WO2002057449A1 - Novel gene tifa - Google Patents

Abstract

A polypeptide selected from the following group: (1) a polypeptide comprising an amino acid sequence represented by SEQ ID NO:1 or 2 in Sequence Listing; (2) a polypeptide comprising at least a fragment or a part of a polypeptide as defined in the above (1); (3) a polypeptide having a polypeptide as defined in the above (1) or (2); (4) a polypeptide having a homology on the amino acid sequence of at least about 70 % with a polypeptide as defined in the above (1) or (2) and having an activity of binding to TRAF; and (5) a polypeptide having an amino acid sequence derived from an amino acid sequence as defined above by deletion, substitution, addition or insertion of one to several amino acids and having an activity of binding to TRAF; and a polynucleotide encoding the above polypeptide.

Description

 Specification

TECHNICAL FIELD The present invention relates to a novel TRAF binding factor, in particular, to a TRAF Interacting protein with a Forkhead—Associated domain (hereinafter sometimes referred to as TIFA). More specifically, a peptide or polypeptide having all or a part of the amino acid sequence of TIFA, a polynucleotide encoding the peptide or polypeptide, a recombinant vector containing the polynucleotide, a recombinant vector containing the polynucleotide, A transformant transformed in step 1, a method for producing a peptide or polypeptide using the transformant, an antibody against the peptide or polypeptide, a method for identifying a compound using the same, a method for identifying the identified compound, Active P and harmful compounds or active and activating compounds acting on the polypeptide or the polynucleotide, a pharmaceutical composition related thereto and a method for producing the same, and a therapeutic method using the pharmaceutical composition , And related methods of diagnosing diseases. Landscape technology

TNF (tumor necrosi sf actor) receptor suha. The stimulatory response mediated by the family or ΤοΙ Ί / IL-1 (intel l euki n-1) receptor family plays an important role in biological reactions such as immunity, inflammation, apoptosis and allergic reactions. It has become clear. These reactions are triggered by the binding of each ligand to the receptor, and the signal is transmitted into the cell, which appears as a corresponding response.However, molecules involved in the signal transduction mechanism and signal transduction in the cell Has not been fully elucidated. Recently developed yeast two-hybrid cDNA screening The establishment of the method has made a great contribution to the study of signal transduction where the interaction between protein and protein is a point, but the TNF receptor superfamily and To Ί 1/1 L-

The present method has been used to identify one molecule of the TRAF (tumor necrosisfactorrrececeptor—as sociated factor) protein family involved in signal transduction from the 1 receptor family.

 Ding 1 ^ proteins include TRAF 1 and TRAF 2 previously identified from 1 "-1I, TRAF3 identified from CD40, TRAF4, CD40 identified as a breast cancer cell-specific expression gene and lymphotoxin /? There are known TRAF 5, which binds to the receptor, and TRAF 6, which is thought to transmit CD40 and IL-1 RI signals. In addition, the function is partially speculated as follows: The activation of NF-<< B is induced by forced expression of TRAF 2, 5, and 6 molecules.

 Conversely, the forced expression of TRAF1 and TRAF3 suppresses the activation of NF-<< B.

 TRAF 2 is involved in signal transduction from type I and type I TNFRs, but its effect is to block the pathway from TNF to caspase, and suppress cell death. In addition, it has been reported that the TRAF 6 molecule is essential for osteoclast differentiation and gain of function, and K0 mice of this molecule show bone dysplasia.

 Since NF-ΛΤΒ activates the entire immune system widely, regulation of NF-Λ: Β activation is physiologically important, and this regulatory system may be a target for drugs in the treatment of various diseases. Significant. For example, the proliferation signal from CD40 is constantly contained and is self-reactive. In autoimmune diseases in which cells are not apoptotic, NF-ΛΓ through TRAF 2, 5, or 6 molecule is used. Activation may be involved. In addition, septic shock, acute hepatitis, osteoporosis, etc. may be related to the TRAF 6 molecule.

It is known that after TRAFF 2,5,6 molecules are multimerized, several molecules are activated stepwise until NF-II: Β is activated. All three types of TRAF molecules activate NIK (NF—; trB Ind ucing Kinase) molecules. NIK activates the I KK (I-κΒ Kinase) molecule, Phosphorylates I, promotes I½B decomposition, and activates NF-. This route is common for TRAFs 2, 5, and 6, and the analysis is relatively advanced. On the other hand, the pathways from the TRAF2, 5, and 6 molecules to NIK are largely unknown, and have not yet been fully elucidated. (EX ρ eri me nta Ί Cel l Research Vol. 254 pp 14-24 2000)

Studies are underway to elucidate the mechanisms of TRAF-mediated signal transduction, but the presence of several proteins associated with TRAF molecules has recently been identified. For example, by screening using the yeast tw oh ybrid system using TRAF3 and TRAF2 as baits, TANK (TR AF—family member—associated NF½B acitivator) and I—TR AF (TRAF—interacti) ng protein) has been found. TANK and I-TRAF are the same substance and are associated with known TRAF molecules other than TRAF 4 via the TRAF_C domain. Also, TRIP (TRAF-interacting protein) has been screened by TRAF1-based yeast two-hybrid system. Although experimental evidence suggests that TRIP-1 interacts with TRAF1 and TRAF2 in animal cells, it activates TNF-α or CD30-induced NF-ΒΒ Functional findings have shown that it suppresses IL-11 but not IL-11 mediated activation. The Peg3 protein, also known as the product of the so-called genomic imprinting gene, is also known to interact with TRAF2, and overexpression of Peg3 can induce NF-II: B activation. It has been reported. Casper, which has sequence similarity to caspase 8, is also a molecule that interacts with the TRAF molecule, and has been reported to interact with TRAF 1 and TRAF 2 via the TRAF-N domain. Casper leads apoptosis, but this reaction competes with c-IAP. This c-IAP has also been reported to be a molecule that interacts with TRAF 2 through the TRAF-N domain It is suggested that the molecule interacting with the F molecule causes cell death and is involved in the control mechanism of cell survival.

Although a RIP molecule has been identified as a Fas-binding protein, studies of this molecule and a group of molecules that interact with this molecule suggest that TRAD D molecules are recruited in the intracellular region of TNFR1 in a signal-responsive manner, and A scheme has been proposed in which the RIP molecule, TRAF2, is recycled to transmit signals to JNK and IKK activity. As described above, TRAF molecules and molecules that interact with TRAF molecules have been discovered one after another, and their functions have been elucidated. However, most recently, T6BP (TRAF6-inte ractinng) has been identified as a molecule that interacts with TRAF 6. protein) was identified. Interaction between T6BP and TRAF6 occurs through the coiled-coil region of T6BP and the N-terminal Ringfinger and Zinccfger domains of TRAF6. Although IL-11 signal transmission involves a TRAF6-mediated mechanism, it has been reported that IL-1 ligand-dependent formation of the TRAF6-specific 6BP complex upon IL-1 stimulation. Have been. According to this report, T6BP does not affect the activation of NF-; ίB or JK during IL-11 stimulation, which is a typical TRAF6-mediated signal response. In other words, the functional aspects of T6BP are not clear. The present invention relates to a novel TRAF-binding factor, in particular, a TRAF-interacting protein forkhead-associated domain (hereinafter sometimes referred to as TIFA), a polynucleotide encoding the same, and a substance related thereto. One of the purposes is to clarify and make these applications possible. This time, the present inventors succeeded in cloning a novel molecule that binds to TRAF 6 by a two-hybrid screening method using mouse TRAF 6 as a bait, and at least NF-; The present inventors have found that the present invention has a function relating to sexualization, and completed the present invention. DISCLOSURE OF THE INVENTION The present invention provides (1) a polypeptide selected from the following group;

 (1) a polypeptide represented by the amino acid sequence of SEQ ID NO: 1 or 2 in the sequence listing,

(2) A fragment of the polypeptide of (1) or a polypeptide comprising at least one portion

 (3) a polypeptide containing the polypeptide (1) or (2),

 ポ リ a polypeptide having at least about 70% homology on the amino acid sequence with the polypeptide of ① or ② and having TRAF binding activity;

and

ポ リ a polypeptide having a mutation such as deletion, substitution, addition or insertion of one or several amino acids in the amino acid sequence and having an activity of binding to TRAF; (2) SEQ ID NO: A peptide having at least about 8 contiguous amino acid sequences of the amino acid sequence of 1 or 2, (3) the polypeptide of 1 or 2 or a polynucleotide encoding the peptide or a complement thereof, (4) a polypeptide of 3 A polynucleotide that hybridizes with a polynucleotide or its sequence under selective conditions; (5) at least about the nucleotide sequence of SEQ ID NO: 3, 4, or 5 or a complementary sequence thereof (1) a polynucleotide represented by 5 consecutive nucleotide sequences; (6) a recombinant vector containing any one of 3 to 5 polynucleotides; (7) (8) the method for producing the polypeptide or peptide according to (1) or (2), which comprises a step of culturing the transformant according to (7); Or (10) an antibody that immunologically recognizes the polypeptide or peptide of (2), (10) the antibody of (9), which inhibits the activity of binding to TRAF6, and (11) that interacts with the polypeptide of (1). A method for identifying a compound that inhibits or activates its activity, and a compound that interacts with Ζ or the polynucleotide S3 or 4 to inhibit or promote its expression, wherein 2 or any one of the above-mentioned 3 to 5 polynucleotides, the above 6 vector, the above 7 transformant, and the above 9 or 10 antibody. (12) a compound that interacts with the polypeptide (1) to inhibit or activate its activity, or a compound (3) that interacts with the polynucleotide (3) or (4) above. A method of identifying a compound that inhibits or enhances expression, comprising screening a polypeptide or polynucleotide under conditions that allow the interaction between the compound and the polypeptide or polynucleotide. The interaction of the compound with the compound to be evaluated (the interaction is a detectable signal in response to the interaction of the compound with the polypeptide or polynucleotide). Associated with the second component that can be provided), and then detecting the presence or absence or a change in the signal caused by the interaction of the compound with the polypeptide or polynucleotide, thereby providing A method comprising determining whether the compound interacts with the polypeptide or polynucleotide to activate or inhibit its activity, (13) identified by the method of 11 or 12 above. (14) a compound that interacts with the polypeptide (1) to inhibit or activate its activity, or a compound that interacts with the polynucleotide (3) or (4) to inhibit or promote its expression (15) | ij | B 1 or 2 polypeptide or peptide, any one of the polynucleotides 3 to 5, the vector 6 and the transformant of till 7 if Among 1 0 of antibody or 1 3 or 1 4 compounds, to pharmaceutical compositions characterized by containing at least any one,

(16) A method for diagnosing a disease associated with the expression or activity of the polypeptide of 1 in an individual, comprising: (a) a nucleic acid sequence encoding the polypeptide; and (b) an individual A method comprising analyzing the polypeptide as a marker in a sample derived therefrom, (17) using the pharmaceutical composition of the above (15) for a disease associated with TRAF, DIFA or NF-Λ: Β. (18) A method for producing the pharmaceutical composition of the above (15). BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a diagram showing Northern routing showing the expression level of TIF AmRΝ in mice.

FIG. 2 shows the results of an experiment in which the binding of TIFA to various TRAFs was confirmed by immunoprecipitation.

FIG. 3 is a graph showing the results of examining the effect of TIFA on NF-IB activation by a function confirmation experiment using a luciferase reporter.

FIG. 4 is a diagram showing the nucleotide sequence of cDNA of human TIFA and the amino acid sequence encoded thereby.

FIG. 5 shows the nucleotide sequence of the cDNA of mouse TIFA and the amino acid sequence encoded thereby.

FIG. 6 is a continuation of FIG. 5 and shows the nucleotide sequence of the mouse DNA DNA and the amino acid sequence encoded thereby.

FIG. 7 is a graph showing electrophoresis by SDS-PAGE showing concentration-dependent promotion of phosphorylation of GST-c-Jun by addition of pME-FLAG-TIFA. FIG. 8 is a diagram showing Western blotting for confirming the binding site of TIF A to TRA F6.

FIG. 9 is a diagram showing wesin blotting for confirming the binding site between TIFAs.

FIG. 10 is a diagram showing Western blotting for confirming the binding site of TRAF6 to TIFA.

FIG. 11 is a graph showing the effect of each region of TIFA on NF-NF: B activation using various mutants of TIFA.

FIG. 12 is a graph showing the effect of each region of TIFA on NF-Λ: B activity using various mutants of TIFA. '

FIG. 13 is a graph showing the inhibition of the function of ΤIΨ / \ by the expression of TRAF6 dominant negative (DN).

FIG. 14 is a graph showing the inhibition of the function of TRAF6 due to the expression of a TIFA dominant negative (DD) body.

Replacement paper FIG. 15 is a graph showing the effect of TIFA dominant negative (DN) on IL-13 activation of NF-—B.

FIG. 16 is a graph showing the effect of TIF A dominant negative (DN) on NF-¾B activation by TN FcH. BEST MODE FOR CARRYING OUT THE INVENTION (TIFA)

The TIFA provided in the present invention is obtained by obtaining its cDNA from a cDNA library as a substance having a novel amino acid sequence. The presence of mRNA in the spleen, heart, brain, liver, lung, kidney, testis, and muscle of the TIFA of the present invention was confirmed by Northern plotting. The TIFA of the present invention has the following properties or activities. Binds to TRAF, preferably TRAF1, TRAF2 or TRAF6. Also, it binds to the 領域 -terminal region of 6 !. The binding to TRAF 6 involves the C-terminal region (C domain) of TIFA, and particularly the 01u residue at position 178 is important. — Regulates the activity of «Β or its activity, promotes JNK phosphorylation, and is involved in AP-1 activation NF-« Β activation involves TIFA FHA domain and C domain are involved, Ser at position 50, Ser at position 66, and Guu residue at position 178. Involved in the activation of NF-<< B via TRAF6 by IL-1 stimulation. It contains a part called protein-associated (FHA) domain that is involved in protein-protein interaction. In humans, it contains the amino acid sequence of SEQ ID NO: 1 in the sequence listing. From the vicinity of the 47th V to the vicinity of the 103rd L, and in mice, the 47th V is added to the amino acid sequence of SEQ ID NO: 2 in the sequence listing. It is considered that this region corresponds to the region including the portion from near to the 103rd L. Phosphor rooster change protein, peptide or amino acid (eg, phosphoserine, phosphothreonine, etc.) via FHA It also binds between TI FA molecules, including the FHA domain and the C domain. Involvement.

(Polypeptide or peptide)

The TIFA of the present invention is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2 in human or mouse, in humans or mice. In addition, the polypeptide or peptide of the present invention is selected from a polypeptide that is at least a part of the polypeptide described in SEQ ID NO: 1 or 2 in the sequence listing, or a polypeptide or peptide containing the same. You. Further, the polypeptide or peptide of the present invention is obtained by combining the polypeptide of SEQ ID NO: 1 or 2 with about 40% or more, preferably about 70% or more, more preferably about 80% on the amino acid sequence. %, More preferably about 90%, particularly preferably about 95% or more. Selection of a polypeptide or a peptide having this homology can be performed based on at least one of the quality or activity of the fijf-owned TIFA or an effect on the quality or activity. Specifically, TRAF, for example, an activity capable of binding to TRAF1, TRAF2, or TRAF6 and Z or NF—the activity of regulating (promoting or inhibiting) B activity, particularly the activity of is there. The above activity can be determined by a known method, for example, immunoprecipitation for TRAF binding activity, and repo overnight assay using luciferase or the like for NF-½B regulatory activity, specifically described in Examples. Can be detected or measured by the method used. Specific examples of these polypeptides are given in the examples. Techniques for determining the homology of amino acid sequences are known per se, and include, for example, a method for directly determining an amino acid sequence, a method for determining a nucleotide sequence of cDNA and then estimating an amino acid sequence encoded thereby. . The polypeptide or peptide of the present invention includes a polypeptide or a peptide having a partial sequence of the polypeptide set forth in SEQ ID NO: 1 or 2 in the Sequence Listing, such as a reagent, a standard, or an immunogen. Also available as With its smallest unit Comprises an amino acid sequence consisting of 8 or more amino acids, preferably 10 or more amino acids, more preferably 12 or more, still more preferably 15 or more consecutive amino acids, and is preferably immunologically Polypeptides or peptides that can be identified as such are the subject of the present invention. These peptides can be used alone or as a carrier (for example, molysin or ovalbumin, etc.) as a reagent or standard, or as an antigen for producing an antibody specific to TIFA as described below. Although they can be used in combination, those to which other kinds of proteins or substances are bound are also included in the scope of the present invention. Further, based on the polypeptide or peptide identified in this manner, at least one of the above-mentioned TIFAs or its activity, or its effect on it, is used as an index, whereby one or more, for example, 1 to 100, Preferably, amino acids having mutations such as deletion, substitution, addition or insertion of several amino acids are preferably 1 to 30, more preferably 1 to 20, still more preferably 1 to 10, particularly preferably 1 to 10. Also provided is a polypeptide or peptide consisting of a noic acid sequence. The means for deletion, substitution, addition, or insertion are known per se. For example, site-directed mutagenesis, homologous recombination, primer extension, or polymerase chain amplification (PCR) can be used alone or separately. Appropriately combined, for example, Sambrook et al. [Molecular Cloning, Laboratory Manual 2nd Edition] Cold Spring Harbor Laboratory, 1989, Masamura Muramatsu [Lab Manual Genetic Engineering] Maruzen Co., Ltd., 1988, Ehrlich, HE Ed. [PCR technology, principle and application of DNA amplification] It can be carried out according to the method described in a written book such as Stoketon Press, 1989, or by modifying those methods. One mer key technique (Science, 219, 666, 1983) can be used. From the viewpoint of not changing the basic properties (physical properties, activity, immunological activity, etc.) of the protein upon introduction of the above mutation, for example, homologous amino acids (polar amino acids, nonpolar amino acids, j¾ ? M raw amino acid, hydrophilic amino acid, positive load Mutations between charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) are easily assumed. The FHA region is considered to be an important part for the expression or regulation of the activity, and it is preferable that the region containing these is well maintained on the primary sequence and / or the three-dimensional structure in order to maintain the TIFA activity. Further, the polypeptide or peptide of the present invention is included in the scope of the present invention regardless of the presence or absence of a sugar chain. Examples of specific mutant polypeptides are given in the Examples. In the present invention, a polypeptide having the same TIFA activity as the polypeptide shown by the amino acid sequence of SEQ ID NO: 1 or 2 in the sequence listing, or a minimum activity unit thereof

(Regions or domains) are provided, as well as polypeptides with altered strength or specificity of activity. These also may be, for example, as TIF A activity-like substances (eg, polypeptides comprising the FA—C domain) or TIFA antagonists (eg, polypeptides comprising the C domain or FA domain), or It is useful in screening for a substance that regulates, for example. Incidentally, homologous gene products of animal species other than human and mouse are naturally included in the scope of the present invention.

Furthermore, in order to facilitate the detection or purification of the polypeptide of the present invention or to add another function, another protein such as alkaline phosphatase is added to the N-terminal or C-terminal side. Monoclonal immunoglobulin Fc fragments such as galactosidase and IgG or peptides such as FLAG-tag (Asp Tyr Lys Asp Asp Asp Asp Lys) (Biotechnol ogy, 6, 1205-1210, 1988) It is easy for those skilled in the art to add indirectly using a genetic technique or the like via a linker peptide or the like, and a polypeptide or the like to which these other substances are bound is also included in the scope of the present invention. Is done.

(Polynucleotide)

In one embodiment, the polynucleotide of the invention and its complement are A polynucleotide encoding the amino acid sequence of the polypeptide or peptide, for example, the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing, and a complementary strand to the polynucleotide. These provide, for example, gene information useful for the production of the above-mentioned TIFA, or can be used as a reagent or standard for nucleic acids. SEQ ID NOs: 3 and 4 in the Sequence Listing showing preferred polynucleotides are nucleotide sequences deduced as coding regions of human or mouse TIFA, respectively. In another embodiment, the present invention relates to a polynucleotide encoding the amino acid sequence of the polypeptide or the peptide of the present invention, for example, a nucleotide encoding the amino acid sequence of SEQ ID NO: 1 or 2 in the sequence listing, preferably SEQ ID NO: 3, 4 or Provided is a polynucleotide that hybridizes to a polynucleotide represented by the nucleotide sequence of No. 5 or a region corresponding to the complementary strand thereof, preferably under stringent conditions, under selective conditions. The method of hybridization is described, for example, in Sambrook et al. [Molecular Cloning, Laboratory Manual 2nd Edition] Cold Spring Harbor Laboratory, (1989), or the method described in Shaw, etc. (Nucl eic Acids Res., Vol. 11, pp. 555-573, 1983), or a partial modification thereof. Here, the selective hybridization condition means that a desired nucleic acid, for example, a nucleic acid having a desired homology with a specific probe (for example, a nucleic acid encoding the TIFA of the present invention of SEQ ID NO: 3, 4 or 5) is selected. Conditions that specifically or specifically hybridize and do not hybridize to other unrelated nucleic acids, eg, nucleic acids of lower homology. Generally, the octopus temperature (Tm) is used as an indicator of the stability of a double-stranded molecule (hybrid) of a nucleic acid. This is based on the length of the strand, the base length, and the chemical conditions (ionic strength, chemical strength, etc.). In general, hybridization is carried out at a temperature of Tm or less. Empirical empirical formulas have been obtained for the Tm values of hybrids of perfect complementarity with DNA, RNA or oligonucleotide probes (Human Molecular Genetics, Tom Strachan. and Andrew P. Read) Supervised by Masanori Muramatsu, Medical-Science International, 1997, etc.) ₀ Estimated value is Tm (° C) = 4 (G + when the probe is smaller than 50 nucleotides). C) +2 (A + T) [A, T, G, and C are the numbers of each base in the probe]. In order to selectively obtain perfect complementarity, the hybridization temperature should be 5 ° C lower or higher than Tm (preferably lower than Tm), and 1 to maintain the stability of the hybrid. It is necessary to lower the hybridization temperature of the pair of unpaired bases by about 5 ° C below Tm. If the probe is 50 nucleotides or more, Tm (° C) = 81.5. C + 16, 61 o gM + 0.41 (% G +% C) -500 / nO. 61 (% formamide) [M is the monovalent cation of the solution ¾J (mo, / shi), n is the base pair Duplex length], and the Tm is reduced by 1 ° C for each 1% unpaired base, so adjust the hybridization temperature accordingly. An indication of the hybridization temperature is, for example, 55 ° C, preferably 40 ° C, more preferably 25 ° C, still more preferably 10 ° C, and particularly preferably 5 ° C lower than the Tm of the perfect complementarity hybrid. By setting the temperature, nucleic acids having desired homology can be selectively hybridized. Specifically, there is a method in which a method such as Shaw is partially modified. That is, a filter (eg, a nylon membrane or a nitrocellulose filter) to which nucleic acid has been bound is washed at 65 ° C for 10 minutes and contains 0.1% SDS. 3 XSSC (Standard Sample Plate: 1 xSSC is 0%). Wash with 15M NaC 1, 0.011 5M sodium citrate), then 5X SSCP containing 50% formamide, 5X Denhalt's solution, 0.1% SDS, 25 OsZml of denatured salmon sperm DNA (1 xSSCP is, 0. 1 5M NaCl, 0. 01 5 M Kuen acid _{sodium, 10mM N aH 2 P0 4J 1} mM EDTA, p H 7.2) in, 42. C, 5 hours (or 65 ° C, 2 hours) Prehybridization Next, a 5X SSCP containing 50% formamide, 1x Denhardt's solution, 0.1% SDS, 100 / ug / ml denatured salmon sperm DNA, and 10% (w / v) dextran sulfate An appropriate amount is added, and the filter is heated at 28 ° C (preferably 37 ° C, more preferably 42 ° C, further preferably 50 ° C, particularly preferably <65 ° C). For 18 hours. Wash the filter twice with 2XSSCP containing 0.1% SDS at room temperature (preferably <37 ° C) for 5 minutes, then use 0.3XSSCP containing 0.1% SDS at 37 ° C Wash three times (preferably 50 ° C, more preferably 65 ° C) three times (1 hour in total). Thereafter, the presence of the probe is specifically detected by using a smart graph or an appropriate detection method. Hybridization conditions, washing conditions, and the like can be appropriately combined depending on the probe used. These polynucleotides need not necessarily be complementary sequences, as long as they hybridize to the target polynucleotide, particularly the polynucleotide represented by SEQ ID NO: 3 or 4 in the sequence listing, or a polynucleotide thereof. O, for example, at least about 40%, for example, about 70% or more, preferably about 80% or more, more preferably about 90%, in homology to the base sequence of SEQ ID NO: 3 or 4 in the sequence listing or its complementary sequence. Or more, more preferably about 95% or more. In addition, the polynucleotide of the present invention comprises 10 or more consecutive nucleotides, preferably 15 or more, more preferably 20 or more, and still more preferably 25 or more, corresponding to an arbitrary or specific region of the designated base sequence. Includes polynucleotides and oligonucleotides consisting of the above nucleotides and their homologous chains. These polynucleotides may be used as a nucleic acid encoding TIFA, for example, as a probe or primer for detecting the gene or mRNA, or for regulating gene expression in the production of the polypeptide or the like of the present invention. It is useful as an antisense oligonucleotide. In that sense, the polynucleotides and oligonucleotides of the present invention include those corresponding to untranslated regions as well as translated regions. For example, in order to specifically inhibit the expression of TIFA by antisense, for example, it is assumed that a nucleotide sequence of a region unique to TIFA other than a motif sequence region such as FHA is used. On the other hand, the use of motif sequences could simultaneously suppress the expression of multiple similar proteins including TIFA. Here, determination of the nucleotide sequence encoding TIFA or a polypeptide having a similar activity is performed, for example, by using a known protein expression system to confirm the expressed protein. The activity can be determined by screening and using the physiological activity, for example, the activity of binding to TRAF, as an index. When a cell-free protein expression system is used, for example, ribosome technology derived from embryos, rabbit reticulocytes, etc. can be used (Nature, 179, 160-161, 195). 7). Further, the polynucleotide of the present invention can be applied to gene therapy in a condition requiring modulation of TIFA activity.

(Transformant)

In addition to the above-described cell-free protein expression system, the present invention also provides a gene recombination technique using a host known per se, such as Escherichia coli, yeast, Bacillus subtilis, insect cells, and mammalian cells. Peptides and polypeptides comprising the TIFA of the invention and its derivatives can be provided. In the embodiment of the present invention, a specific cell was used, but it is needless to say that the present invention is not limited to this. Transformation is performed by a known method, for example, by using a plasmid, chromosome, virus, or the like as a replicon to transform a host. A more preferable system is an integration method into a chromosome in consideration of the stability of the gene, but a simpler method is the use of an autonomous replication system using an extranuclear gene. The vector is selected according to the type of the selected host, and includes a gene sequence to be expressed and a gene sequence carrying information on replication and control as components. Combinations are classified according to prokaryotic cells and eukaryotic cells, and promoters, ribosome binding sites, terminators, signal sequences, enhancers, and the like can be used in combination by a method known per se. In a specific embodiment of the present invention, a mammalian cell and yeast expression system was used, but is not limited thereto. The transformant is cultured under conditions suitable for the culture conditions of each host known per se. Culturing may be performed using the activity of peptides and polypeptides composed of TIFA and its derived products expressed as an index, but may be performed by subculturing using the amount of transformants in the soil as an index. It may be produced by a customer. (Recovery of TIFA and its derivatives)

 The recovery of peptides and polypeptides consisting of TIFA and its derivatives from the culture medium can be performed using molecular sieves, ion column chromatography, affinity chromatography, etc., using the activity of TIFA, for example, the activity of binding to TRAF, as an index. It can also be purified or recovered by means of fractionation of ammonium sulfate, alcohol, etc. based on the solubility difference. Preferably, a method is used in which an antibody against the amino acid sequence is prepared based on the information on the amino acid sequence, and the antibody is specifically adsorbed and collected using a polyclonal antibody or a monoclonal antibody.

(Antibody)

Antibodies are prepared by selecting antigenic determinants of peptides or polypeptides comprising the TIFA of the present invention and its derivatives. The antigen may be TIFA or a fragment thereof and is composed of at least 8, preferably at least <10, more preferably at least 12, and even more preferably <15 amino acids. In order to produce an antibody with increased specificity to TIFA, a region consisting of a sequence unique to TIFA other than a motif region such as FHA may be used. This amino acid sequence does not necessarily need to be homologous to SEQ ID NO: 1 or 2 in the sequence listing, and is preferably a site that is exposed to the outside in the three-dimensional structure of the protein. It is also effective that the amino acid sequence has a continuous position. The antibody is not particularly limited as long as it immunologically binds or recognizes a peptide or polypeptide consisting of TIFA and its derivative. The presence or absence of this binding or recognition is determined, for example, by a known antigen-antibody binding reaction. In order to produce antibodies, a peptide or polypeptide comprising the TIFA of the present invention and a derivative thereof is used alone or in the presence of an adjuvant in the presence or absence of an adjuvant to produce a body fluid. It is performed by inducing immunity such as sexual response and Z or cellular response. The carrier itself has deleterious effects on the host. Otherwise, there is no particular limitation, and examples thereof include cellulose, polymerized amino acids, albumin and the like. As the animal to be immunized, a mouse, a rat, a heron, a sheep, a goat, a horse, and the like are preferably used. The polyclonal antibody is obtained by a known antibody recovery method from serum. A preferable means is an immunoaffinity mouth method. In order to produce a monoclonal antibody, antibody-producing cells (for example, spleen or lymph node-derived) are collected from an animal to which the above-mentioned immunization method has been applied, and a permanently proliferating cell (for example, P 3 X 6 Hybridomas are prepared by fusion with 3 Ag 8 strains such as myeloma B-cells). After further cloning, a hybridoma producing an antibody that specifically recognizes the TIFA of the present invention is selected, and the antibody is recovered from the culture solution of the hybridoma. Among the antibodies of the present invention, a polyclonal antibody or a monoclonal antibody that can suppress TIFA activity is particularly preferred, and can control the activity of TIFA, for example, is involved in the NF-¾B5 tongue signal. The activity can be easily controlled.

(Identification of compound. Screening method)

Peptides or polypeptides comprising TIFA and its derivatives, polynucleotides encoding them and their complementary chains, cells transformed based on their amino acid sequences and base sequences, or cells transformed therefrom Antibodies that immunologically recognize the protein synthesis system used and the peptide or polypeptide consisting of TIFA and its derivatives can be used alone or in combination of two or more to produce peptides, polypeptides or polynucleotides consisting of TIFA and its derivatives. The present invention provides an effective means for a method of identifying or screening a modulator or modulator of activity against, for example, an activity inhibitor or an activator. In these methods and systems, information on the nucleotide sequence of the amino acid sequence or nucleotide sequence of the polypeptide of the present invention and a computer-readable storage medium storing the same are stored. Are effectively used, for example, for predicting a three-dimensional structure or estimating an epitope, and these are also included in the scope of the present invention. For example, selection of antagonists by drug design based on the three-dimensional structure of peptides or polypeptides using a computer, selection of expression regulators at the gene level using a protein synthesis system, antibody recognition using antibodies Substance selection and the like can be used in a drug screening system known per se. Here, the above-mentioned regulation includes inhibition, antagonism, activation, activity promotion, activity activation and the like. Further, a peptide or polypeptide comprising the TIFA of the present invention and a derivative thereof, or a polynucleotide or a transformant of the present invention, comprises a compound between a screening compound and such a peptide or polypeptide. The conditions that allow the interaction of the signal are selected, and a system that uses a signal (marker 1) that can detect the presence or absence of this interaction is introduced. The presence or absence of this signal (marker 1) or By detecting a change in the amount of the signal, a compound that activates or inhibits the activity of the peptide and polypeptide comprising the TIFA of the present invention and a derivative thereof, or inhibits or inhibits the expression of the polynucleotide of the present invention. Compounds that promote it can be identified. As a system using a signal, the binding between the polypeptide of the present invention and another protein (for example, a D, preferably a D6, and particularly a D-C domain) is measured. And a system for measuring the activity of the polypeptide of the present invention, for example, an activity involved in NF-κΒ activation or a system for measuring the expression level of a polynucleotide. The specificity of the action of the compound can be confirmed by combining with various TRAF expression systems and comparing the reactions in each system. In addition, each transformant may be replaced with a cell line in which the expression of the corresponding gene has been confirmed, etc. Furthermore, according to the present invention, the nucleotide or polynucleotide of the present invention or the peptide or polypeptide of the present invention Transgenic animals containing peptides (especially mice, Rat, pig, and mammal). Still, according to the present invention, cells or animals in which all or at least a part of the original TIFA gene has been mutated, particularly deleted, such as knockout animals (particularly mammals such as mice, rats, pigs, and maggots) can be used. Can be made. These cells or animals are also useful in the above-mentioned screening and the like, and are included in the present invention.

(Compound, pharmaceutical composition)

The compounds thus identified are candidates for inhibitors or antagonists, antagonists, activators, accelerators or activators of the activity or action of peptides and polypeptides consisting of TIFA and its derivatives. Available. It can also be used as a candidate compound for expression inhibitors, expression antagonists, expression activators, expression promoters, and expression activators for TIFA and its derivatives at the gene level. These compounds can be used as modulators or modulators of the activity, action or function of TRAF, TIFA or NF- to prevent and / or treat various pathological symptoms associated with them. The candidate compound thus selected can be prepared as a pharmaceutical composition by selecting in consideration of the balance between biological utility and toxicity. Further, a peptide or polypeptide comprising the {F} of the present invention and a derivative thereof, a polynucleotide encoding the same and a complementary strand thereof, a vector containing the nucleotide sequence thereof, and a peptide comprising a TIFA and a derivative thereof Alternatively, an antibody that immunologically recognizes a polypeptide itself can be used as a disease diagnostic means such as a diagnostic marker or a reagent, or inhibits, antagonizes, activates, or promotes the expression, activity, function or action of TIFA. It can be used as a medicament such as a therapeutic agent utilizing the activity, action or function to activate. In the preparation, a known formulation means such as a peptide or polypeptide, a protein, a polynucleotide, and an antibody may be introduced according to each subject. The above-mentioned pharmaceutical thread is the peptide or polypeptide of the present invention, , A vector, a transformant, an antibody, or the above-mentioned compound of the present invention. The drug can be used to treat diseases associated with TIFA, DAF RAF or NF-;, especially TIFΑ, such as autoimmune disease, shock (such as septic shock) and bone disease (such as osteopathy). Or it is useful for prevention. As a diagnostic means, it is useful as a diagnostic means for a disease associated with the expression or activity of a peptide or polypeptide comprising the TIFA of the present invention and its derivative, and the diagnosis is carried out, for example, by a nucleic acid sequence encoding the peptide. Using the interaction or reactivity with the peptide to determine the amount of the corresponding nucleic acid sequence and / or determining the biodistribution of the peptide in an individual; and / or The determination is performed by determining the presence of, the amount present in a sample derived from an individual, or the amount of activity. That is, TIFA is tested as a diagnostic marker. The measurement may be carried out using a known antigen-antibody reaction system, enzyme reaction system, PCR reaction system, or the like. Further, detecting a single nucleotide polymorphism (SNP) by a known method is also a useful diagnostic means. Example

 Hereinafter, the present invention will be specifically described based on examples, but the present invention is not limited to the following examples. Example 1 Obtaining DNA Encoding Mouse TIFA

Screening was performed by yeast two-hybrid screening method in order to clone the cDNA of a protein that binds to mouse TRAF6. That is, first 7 a cZ _N H is 3 and e 2 gene in yeast is incorporated as reporter one coater gene, form that combines TR AF 6 transcription factor GAL 4 DNA binding region on a yeast expression base Kuta one The gene was constructed and introduced. Subsequently, a cDNA library was constructed on the expression rooster vector, in which the ACTIPETA domain of GAL4, which is a transcription factor, and the cDNA product were fused and expressed. Then, screening was performed using the index of change in yeast expression form as an index when binding between TRAF6 and the cDNA expression product was observed. The details are described below. Preparation of c DNA library

1 Tota RNA was extracted from bone marrow cells prepared from the femurs of five ICR mice (8 weeks old) according to a conventional method, and poly-A-RNA was extracted from the cells using Rigo dT-Latex (Daiichi Pure Chemicals). did. Using this poly A-RNA, Hybri ZAP-2.1 XR Library Construction on Kit (Stratagene), and HybriZAP-2.1 XR cDNA Synthetics is Kit (Stratagene), Thus, a cDNA library was synthesized. First, in order to synthesize First Strand cDNA, a predetermined First Strand synthesis reaction reagent containing polyA-RNA5 X9 equivalent, the Xho I linker primer attached to the kit, and 5-methy 1 dCTP is used. The mixture was mixed and allowed to stand at room temperature for 10 minutes. Then, 1.5 ^ L of MMLV-RT (50 \} / (± L) was added to this reaction system, mixed gently, and reacted at 37 ° C for 1 hour. To perform the synthesis, add the second strand synthesis reaction reagent attached to the kit to the rst-strand synthesis reaction system, and further add 2 L of RNaseH (1.5 U / ML) and 11 L of DNA polymerase I ( 9.0 U / μL) was added and mixed, and reacted for 2.5 hours at 16 ° C. After the second-stran d synthesis reaction, 23 dNTPmix and 2yCtL of Pfu DNA polymerase were synthesized. The mixture was added, mixed quickly, and reacted for 30 minutes at 72 ° C to perform b1 unting of the cDNA end.After B1 unting reaction product was subjected to phenolic mouth-mouth form extraction and ethanol precipitation as usual. The pellet (pellet) was dissolved in a 9 ^ L EcoRI adapter and incubated at 4 ° C for 30 minutes. Was added, followed by the addition of 4 DN A1 igase (4 U / L), followed by overnight reaction at 8 ° C. The ligation reaction of this EcoRI adapter was carried out at 70 ° C. for 30 minutes. After stopping, bring to room temperature Add 10 mM ATP2 / and a phosphorylation terminator for phosphorylation of the EcoRI adapter end, and add 1 μΙ_ of T4 pol ynucl eotide kinase (10 U / μ し), . C was anti-ifced for 30 minutes. After stopping the reaction by treatment at 70 ° C for 30 minutes, returning to room temperature, add the restriction enzyme Xhoi and the reagent for the restriction enzyme reaction to this reaction system, and mix at 37 ° C for 1.5 hours. The reaction was performed. This reaction product was subjected to gel filtration chromatography using Sepharose CL-2B, and a fraction containing an XhoI digest of EcoRI adapter-added cDNA was recovered. The collected fractions were subjected to phenol-monochrome form extraction and ethanol precipitation as usual. 1 OOng of the recovered cDNA and 1 g of Hyb “iZAP—2.1 vector EcoRI—XhoI (Stratagene)” were subjected to a ligation reaction system and incubated at 12 ° C. The ligation reaction product, 4 yLiL, was mixed with a packaging extract (Stratagene) and subjected to a packaging reaction at room temperature for 2 hours.After the reaction, an SM buffer was added. A small amount of black-mouthed form was added and mixed, and the aqueous phase after centrifugation was stored in a cold place in a cold place .. About 10 ⁶ pfu of this phage was added to E. coli XL 1 -B1 ue MR F, strain 0. 30D combined (about 10 ⁸ ce Ί 1) and mixed, after incubation for 15 minutes a further Ex a ssist helper phage to at添力□ to 37 ° C, at 37 ° C by addition of LB broth Then, the cells were incubated at 70 ° C for 20 minutes, centrifuged, and the supernatant was collected. It was stored in a cold place as § one Jimido c DNA library. '

Creating a Bai t vector

The expression vector in which the cDNA encoding FLAG-tag is encoded on the plasmid pME18S at the full length of mouse TRAF6, and the N-terminal of the mouse is expressed in pME-FLAG-TRAF6 (PNAS 96 , 1234-1239 (99)) were digested with restriction enzymes EcoR I and Stu I according to a conventional method to prepare a cDNA fragment encoding mouse TRAF6 full-length protein. On the other hand, pGBT9 vector The Yuichi plasmid (CI ontech) was digested with EcoRI and SmaI, and the cDNA fragment (EcoRI-StuI) encoding the full-length mouse TRAF6 protein prepared above was ligated. After the ligation reaction, E. coli DH5 strain was transformed to obtain a transformant. Plasmids were recovered from the transformed Escherichia coli by a conventional method and used as a Bait vector (pGBT9-TRAF 6). In this expression vector, gene expression occurs in a form in which the DNA binding domain of GAL4 protein and the full-length protein of TRAF6 are fused. Transformation and Screening

The yeast strain PJ69-4A (Genetics 144: 1425-1436 (1996)) has the 1 ac Z, f / 7s3, and de2 genes integrated into its genome, When the GA L4DN A binding domain / TRAF 6 fusion protein and the GAL 4 active domain Zc DNA expression product fusion protein are combined, they can grow on a medium lacking histidine and adenine, and are positive for one galactosidase activity. PG BT9—TRAF 6 was introduced into the Toriken mother PJ 69-4A strain by the lithium acetate method, and then spread on Trp-free SC ±± ya at 30 ° C for 3 days. Then, the 3 × 10 ⁶ clone of the cDNA library described above was introduced into the yeast by the lithium acetate method, and SC / —His / −T r ρ / —L eu / Seed on -Ad e medium and cultured for 7 days at 30. C. The emerged colonies were shaken on SCZ-His / -Trp / -Leu ± § ground (2 mL) ± After overnight (30 ° C), the yeast cells are disrupted with a glass bead s, and the lysate O OyOtL contains ON ACL (2 mg / mL) 160 ACL, which is a substrate for yS-Gal. The mixture was incubated at 30 ° C for 1 hour, and the one showing a clear yellow color was selected as positive.Plasmids were extracted from each of the obtained clones, transformed into large bacteria, and the plasmids were collected again After recovery, this plasmid was introduced into yeast PJ69-4A strain which had been transfected with pGBT9-TRAF6 in advance, and confirmed to be a positive clone. CDNA was recovered from this plasmid, introduced into the closing site BamHI / XhoI of pBluescript SK-, and the nucleotide sequence was confirmed by a conventional method. The plasmid having the cDNA (SEQ ID NO: 5) inserted in ρΒuescript was named pB1uescript-mTIFA. The obtained plasmid was placed on January 1, 2001 at 1-3 1-3 Higashi, Tsukuba City, Ibaraki Prefecture, Japan. Biotechnology Industrial Technology Research Institute) (Accession No. FERM P-18154). This was followed by the international approval of the deposit of microorganisms in the patent procedure at the Patent Organism Depositary at the National Institute of Advanced Industrial Science and Technology at 1-1-1 Tsukuba East, Ibaraki, Japan. It was transferred to the International Deposit under the Budapest Treaty on February 27, 2001 and given the accession number FERM BP-7842. Example 2 Northern blotting (gene expression profiling)

Northern blotting was performed to analyze the expression level of TIFA in each tissue. A probe was prepared using a portion corresponding to the coding region of the cDNA sequence of mouse TIFA. Using primer A containing an initiation codon (sequence: 5, -GCCTCGA GATGTCCACCTTTGAAGACGCTG—3 ') and primer B containing a termination codon (sequence: 5'-GCGCGGCCGCTCACAGTTCGTT TTC ATCC ATTTC-3') as a template, mouse TIFA A PGR reaction was performed at the following temperature and time using the plasmid (pBluescript-mTIFA) into which the cDNA was inserted. One cycle of 95 ° C for 2 minutes, 25 cycles of 95 ° C for 1 minute, 52 ° C for 1.5 minutes, and 72 ° C for 2.5 minutes were performed for 25 cycles, and a cycle of 72 ° C for 10 minutes was performed. Separately, a plasmid pME18S for mammalian cells (MoT. Cell Biol. 8: 466-472, 1988, Laboratory for Neonatal Chemistry (edited by the Biochemical Society of Japan, Tokyo Kagaku Dojin), Volume 7: 123-1 (P. 26, 1990), a nucleotide sequence encoding a FLAG-tag and an EcoRI_XhoI-Notl recognition sequence inserted on its 3 side into the EcoRI site. Sumid pME-FLAG was prepared. The obtained amplified fragment was extracted, treated with restriction enzymes XhoI / NotI, and introduced into plasmid pME-FLAG to prepare an expression plasmid pME-FLAG-mTIFA. This plasmid was prepared in a large amount and treated again with the restriction enzymes XhoI / NotI to obtain a DNA fragment encoding TIFA, which was labeled. Labeling was performed using Megaprime DNA labeling system (Amersham Pharmacia Biotech). The DNA obtained above and the random primer are mixed, treated at 100 ° C for 3 minutes, transferred to ice water and quenched, and then added with buffer 1, [Hi-32P] d CTP, and K 1 enow DNA synthase. Gently mixed. After the mixture was reacted at 37 ° C. for 15 minutes, unreacted labeled nucleotides were removed using a Nick column (Amersham Pharmacia Biotech) to prepare a labeled DNA probe. Messenger RNA (mRNA) from mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis tissues, each of which is blocked by 2 / Ug (MTN Blots Mouse Clontech) ) Was treated at 68 ° C. for 30 minutes in a hybridization solution (manufactured by Express Hyb C 1 on tech) 5 m away. The probe obtained above was treated at 100 ° C for 3 minutes, transferred to ice water and quenched, added to a 5 mL hybridization solution, and added to the membrane. The membrane was incubated for 1 hour at 68 ° C and the probe was hybridized to RNA. After the hybridization, the membrane was washed with a washing solution having a composition of 2XSSC and 0.05% SDS at room temperature for 40 minutes. Subsequently, washing was performed at 50 ° C. for 40 minutes with a washing solution having a composition of 0.1 × SSC and 0.1% SDS. Wrap the washed membrane in a wrap and adhere to the X-ray film. The samples were exposed to light for 1 week in the C dip-fridge and developed.

As a result, mRNAs having three kinds of chain lengths of 2.4 kb, 3.2 kb and 4.0 kb were detected. Among them, the 2.4 kb band was confirmed as the strongest band. In mice, TIF AmRNA was highest in the spleen among the identified organs, followed by heart, brain, liver, lung, kidney, testis, and muscle (Fig. 1). Example 3 cDNA expression (1) Construction of expression vector

 A plasmid vector that expresses the cDNA cDNA, which is a fusion protein in which My c epitope tag (amino acid sequence: MEQKL I SEEDL) and mouse TIFA are linked under the control of the S Ra promoter based on the plasmid pME 18 S pME—My c — MTIFA was prepared.

 (2) Gene expression experiment

Mixing the plasmid pME- Myc- mTIFA5Atg and pME- F LAG- TRAF6 5 ^ 9 prepared in the above (total volume 225 Mr), 2X HBS solution _{250μΙ_ (42mM HEPES S 290mM NaCl,} 70mM N a 2 H P0 ₄ pH 7. 1) were mixed, further 2. 5M C a C Ί ₂ 25 teeth and the mixture was mixed gently Yakani 30 seconds to give a calcium phosphate DN a co-precipitate at room temperature 30 minutes stand. The calcium phosphate DNA coprecipitate was gently added dropwise to a 293 T cell culture system (ø6 cm dish). Then, 5% C0 ₂ under at 37 ° C, after one cane down base 4 hours incubator, remove the ±咅養solution, by adding again the growth medium, 40 hours at 5% C0 ₂ under 37 ° C Cultured. In addition, a D-MEM medium supplemented with 10% FCS was used for the culture. After the culture, the cells were washed with PBS. Next, the cells were scraped off using a scraper, and this was added to TNE buffer (1 OmM Tris-HC1, pH 7.8 / 1% NP 40 / 0.1.15 M NaCl / 1 mM EDTA / 1 Oj mL aprotinin ) 400 μL was added to solubilize the meniscus. After solubilization, centrifugation was performed at 15000 xg for 5 minutes, and the supernatant was recovered.

 A similar procedure was performed in place of pME-FLAG-TRAF6, and similarly prepared expression vectors pME-FLAG-TRAF1, pME-FLAG-TRAF2, pME-FLAG-TRAF3, or pM Performed using E—FLAG—TRAF 5.

 (3) Immunoprecipitation experiment

A suspension of Protein G-Sepharose was added to the cell lysate 3 OOAtU prepared in the previous section, and the mixture was inverted and stirred at 4 ° C for 30 minutes. Then, after centrifugation at 5,000 xG for 5 minutes, the supernatant was collected and added to the mouse anti-FLAG antibody (X Bear (1) and 10 L of Rotating G-sepharose suspension were added, and the mixture was stirred at 4 ° C for 1 hour with ## j. After the treatment, the immune complex / resin was washed with a NE buffer and collected. Suspend in 2X sample buffer (20% glycerol / 10% 2-mercaptoethanol / 6% SD SZ13 OmM Tris-HC1, pH 6.8) equal to immune complex / resin and immunize After dissolving the complex, SDS-PAGE was performed on a 12.5% acrylamide gel. After the electrophoresis, the cells were transferred to a PVDF membrane using a semi-dry blotting apparatus (TRAS—BLOT SD SEMI-DRY TRANSFERCE LL). After the transfer, the membrane was subjected to a blocking treatment using a PBS-T buffer containing 3% skim milk, followed by a primary antibody reaction with a mouse anti-My'c antibody (diluted at 1000: SantaCruz), and further with an HRP-labeled anti-mouse I 9 A secondary antibody reaction with an antibody (Amersham Pharmacia Biotech) was performed. After washing the membrane thoroughly with PBS-T, detection by ECL (Amersham Pharmacia Biotech) was completed.o

 As a result, in an experiment using a lysate of cells expressing both TRAFs 1, 2, and 6, mouse TIFA protein was detected, and was particularly remarkable in TRAFs 2 and 6. This revealed that the mouse TIFA protein binds to TRAF1, 2, and 6 molecules (Fig. 2). On the other hand, under the same conditions, mouse TIFA protein was not detected in experiments using crushed cells of cells expressing both TRAF3 or TRAF5, and it was considered that TRAF3 and 5 molecules did not bind to mouse TIFA protein. . Example 4 Function confirmation experiment using Lucifera reporter

The effect of TIFA on NF-κB activation was confirmed by expressing the NF-B repo overnight gene and TIFA in the same cells. Ν F— Λ: NF NF — K と し て responsive elements are connected in series as a reporter gene, and the HSV-TK promoter sequence is connected downstream of them, and a reporter gene that expresses the luciferase gene under their control. Tarplasmid 3x «BLuc was used. NF— «as a negative control B-response 3XM-B uc was used that introduced a mutation in the sequence and did not respond to NF-B. Using 10 ng of this reporter plasmid and the expression plasmid containing the mouse TIFA gene prepared in Example 3 (1), varying the concentration from 1 Ong to 10 g, transfect 293T cells ( ⁵ × 10 ⁵ ) by calcium phosphate method. Formed. Similarly, transformation was performed using 3 XM¾BLuc1 Ong and ρΜΕ—Myc—mTIFA with varying concentrations from 3 ng to 3 μg. Twenty-four hours before the gene transfer, ⁵ × 10 5 293 T cells were seeded on a 6 cm plate. The cells were cultured at 37 ° C. in a 5% C02 environment.

225 DNA dissolved in L of distilled water and 250 L 2XHBS solution (42mM HEPES, 290mM NaCl, 70mM Na 2 HP0 4 pH7. 1) were mixed, further 2. 5 M CaCl 2 was added and gently mixed 30 seconds Thereafter, the mixture was allowed to stand at room temperature for 30 minutes to obtain a calcium phosphate DA coprecipitate. The calcium phosphate DNA coprecipitate was added evenly over the 293 T cells in the entire 6 cm plate, allowed to stand for 4 hours, the medium was aspirated off, and 4 ml of ± ground was added to continue the culture. After 36 hours from the transformation, the cells were washed once with PBS, and a cultured cell lysate LC? (Manufactured by Toyo Ink) was added and treated at room temperature for 15 minutes to lyse. The lysate was centrifuged at 15000 rpm for 2 minutes at 4 ° C, and only the supernatant was obtained as an enzyme source. 10 L of enzyme solution was coated with a luminescent substrate (manufactured by Toyo Ink Co., Ltd.) for 1 OOyUL each, and the luminescence was measured using a luminometer.

As a result, T1s "gene emission amount increases as the introduced amount up to 0.3 ₉ becomes 14.4 times the maximum to increase Caro. Emission amount reversed in more introduction amount is reduced (FIG. 3). At this time, the control of 381_110 and 1 \ / 1m-1 \ / 10— [10-c1 transfected cells, pME-Myc-mTIFA showed almost no effect between 3ng and 3yctg. (Fig. 3) Similar results were obtained when HepG 2 $ cells were used instead of 293 T cells Example 5 Cloning of human TIFA cDNA

Cloning of DNA of human TIFA based on the sequence of mouse TIFA Was. A draft sequence of human genomic DNA was searched based on the mouse sequence, and a sequence assumed to be a homolog was obtained. Based on this sequence, primers hT IFA—S 1 (sequence; ATG ACC AGT TTT GAA GAT GCT G) and h TIFA—F 1 (sequence; TCAT GA CTC) to amplify the coding region of human TIFA ATT TTC ATC), and a PCR reaction was performed using KOD polymerase (manufactured by Toyobo) at the following temperature and time, using human B microbeta-encapsulated cDNA library (manufactured by Clontech) as a template. Was. One cycle at 94 ° C for 1 minute, 40 cycles at 92 ° C, 40 seconds at 60 ° C, 30 cycles at 1.5 minutes at 75 ° C, and one cycle at 5 minutes at 75 ° C. PCR was performed again using the obtained reaction solution as a template. The primers were hETIFA-S 1 (sequence; AAG AAT T CA TGA CCA GTT TTG AAG ATG CTG) with Eco RI site and Xhol site attached to both ends of the amplification region, respectively. h XT I FA-F 1 (sequence; A AC TCG AGT CAT G AC TCA T TT TCA TC) was used, and the other reaction conditions were the same as before. The obtained fragment was extracted, treated with restriction enzyme EcoRI / XhoI, introduced into the EcoRI / XhoI site of plasmid pBLuescriptltlSK +, and the plasmid pBLuescript— hTI FA was prepared. When the nucleotide sequence was determined by a conventional method, the nucleotide sequence of SEQ ID NO: 3 was confirmed. When compared with the above-mentioned human genome sequence, A at the start codon was base number 1 and T at base number 498 was C. In another clone, C at base number 300 was T as well as the base position. The deduced amino acid sequences were the same in each case. The obtained plasmid pBluescript® hTIFA was released on January 5, 2001 at the Institute of Biotechnology, Ministry of Economy, Trade and Industry (METI) at 1-3 1-3 Tsukuzuhigashi, Ibaraki, Japan. Deposited at the National Institute of Advanced Industrial Science and Technology (AIST) (accession number FERM P-18153). This was followed by international approval for the deposit of microorganisms in the patent procedure at the National Institute of Advanced Industrial Science and Technology Terakyo Biological Depositary located at 1-1-1 Tsukuba East East, Ibaraki Prefecture, Japan. International deposit under the Budapest Treaty December 27, 2001 Transferred as of date with accession number F ERM BP-7841. A cDNA having the nucleotide sequence of SEQ ID NO: 3 has been inserted into the plasmid. Furthermore, this plasmid was treated with the restriction enzyme XhoI / No11 to obtain a DNA fragment encoding hTIFA, which was introduced into the plasmid pME-FLAG and expressed in the plasmid pME-FLAG-hTIFA. Was prepared. Example 6 Function estimation

The function of TIFA protein was deduced from the amino acid sequence. A motif search was performed on the protein using a database (PROS I TE MOT IF, PROS I TE PROF I LE, PR I NTS). The protein is structurally similar to the forkhead-as-sociated (FHA) domain from the 47th V to the 103rd L in the amino acid sequence of SEQ ID NO: 1 or 2. It is suggested that there is great similarity. The FHA domain, which was discovered as a motif for eukaryotic nuclear proteins, is also conserved in prokaryotes. Rad53p, a yeast protein, also has an FHA domain, but this protein is a kinase that controls the cell cycle and has the property of binding to phosphorylated peptides. Recent studies have shown that the FHA domain has a function to bind to the phosphorylated peptide of Rad53p (Moeccu! ArCell, Vol. 4 pp387-394, 1999). In addition, it was confirmed that some FHA domains of other proteins have a function of binding to a phosphorylated peptide similarly to that of Rad53p. Having an FHA domain strongly suggests that TIFA may be involved in intracellular signal transduction via phosphorylation. On the other hand, the TRAF molecule itself is not expected to transmit signals by phosphorylation. The TI FA molecule binds to phosphorylated proteins, strongly suggesting that the TI FA molecule may be involved in the signaling by transferring phosphate downstream of the signal transmission from the TAF molecule. This fact is strongly suggested by the fact that overexpression of the transcription factor NF-II: B has confirmed the activation and suppression of B. Example 7 JNK Atsushi

Plasmid pEF-His-JNK, which expresses Jun-N-Terminal kinase (hereinafter referred to as JN), which is connected to the histidine tag, under the control of the fermentation factor-promoter. A region containing the coding sequence of His-JNK was excised from His-JNK (Oncogene 1996; 12 (3): 641-50), and a plasmid having an elonge-gene factor-1 promoter (Nucleic Acids Res 1990; 18: 5322). 1 O / Lt g from 0.01 to 1 Oy g from pME-F to AG-mTIFA (In Examples 7 to 15, mouse TIFA (mTIFA) was used unless otherwise specified. , MT IFA may be referred to as TIFA) and transfection was performed on ⁵ × 10 ⁵ cells by the calcium phosphate method. 19 or 109 pME-FLAG-TRAF6 was used as a control instead of pME-FLAG-mTIFA. After incubation for 36 hours after the transformation, the ± nutrient solution was removed, and the cells were washed with PBS. To this, 400 L of TNE buffer was added to solubilize the cells, and the cells were scraped using a scraper and collected in a 1.5 mL tube. This was left on ice for 15 夯, then rotated for 15 5 and stirred. Thereafter, centrifugation was performed at 14000 Xg for 15 minutes, and the supernatant was recovered.

To this supernatant, an anti-T7 antibody (Novagen) for recognizing histidine was added, followed by 20 L of ProteinG-Sepharose suspension, followed by ttU stirring at 4 ° C for 30 minutes. After the treatment, centrifugation was performed at 14,000 9 for 15 minutes to recover the immune complex Z resin. Wash the immune complex / resin three times with TNE buffer and add Kinase Buffer (2 OmM HEP ES-KOH, pH 7.9 20 mM MgCl 2 150 mM NaC Ί 0.5 mM Na F 0.1 mM Na 2 VO 3 2 OmM Glceropho sphate 2 mM DTT). After suspending the immune complex / camphor fat in 50% of 1 <ぅ nase Buf fer, treating 10 of them and decomposing the immune complex, perform SDS-PAGE on a 10% acrylamide gel. Confirmation of immunoprecipitation was performed.

The remaining sample was supplemented with 92.5 kBq (2.5 C i) α ³² P—ATP and 2 g GST—c—j un (Cel 1 Signal Engineering) 30. C Treated for 20 minutes. After adding 20 μl_ of 3 × sample buffer to the sample and boiling for 3 minutes, SDS-PAG was performed on a 10% acrylamide gel. The gel was dried with a gel dryer and subjected to radiography.

 As a result, when pME-FLAG-TIFA was added from 0.01 g to 1 Atg, phosphorylation of GST-c-Jun was promoted in a concentration-dependent manner with an increase in the amount introduced. The TI FA protein was thought to activate JNK. Conversely, when the amount of TIFA introduced into the expression vector was more than the optimal amount, the promotion of c-Jun phosphorylation decreased (Fig. 7). Example 8 Preparation of various mutants

 In order to identify the active region of TIFA protein and to analyze the function of each domain, various partial deletion mutants of TIFA were generated. For convenience, each domain of mouse TIFA is defined as follows (see also the schematic diagram of FIG. 8).

table 1

これらのドメィンまたはドメィンの組合せを G S Tとの融合蛋白質として発現 させる発現べクターを以下のように作製した。

An expression vector for expressing these domains or a combination of domains as a fusion protein with GST was prepared as follows.

By inserting the DNA fragments encoding these domains into a vector constructed based on pME18S, which expresses a GST fusion protein under the control of the SR promoter, together with GST and frame, Each subclaw Ft.

 S

 Hereinafter, the expression vectors and the proteins encoded thereby are shown in Table 2 below (this is also abbreviated (see also the schematic diagram in FIG. 8).) The COA that was presumed to be involved in the binding to TRAF6 in TIFA was A vector expressing a mutant in which glutamic acid at position 178 in the sequence listing SEQ ID NO: 2 was converted to alanine was constructed (Table 2) .FHA domain is also important for maintaining the activity of TIFA. Since it is assumed to be present, it is well conserved in proteins having various FHA domains, and is considered to be important for maintaining the function of the FHA domain.

 1

Of serine glycine and 66 amino acids is the amino acid, respectively to prepare a base Kuta one expressing both converted variants glutamic acid and Aranin (Table 2) _(also, the fusion protein of FLAG and MTI FA full length PME-FLAG-TIFA was used.

Table 2 Expression vector protein Composition of fusion protein

 pME-G ST -T G S T -T I G S T tag 1 mT I FA Full length I F A FA

 pME-GST-NGST-N-GS T tag N domain F-FA FA HA domain

 pME-GST-N GST-N GST tag-N domain

 G ST -FA GST evening FHA domain One C one C domain

 pME-GS T -F GS T-FA GST Evening FHA domain A

 pME-GST-C GST—C GST

 GST and mT I F A where 178th G 1 u is replaced with A 1 a

 pME-GST — GGST — G5GST and the 50th G1y and the 50Es66A0ES66A and the 66th Ser are replaced by Glu and A1a, respectively, mTIFFA

 pME—FLAG—FLAG-TFFLAG-mTIFA

TIFAI FA On the other hand, in order to determine the binding region of the TRAF6 protein with TIFA, a vector that encodes a fusion protein of the FLAG tag peptide and various partially deleted mutants of mouse TRAF6 was selected. Produced. Hereinafter, the expression vectors and the proteins encoded thereby are abbreviated as shown in Table 3 below (see also the schematic diagram of FIG. 10). The amino acid number follows the known amino acid number of mouse TRAF 6 (J. Biol. Chem. Vol. 271, No. 46, pp. 28745-28748, 1996). Table 3

Example 9 Confirmation of binding site of TIFA to TRAF6

The binding site of TIFA to TRAF 6 was confirmed. 5yctg of each of the plasmids encoding the mTIFA mutant prepared in Example 8 and pME-FLAG-TRAF65 / g were mixed and transfected into 293T cells by the calcium phosphate method. Project. After culturing for 40 hours, TNE buffer was added to solubilize the cells. After solubilization, centrifugation was performed, and the supernatant was collected. Protein G—Separose suspension was added thereto, and the mixture was stirred with a car. Then, after centrifugation, the supernatant was collected, and a glutathione-separation suspension was added thereto, and the mixture was inverted and stirred. After the treatment, the protein / resin complex was washed with a TNE buffer, centrifuged, and the precipitate was collected. After suspending the protein in a 2X sample buffer equivalent to the protein / resin complex and decomposing the immunocomplex by a manual process, SDS-PAGE was performed on an acrylamide gel. After electrophoresis, it was transferred to a PVDF membrane. After the transfer, the membrane was blocked, then a primary antibody reaction with an anti-FLAG antibody (1000 dilution: Sigma), and further with an HRP-labeled anti-mouse Ig antibody (Amersham Pharmacia Biotech) A secondary antibody reaction was performed. After the membrane was sufficiently washed with PBS-T, detection was performed using ECL Western Blotting Detection Formulation (Amersham Pharmacy Abyssai Tech).

 As a result, GST—N—FA, GST—N, GST—FA, and GST—E178A did not co-precipitate with RAF 6, and GST—TI FA, GST—FA—C, GST—C, and G ST-G50ES 66A was confirmed to co-precipitate with TRAF6. From this, the C-terminal region of TIFA is important for the binding of TIFA and TRAF6 molecules, and among them, glutamic acid of amino acid number 178 shown in the sequence listing is very important for the binding to TRAF6. Was thought to be. On the other hand, the N domain and FA domain of TIFA were not considered to be important for binding to TRAF6 (FIG. 8). Example 10 Confirmation of binding site between 0 TIFA

 The fact that TRAF transmits a signal through multimerization suggests that TIFA may also multimerize. Each plasmid 5Atg encoding a mutant of mTIFA prepared in Example 8 was mixed with pME-FLAG-mTIFA5, and gene transfer was performed by the calcium phosphate method in the same manner as in Example 9, and anti-GS antibody An immunoprecipitation experiment was performed, and detection was performed using an anti-FLAG antibody.

As a result, GS TN-FAs GST-N, GST-FA and GST-C GST-TIFA, GST-FA-C, and GST-G50ES66A did not co-precipitate with LAG-TIFA, and coprecipitation with TRAF6 was confirmed. This suggests that the binding between TIFA molecules is important for both the FA domain and the C domain of TIFA, and that the G50ES66A mutation in the FA domain is not important for the binding between TIFA molecules. The N domain of TIFA was not considered to be relatively important for binding between TIFAs (Figure 9).

 In addition, two bands with different molecular weights were observed in TIFA, but only low-molecular-weight bands were obtained by treatment with bacterial alkaline phosphatase (BAP, Takara Shuzo), so phosphorylated and dephosphorylated forms were observed. It was thought that there was. Example 11 Confirmation of Binding Site of TRAF6 to 1 TIFA

 Each of Brasmid 5 encoding the mutant of TRAF 6 prepared in Example 8 was mixed with PME-Myc-mTIFA 5μ9, and gene transfer was performed by the calcium phosphate method in the same manner as in Example 9. An immunoprecipitation experiment was performed with a LAG antibody, and detection was performed with an anti-Myc antibody.

As a _{result, TRAF6, T 6AR S Τ 6ΔΖ} Κ Τ6ΔΖ3, Τ6厶Zeta5, coprecipitation of My c- T IF Alpha all及beauty T 6 T is δ Che認. This suggests that binding to the {IF} molecule only requires the presence of at least the coiled-coil portion and the C domain of the TRAF molecule (Fig. 10). Example 12 NF- «B activation experiment of each mutant

The effect of each region of TIFA on NF-B activation was confirmed by expressing the NF-ΛΓΒ repo overnight gene and various mutants of TIFA in the same cell. NF-II: Three NF-II-response elements are connected in series as a B repo overnight gene, and the HSV-II-promoter sequence is connected downstream of them. The luciferase gene is expressed under their control. The reporter plasmid 3 × ΔB Luc was used. As a negative control, 3XM¾B Luc which was mutated into the NF-ΛΓB responsive sequence and did not respond to NF-<< B was used. This reporter plasmid 1 Ong and prepared in Example 8 The expression plasmid containing the TIFA mutant gene was transfected into 293T cells (5 × 10 ⁵ cells) using the calcium phosphate method using 10 ng and 1 O 9 of each. 36 hours after the transformation, the cells were washed once with PBS, and ± cultured cell lysate LC (manufactured by Toyo Inki) was added, and treated for 15 minutes at room temperature to dissolve. Dissolve the lysate at 15000 rpm for 2 minutes 4. After centrifugation at C, only the supernatant was obtained as an enzyme source. 100 liters of Pitka Gene luminescent substrate (manufactured by Toyo Ink Co., Ltd.) was added to each of the solutions, and the amount of luminescence was measured over a luminometer.

 As a result, concentration-dependent activation of NF — << B could not be confirmed with the introduction of GST—N, GST—N—FA, GST-FA, or GST—C, but GST—FA—C and GST— In TIFA, the luminescence increased up to 10 μg as the amount of vector introduced increased (Fig. 11). GST-E178A and GST-G50ES 66A did not show concentration-dependent activation of NF-B (FIG. 12). From these facts, both the FA and C domains of TIFA are important for signal transduction by TIFA, and G1y50 and Ser66 of FHA domain bind to TRAF6 and bind to TIFA molecules. It was found to be insignificant, but important for signal transmission. The mutant E178A failed to bind TIFA to the TRAF6 molecule, but also failed to activate NF-κΒ, indicating that this residue is important for signal transduction. . It was also found that the I domain of T IF 相 対 的 に was relatively unimportant for T IF A signaling. Example 13 TIFA by Expression of TRAF6 Dominant Negative (DN) Body

By expressing the NF-; trB reporter gene and a certain amount of pME-FLAG_mT IFA and pME-T6 DZ5 in the same cell, the effect of TIFA on NF-¾B activation was confirmed. 10 ng of 3x «BLuc, 1 μg of pME—FLAG—mTIFAO. And the expression plasmid pME—T6DZ5 (from 1 Ong to 10 μg) prepared in Example 2 were transfected into 293T cells by the calcium phosphate method. As a result, depending on the dose of PME—T6DZ5, the NF of TIFA—Λ: Β activation Performance was impaired. This revealed that at least part of the NF-activating effect of TIFA was dependent on TRAF6 (Fig. 13). Example 14 TRAF 6 by Expression of TI FA Dominant Negative (DN) Body

The expression of the NF-reporter gene and a certain amount of pME-Myc-TRAF6 and pME-GST-C in the same cells was used to confirm the effect of TRAF6 on NF-wB activity. 3 x / iBL uc 10 ng, pME—My c-TRAF 60.3 μg and the expression plasmid ρΜΕ—GST—C (from 1 ng to 10 g) prepared in Example 8 were used in 293 T cells. Was transfected by the calcium phosphate method.

 As a result, the ability of TRAF6 to activate NF-C: B was inhibited depending on the dose of pME-GS T-C. This suggests that at least part of the NF-— activation of TRAF 6 depends on TIF F (Fig. 14). Example 15 Experiment of Inhibition of TRAF6 Function by IL-1β Stimulation by Expression of 5 TIFA Dominant Negative (DN) Body

 The effect of TRAF 6 on NF-½B activity was confirmed by expressing the NF- «B reporter gene and pME-GST-C in the same cells. 10 ng of 3 × ΔB Luc and the expression plasmid pME—GST—C (from 1 g to 1 Og) prepared in Example 8 were transfected into 293T cells by the calcium phosphate method. The cells were spiked with ± 1.50 ng / mL or TN Fa to 20 ng / mL in a ± nutrient solution of the cells.

 As a result, the ability of IL-11 to activate NF-¾B was inhibited depending on the dose of pME-GST-C (Fig. 15). On the other hand, the ability of TNF to activate NF-B was hardly inhibited (Fig. 16). This suggests that NF-Λ through TRAF 6 stimulated by IL-1 stimuli; 少 な く と も B-activating activity depends at least in part on TIF 、, but NF- Λ BB activity via TRAF 2 stimulated by TNF α The effect was not considered to be dependent on TIFII.

Claims

The scope of the claims  1. A polypeptide selected from the group consisting of:  ① a polypeptide represented by the amino acid sequence of SEQ ID NO: 1 or 2 in the sequence listing, (2) A fragment of the polypeptide of (1) or a polypeptide comprising at least one portion  (3) a polypeptide containing the polypeptide (1) or (2),  ポ リ a polypeptide having at least about 70% homology on the amino acid sequence with the polypeptide of ① or か つ and having an activity of binding to TRAF; and  ぺ A polypeptide having a mutation such as deletion, substitution, addition or insertion of one or several amino acids in the amino acid sequence and having an activity of binding to TRAF. 2. A peptide having at least about 8 consecutive amino acid sequences of the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing.  3. A polypeptide encoding the polypeptide according to claim 1 or 2, or a polynucleotide encoding the peptide, or a complementary strand thereof.  4. A polynucleotide which selectively hybridizes with the polynucleotide of claim 3 or a complementary strand thereof under a specific condition.  5. A polynucleotide represented by at least about 15 consecutive nucleotide sequences of the nucleotide sequence set forth in SEQ ID NO: 3, 4, or 5 in the Sequence Listing, or a complementary sequence thereof. 6. A recombinant vector comprising the polynucleotide according to any one of claims 3 to 5.  7. A transformant transformed with the recombinant vector according to claim 6. 8. The method for producing a polypeptide or a peptide according to claim 1 or 2, comprising a step of culturing the transformant according to claim 7.  9. An antibody that immunologically recognizes the polypeptide or the peptide according to claim 1 or 2. 10. The polypeptide or peptide according to claim 1 or claim 2. 10. The antibody according to claim 9, which suppresses the activity of binding to RAF.  11. A compound that interacts with the polypeptide according to claim 1 to inhibit or inhibit the activity thereof and / or interacts with the polynucleotide according to claim 3 or 4. A method for identifying a compound that acts to inhibit or promote its expression, comprising the polypeptide or the peptide according to claim 1 or 2, and the claims 3 to 5. Any one of the polynucleotides according to claim 6, the vector according to claim 6, the transformant according to claim 、, the antibody according to claim 9 or 10. A method characterized by using at least one of the following.  12. A compound that interacts with the polypeptide described in claim 1 to inhibit or activate the activity thereof, or interacts with the polynucleotide described in claim 3 or 4. A method of identifying a compound that acts to inhibit or enhance its expression, wherein the polypeptide or polynucleotide is screened under conditions that allow the interaction between the compound and the polypeptide or polynucleotide. Assessing the interaction of the compound with the compound to be contacted (such interaction is related to a second component that can provide a detectable signal in response to the interaction of the compound with the polypeptide or polynucleotide). And then the presence or absence or alteration of the signal resulting from the interaction of the compound with the polypeptide or polynucleotide. How by detecting comprises compound interacts with Poribe peptide or Porinuku les old tide, to determine whether the activity of the active I spoon or inhibit.  13. A compound identified by the method according to claims 11 to 13. 14. A compound that interacts with the polypeptide described in claim 1 to inhibit or activate the activity thereof, or interacts with the polynucleotide described in claim 3 or 4. A compound that acts to inhibit or promote its expression. 15. The polypeptide or peptide according to claim 1 or 2, the polynucleotide according to any one of claims 3 to 5, the vector according to claim 6. The transformant according to claim 7, wherein the transformant A pharmaceutical fiber comprising at least one of the antibody according to claim 9 or 10, or the compound according to claim 14 or 15.  16. A method for diagnosing a disease associated with the expression or activity of the polypeptide according to claim 1 in an individual, comprising: (a) a nucleic acid sequence encoding the polypeptide; and And / or (b) a method comprising analyzing the polypeptide in a sample derived from an individual as a marker.  17. A therapeutic method comprising using the pharmaceutical composition according to claim 15 for a disease associated with TRAF, TIFA or NF- -B.  18. A method for producing the pharmaceutical composition according to claim 15.