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WO2002064786A1 - Nouveau gene tcif - Google Patents

Nouveau gene tcif Download PDF

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Publication number
WO2002064786A1
WO2002064786A1 PCT/JP2002/001207 JP0201207W WO02064786A1 WO 2002064786 A1 WO2002064786 A1 WO 2002064786A1 JP 0201207 W JP0201207 W JP 0201207W WO 02064786 A1 WO02064786 A1 WO 02064786A1
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Prior art keywords
polypeptide
activity
compound
polynucleotide
traf
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Japanese (ja)
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Jun-Ichiro Inoue
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Mochida Pharmaceutical Co Ltd
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Mochida Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a novel TRAF binding factor, in particular, to a TRAF—Cerminal Inte lectin ng Fac tor (hereinafter sometimes referred to as TCIF).
  • TCIF TRAF—Cerminal Inte lectin ng Fac tor
  • TCIF TRAF—Cerminal Inte lectin ng Fac tor
  • TCIF TRAF—Cerminal Inte lectin ng Fac tor
  • TN F tumor necrosisf actor
  • Stimulatory responses mediated by one family and To11 / IL-1 (intelleukin-1) receptor family play important roles in biological reactions such as immunity, inflammation, apoptosis and allergic reactions It has become clear that it is fulfilling. In these reactions, the binding of each ligand to the receptor triggers and the signal is transmitted into the cell, and appears as a corresponding response, but is involved in the signal transmission mechanism and signal transmission in the cell. The molecule has not been fully elucidated.
  • TRAF proteins include TRAF 1 and TRAF2 previously identified from TNFR-II, TRAF3 identified from CD40, TRAF 4, CD40 identified as a breast cancer cell-specific expression gene, and TRAF binding to lymphotoxin receptor 5, and TRAF 6, which is thought to transmit a signal of 1 to 11 RI, is known.
  • TRAF2 5, and 6 molecules induce NF-B activation by forced expression.
  • forced expression of TRAF 1 and TRAF3 suppresses the activation of NF-1 ⁇ 2B.
  • TRAF 2 is also involved in signal transduction from type I and type I TNFR, but its effect is to block the pathway from TNF to caspases, and suppress cell death.
  • TRAF 6 molecule is essential for osteoclast differentiation and gain of function, and K0 mice of this molecule show bone dysplasia.
  • NF-B Since NF-B widely activates the entire immune system, the control of NF-3 ⁇ 4B activity is physiologically important, and this control system is also significant as a drug target in the treatment of various diseases. Is also big. For example, in autoimmune diseases in which autoreactive B cells do not apoptosis due to the constant entry of proliferation signals from CD40, NF-mediated by any of TRAF2, 5, or 6 molecules; It is possible that iB's activity is involved. TRAF 6 molecules may be involved in septic shock, acute hepatitis, osteopathy, etc.
  • TRAF TRAF
  • IKK I-3 ⁇ 4B Kinase
  • IKK I-3 ⁇ 4B Kinase
  • IKK phosphorylates IB, promotes ⁇ -decomposition, and activates NF-B.
  • the road is common for TRAF2, 5, and 6, and the analysis is relatively advanced.
  • the pathway from the TRAF 2, 5, 6 molecules to NIK is largely unknown and has not yet been fully elucidated. ( ⁇ X peri me nta ⁇ Cel l Research Vol. 254 pp 14-24 2000)
  • TRAF TRAF-associated NF B acitivator
  • I TRAF
  • TRIP TRAF-inte ractin ng protein
  • TR IP-1 interacts with TRAF 1 and TRAF 2 in animal cells, but suppresses the activation of NF-1 ⁇ 2B induced by TNF- or CD30.
  • functional findings indicate that IL-1 mediated activation is not suppressed.
  • the Peg3 protein which is a product of a so-called genomic imprinting gene, also interacts with TRAF 2, and that overexpression of Peg3 induces NF-; ⁇ B activity.
  • 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.
  • c-IAP has also been reported to be a molecule that interacts with TRAF 2 via the TRAF-N domain, and that the TRAF molecule and the molecule that interacts with the TRAF molecule are responsible for cell death or cell survival. Included in the control mechanism It is suggested that
  • TRAF2 jl-direct RIP molecule
  • T6BP The interaction between T6BP and TRAF6 occurs through the coi 1 ed—coi 1 region of T6BP and the N-terminal Ringfnger domain and Zinccfnger domain of TRAF6.
  • IL-1 signal transmission involves a TRAF6-mediated mechanism, but it has been reported that upon IL-1 stimulation, a TRAF6-T6BP complex is formed in an IL-1 ligand-dependent manner. I have. According to this report, it is reported that T6BP does not affect NF-B activation or JNK activation during IL-1 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-C terminal interaction factor (hereinafter sometimes referred to as TCIF), a polynucleotide encoding the same, and substances related thereto.
  • TRAF-C terminal interaction factor hereinafter sometimes referred to as TCIF
  • One of the objectives is to clarify and make these applications possible.
  • the present inventors succeeded in cloning a novel molecule that binds to TRAF6 by a two-hybrid screening method using mouse TRAF6 as a vector, and at least demonstrated a function involved in NF-B activation.
  • the present invention has been completed.
  • DISCLOSURE OF THE INVENTION The present invention provides (1) a polypeptide selected from the following group;
  • ⁇ ⁇ a polypeptide having at least about 70% amino acid sequence homology with the polypeptide of 1 or ⁇ ⁇ and having an activity of binding to TRAF;
  • ⁇ 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;
  • the TF allows interaction between the polypeptide or polynucleotide and the compound to be screened to evaluate the interaction of the compound (eg, such an interaction is Associated with a second component that can provide a detectable signal in response to the interaction of the compound with the peptide or polynucleotide), and then with the compound and the polypeptide. Or the compound interacts with the polypeptide or polynucleotide to activate or inhibit its activity by detecting the presence or absence or a change in the signal resulting from the interaction with the polynucleotide or the polynucleotide. (13) a compound identified by the method of 11 or 12 above,
  • FIG. 1 shows the nucleotide sequence of cDNA of mouse TCIF and the amino acid sequence encoded thereby.
  • FIG. 2 is a continuation of FIG. 1 and shows the nucleotide sequence of mouse TCIF cDNA and the amino acid sequence encoded thereby.
  • FIG. 3 is a continuation of FIG. 2 and shows the nucleotide sequence of the mouse TC IF cDNA and the amino acid sequence encoded thereby.
  • FIG. 4 is a Northern plot showing the expression level of TCIFmRNA in each tissue in mice.
  • FIG. 5 is a diagram showing the results of an experiment in which the binding of TCIF to N-terminal deletion mutants of TRAF6 or various TRAFs was confirmed by immunoprecipitation.
  • FIG. 6 is a graph showing the results of examining the effect of TCIF alone on NF—; ⁇ B activity in a function confirmation experiment using a luciferase reporter.
  • FIG. 7 is a graph showing the results of examining the effects of various doses of TCIF on NF- ⁇ : B activity under TRAF coexpression in a function confirmation experiment using a luciferase reporter.
  • FIG. 8 is a graph showing the results of examining the effect of TCIF on NF_ ⁇ B activity under various co-expressions of TRAF in a function 5 experiment using a luciferase reporter.
  • ⁇ 2 and ⁇ 6 indicate TRAF2 or TRAF6 expression vectors, respectively.
  • the TC IF provided in the present invention is obtained by obtaining its cDNA as a substance having a novel amino acid sequence from a cDNA library.
  • the presence of mRNA in the TC IF of the present invention was confirmed in the liver, testis, heart, brain, kidney, muscle, spleen, and muscle by Northern blotting.
  • the TC IF of the present invention has the following properties or activities.
  • TRAF molecule preferably Binds to TRAF2 or TRAF6, especially TRA F6. Forced expression of the TCIF molecule alone in 293 T cells has little effect on NF-B activity or its activation. However, if TCIF is forcibly expressed in a state where low levels of TRAF2 or TRAF6 are expressed, NF-B is activated according to the expression level of TCIF.
  • Structural features include two C3 ⁇ Zs located in the amino acid sequence of SEQ ID NO: 1 from the 105th position to the 123rd position and from the 180th position to the 208th position in the mouse.
  • the TC IF of the present invention is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 in mice in mice.
  • a sequence considered to be a homologue in a human is shown in the public database Gen Bank with Accession number AAF67649.
  • 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 of the sequence listing, or a polypeptide or a polypeptide containing the same.
  • polypeptide or peptide of the present invention is obtained by combining the polypeptide of SEQ ID NO: 1 with about 40% or more, preferably about 70% or more, more preferably about 80% or more on the amino acid sequence. More preferably, it has about 90% homology, particularly preferably about 95% or more. Selection of a polypeptide or peptide having this homology can be carried out by using at least one property or activity of TCIF as described above or an effect on the property or activity as an index.
  • TRAF for example, TRAF 2
  • the above activity can be determined by a known method, for example, immunoprecipitation for TRAF binding activity, and reporter gene assay using luciferase or the like for the regulatory activity of NF-II: ⁇ . It can be detected or measured by the methods described 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 the amino acid sequence, a method for determining the nucleotide sequence of cDN ⁇ and then estimating the amino acid sequence encoded thereby.
  • the polypeptide or the 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 in the sequence listing, such as a reagent, a standard substance, or an immunogen. Also available as The minimum unit is an amino acid sequence composed of 8 or more amino acids, preferably 10 or more amino acids, more preferably 12 or more amino acids, still more preferably 15 or more consecutive amino acids. Is an immunologically identifiable polypeptide or peptide as the dimension of the present invention.
  • peptides can be used alone or in combination with a carrier (eg, keyhole limpet mosquisin or ovalbumin, etc.) as a reagent or standard, or as an antigen to generate antibodies specific to TCIF, as described below.
  • a carrier eg, keyhole limpet mosquisin or ovalbumin, etc.
  • those bound with other kinds of proteins or substances as described above are also included in the scope of the present invention.
  • at least one of the above-mentioned TCIF 'I is based on the polypeptide or peptide identified in this way, at least one of the above-mentioned TCIF 'I.
  • 1 to 100 preferably 1 to 30, more preferably 1 to 20, still more preferably 1 to 10, particularly preferably 1 to several amino acid deletions, substitutions,
  • a polypeptide or peptide comprising an amino acid sequence having a mutation such as addition or insertion is also provided.
  • the means for insertion is known per se, for example, site-specific mutagenesis, homologous recombination, primer extension or polymerase chain amplification (PCR) alone or in combination as appropriate. Brooke et al.
  • 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.
  • polypeptides having altered activity intensity or specificity are provided. These are also useful, for example, as TCIF activity-like substances or TCIF antagonists, or for screening substances that regulate TCIF activity.
  • animal species other than mice, for example, human homologous gene products are naturally included in the scope of the present invention.
  • another protein such as alkaline phosphatase or galactose is added to the N-terminal side or the C-terminal side.
  • Immunoglobulin F c cleavage such as U.S.
  • Fragment or F LAG—tag (Asp Tyr Lys Asp Asp Asp Asp Lys) (Botechnol ogy, 6, 1205-1210, 1988) or My cepitope tag (amino acid sequence: MetGluG1nLysLeuIl It is easy for those skilled in the art to add peptides such as eSerGI uG1uAspLeu) directly or indirectly via linker peptides using genetic engineering techniques. Polypeptides and the like to which another substance is bound are also included in the scope of the present invention.
  • the polynucleotide of the present invention and its complementary strand are a polynucleotide encoding the amino acid sequence of the polypeptide or peptide of the present invention, for example, the amino acid sequence described in SEQ ID NO: 1 in the sequence listing, and the polynucleotide. Means the complementary strand.
  • SEQ ID NOs: 2 and 3 in the Sequence Listing showing preferred polynucleotides are the nucleotide sequences of nucleic acids and cDNAs, which are predicted to be mouse TCIF coding regions, respectively.
  • sequence considered to be a homologue in humans is identified by the public data database Accession number AF220184 (mRNA sequence) as well as the accession number ACO in the human genome draft sequence database of GenBank. Present in 15585 and A CO 18867. These encoded polypeptides have high sequence homology with mouse TCIF and retain the above-mentioned characteristic sequences, and thus are considered to have the same activity as mouse TCIF. Therefore, polynucleotides of human homologs are also included in the subject of the present invention, and can be used for the same uses as mouse polynucleotides.
  • the present invention relates to a nucleotide sequence encoding the amino acid sequence of the polypeptide or the peptide of the present invention, for example, the amino acid sequence of SEQ ID NO: 1 in the sequence listing, preferably the nucleotide of SEQ ID NO: 2 or 3 in the sequence listing.
  • the method of high predication is described in, for example, Sambrook et al. [Molecular Cloning, Laboratory Manual, Second Edition] Cold Spring Harbor Laboratory, (1989) or Shaw, etc. (Nucleic Acids Res., Vol. 11, pp.
  • the selective hybridization is defined as a nucleic acid having a desired homology with a desired nucleic acid, for example, a specific probe (for example, a nucleic acid encoding the TCIF of the present invention of SEQ ID NO: 2 or 3).
  • Conditions that selectively or specifically hybridize and do not hybridize to other unrelated nucleic acids for example, nucleic acids of lower homology.
  • the critical temperature (Tm) is used as an index of the stability of a double-stranded molecule (hybrid) of a nucleic acid. This is based on the strand length, base composition, and chemical conditions (ionic strength, chemical denaturation).
  • hybridization is performed at a temperature below Tm.
  • Tm The empirical formula has been obtained for the Tm value of hybrids of perfect complementarity in DNA, RNA, or natural nucleotide probes (Human Mo ecu ar Genetics, Tom St. Rachan and Andrew P. Read) Supervised by Masanori Muramatsu, Medical 'Science-International, 1997, etc.).
  • the hybridization temperature should be 5 ° C lower or higher than Tm (preferably lower than Tm), and in order to maintain the stability of the hybrid. Requires a hybridization temperature of about 5 ° C below Tm for each pair of unpaired bases.
  • Tm (° C) 81.5 ° C + 1 6.61 o g M + 0.41 (% G +% C) -50 O / n-0.61 (% Formamide)
  • M is the monovalent cation strength of the solution (mo 1)
  • n is the length of the duplex in base pairs
  • each time 1% unpaired base is included Tm is reduced by 1 ° C, so the hybridization temperature Adjust accordingly.
  • the standard of the hybridization temperature is calculated from the Tm of the fully-phased neoplastic hybrid, for example, 55 ° C, preferably 40 ° C, more preferably 25 ° C, still more preferably 10 ° C, particularly preferably 5 ° C.
  • a method such as Shaw is partially modified. That is, a filter (eg, a nylon membrane or a nitrocellulose filter) to which nucleic acid is bound is filtered at 65 ° C. at 3 ° C. with 3% SDS containing 0.1% SDS.
  • SC was washed with 0.15M NaC1, 0.015M sodium citrate), then denatured salmon sperm of 50% formamide, 5X Denhardt's solution, 0.1% SDS, 250 / gZm 5xS SCP containing DNA in 5xS SCP (1xSSCP is 0.15M NaC1, 0.015M sodium citrate, 10mM NaH2P04, 1mM EDTA, pH 7.2) at 42 ° C for 5 hours (Or 65 ° C, 2 hours) Prehybridization. Next, 50% formamide, 1 X de Nharu Bok solution, 0.
  • the polynucleotide of the present invention has 10 or more consecutive nucleotides, preferably 15 or more, more preferably 20 or more, corresponding to an arbitrary or specific region of the specified nucleotide sequence. More preferably, it includes polynucleotides consisting of 25 or more nucleotides, oligo nucleotides and their complementary strands.
  • polynucleotides are used as probes or primers for detecting TCIF-encoding acid, for example, the gene or mRN, in the production of the polypeptide or the like of the present invention, or for regulating gene expression. 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. On the other hand, using a motif sequence could simultaneously suppress the expression of multiple similar proteins including TCIF.
  • the nucleotide sequence encoding TCIF or a polypeptide having a similar activity is determined by, for example, confirming the expressed protein using a known protein expression system and determining its physiological activity, for example, when utilizing c cell-free protein expression system can be performed by selecting by the activity of binding to a TRAF to index, available for example embryo, a re tailed one ⁇ techniques from rabbit reticulocytes and the like ( Nature, 1 79, 1 60-1 61, 1 9
  • the present invention also relates to a gene recombination technique using a known host such as Escherichia coli, yeast, Bacillus subtilis, insect cells, and mammalian cells.
  • a known host such as Escherichia coli, yeast, Bacillus subtilis, insect cells, and mammalian cells.
  • peptides and polypeptides comprising the TCIF of the present invention and its derivatives can be provided.
  • 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 means known per se, for example, as plasmid, as plasmid. Transformation of a host is carried out by using a gene, a chromosome, a virus, or the like.
  • 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.
  • the combination is classified into prokaryotic cells and eukaryotic cells, and a promoter, a ribosome binding site, a terminator, a signal sequence, an enhancer, and the like can be used in combination by a method known per se.
  • 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.
  • the ⁇ culture can be performed using the activity of peptides and polypeptides composed of TCIF and its derived products expressed as an index, but it can be subcultured using the amount of transformants in the soil as an index. Or you may produce by batch.
  • the recovery of peptides and polypeptides consisting of TCIF and its derivatives from the soil is performed by molecular sieve, ion column chromatography, affinity chromatography, using the activity of TCIF, for example, the activity of binding to TRAF, as an index. Purification and recovery can be achieved by combining chromatography, etc., or by means of fractionation of ammonium sulfate, alcohol, etc. based on the solubility difference.
  • 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.
  • An antibody is prepared by selecting an antigenic determinant of a peptide or polypeptide comprising the TCIF of the present invention and its derivative.
  • the antigen may be TCIF or a fragment thereof; at least 8, preferably at least 10, more preferably at least 12 And more preferably 15 or more amino acids.
  • This amino acid sequence does not necessarily need to be homologous to SEQ ID NO: 1 in the sequence listing, and is preferably a site exposed to the outside of the three-dimensional structure of the protein.
  • a continuous amino acid sequence is also effective.
  • the antibody is not particularly limited as long as it immunologically binds or recognizes a peptide or polypeptide consisting of TCIF and its derivatives.
  • a peptide or polypeptide consisting of TCIF of the present invention and a derivative thereof, alone or in combination with a carrier is humoral to an animal in the presence or absence of an adjuvant. It is performed by inducing immunity such as a response and / or a cellular response.
  • the carrier is not particularly limited as long as it does not cause harmful effects on the host, and examples thereof include cellulose, polymerized amino acids, albumin and the like.
  • 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.
  • antibody-producing cells for example, spleen or lymph node-derived
  • a permanently proliferating cell for example, P 3
  • a hybridoma producing an antibody that specifically recognizes TCIF of the present invention is selected, and the antibody is recovered from a culture of the hybridoma.
  • polyclonal antibodies or monoclonal antibodies capable of suppressing TCIF activity are particularly preferred, and can control the activity of TCIF. The activity involved can be easily controlled. (Compound identification and screening method)
  • Peptides or polypeptides comprising TCIF and its derivatives, polynucleotides encoding them, and their corresponding heavy chains, cells transformed based on their amino acid sequence and base sequence information, or Antibodies that immunologically recognize a protein synthesis system using them and a peptide or polypeptide consisting of TCIF and its derivatives can be used alone or in combination of two or more to produce peptides or polypeptides consisting of TCIF and its derivatives.
  • the present invention provides an effective means for a method for identifying or screening for a modulator or a modulator of activity against a polynucleotide, for example, an activity inhibitor or an activator.
  • the information on the amino acid sequence of the polypeptide or the nucleotide sequence of the nucleotide of the present invention and the computer-readable storage medium storing the information can be used, for example, for predicting the tertiary structure or estimating the epitope. It is effectively used for such purposes, and these are also included in the scope of the present invention.
  • using a computer to select antagonists by drug design based on the three-dimensional structure of peptides or polypeptides selecting expression regulators at the gene level using a protein synthesis system, and recognizing antibodies using antibodies Substance selection and the like can be used in a drug screening system known per se.
  • a peptide or polypeptide comprising the TCIF of the present invention and a derivative thereof, or a polynucleotide or a transformant of the present invention, comprises a screening candidate compound and a peptide or polypeptide or the like.
  • the conditions that enable the interaction are selected, and a system that uses a signal (marker 1) that can detect the presence or absence of this interaction is introduced.
  • this signal or the amount of signal A compound that activates or inhibits the activity of the peptide and polypeptide comprising the TCIF of the present invention and a derivative thereof, or a compound that inhibits or promotes the expression of the polynucleotide of the present invention, by detecting a change in Identify be able to.
  • the system using a signal include a system for measuring the activity of the polypeptide of the present invention, for example, an activity involved in NF-B activation, or a system for measuring the expression level of a polynucleotide. This is specifically illustrated in the examples. These may apply a well-known method.
  • each transformant may be replaced with a cell line in which the expression of the corresponding gene has been confirmed.
  • a transgenic animal particularly, a mammal such as a mouse, a rat, a pig, a mouse, etc.
  • a transgenic animal containing the nucleotide or polynucleotide of the present invention or the peptide or polypeptide of the present invention can be produced.
  • cells or animals in which all or at least a part of the original TCIF gene has been mutated, particularly deleted, such as knockout animals (particularly, mammals such as mice, rats, puta, and mice) can be produced.
  • knockout animals particularly, mammals such as mice, rats, puta, and mice
  • These cells or animals are also useful in the above-mentioned screening and the like, and are included in the present invention.
  • the compound thus identified is a candidate compound for an inhibitor of activity or action, an antagonist, an activator, an accelerator, or an activator for peptides and polypeptides composed of TCIF and its derivatives. , Available. It can also be used as a candidate for expression inhibitors, expression antagonists, expression activators, expression promoters, and expression activators for TCIF and its derivatives at the gene level. These compounds can be expected to prevent and / or treat various pathological symptoms related to TRAF, TCIF or NF-II: ⁇ as modulators or modulators of the activity, action or function of NF- ⁇ : ⁇ .
  • the candidate conjugate thus selected can be prepared as a pharmaceutical composition by selecting in consideration of the balance between biological utility and toxicity.
  • the TC of the present invention Peptides or polypeptides consisting of IF and its derivatives, polynucleotides encoding them and their nucleotide chains, vectors containing these nucleotide sequences, and peptides or polypeptides consisting of TCIF and its derivatives
  • 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 pharmaceutical composition can be produced using the peptide or polypeptide of the present invention, polynucleotide, vector, transformant, antibody, or the compound of the present invention.
  • the pharmaceutical composition is useful for treating or preventing TCIF, TRAF or NF-B, particularly diseases associated with TCIF, for example, autoimmune diseases, shock (such as septicemia and sucrose) and bone diseases (such as osteoporosis). is there.
  • a diagnostic means it is useful as a diagnostic means for a disease associated with the expression or activity of the peptide or polypeptide consisting of the TCIF of the present invention and a derivative thereof.
  • the diagnosis is, for example, a nucleic acid encoding the peptide. Utilizing the interaction or reactivity with the sequence to determine the abundance 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 peptide, the amount of the peptide in the sample derived from the individual, or the amount of the activity. In other words, TCIF is tested as a diagnostic tool.
  • Example 1 Obtaining DNA encoding mouse TCIF
  • a predetermined first-strand synthesis reaction reagent containing polyA-RNA5Atg equivalent, the XhoI linker primer provided with the kit, and 5-methy ⁇ dCTP is mixed. And allowed to stand at room temperature for 10 minutes. Then add 1.5 L of MMLV-RT (50 U / L) to the reaction and mix gently. The reaction was performed at C for 1 hour.
  • the seco attached to the kit was added to the first-strand synthesis reaction system.
  • 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 fraction was subjected to phenol one-mouth mouth extraction and ethanol precipitation as usual. Equivalent to 100 ng of the recovered cDNA and Hybr ZAP-2.1 vector, EcoRI—XhoI (Stratatagene eg) were subjected to a (4) Reaction A mixture of the ligation reaction products was mixed with a packaging extract (Stratagene) and subjected to a packaging reaction at room temperature for 2 hours.
  • a packaging extract Stratagene
  • the aqueous phase after centrifugation was stored cold place as phage solution.
  • the phage about 10 6 pf u corresponding E. coli XL 1- B, ue MR F 'strain 0. 30D (approximately 1 0 8 ce l,) and mixed, to further ExA Ssist thereto
  • LB broth was added and the mixture was further incubated at 37 ° C.
  • centrifugation was performed, and the supernatant was recovered and stored in a cold place as a phagemid cDNA library.
  • Yeast PJ69-4 strain 6 ⁇ 1 1 '03144: 1425-1436 (1996) has a lacZ, His3, and Ade2 gene incorporated into its genome, and has a GA L4D A binding domain /
  • TRAF6 fusion protein and the GAL4 active domain Zc DNA expression product fusion protein bind, they can grow on a medium lacking histidine and adenine, and have a positive galactosidase activity.
  • Has the property of pGBT9-TRAF6 was introduced into the yeast strain PJ69-4A by the lithium acetate method, spread on a Trp-free SC medium, cultured at 30 ° C for 3 days, and recovered colonies.
  • Plasmids were extracted from each of the obtained clones, and transformed into Escherichia coli to collect the plasmids again. After recovery, this plasmid was introduced into yeast PJ69-4A strain, which had been transformed with PGBT9-TRAF6 in advance, and confirmed to be a positive clone. The cDNA was recovered from this plasmid, introduced into the EcoRI / XhoI cloning site of pBluescript SK—, and the nucleotide sequence was self-identified by a conventional method.
  • the plasmid in which the cDNA (SEQ ID NO: 3) was inserted into ⁇ uescript was named pB ⁇ uescrip t-mTC IF.
  • the obtained plasmid was placed on February 8, 2001 at Tsukuba-Higashi 1-chome, Chuo 6th, Ibaraki, Japan 305-8566, Japan Deposited at the Institute of Biotechnology, Ministry of International Trade and Industry (Accession No. FERM P-18199). Further, the plasmid was transferred to an international deposit in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms in Patent Procedures on January 31, 2002, with accession number FERM BP- 7871 is attached.
  • Example 2 Northern blotting (gene expression profiling)
  • Northern blotting was performed to analyze the expression level of TCIF in each tissue.
  • a probe was prepared using a portion corresponding to the coding region of the cDNA sequence of mouse TCIF.
  • Primer A containing the start codon following the Eco RI recognition site (sequence: 5'- ' TC-3 ') and a primer B containing a stop codon following the XhoI recognition site (sequence: 5'-GCCTCGAGTCATTCTTCTAAGTCAAGAGTGT TC-3') and a plasmid (mouse TCI FcDNA inserted as template)
  • a PCR reaction was carried out using pBluescript (m-TCTC) at the following temperature and time.
  • IF was prepared. This plasmid was prepared in a large amount and treated again with the restriction enzymes EcoRI / XhoI to obtain a DNA fragment encoding TCIF, which was labeled. Labeling was performed using a megaprime DNA labeling system (manufactured by Amersham Pharmacia Biotech). After top obtained in DNA and random primers scratch mixture was 100 ° C3 minutes treatment, after quenching transferred into ice-water, buffer, Non - 32 P] dCTP, and K 1 en ow DN A synthetase was added, gently Was mixed. After the mixture was reacted at 37 ° C.
  • RNA messenger RNA
  • the washed membrane was wrapped in a wrapper, adhered to an X-ray film, placed in a 80 ° C deep-fiber for one week, exposed and developed.
  • Plasmid vector pME—Myc—mTC which expresses the cDNA of the fusion protein in which My c-top tag (amino acid sequence: MEQKLISEEDL) and mouse TC IF are linked under the control of the SR promoter based on the plasmid pME 18 S IF was prepared.
  • an expression vector for an N-terminal-deleted TRAF6 mutant was constructed based on pME-FLAG-TRAF6 by the method of site-directed mutagenesis of Kunke 1 (Kunkel, TA (1985) Proc. Natl. Acad. Sci.
  • T6 ⁇ Ring (sometimes referred to as T6dRing; the same applies hereinafter) (amino acids 109 to 530; hereinafter referred to as AA109-530), T6mRZ1 (AA1 55—530), T6m RZ3 (AA 209—530), T6ARZ5 (AA 275—530), T6T (AA359-530).
  • Plasmid pME-Myc-171 ⁇ 15; 0: 9 prepared in the previous section was mixed with
  • NF—; iB reporter gene a NF-B responsive element consisting of three sets of NF-B responsive elements connected in tandem, and an HSV-TK promoter sequence connected downstream thereof, and a luciferase gene expressing under the control of these elements Yuichi Plasmid 3X / iBLuc was used.
  • 1 Ong of this reporter plasmid was used in each experiment.
  • 3 ⁇ BL uc in which a mutation was introduced into the NF-; ⁇ -responsive sequence and did not respond to ⁇ F- ⁇ : ⁇ was used.
  • This negative control reporter plasmid was used in the experiment described in FIG. ng was used, and 100 ng was used in the experiment described in FIG.
  • the above-mentioned reporter plasmid and the expression plasmid containing the mouse TCIF gene prepared in Example 2 were independently introduced into cells from 10 ⁇ 9 to 10 9.
  • various doses (from 1 Ong to 99) of mouse TCIF were added together with 50 ng each of the reporter plasmid and the expression plasmid containing the TRAF2 or TRAF6 gene.
  • An expression plasmid containing the gene was introduced into the cells.
  • an expression plasmid containing the mouse TCIF gene and an expression plasmid containing the TRAF 2 or TRAF 6 gene were used in appropriate combinations in the figure. did.
  • 225 L were dissolved in distilled water DN A and 250 Shino 2 xH BS solution (42mM HEPES N 290mM N aC 1 7 OmM N a 2 H P0 4 pH7. 1) were mixed, and further 2. 5 M CaC 1 2
  • the mixture was gently mixed for 30 seconds, and then allowed to stand at room temperature for 30 minutes to obtain a calcium phosphate DNA coprecipitate.
  • the calcium phosphate DNA coprecipitate was added evenly over the 293 T cells in the entire 6 cm plate. After allowing to stand for 4 hours, the medium was removed by suction, and 4 mL of ⁇ ground was added, and the culture was continued.
  • the cells were washed once with PBS, added with a cultured cell lysate LC (manufactured by Toyo Ink), 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.
  • 1 OL 1 ⁇ L of Picagene luminescent substrate (manufactured by Toyo Ink) was added, and the luminescence was measured using a luminometer.
  • TCI F protein The function of TCI F 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, and PR I NTS). The protein must have structural similarity to the zinc finger domain in the amino acid sequence from the 105th C to the 123rd H and from the 180th C to the 209th H. Was suggested.
  • the DEVD sequence present at amino acid 31 2 can be a substrate for protease, but before and after this sequence, the abundance ratio of basic amino acid and acidic amino acid changes greatly, so that TRAF 2 and TRAF 6 and After undergoing cleavage modification in a peripheral stimulus-dependent manner, there is a possibility that the home conformation can be changed to an active dandelion. This is also suggested by increased 3XB transcriptional activity when a large excess of TCIF is co-expressed with small amounts of TRAF2 and / or TRAF6o

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Abstract

L'invention concerne la clarification de nouveaux facteurs se liant au TRAF, en particulier d'un facteur interagissant avec le TRAF-C terminal (TCIF), de polynucléotides codant lesdits facteurs et de substances associées, ainsi que l'application de ceux-ci. L'invention concerne également des polypeptides choisis parmi les polypeptides suivants: (1) un polypeptide comportant la séquence d'acides aminés représentée par SEQ ID NO:1 dans la liste des séquences; (2) des polypeptides comportant un fragment ou au moins une partie du polypeptide décrit ci-dessus en (1); (3) des polypeptides contenant un polypeptide décrit ci-dessus en (1) ou en (2); (4) des polypeptides présentant au moins une homologie d'environ 70 % de la séquence d'acides aminés avec un polypeptide décrit ci-dessus en (1) ou en (2), et présentant une activité de liaison au TRAF; et (5) des polypeptides présentant une séquence d'acides aminés dérivée de la séquence d'acides aminés décrite ci-dessus, par délétion, substitution, addition ou insertion d'un à plusieurs acides aminés, et présentant une activité de liaison au TRAF; ainsi que des polynucléotides codant ces polypeptides.
PCT/JP2002/001207 2001-02-15 2002-02-13 Nouveau gene tcif Ceased WO2002064786A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006057217A1 (fr) * 2004-11-24 2006-06-01 The University Of Tokyo Proteines dfrp regulant les proteines drg et utilisation de celles-ci

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6060303A (en) * 1995-08-17 2000-05-09 Genetech, Inc. TRAF inhibitors
EP1074617A2 (fr) * 1999-07-29 2001-02-07 Helix Research Institute Amorces pour la synthèse de cADN de pleine longueur et leur utilisation
WO2001053312A1 (fr) * 1999-12-23 2001-07-26 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US6060303A (en) * 1995-08-17 2000-05-09 Genetech, Inc. TRAF inhibitors
EP1074617A2 (fr) * 1999-07-29 2001-02-07 Helix Research Institute Amorces pour la synthèse de cADN de pleine longueur et leur utilisation
WO2001053312A1 (fr) * 1999-12-23 2001-07-26 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides

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Title
CHIN A.I. ET AL.: "TANK potentiates tumor necrosis factor receptor-associated factor-mediated c-Jun N-terminal kinase/stress-activated protein kinase activation through the germinal center kinase pathway", MOL. CELL BIOL., vol. 19, no. 10, 1999, pages 6665 - 6672, XP002952621 *
LING L., GOEDDEL D.V.: "T6BP, a TRAF6-interacting protein involved in IL-1 signaling", PROC. NATL. ACAD. SCI. USA, vol. 97, no. 17, 2000, pages 9567 - 9572, XP002952620 *
SANZ L. ET AL.: "The atypical PKC-interacting protein p62 channels NF-kappaB activation by the IL-1-TRAF6 pathway", EMBO J., vol. 19, no. 7, 2000, pages 1576 - 1586, XP002952619 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006057217A1 (fr) * 2004-11-24 2006-06-01 The University Of Tokyo Proteines dfrp regulant les proteines drg et utilisation de celles-ci

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