame for ~250 amino acids. Fig.2 depicts the preliminary nucleotide (SEQ ID No. 1) and the decoded amino acid sequence (SEQ ID No. 2) MORT-1, which emphasize theme of "domain of death", as well as the possible initial Met residue (position 49; bold, underline, M) and a stop codon broadcast (asterisk under the codon in position 769-771). This motif of the "domain of death" has homology with known motifs "domain of death" p55R and FAS-R (p55-DD and FAS-DD). To determine the exact With the end of the MORT-1 and to obtain evidence about the exact N-Terminus (initial Met) MORT-1 conducted the following additional experiments.
Using methods described previously designed a number of structures that encode molecules MORT-1, fused at their N-end with oktapeptidom FLAG (FLAG-MORT-1), and expressed them in a tile is coded in the following cDNA, containing different parts of the coding MORT-1 sequence:
i) cDNA of oktapeptid FLAG, which is connected to the 5`-end cDNA MORT-1, which were deleterows nucleotides 1-145 SEQ ID No. 1 (see Fig.2);
ii) the cDNA of oktapeptid FLAG, which is connected to the 5`-end cDNA full MORT-1;
iii) FLAG cDNA, linked to the 5`-end cDNA MORT-1, which were deleterows nucleotides 1-145, and nucleotides 832-1701 SEQ ID No. 1 (Fig.2), and the codon GCC in position 142-144 was mutated in TCC to prevent the beginning of broadcast in this site.
After the expression of the above fused products FLAG-MORT-1 were immunoprecipitated, as described above, using monoclonal antibodies against FLAG (M2) or antibodies against TNF-R P75 as a control (No. 9), followed by electrophoresis in LTO-page (10% acrylamide) and autoradiography. The results of this analysis with the above-described fused products FLAG-MORT-1 confirmed With the end of the MORT-1 and provided evidence that the N-end of the MORT-1 may be in position 49 of the sequence shown in Fig.2.
Indeed, it has been shown by additional experiments on the expression of MORT-1 without oktapeptid FLAG, merged with its 5`-end that Met49operates as an effective site of translation initiation.
Search, provide sequence MORT-1. Thus, MORT-1 represents a new specific FAS-IC-binding protein.
High expression of the P55-IC starts causing cell death actions (Boldin et al., 1995). Expression of FAS-IC in HeLa cells also has the same effect, though in a lower degree, which can be detected only with the use of sensitive test. Conducted analysis of ligand-independent run citizeny effects in cells transfected with MORT-1, and the P55-IC and FAS-IC man. The effect of temporal expression MORT-1, FAS-IC man, the P55-IC man or luciferase (control) viability of HeLa cells was evaluated using controlled tetracycline expressing vector. Cell viability was evaluated at 40 min after transfection of these cDNA in the presence or in the absence of tetracycline (1 μg/ml for inhibition of expression), together with cDNA coding secreterial placental alkaline phosphatase. Cell viability was determined by absorption test neutral red or (for specific definitions of viability of specific cells expressing transtitional DNA) by measuring the amounts of alkaline phosphatase placental secreted into the culture medium.
the than cell death, induced expression of FAS-IC. These cytotoxic effects of all proteins P55-IC, FAS-IC and MORT-1, seems to be connected with areas of the "death domains" that are present in all of these proteins and have a tendency to self and thereby may enhance the cytotoxic effects.
In connection with the above properties MORT-1, namely, specific binding to MORT-1 with a specific area of FAS-R, which is involved in the induction of cell death, and due to the fact that even a small change in the structure of the area, preventing the transmission signal (mutation lrDM], abolishes the binding to MORT-1, one might think that this protein plays an important role in the transmission signal or the start cell death. This is further confirmed by the observed ability of MORT-1 independently run cytosine action. Thus, MORT-1 may function as (i) a modulator of self-FAS-R, thanks to its own ability to contact FAS-R, as well as with yourself, or (ii) to serve the website docking for additional proteins involved in the signal transmission FAS-R, i.e., MORT-1 may be "joining" the protein and, therefore, can bind other receptors, in addition to FAS-R, or (iii) MORT-1 may be part of a separate system of signal transmission, which the modulation mediated FAS-R cellular effects (cytotoxicity), the above experiments were performed using vectors encoding part of MORT-1 ("head MORT-1, amino acids 1-117, and "MORT-1-dd, amino acids 130-245) (separately), with the vector coding for FAS-R man for cotransfected in HeLa cells. In these experiments, various proteins and combinations of proteins temporarily expressed in HeLa cells containing controlled by the tetracycline transactivator (HtTA-1), by embedding sequences encoding these proteins, controlled by tetracycline expressing vetor pUHD10-3. In the control transfected used vectors encoding only FAS-R, and vectors encoding the protein FLAG-55.11 (protein 55.11 represents specifically the P55-IC-binding protein, part of which, containing amino acids 309-900 was merged (N-end) with oktapeptidom FLAG).
After periods of transfection and incubation transfetsirovannyh cells were treated with various concentrations of monoclonal antibodies against FAS-R (CH-11), which binds specifically to the extracellular domain of FAS-R expressing cells. This binding antibodies against FAS-R induces the aggregation of FAS-R on the cell surface (as well as ligand FAS-R) and induces an intracellular cascade of reactions signal, mediarray FAS-IC, privaie monoclonal antibodies against FAS-R (SN-11) in the range of 0.01-10 μg/ml, usually the concentration of 0,005; of 0.05, 0.5 and 5 μg/ml Cells were treated with antibody against FAS-R in the presence of 10 μg/ml cycloheximide.
The results of this analysis show that the expression of FAS-R transfected cells reported increased sensitivity to cytocidal effects of antibodies against FAS-R (compare "fas" and "55.11"). Further, coexpressed district MORT-1, contains a region of homology "domain of death" and FAS-R ("fas + MORT-1 dd), strongly inhibits inducible FAS (i.e., mediated FAS-R) cell death, as was to be expected on the basis of the ability of the district "death domain" (DD) to bind with the "domain of death" FAS-R (FAS-DD). In addition, coexpression N-terminal part of MORT-1 and FAS-R ("fas + MORT-1 he") does not preclude mediated FAS-R cell death and, if at all valid, slightly increases the cytotoxicity (i.e., slightly increases cell death).
Thus, the above results clearly show that the protein MORT-1 has two separate area related to binding to FAS-IC and mediareview cytotoxicity of FAS-R.
Therefore, these results also provide the basis for the use of various parts (i.e., active fragments or analogs) protein MORT-1 for various pharmaceutical applications. For example, analogs or fragments or p is ibili", can be used for inhibition mediated FAS-R cytotoxic effects in containing FAS-R cells or tissues and, therefore, to protect these cells or tissue from the harmful effects of ligand FAS-R in such cases, such as acute hepatitis. Alternatively, analogs or fragments or derivatives thereof of protein MORT-1, which contain essentially only the N-terminal part of MORT-1, can be used to enhance mediated FAS-R cytotoxic effects in containing FAS-R cells and tissues and thus cause increased destruction of these cells or tissues, if it is desirable, in such cases, as, for example, tumor cells and self-reactive T and b cells. As described above, the application of different areas of the MORT-1 can be performed using various recombinant virus (e.g. vaccinia virus) for doing the coding region MORT-1 sequence in specific cells or tissues, which must be processed.
In addition, it is also possible to prepare and use various other molecules, such as antibodies, peptides and organic molecules that have sequence or molecular structure corresponding to the above areas of MORT-1, to achieve the identification, selection and characteristics of other proteins, is able to connect with MORT-1 (i.e., MORT-1-binding proteins), see Examples 2 and 3.
Example 2: allocation of the MORT-1-binding protein
(i) Dvuhserijnyj screening and dvuhserijnyj test expression-galactosidase
Similarly to the procedure described in Example 1, using the intracellular domain of the P55 TNF-R (P55-IC) and MORT-1 as "baits" and screening libraries of human cells were obtained two clones of cDNA that encode a protein product that can communicate both with MORT-1, and with the P55-IC. Both clone have identical nucleotide sequences at the 5`end, as shown in Fig.3 (SEQ ID NO. 3).
(ii) Binding properties of new cloned cDNA in twohybrid the screenings
Using the aforementioned yeast twohybrid procedure construct containing cDNA new MORT-1-binding protein, was used as "production", to which were added some "bait" in separate reactions, to determine the binding specificity MORT-1-binding protein encoded by this cDNA. These "bait" consisted of design, coding MORT-1, part of MORT-1 ("head MORT-1, amino acids 1-117, "tail of the MORT-1, amino acids 130-245), the P55-IC (206-426 P55) or part ("domain GMI src="https://img.russianpatents.com/img_data/82/820860.gif">
The above results twohybrid test expression-galactosidase to assess the binding of clone with a large set of "lures" confirmed that the protein encoded by this clone, specifically binds to the domains of death as TNF-R p55 and MORT-1.
In General, MORT-1-binding protein can be used directly to modulate or meditirovaniya associated with MORT-1 action on the cells or indirectly, to modulate or meditirovaniya actions ligand FAS-R cells, code this action is modulated or mediasuite MORT-1. The same is true in other intracellular proteins or intracellular domains of transmembrane proteins, as has been specifically demonstrated for TNF-R p55 here.
MORT-1-binding proteins include proteins that are specifically associated with the whole protein MORT-1, or proteins that are associated with different regions of the protein MORT-1, for example, with the above N - and C-terminal regions MORT-1. MORT-1-binding proteins that are specifically associated with such areas, can be used to modulate activity in these areas and, consequently, the specific activity of MORT-1, defined in these areas.
Example 3: selecting and /img.russianpatents.com/chr/946.gif">-galactosidase test, sequencing and sequence analysis
Using the procedures described in Examples 1 and 2, a full-sized design, protein-coding MORT-1 person, used as "bait" in a yeast twohybrid the system to allocate a clone cDNA encoding another new MORT-1-binding protein. This new protein was originally identified as MORT-2, now renamed and called IAIS (i.e., MORT-1-associated homolog CED3) on the basis of its characteristics, described in detail below.
This cDNA clone sequenced by standard methods described in Examples 1 and 2 above. The sequence analysis using standard procedures and computer programs (see Examples 1 and 2) revealed that this cDNA is a new sequence and encodes a novel protein (or DNA sequence or amino acid sequence not found in the databases of GENBANK or PROTEINBANK). Next, cDNA encoding MACH, found an open reading frame ORF-B, which has strong homology with the above-described region (5` progress against transcription) of the motif of "death domain" of a protein MORT-1 (see Example 1). In Fig.4A-C shows the structure of a part of the cDNA clone MACH, which contains the ORF-B (235 amino acid residues; the activity (SEQ ID No. 4) of the cDNA molecules MACH (Fig.4C). In Fig.4A shaded region ORF-B is a region having high homology with a region of the MORT-1 progress against transcription from the motive of "domain of death" MORT-1, and this region of homology ORF-B MACH consists of the amino acid residues underlined in Fig.5V.
Next, yeast dvuhserijnyj test was used to assess the binding specificity MACH with MORT-1, in particular, to determine the area of MORT-1, associated with MACH, as well as to determine which of the ORF MACH interacts with MORT-1, and the procedure described here above in Examples 1 and 2. Briefly, prepared various designs MORT-1 and MACH to test the interaction of proteins encoded by the structures of the DNA-binding domain and an activating domain of GAL4, in transfected SY526-yeast cells with the evaluation using the test filter expression-galactosidase. Design with the DNA-binding domain prepared in vectors pGBT9 and designs activating domain was prepared in the vector pGAD-GM. For designs activating domain used cDNA full-MACH (MACH), as well as design, coding only the area of the ORF-B (IAIS). Control structures activating domain were constructs containing the coding sequence polnorazmernogo the rd DNA-binding domain used cDNA full MORT-1 (MORT1), as well as design, coding only the area of the MORT-1 progress against transcription (MORT-1-DD, amino acids 130-245). The control structure of the DNA-binding domain, which were designed to determine the binding specificity IAIS included design, encoding Lamin (Lamin), remains 287-461 intracellular domain of TNF-R P75 man (human h75 IC), cyclin D (cycD), SNF1, the remains 206-426 intracellular domain of TNF-R P55 man (human h55 IC), the "domain of death" intracellular domain of FAS-R man (human Fas DD), remains 216-277 intracellular domain of human CD40 (human CD40 IC), vectors without inserts or "empty" vectors pGBT9 (pGBT9, negative control) and the design, the coding region ORF-B IAIS (IAIS). This analysis determined the development of the color, the higher was the development of color, the greater was the interaction between the constructs encoding the DNA-binding domain and an activating domain. The development of painting depicted by symbols, where "+++" and "+" indicate the development of a strong staining for 30 and 90 min of the test, respectively, and "---" indicates no development of color within 24 hours of the test. In cases where the interaction was not determined, there is no character. The results of the various interactions described above for the case presented in tab is img>
Thus, as can be seen from the results shown in table 3 above, it is clear that:
(a) IAIS binds to MORT-1 very strong and specific;
(b) the binding site IAIS in MORT-1 is in front (against the course of transcription) motif of the "domain of death" in MORT-1, i.e. it is in the area of MORT-1, defined by amino acids 1-117 MORT-1;
(c) District ORF-B IAIS is interacting with MORT-1 region of the protein IAIS; and
(d) District ORF-B IAIS able to samonazvanie (self).
(ii) Cell cytotoxic effects mediated by the ability of the self-protein IAIS
The observation that IAIS can coassociativity, in particular, that the district ORF-B IAIS samoassotsiiruyutsya, and cellular cytotoxicity observed for the intracellular domain of TNF-R P55 and FAS-R and observed for MORT-1 (see Example 1), it was assumed that the self-Association IAIS may also participate in cellular cytotoxicity.
To study this possibility was preparing design, coding IAIS using controlled tetracycline expressing vector (see Example 1). These constructs were used for transfection of HeLa cells, in which these vectors are temporarily expressed. In addition to the structures IAIS and other control structures primenyaleya structures IAIS. These other designs included MORT-1, FAS-IC man and the luciferase (Luc). In addition, it was also tested cotransfection of HeLa cells using constructs MORT-1 and IAIS to determine what effects could cause an interaction between these proteins. After transfection of HeLa cells incubated and cell viability was assessed after 48 h after transfection in the presence or in the absence of tetracycline (1 μg/ml) for inhibition of expression. Cell viability was determined by the test of absorption of neutral red.
These results are shown in Fig.6, which shows in chart form the ligand-independent start citizeny effects in cells transfected with the IAIS, in comparison with cells, transfitsirovannykh constructs encoding other proteins, as well as cotranslationally cells (MORT 1 + IAIS). The results are presented in the form of viable cells in units of OD at 540 nm for each design, and each design is shaded column represents the incubation of the cells after transfection in the absence of tetracycline, and the solid column represents the incubation of the transfected cells in the presence of tetracycline.
From the results shown in Fig.6, it is seen that the IAIS induce, leading to a dramatic cytotoxic effect. This cytotoxic effect is probably related to the ability IAIS to self.
(iii) Northern analysis
Using well-known procedures (see Example 1) was performed Northern analysis of some cell lines using cDNA IAIS as a probe. The results of this analysis show that in a large number of cell lines, in particular, cell lines CEM, Raji, Daudi, HeLa, Alexander, Jurkat and A there are two hybridization transcript with a size of approximately 3,2 T. p. N.
In connection with the foregoing, protein IAIS, in particular, protein MACH1 (ORF-B IAIS), can be used directly to modulate or meditirovaniya actions ligand FAS-R cells when this action is modulated or mediasuite MORT-1. The fact that the IAIS specifically binds to MORT-1, provides the specific way in which the IAIS or ORF-B IAIS can be used for modulation of this specific area of MORT-1 and, consequently, the specific activity of MORT-1, is defined by the area located against the course of transcription. Further, the IAIS or ORF-B IAIS can be used as a modulator or mediator of intracellular effects similar to itself is knosti.
Further analyses of protein IAIS and DNA sequences encoding it, were performed as described below. Further, it was found that the ORF-B IAIS represents only one of the isoforms of the IAIS. Therefore, protein IAIS and encoding its DNA sequences were renamed, as will become clear from the following description.
(a) Dvuhserijnyj screening for proteins that bind to MORT-1, have discovered a new protein that contains a common motif sequence with MORT-1
As mentioned above, for the identification of proteins that are involved in the induction of cell death MORT-1, used dvuhserijnyj method for screening cDNA libraries for proteins that are associated with MORT-1. Dvuhserijnyj screening of libraries In human cells (Durfee et al., 1993) using cDNA MORT-1 as "bait" gave the cDNA clones of the MORT-1, which reflects the ability of this protein to self, as well as clones TRADD, which effectively binds to MORT-1 (see Example 2). The screening gave the cDNA clones of the new sequence, the product of which specifically binds to MORT-1. This protein, which was originally named the IAIS, and later, after the discovery that it occurs in the form of multiple isoforms (see below), was renamed and called IAISIn Fig.5 shows the results of the interaction of MORT-1 and IAIS in transfected yeast cells. Briefly, MORT-1 and MACH1 and deletion constructs, as well as MACH1, the mutant MACH1, in which the catalytic s360replaced by Ser (MACH1 (C360S)), and the intracellular domain of FAS-R man (FAS-IC) expressed in transfected SFY526 yeast in the construction of DNA-binding domain and an activating domain of GAL4 (pGBT9 and pGAD-GH). Their interaction was evaluated in the test expression-galactosidase filter, as described Boldin et al. (1995b). The results are presented as the time required for the development of a strong color. ND indicates that the test was not performed. None of the tested inserts not interacted with a number of tested negative controls, including with the intracellular domain of TNF-R P55 human TNF-R P75 and CD40 and designed, cyclin D and "empty" vectors GAL4. MACH1 cloned twohybrid screening labeled GAL4 AD libraries In human cells (Durfee et A1., 1993) proteins that are associated with MORT-1, using a reporter strain HF7c yeast. Except when there are other indications, all of experiments, 995). Deletion analysis showed that MACH1 associated with the N-terminal part of MORT-1, which is involved in the induction of cell death (Chinnaiyan et al., 1995). MACH1 also coassociativity in transfected yeast. However, he was not in touch with some of the control protein and, in contrast to MORT-1, was not able to contact FAS-R (Fig.5). The expression of molecules IAISl in mammalian cells gave a protein of 34 kDa, which was associated with molecules of MORT-1, coexpression with him. He was also able to contact fused protein GST-MORT-1 in vitro.
Comparison of amino acid sequences in MACH1 and MORT-1 revealed the common motif sequence (designated as "Mort module" (module MORT)) in these two proteins, different from the motif of death, through which the MORT-1 binds to FAS-R. This motif occurs once in MORT-1 and twice in MACH1. The same motif is also found in REA-15, the phosphoprotein of astrocyte unknown function. Preliminary data suggest that the motif of the MORT-1 is involved in the binding MACH1 (and other isoforms IAIS) with MORT-1.
Fig.7A depicts the decoded amino acid sequence mutants MACH1 (figs.7) are indicated by asterisks. Fig.7B shows the sequence homology of these modules in IAIS(labeled IAIS in Fig.7B), MORT-1 and gene REA-15 (access number H. Identical and similar residues are indicated by blocks and shaded areas, respectively.
Fig.8 gives a view of the chart domain of death and MORT modules and district homology CED3/ICE in FAS/APO1, MACH1 and MACH1.
It was shown that the area in MORT-1, which contains a "module MORT", participates in the induction of cell death by this protein (see Example 1 above). It was also shown that it helps, though not a sufficient, self-MORT-1 (see Example 1). As shown in Fig.5, analysis of the binding properties of deletion constructs MACH1 in transfected yeast found a similar part of the modules MORT in self-MACH1, and its binding to MORT-1: analysis of structures in which the area below (in the direction of transcription from) module MORT was absent, were unable to bind to each other, although they retained the ability to bind to full-MORT-1 and with a full MACH1. Further korepodobnoj these proteins. To further assess the involvement of MORT modules in these interactions deletion mutants MACH1, merged with oktapeptidom FLAG (FLAG-MACH1), expressed in HeLa cells and evaluated for their binding in vitro with produced in bacteria fused protein glutathione-S-transferase-MORT-1 (GST-MORT-1). As shown in Fig.9A-C, it was found that like the binding observed in yeast twohybrid test, this binding in vitro depends on the interaction area in the modules MACH1. Fig.9A and 9B show the results (autoradiogram) interaction in vitro IAIS1 and its deletion mutants with MORT-1. Briefly, [32S]-labeled metabolic MACH1, MACH1, fused at its N end with oktapeptidom FLAG (FLAG-MACH1) shorter-end of the mutants FLAG-MACH1 and, as a control, luciferase were obtained in transfected HeLa cells. Expression was performed using a controlled tetracycline expressing vector, clone of HeLa cells (HtTA-1), which expresses controlled by the tetracycline transactivator.
Fig.9A shows the expression of these proteins and their m the following antibodies: Rabbit antisera against MACH1 and against the MORT-1 received against the fused protein GST-MACH1 and GST-MORT-1. Mouse antibodies against oktapeptid FLAG (M2) and against FAS/APO1 (SN, Yonehara et al., 1989) bought by Eastman Kodak and Oncor (Gaithesburg, MD), respectively. Mouse monoclonal antibody against epitope (S, Field et al., 1988) and antibody against TNF was obtained in our laboratory according to conventional methods, well known in this field. Fig.9B shows affinity binding of these proteins with GST-MORT-1 adsorbed to glutathione-agarose pellets (or, as control, with GST or GST fused to the intracellular domain of FAS/APO1). Fig.9C shows the results of immunoprecipitate merged various designs MORT-1 and IAIS using a variety of specific antibodies.
(b) MACH occurs in multiple isoforms
Northern analysis using cDNA IAIS1 as a probe detected not detected a large number of transcripts (transcripts) of approximately 3 M. p. H. in some different cell lines. Briefly, Northern blotting total RNA (14 µg per track) or poly (A+-PHK (2 µg) from multiple cell lines was performed using cDNA MACH1 as a probe. Researched kletochnye gland, acute lymphoblastic T-cell leukemia, Burkitt lymphoma, epitheloid carcinoma, hepatoma human acute T-cell and rhabdomyosarcoma, respectively. Quite diffuse outlines hybridization bands on Northern-blots suggest that these transcripts are heterogeneous sizes in the range from 2.85 to 3.5 T. p. N. Both the quantity and the sizes of these transcripts were varieeruvusi among various human tissues and did not correlate with expression of MORT-1 (Chinnaiyan et al., 1995) or FAS/APO1 (Watanabe et al., 1992). The cDNA probes radioactively marked with a set for random priming (Boehringer Mannheim) and used for analysis of multiple tissue blotto person (Clontech) according to the manufacturer's instructions. For example, in the testis and skeletal muscle transcripts MACH were barely detectivesyme, although these tissues Express a significant amount of MORT-1. In contrast, it was found that resting mononuclear peripheral blood leukocytes, in which the expression of MORT-1 is very low, Express IAIS at high levels. Activation of the lectin these leukocytes leads to a noticeable change in the size distribution of transcripts IAIS, together with the induction of MORT-1.
Exploring perods cDNA probe MACH1. MACH1 and IAIS2 cloned from a cDNA library Charon BS derived from mRNA of human thymus. This library was subjected to screening under strict conditions cDNA probe MACH1, labeled with a set of random priming (Boehringer Manngeim). Other isoforms IAIS cloned using RT-PCR carried out on total RNA from Raji (MACH1,2,3,3,4 and5) and Daudi (IAIS2,2,3,4 and5) lymphoblastoid cells. The reaction of reverse transcriptase (RT) was performed with oligo-dt-adapternum primer (5`-GACTCGAGTCTAGAGTCGAC (T)17-3`; SEQ ID No. 26) and the reverse transcriptase Superscript II (GIBCO-BRL), used in accordance with the manufacturer's instructions. The first cycle of PCR was performed with the system Expand Long Template PCR (Boehringer Mannheim) using the following sense and antisense primers: 5`-AAGTGGAGCAGATCAGAATTGAG-3`, corresponding to nucleotides 530-551 cDNA MACH1 (SEQ ID No. 4), and 5`-GGGGGG-3` (SEQ ID No. 27), respectively. The second C is th" type: 5`-GAGGATCCCCAAATGCAAACTGGATGATGAC-3` (SEQ ID No. 28) and 5`-GCCACCAGCTAAAAACATTCTCAA-3` (corresponding to nucleotides 962-939 SEQ ID No. 4) cDNA MACH1, respectively.
To confirm that the IAIS3 and IAIS4 have the initiation codons, cloned most 5`sequence of these isoforms from RNA of Raji cells. The reaction RT-PCR performed using oligo-dt-adapting primers described above were accompanied by two cycles of PCR with Vent polymerase (NEB)) using the following sense and antisense oligonucleotides: 5`-TTGGATCCAGATGGACTTCAGCAGAAATCTT-3` (SEQ ID No. 29) and 5`-ATTCTCAAACCCTGCATCCAAGAG-3` (corresponding to nucleotides 946-923 SEQ ID No. 4) MACH1. The last oligonucleotide is specific for-isoforms. Among the clones obtained in this way, the clones that were found, contain the nucleotides encoding amino acid "unit 2" (the presence of which distinguishes MACH3 and IAIS4 from MACH1 and MACH2, as discussed below), were completely sequenced. Nucleotide sequences of all cloned isoforms was determined in both directions using the method of dideoxyadenosine chain. Were obtained only partial cDNA clones IAIS3 and IAIS of these isoforms was investigated in detail. These results are presented in the form of diagrams in Fig.12 and shown as examples in Fig.13, where the amino acid sequences of three of these isoforms was compared with known homologues.
Fig.10 shows in chart form the various isoforms of the IAIS. Coding regions are represented in the form of prisoners in units of zones. Different domains in the coding regions are marked by different shades: modules MORT-1 (); three blocks of amino acid sequences that are found in various combinations in these isoforms. Shows the position of the residues in the region of homology CED3/ICE, which is considered in determining the catalytic activity of ICE, on the basis of x-ray (crystalline) structure. Those portions of the nucleotide sequence of MACH1, which are absent from the sequences of other isoforms are indicated in the diagrams of these isoforms of V-shaped connecting lines. Length districts cDNA, which may correspond to the individual exons are listed below the chart MACHl. No 65 nucleotides that MACHl code "block 2", causes a change in the IAIS1 and MACH2 RA other amino acids, which together comprise their unique C-terminal region. On the other hand, in IAIS3 and IAIS4 saved frame read "unit 3", but the absence of nucleotides that encode the district CED3/ICE, and part of the 3`non-coding region leads to a change in reading frame nucleotide located further in transcription. Because of this change, large 5`-part of this non-coding region downstream of the transcription actually encodes 10 amino acids, which constitute the C-terminal region unique to these two isoforms (shaded). As indicated in Fig., were obtained only partial cDNA clones IAIS3 and IAIS2.
Isoforms cloned from cDNA library of human cells (IAIS1), from a cDNA library of human thymus (MACH1 and2) and of mRNA lymphoblastoid Raji cells human (MASN1,2,3,3,4 and5) and Daudi (IAIS2,2,3,4 and1. The initiating codon of the clones selected in such way, is located inside the second module MORT. The cDNA sequence and amino acid sequence of the isoform IAIS represented in the list of sequences and identified as follows in table 4.
In the sequence of the different isoforms are related to each other as follows: (a) all isoforms IAIS have shared consisting of 182 amino acids N-terminal site, which covers modules MORT, although variious carboxy-terminal (3`- downstream of the transcription) on these modules, as well as in their non-coding regions. (b) On the basis of their C-terminal sequences of these isoforms are divided into two subgroups: four isoforms, defined as the subgrouphave different-all because of a change in reading frame. Two (MACH1 and IAIS2) have in common With the end of the detected isoforms, originally cloned in twohybrid screening, and two (IAIS3 and IAIS4) have different the C-terminal region, which is very similar to protease family CED3/ICE (see below); (C) Areas extending between the area of the module MORT and C-terminal region, which defines these subgroups, variious from one isoform to another. However, careful research has shown that these intermediate areas are composed of various combinations of the same three amino acid blocks (blocks 1, 2 and 3). Variations in amino acid sequence among different clones represent two types of variations in nucleotide sequence, which, most likely, are formed by alternative splicing: (a) inserting or absence of any of the two nucleotide sequences, one of 45 nucleotides and the other from 65 nucleotides, or both, below nucleotides encoding Lys184; (b) the presence of additional inserts in the area, which in IAIS1 is 3s-non-coding part. These variations affect both the reading frame, and the length of the protein.
Part isoforms IAIS includes homolog of the CED3/ICE. Search in the data Bank revealed that the C-terminal region isoforms IAISincludes unit 3, and the sequence stretches in the course of transcription from him, closely similar to protease family CED3/ICE. PCIe ced3 protein Caenohabditis elegans, CED3 (Ellis and Horvitz, 1986; Yuan et al., 1993) and the known protease family CED3/ICE: SRR (Fernandes-Alnemri et al., 1994), also called apopain (Nicholson et al., 1995) and Yama (Tewari et al., 1995b), Mch2(Fernandes-Alnemri et al., 1995), Ich-1 (Wang et al., 1994; human homolog of the murine protein Nedd2, Kumar et al., 1994), ICErelII (Munday et al., 1995), also called TX and Ich2 (Faucheu et al., 1995; Kamens et al., 1995), and ICE (Thornberry et al., 1992; Cerretti et al., 1992). Fig.11 schematically depicts kulinarne alignment (analysis of primary structure) of the amino acid sequences of isoforms IAIS and various well-known members of the family proteases CED3/ICE. Shows the amino acid sequence MACH1, MACH1, IAIS3 and CED3 protease Caenohabditis elegans and known human protease family of proteases CED3/ICE.
The above C-terminal region IAIS most closely similar to SRR (41% identity and 62% homology) and CTD3 (34% identity and 56% homology). He finds much less similarity with ICE (with 28% identity and 50% homology) and its closely related homologues ICErelII (also known as TX and Ich) and ICErelIII. The similarity observed almost throughout the area, starting from Tug block 3 to the end of the isoforms IAIS1. However, there is one exception - conservative substitution of Ser to Thr at the site corresponding to Ser347 ICE. Another small but potentially important difference sequence between isoforms IAISand other members of this family of proteases is to replace the AGD on Gln residue corresponding to ID ICE. This residue, which is adjacent to the putative catalytic cysteine residue is completely conservative in all other members of the family CED3/ICE. Also part of the residues at sites located near astatke the src="https://img.russianpatents.com/chr/945.gif">from found in other members of the family CED3/ICE.
(b) Protease family CED3/ICE contain sites camerasmobile. It is known that some of these proteases are really smarasderagd and manifestation of maximum activity depends on this processing. Their full bioactive form consists of two ecovalence related products splitting, which differ in size (P20 and R17 in ICE; p17 and R in SR, as shown by the arrows in Fig.11). The presence of potential sites authorssale other members of this family suggests that they also be subject to similar processing, which depends on the manifestation of maximum activity. These potential sites authorssale found in MACH1 almost in the same locations as in SR (see shaded blocks in Fig.11). The website corresponding to the N-end of the subunit R17 SRR, is the second conservative block of amino acids, at a distance of only a few amino acids against the course of transcription relative to the N-Terminus region of homology CED3/ICE (below Asp216). The website that corresponds to the point of cleavage between the two subunits SRR, is, like all other members of the family SRR known that they Russiain (below Asp374). This conservatism assumes that the region of homology CED3/ICE MACH1 is subject to proteolytic processing. The sizes of the two expected products of this cleavage is very close to the dimensions of the two subunits protestirovanny molecules SRR.
(C) a Region of homology CED3/ICE in the IAIS has proteolytic activity
To determine whether the region of homology CED3/ICE in IAISproteolytic activity, applicants expressed the area extending from potential customers cleavage progress against transcription of this area, between Asp216 and Ser217, to the end of this protein in bacteria as a fusion with GST protein. Bacterial lysates were tested for the ability to split fluorogenic peptide substrates, which, as has been shown before that split other homologues CED3/ICE. Used two substrate peptide: First, Acetyl-Asp-Glu-Val-Asp-a-(4-methylcoumarin-7-amide) (Ac-DEVD-AMC), corresponds to the sequence in poly(ADP-ribose)polymerase (PARP), a nuclear protein, which is cleaved in the cells before stimulation of the FAS-R (Tewari et al., 1995b), as well as in other processes of apoptosis (Kaufiaann, 1989; Kaufmann et al., 1993; Lasebnik et al., 1994). This fluorogenic substrate efficiently cleaved SRR. The WTO is the IR IL-1. This fluorogenic substrate is cleaved ICE. As shown in Fig.12A-F and 13A-B, lysates of bacteria expressing a region of homology CED3/ICE MACH1, effectively uncoupled produced from a sequence of PARP fluorogenic substrate. Although they are not measured proteolytic activity against produced from the sequence of precursor IL-1fluorogenic substrate (controls), Ac-YVAD-AMC, which is the site of cleavage ICE in the precursor of IL-1(Thornberry et al., 1992). Proteolytic activity inhibited iodixanol acid (5 mm), which confirms that it is mediated thiol protease. Cleavage was not observed with lysates containing fused with GST district homology CED3/ICE IAIS, in which the catalytic cysteine residue s360was replaced by Ser. Also, lysates from bacteria expressing the full-size protein MACH1 in the form of a fusion with GST protein did not split Ac-DEVD-AMC, possibly due to the absence of bacterial enzymes capable of processing full-sized molecules. Cleavage was not observed with lysates containing either of the two potential products of the cleavage area homologo substrate, Ac-DEVD-AMC (50 μm), extracts of E. coli expressing a fusion with GST protein region of homology CED3/ICE MACH1 (Ser217 to-end of the protein), compared with the absence of cleavage extracts of bacteria expressing GST-fused proteins with full-length molecule MACHl or either of the two potential proteolytic products of the region of homology CED3/ICE (Ser217 to Asp374 and Asp374 to-end of the protein).
It also shows a dependence on the concentration of substrate cleavage of Ac-DEVD-AMC, incubated for 180 min with extracts of bacteria expressing a region of homology CED3/ICE MACH1 in fused with GST protein (see Fig.13B). Splitting is not observed in the presence of iodixanol acid (5 mm). The extracts had no activity on Ac-YVAD-AMC, fluorophenol the substrate corresponding to the substrate site ICE in the precursor of IL-1.
In short, fused with GST proteins were received in bacteria XL1-blue using the expressing vector pGEX3. Bacteria were literally by sonication in buffer containing 25 mm HEPES (pH 7,5), 0,1% 3-[3-cholamidopropyl)dimethylamino]-1-propanesulfonate, 5 mm EDTA and 2 mm DDT, followed by centrifugation at 16000 g for 10 min electrophoresis Analysis the quota of 50 µl of the extracts (4 mg/ml total protein) were incubated at room temperature for specified time periods in a total reaction volume of 500 μl with fluorogenic substrates at the indicated concentrations. The release of AMC was measured spectrofluorometry an excitatory wavelength of 380 nm and the wavelength of emission of 460 nm. The concentration of AMC was determined using curve standards. Both fluorogenic substrate was obtained from Peptide Institute Inc. (Osaka, Japan). It has been shown that other protease CED3/ICE show full activity only after proteolytic processing, which is performed either by camaraderie, either through their cleavage by other proteases (review Kumar, 1995; Henkart, 1995). The observation that the applicants lysates of bacteria expressing molecules GST-MACH1, do not possess enzymatic activity, in contrast to the activity observed in the lysates of bacteria expressing a region of homology CED3/ICE, suggests that activity IAISalso requires processing. The way in which the processing IAISis carried out in a cell of a mammal, and how this processing is invoked by the run FAS-R or p55-R, unknown. It was shown that the MORT-1 binds to cells with activated FAS-R, together with other proteins (Kischkel et al., 1995). These proteins probably include IAIS1 and other Neoforma IAIS. It seems likely that binding to MORT-1 in the Association shall modify or run autolytic processing IAISor do IAISsusceptible to cleavage by other proteases. Stimulation of the p55-R can run the self-processing IAISsimilar, though less direct way, by combining several molecules TRADD or induction of conformational changes in them, which, in turn, induces a change in education or the state of aggregation of MORT-1 and related molecules IAIS.
The substrate specificity of the IAISis, apparently, quite focused on loss. Although the IAIScould cleave the substrate peptide corresponding to the site of cleavage of the substrate death PARP (Ac-DEVD-AMC), IAISnot found proteolytic activity against peptide corresponding to the site of processing of precursor IL-1ICE (Ac-YVAD-AMC). Identification of cellular proteins that serve as substrates for cleavage IAISwill be able to clarify the deeper events in the induction of cell death by this protease. Possible substrates for cleavage IAISare other family members CED3/ICE, such SRR and ICE. Some of these proteases discutez, which do not belong to the family of CED3/ICE are also activated IAIS, either directly or through the actions of other proteases CED3/ICE. The participation of multiple proteases in cell death is consistent with the reported ability of inhibitors of various proteases, including serine proteases and inhibitors splitting ICE, as well as antisense cDNA ICE, to protect the cells from induced FAS-R and TNF receptor toxicity (Weitzen and Granger, 1980; Ruggiero et al., 1987; Enari et al., 1995; Los et al., 1995).
Many other enzymes, including phospholipase, sphingomyelinase and protein, may participate in the induction of cell death TNF-R and FAS-R (see Eischen et al., 1995; Vandenabeele et al., 1995; Cifone et al., 1995 and references therein). Some of these enzymes become activated as the result of proteolytic cleavage initiated IAIS. However, it is also likely that at least some of these other related cell death activity is stimulated by different signal transduction pathways, regardless of the stimulation IAIS. The participation of more than one cascade of reactions in the induction of cell death, some common to the p55-R and FAS/APO1, and some induced by only one of them, would be consistent , two receptors (Grell et al., 1994; Schulz-Osthoff et al., 1994; Wong and Goeddel, 1994; Clement and Stamenkovic, 1994).
(d) MACH1 binds to MORT-1, as well as with MACH1
To determine whether MACH1 contact MORT-1, as IAISfirst investigated the interaction of these proteins in transfected yeast. MACH1, apparently, has a significant cytotoxic effect on yeast. This effect was manifested as a marked reduction of the yield of colonies in yeast that expressed this protein in the vector with the activating domain (AD-vector) (the expression level of which is higher than the level of expression vector DNA-binding domain (DSD)). On the other hand, MACH1, in which the catalytic cysteine residue, s360was replaced by Ser (MACH1 (C360S)) was not cytotoxic for mammalian cells (see below), nor for the yeast. Like MACH1, MACH1 (C360S), was associated in transfected yeast with MORT-1, as well as with yourself. He also contacted MACH1. Also yeast expressing MACH1 wild type together with MORT-1 or MACH1, found the th yeast; in yeast transfected MACH1 as HELL-and DSD-vector, observed a colored product, possibly due to the cytotoxic effect of MACH1 wild type. Yet, despite this Valerevna coloring, yeast expressing MACH1 or in combination with MORT-1, or in combination with MACH1, gave clearly positive indication of the interaction of the transfected proteins. In contrast to MACH1, MACH1 was not found samozaodrasle in twohybrid test (Fig.5).
As MACH1 (C360S), and IAIS1 coimmunoprecipitation with MORT-1 from lysates of embryonic human kidney cells 293-EBNA, indicating that they are associated with MORT-1 in mammalian cells. To further test whether MACH1 contact MORT-1 in mammalian cells, MACH1 or MACH1, merged with oktapeptidom FLAG, expressed together with labeled epitope ON the molecule MORT-1. [32S]-labeled metabolic MACH1 and MACH1, the slit their the th epitope (Field et al., 1988), expressed in HeLa cells. Immunoprecipitation these proteins from lysates of cells was performed using mouse monoclonal antibodies against oktapeptid FLAG (M2; Eastman Kodak), epitope (S, Field et al., 1988) or TNF receptor p75 (No. 9, Bidga et al., 1994) as a control. Proteins were analyzed by electrophoresis in LTO-page (12% acrylamide) followed by autoradiography. As MACH1 and MACH1 coimmunoprecipitation with MORT-1 from lysates of these cells, suggesting that they are associated with MORT-1. The effectiveness of the interaction MACH1 with MORT-1, seems to be lower than the interaction MACH1.
(e) Molecules IAIS, containing the region of homology CED3/ICE, can mediashout cell death
To clarify the participation of the IAIS in the induction of cell death was investigated the effect of overexpression of different isoforms IAIS on cell viability. The test was performed by transferowania expressing vectors IAIS together with expressing vector-galactose as a marker for transfection into embryonic stem cells of human kidney 293-EBNA cells and breast carcinoma MCF7.
Briefly, cells 293EBNA cells, breast carcinoma man fetal calf serum, non-essential amino acids, 100 U/ml penicillin and 100 μg/ml streptomycin. Cups for tissue culture (5105cells 293-EBNA, 3105cells MCF7 or 3105HeLa cells in cups with a diameter of 6 cm) was temporarily transfusional in one way by precipitation of calcium phosphate, using the cDNA of these proteins, together with expressing vector-galactosidase. In the experiments shown in Fig.14A-D and 15, each Cup was transfusional 3.5 g of the specified design IAIS and 1.5 μg pSV--gal. In the experiments shown in Fig.16A-D and 17-19, each Cup was transfusional 2.5 mg of the indicated construction IAIS or MORT-1 (or, as a control, empty vector) and 1.5 μg pSV--gal. Cells were rinsed through 6-10 h after transfection. Cells 293-EBNA and MCF7 were incubated for another 18 h without additional processing. HeLa cells were incubated for 26 h after transfection and then for 5 h in the presence of either antibodies against FAS/APO1 (SN, 0.5 μg/ml) or TNF (100 ng/ml), together with cycloheximide (10 µg/ml). The degree of cell death at the end of the incubation periods were assessed by determining the expression1 or IAIS2, revealed extensive cell death, manifested by rounding of cells, formation of bubbles, compression, and finally the separation of cells from the Cup (Fig.14V). At 20 h after transfection large part of transfected cells, identified by-galactosidase staining (X-Gal), found condensed morphology typical of apoptosis (Fig.14V). In contrast, cells expressing an empty vector, remained viable.
In particular, Fig.14A-D show the morphology of the embryonic human kidney cells 293-EBNA, temporarily expressing these isoforms IAIS. The arrows (Fig.14B) indicate detected the apoptosis of cells. Forografii did through 26 h after transfection. Fig.15 shows quantification of induced IAIS cell death 293-EBNA (light rectangles) and MCF7 cells (black rectangles are by definition part of expressing-galactosidase cells, showing the morphology of apoptosis after 20 h after transfection with the indicated constructs. Data are taken from three independent experiments with cells 293-EBNA and two independent experiments with MCF7 cells. Data expressed as cf is 0 cells per sample).
To study the participation of the region of homology CED3/ICE isoforms IAISin their apoptopic the cells were transfusional expressing vector for isoforms IAIS1, which does not have a region of homology CED3/ICE, as well as expressing vectors for isoforms IAIS3, which has no N-terminal part of the region, and expressing vectors for MACH1 (C360S) and C-terminal truncated mutant MACH1 (IAIS(1-415)), which has one of the residues that are deemed to be crucial for by the functions of the CED3/ICE (corresponding Sr347in ICE). Cell death did not occur (except for small amounts observed in cells 293 EBNA and MCF7, transfected expressing empty vector) cells 293-EBNA and MCF7, expressing transfected with vectors for IAIS3, MACH1 (1-415) or MACH1 (C360S). In addition, cells, transfetsirovannyh MACH1 together with these vectors, showed very little cell death, suggesting that the molecules of the IAIS, which contain rezerwuj IAIS1, which does not contain any district CED3/ICE really found some cell death (Fig.15). This effect MACH1, which, most likely, is the result of activation of endogenous molecules MACH1, was for some reason more pronounced in transfected HeLa cells. Furthermore, in HeLa cells IAIS3, MACH1 (1-415) and IAISl (C360S) were also slightly cytotoxic (Fig.19).
Fig.19 is a chart of the interaction between the receptor and the protein target involved in the induction of cell death by FAS/APO1 (FAS-R and p55-R Activity IAISapparently is the earliest enzymatic stage in the cascade signal for citizeny actions FAS/APO1 and p55-R. the Ability MACH1 to contact with MORT-1 and MACH1 suggests that this isoform enhances the activity of enzymatically active isoforms.
It is possible that some of the isoforms IAIS serve additional functions. The ability MACH1 to contact with MORT-1 and MACH1 suggests that this isoform may enhance the activity fnih this isoform and quite a significant cytotoxic effect, which it manifests in HeLa cells might reflect the activation of endogenously expressed molecules IAISwhen linking with transfitsirovannykh molecules MACH1. It is clear that some isoforms could also act as home sites for molecules involved in other, non-toxic effects of FAS/APO1 and TNF receptors.
(f) Inhibition functions IAISprevents the induction of cell death by FAS/APO1 and receptor P55
To assess the contribution of IAISin the cytotoxicity of FAS/APO1 (FAS-R and p55-R, IAIS3, as well as non-functional mutants MACH1, MACH1 (1-415) and MACH(C360S), expressed in cells that were induced for the manifestation of this cytotoxicity. Induced p55-R cytotoxicity was launched in cells 293-ENBA transient overexpression of this receptor and induced FAS/APO1 cytotoxicity by overexpression of a chimeric molecule consisting of the extracellular domain of the p55-R and the transmembrane and intracellular domain of FAS/APO1. For some reason, this Chimera had a much more strong which was ndesirable processing them or TNF antibody against FAS/APO1 in the presence of an inhibitor of protein synthesis cycloheximide. HeLa cells were made sensitive to FAS/APO1 temporal expression of this receptor. All tested systems IAIS3 and nonfunctional mutants MACH1 provides effective protection against cytotoxicity induced by the start signal FAS/APO1 or p55-R (Fig.16-19). This protection was also observed, as previously reported (Hsu et al., 1996; Chinnaiyan et al., 1996), in cells transfected with N-terminal deletion mutant of MORT-1, which has no IAIS-binding region (MORT-1 (92-208)). These protective effects indicate that the IAISis a necessary component induced as FAS/APO1, and p55-R cascade signal for cell death.
In particular, Fig.16A-D show the morphology of the cells 293-ENBA, in which cell death was induced transient expression of chimeras consisting of the extracellular domain of the p55-R (amino acids 1-168), fused with the transmembrane and intracellular domains of FAS/APO1 (amino acids 153-319) (chimeras P55-R-FAS) (Fig.16A and 16B), or the expression of the p55-R (Fig.16C and 16D), and cells that were protected from these cytotoxic effects of simultaneous transfection their MACH1 (C360S) (Fig.16B and 16D). The pictures were taken 24 h after transfection.-FAS or p55-R, together with an empty vector, deletion mutant MORT-1, without IAIS-binding region (MORT-1 (92-208)), or molecules IAIScontaining a non-functional area of the CED3/ICE. Fig.18 shows the death of HeLa cells, which temporarily Express FAS/APO1, induced by treatment with antibody against FAS/APO1 (aFas) and cycloheximide (CHI), and its prevention by cotransfection MORT1DD (98-208), IAIS(C360S) or IAIS3.
IAIS is expressed in various tissues at markedly different levels and, obviously, with different distribution of isotypes. These differences may contribute to tissue-specific features of the response to the ligand, FAS/APO1, and TNF. As in the case of other homologues of the CED3/ICE (Wang et al., 1994; Ainemri, 1995), found that the isoforms IAIS containing incomplete areas of the CED3/ICE (e.g., IAIS3), inhibit the activity coexpression molecules MACH1 or IAIS2; also found that they inhibit the induction of cell death caused by FAS/APO1 and p55-R. Expression of these inhibitory isoforms may be the mechanism of cellular defense against mediated by FAS/APO1 and TNF cytotoxicity. A wide heterogeneity isoforms IAIS, the cat is to make possible a particularly refined the configuration of the active isoforms of IAIS.
After a complete description of this invention, the skilled in the art, it should be clear that the same thing can be performed in a wide range of equivalent parameters, concentrations, and conditions without departing from the idea and scope of this invention and without undue experimentation.
Although this invention has been described in connection with its distinctive options, it should be clear that further modification. This proposal should cover any variations, use or adaptation of these discoveries in accordance, generally, with the principles of this invention and such departures from the present invention, which is permissible within known and customary practice in the field to which this invention relates, in accordance with its basic features set forth in the scope of the attached claims.
All cited here references, including journal articles or abstracts, published or corresponding patent application U.S. or other countries, published U.S. patents and foreign patents, or any other references are fully incorporated by reference here, including all data, tables, drawings, and text presented in the cited reference the AI method, stage conventional methods, known methods or conventional methods in any case are not a recognition that any aspect of the description or variant of the present invention is described, reported or suspected in the relevant field of knowledge.
The above description of the characteristic options so fully reveals the General nature of this invention that others can, by applying knowledge of common school in this area (including the content cited here for reference), it is easy to modify and/or adapt for various applications these characteristic ways, without undue experimentation, without departing from the General concept of the present invention. Therefore, such adaptations and modifications are within ideas and range of equivalents of the described variants presented here is based on the description and guidelines. It should be understood that the phraseology or terminology used here for the purpose of description and not of limitation, so that the terminology or phraseology of this application should be interpreted by a specialist in light of the description and guidance presented herein, in combination with the knowledge of a specialist with the usual(1993) Science 262:1512-1514.
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1. The DNA sequence encoding a polypeptide that binds to MORT-1, where MORT-1 is a protein that binds to the intracellular domain of FAS-R, and which is associated with protein TRADD, which binds to the intracellular domain of the P55 TNF-R, and determining the amino acid sequence of the specified polypeptide, consisting of (a) residues 1-182 of the sequence SEQ ID No. 5; (b) a fragment of the sequence (a) that binds to MORT-1; or (C) analog (a) or (b) having no more than ten conservative substitutions in the amino acid sequence of (a) or (b) where each of the specified substitution is a substitution, the polypeptide according to (a) p. 1 has the sequence the identical sequence of a native protein that binds to MORT-1, and affects the intracellular process signal initiated by binding of FAS ligand to its receptor or binding c TNF p55-TNF-R.
3. The DNA sequence under item 1, where the polypeptide according to (a) p. 1 is the isoform protein IAIS selected from the group comprising IAIS1 (SEQ ID NO:7), IAIS2 (SEQ ID NO:18), IAIS3 (SEQ ID NO:20), IAIS1 (SEQ ID NO:5), IAIS2 (SEQ ID NO:22), IAIS3 (SEQ ID NO:8), IAIS4 (SEQ ID NO:25) and IAIS5 (SEQ ID NO:34).
4. The DNA sequence under item 3, where the specified polypeptide having a sequence containing (a) is the IAIS1, IAIS1 or IAIS3.
5. The DNA sequence under item 3, where this isoform protein IAIS IAIS is1.
6. The DNA sequence under item 3, where this isoform protein IAIS IAIS is1.
7. The DNA sequence under item 3, where this isoform protein IAIS IAIS is3.
8. The sequence Vetsa with MORT-1.
9. The DNA sequence under item 3, encoding the isoforms of the protein IAIS selected from the group comprising IAIS1, IAIS2, IAIS3, IAIS1, IAIS2, IAIS3, IAIS4 and IAIS5.
10. The DNA sequence under item 9, where this isoform protein IAIS IAIS is1, IAIS1 or IAIS3.
11. The DNA sequence under item 9, where this isoform protein IAIS IAIS is1.
12. The DNA sequence under item 9, where this isoform protein IAIS IAIS is1.
13. The DNA sequence under item 9, where this isoform protein IAIS IAIS is3.
14. The DNA sequence encoding a polypeptide that is associated with ORT-1, where MORT-1 is a protein that binds to the intracellular domain of FAS-R and which is associated with protein TRADD, which binds to the intracellular domain of the P55 TNF-R, and the polypeptide affects the intracellular process signal initiated by binding of FAS-ligand with its recipe is containing a series of (a) residues 1-182 and 221-479 sequence SEQ ID No. 7; b) a fragment of (a), which binds to MORT-1 and affects the intracellular process signal initiated by binding of FAS ligand to its receptor or binding c TNF p55-TNF-R; or (C) analog (a) or (b) having no more than ten conservative substitutions in the amino acid sequence of a) or b), where each of the specified substitution is a substitution, deletion or insertion of amino acids, while the analogue binds to MORT-1 and affects the intracellular process of signal transmission, initiated by the binding of FAS ligand to its receptor or binding c TNF p55-TNF-R.
15. The DNA sequence for p. 14, where the polypeptide according to (a) p. 14 is a native protein that binds to MORT-1 and affects the intracellular process signal initiated by binding of FAS ligand to its receptor or the binding of TNF to P55 TNF-R.
16. The DNA sequence for p. 14, where the polypeptide according to (a) p. 14 is isoform protein IAIS selected from the group comprising IAIS1 (SEQ ID NO:7) and IAIS2 (SEQ ID NO:18).
17. The DNA sequence for p. 14, native encoding a protein that binds to MORT-1 and affects the intracellular process signal initiated by binding of F is LM IAIS, selected from the group comprising IAIS1 (SEQ ID NO:7) and IAIS2 (SEQ ID NO:18).
19. A vector for expression, comprising a regulatory sequence operatively associated with a DNA sequence under item 1.
20. Vector for p. 19, is able to be expressed in eukaryotic cells is the master.
21. Vector for p. 19, is able to be expressed in prokaryotic cells is the master.
22. A method of obtaining a polypeptide that binds to MORT-1, including growing host cells transformed by the vector according to p. 19, under conditions suitable for expression of the specified polypeptide, and the allocation of the expressed protein.
23. Polypeptide that binds to MORT-1, where the specified polypeptide has a sequence comprising (a) residues 1-182 of the sequence SEQ ID No. 5; (b) a fragment of (a), which binds with protein ORT-1; or (C) similar to a) or b), deviating not more than ten conservative amino acid substitutions, with the analogue binds with protein MORT-1.
24. Polypeptide p. 23 where a specified sequence containing (a) is an isoform of protein IAIS selected from the group comprising IAIS1 (SEQ ID NO:7), IAIS2 (SEQ ID NO:18), IAIS3 (SEQ ID NO:8), IAIS4 (SEQ ID NO:25) and IAIS5 (SEQ ID NO:34).
25. The polypeptide according to p. 23 where a specified sequence containing (a) is the IAIS1 (SEQ ID NO:7), IAIS1 (SEQ ID NO:5) or IAIS3 (SEQ ID NO:8).
26. The polypeptide according to p. 23 where a specified sequence containing (a) is the IAIS1 (SEQ ID NO:7).
27. The polypeptide according to p. 23 where a specified sequence containing (a) is the IAIS1 (SEQ ID NO:5).
28. The polypeptide according to p. 23 where a specified sequence containing (a) is the IAIS3 (SEQ ID NO:8).
29. The polypeptide according to p. 23 having a sequence containing a).
30. The polypeptide according to p. 23 having a sequence containing b).
31. The polypeptide according to p. 23 having a sequence containing C).
32. The polypeptide according to p. 23, where the specified equivalent) different from a) or b) not more than five amino acid substitutions.
33. The polypeptide according to p. 23, where the specified equivalent) different from a) or b) no more than three amino acid substitutions.
34. Polypeptide that binds to MORT-1, where MORT-1 is a protein that binds to the intracellular domain of FAS-R, and which is associated mocny the process of signal transmission, initiated by the binding of FAS ligand to its receptor or binding c TNF p55-TNF-R, where the specified polypeptide contains a) residues 1-182 and 221-479 sequence SEQ ID No. 7; (b) a fragment of (a), which binds to MORT-1 and affects the intracellular process signal initiated by binding of FAS ligand to its receptor or binding c TNF p55-TNF-R; or (C) analog (a) or (b) having no more than ten conservative substitutions in the amino acid sequence of a) or b), where each of the specified substitution is a substitution, deletion or insertion of amino acids, while the analogue binds to MORT-1 and affects the intracellular process signal initiated by binding of FAS ligand to its receptor or binding c TNF P55-TNF-R.
35. The polypeptide according to p. 34, where the polypeptide according to (a) p. 34 is identical to the native protein, that is associated with ORT-1 and affects the intracellular process signal initiated by binding of FAS ligand to its receptor or the binding of TNF to p55-TNF-R.
36. The polypeptide according to p. 34, where the polypeptide according to (a) p. 34 is isoform protein IAIS selected from the group comprising IAIS1 (SEQ ID NO:7) and IAIS2 (SEQ ID NO:18).
37. The polypeptide according to p. 34, cotoneaster on the intracellular process of signal transmission, initiated by the binding of FAS ligand to its receptor or binding c TNF p55-TNF-R.
38. The polypeptide under item 37, which has a sequence identical to the sequence protein isoforms IAIS selected from the group comprising IAIS1 (SEQ ID NO:7) and IAIS2 (SEQ ID NO:18).
39. The method of modulating the action of the ligand FAS-R or TNF on cells carrying FAS-R or p55-R capable of handling these cells the polypeptide according to p. 34.
40. The method according to p. 39, where this treatment of these cells involves the introduction into the cells of one or more of these polypeptides in a form suitable for intracellular introduction, or the introduction into the cells a DNA sequence that encodes one or more of these polypeptides in the form of a suitable vector carrying the specified sequence, and this vector is able to carry out the introduction of this sequence into these cells in such a way that this sequence is expressed in the cells.
41. The method of modulating the action of the ligand FAS-R or TNF on cells by p. 40, where the specified processing cells includes the introduction into the cells of the specified polypeptide or a DNA sequence that encodes the specified polypeptide in the form of approach is th sequence in these cells thus this sequence is expressed in the cells.
42. The method according to p. 40, in which the specified processing of these cells represents the transfection of these cells with recombinant viral vector (virus animal), including the stage of (a) constructing a recombinant viral vector (virus animal) carrying a sequence encoding a viral surface protein (ligand) that is capable of contact with a specific cell surface receptor on the surface of the carrier FAS-R or p55-R cells, and a second sequence encoding a specified polypeptide, which when expression in these cells capable of modulating/mediaremote activity of the FAS-R or p55-R, and (b) infection of these cells by the vector (a).
43. The method according to p. 39, in which the MORT-1-binding protein or specified polypeptide, one of which can specifically bind with MORT-1, which, in turn, specifically binds to FAS-IC, or which is able to connect with MORT-1, which, in turn, binds to TRADD, which, in turn, communicates with the p55-IC.
44. The method according to p. 39, where the specified polypeptide is isoform IAIS designated as IAIS1 (SEQ ID NO:7).
45. The method according to p. 39, where specified the polyp is I steps ligand FAS-R or TNF on cells, bearing FAS-R or p55-R capable of handling these cells oligonucleotide sequence that encodes the antisense sequence complementary to at least part of the mRNA sequence that encodes a MORT-1-binding protein in p. 34, and this oligonucleotide sequence capable of inhibiting expression of MORT-1 binding protein.
47. The method according to p. 46, in which the specified oligonucleotide sequence is entered in the specified cell with a recombinant viral vector (virus animal).
48. Method of treatment of tumor cells or HIV-infected cells or other abnormal cells, comprising (a) constructing a recombinant viral vector (virus animal) carrying a sequence encoding a viral surface protein that can bind to specific receptor surface of tumor cells, or the receptor surface of HIV-infected cells, or a receptor located on the surface of other abnormal cells, and the sequence encoding the polypeptide under item 34, which when the expression in the tumor, HIV-infected or other pathological cell can kill the cell; and (b) with the simulation steps ligand FAS-R or TNF on cells, involving ribozymes the way in which a vector encoding the sequence of the ribozyme capable of interacting with a cellular mRNA sequence that encodes a polypeptide according to p. 34, is introduced into these cells in a form that allows you to Express the sequence of the ribozyme in these cells, and expression of the ribozyme sequence in these cells leads to inhibition of expression of the specified polypeptide in these cells.
50. Isolation and identification of proteins by p. 23, the ability to communicate with ORT-1, involving the application of yeast twohybrid the way in which the sequence encoding a protein MORT-1, is carried by one hybrid vector and sequence from a cDNA library or genomic DNA is transferred to the second hybrid vector, and these vectors are then used to transform yeast host cells and the positive transformed cells release with subsequent extraction of the second hybrid vector to obtain a sequence that encodes a protein that is associated with the specified protein MORT-1, and this protein is the MORT-1-binding protein.
51. The method of modulation induced MORT-1 action or induced MORT-1 is a sequence encoding the polypeptide under item 23 or 34, and this processing leads to the enhancement or inhibition mediated MORT-1 action and consequently also mediated FAS-R or p55-R steps.
52. The method according to p. 51, wherein said polypeptide is a part of MORT-1-binding protein, which specifically involved in binding to MORT-1 or by MORT-1-binding protein or the MORT-1-binding protein is encoded by a sequence that encodes part of MORT-1-binding protein, which specifically involved in binding to MORT-1 or by MORT-1-binding protein.
53. The method according to p. 51, wherein said polypeptide is one of the isoforms IAIS selected from the IAIS1 (SEQ ID No:7), IAIS1 (SEQ ID No:5) and IAIS3 (SEQ ID No:8), and isoforms IAIS can enhance associated with MORT-1 action on the cells and therefore also associated with FAS-R or p55-R action on the cells.
54. The method according to p. 51, wherein said polypeptide is any of the isoforms IAIS selected from the IAIS2 (SEQ ID No:18) or IAIS3 (SEQ ID No:20), and these isoforms IAIS is able to inhibit the activity of isoforms IAIS intracellular and sleds is a journey of cells.
55. Pharmaceutical composition for modulation of the action of the ligand FAS-R or TNF on cells containing as an active ingredient the polypeptide according to p. 34 in an effective amount to bind MORT-1, thereby decreasing the effect of ligand FAS-R or TNF on cells.
< / BR>as a substrate for the determination of thrombin