JP4236852B2 - TRAF3-binding B cell specific receptor - Google Patents

Description

【００９９】

【図面の簡単な説明】
【図１】図１Ａは、Ｔ３ＢＰの推定アミノ酸配列、図１ＢはＴ３ＢＰ ｍＲＮＡのノーザンブロット分析の結果を示す。
【図２】図２Ａは、ＧＳＴ融合タンパク質（ＧＳＴ−クローン３，ＧＳＴ−クローン１０）のＴＲＡＦ２，ＴＲＡＦ３に対する結合特性のＧＳＴプルダウンアッセイによる分析結果を示す。
【図３】図２Ｂは、ＧＳＴ−Ｔ３ＢＰのＴＲＡＦ２，ＴＲＡＦ３，ＴＲＡＦ５，ＴＲＡＦ６に対する結合特性のＧＳＴプルダウンアッセイによる分析結果を示す。
【図４】図２Ｃは、ＴＲＡＦ３とＴ３ＢＰとの相互作用の免疫沈降アッセイによる分析結果を示す。
【図５】図３Ａ及び図３Ｂは、ＴＲＡＦ３中のＴ３ＢＰ結合領域のマッピングの結果を示す。
図３Ｃは、マウスＴＲＡＦ３の一次構造の概略を示す。
【図６】図４Ａは、ＧＳＴ融合Ｔ３ＢＰトランケーション突然変異体のＴＲＡＦ３に対する結合特性のin vitroアッセイによる分析結果を示す。
図４Ｂは、ＧＳＴ融合Ｔ３ＢＰトランケーション突然変異体のＴＲＡＦ３に対する結合特性のＧＳＴプルダウンアッセイによる分析結果を示す。
図４ＣはＴ３ＢＰの一次構造とそのＴＲＡＦ３結合領域を示す。
【図７】図５Ａは、Ｔ３ＢＰの発現による細胞の形態変化を示す。
図５Ｂは、ＴＲＡＦ３の細胞分布に及ぼすＴ３ＢＰ発現の影響を示す。
【図８】図６Ａは、Ｔ３ＢＰによる細胞性Ｆアクチン含量の増大を示す。
図６Ｂは、Ｔ３ＢＰを発現する細胞中におけるＦアクチンの細胞分布を観察した結果を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel TRAF3-binding protein that is a member of a B cell specific receptor. The present invention also provides a gene encoding the protein, a vector having the gene, a transformant having the gene, an antibody recognizing the protein, a method for screening a drug using the protein, and a diagnosis using the protein Regarding the method.
[0002]
[Prior art]
Biological effects are controlled by responses to certain stimuli. That is, many cytokines bind to receptors and cause intracellular reactions. For example, tumor necrosis factor (TNF) acts through the TNF receptor and regulates biological processes such as infection and induction of shock and protection against inflammatory diseases. TNF belongs to the TNF-ligand superfamily and acts with its ligand, the TNF receptor superfamily. Known ligands include TNF-α, lymphotoxin (LT) -α, LT-β, FasL, CD40L, CD27L, CD30L, OX40L, and neurotrophic factor (NGF). As the TNF receptor superfamily, p55TNF receptor, p75TNF receptor, TNF receptor-related factor, Fas antigen, CD40, CD27, CD30, OX40, low-affinity p75 and NGF receptor are known. The function of several TNF receptor families has been elucidated, for example, Fas antigen, low affinity NGF receptors and their ligands are involved in programmed cell death, and CD40 ligand is a T cell-dependent B cell. Involved in activation.
[0003]
Programmed cell death, one of the physiological processes, is essential for normal development and homeostasis of multicellular organisms, and disruption of apoptosis includes cancer, neurodegenerative diseases, and acquired immune deficiency syndrome (AIDS) Involved in the pathogenesis of human disease. Activation of Fas causes death domain-containing adapter molecule FADD / MORT1 to bind to the intracellular domain of Fas, which in turn activates caspase 8, a pro-apoptotic protease, and finally executes apoptosis. On the other hand, TNFR-1 transmits a series of signals of a wide range of biological activities mainly by activating NF-κB. TNFR-1 binds to the intracellular domain of TRADD and TNFR-1 containing the death domain, and through association with a number of signaling molecules such as FADD, TRAF2, and RIP, signals for both apoptosis and NF-κB activation To communicate. Thus, the actions of TNF family ligands and TNF family receptors are diverse and affect many biological processes, so identifying and analyzing these receptors, ligands and their signaling molecules It is important in establishing a cure for the disease.
[0004]
TNF receptor-related factor (TRAF) is a protein family first discovered as a downstream signaling factor in the TNF receptor superfamily [Rothe, M. et al. (1994) Cell 78, 681-692; Hu, HM (1994) J. Biol. Chem. 269, 30069-30072; and Cheng, G., et al. (1995) Science 267, 1494-1498]. To date, six factors have been identified for the TRAF family [Rothe, M. et al. (1994) Cell 78, 681-692; Hu, HM, et al. (1994) J. Biol. Chem. 269 , 30069-30072; Cheng, G., et al. (1995) Science 267, 1494-1498; Cao, Z., et al. (1996) Nature (London) 383, 443-446; Mosialos, G., et al. ( 1995) Cell 80, 389-399; Sato, T., et al. (1995) FEBS Lett. 358, 113-118; Regnier, CH, et al. (1995) J. Biol. Chem. 270, 25715-25721; Ishida , TK, et al. (1996) Proc. Natl. Acad. Sci. USA 93, 9437-9442; Nakano, H., et al. (1996) J. Biol. Chem. 271, 14661-14664; and Ishida, TK, Et al. (1996) J. Biol. Chem. 271, 28745-28748]. Each of TRAF1-6 has a coiled-coil TRAF-N domain and a C-terminal TRAF-C domain is conserved. [Cao, Z., et al. (1996) Nature (London) 383, 443-446]. The TRAF-C domain mediates the interaction between TRAF proteins, as well as the binding of the protein to TNFR superfamily factors [Cheng, G., et al. (1995) Science 267, 1494-1498; Takeuchi, M. (1996) J. Biol. Chem. 271, 19935-19942; and Hsu, H., et al. (1996) Cell 84, 299-308].
[0005]
TRAF3 was first identified as a molecule bound to the cytoplasmic domain of CD40, a B cell surface marker, and LMP-1, an Epstein-Barr virus latent membrane protein [Mosialos, G., et al. (1995) Cell 80 , 389-399; Sato, T., et al. (1995) FEBS Lett. 358, 113-118; Hu, HM, et al. (1994) J. Biol. Chem. 269, 30069-30072; and Cheng, G. , Et al. (1995) Science 267, 1494-1498]. TRAF3 has also been shown to interact with the cytoplasmic terminal portion of other factors in the TNFR family such as the lymphotoxin β receptor [Force, WR, et al. (1997) J. Biol. Chem. 272, 30835-30840]. TRAF family proteins including TRAF3 are cytoplasmic proteins that are thought to lack catalytic activity and are generally considered to function as adapter proteins. In this context, TRAF3 has been found to interact with other signaling molecules such as NIK, ASK and TANK [Song, HY, et al. (1997) Proc. Natl. Acad. Sci. USA 94 , 9792-9796; Nishitoh, H., et al. (1995) Mol. Cell 2, 389-395; and Regnier, CH, et al. (1997) Cell 90, 373-383]. However, the mechanism by which TRAF3 mediates signal transduction has not been fully elucidated.
[0006]
[Problems to be solved by the invention]
An object of the present invention is to identify a signaling molecule that binds to TRAF3. Another object of the present invention is to clone a gene encoding a signaling molecule that binds to TRAF3 and to elucidate the structure and function of the gene. Another object of the present invention is to provide a novel pharmaceutical or diagnostic method using a gene encoding a signaling molecule that binds to TRAF3.
[0007]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors performed screening of protein-protein interaction using yeast. This screening utilizes the fact that the yeast RAS signal transduction pathway is activated by recruiting human-derived Sos (hSos) protein to the cell membrane to complement the yeast cdc25 gene deficiency. The present inventors identified a novel TRAF3-binding protein (hereinafter also referred to as T3BP) by the above screening method using a cDNA expression library derived from WEHI231 cells and full-length TRAF3 fused to hSos, and further, this T3BP protein The structure and function were elucidated. The present invention has been completed based on these findings.
[0008]
That is, according to the present invention, a protein having any of the following amino acid sequences is provided.
(A) the amino acid sequence set forth in SEQ ID NO: 1;
(B) an amino acid sequence obtained by deleting, substituting and / or inserting one to several amino acids in the amino acid sequence set forth in SEQ ID NO: 1 and having binding activity to TRAF3; or
(C) an amino acid sequence having 60% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and having an activity of binding to TRAF3:
[0009]
Furthermore, according to the present invention, a gene encoding the protein of the present invention is provided.
Furthermore, according to the present invention, a gene having any of the following base sequences is provided.
(A) the nucleotide sequence set forth in SEQ ID NO: 2;
(B) a base sequence in which 1 to several bases are deleted, added or substituted in the base sequence shown in SEQ ID NO: 2 and encoding a protein having a binding activity to TRAF3; or
(C) a base sequence that hybridizes with the base sequence described in SEQ ID NO: 2 under stringent conditions and encodes a protein having binding activity to TRAF3:
[0010]
Furthermore, according to the present invention, a vector containing the gene of the present invention is provided.
Furthermore, according to the present invention, a transformant having the gene of the present invention or the vector of the present invention is provided.
Furthermore, according to the present invention, there is provided a method for producing a protein having a binding activity to TRAF3, characterized by using the transformant of the present invention.
Furthermore, the present invention provides an antibody that recognizes the protein of the present invention.
[0011]
Furthermore, according to the present invention, there is provided a method for screening for a substance that promotes or suppresses the function of the protein or antibody using the protein or antibody of the present invention. Preferably, the substance that promotes or suppresses the function of the protein or antibody of the present invention is a substance that promotes or suppresses intracellular signal transduction via TRAF3.
Furthermore, the present invention provides a substance that promotes or suppresses the function of the protein of the present invention obtained by the screening method of the present invention.
Furthermore, according to this invention, the pharmaceutical which contains the said substance as an active ingredient is provided. The medicament of the present invention is preferably used for the prevention or treatment of diseases associated with abnormalities in intracellular signaling via TRAF3.
Furthermore, according to the present invention, there is provided a method for diagnosing a disease associated with an abnormality in intracellular signal transduction via TRAF3, comprising measuring the level of the protein of the present invention in a biological component.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments and methods of the present invention will be described in detail.
(1) Protein and gene of the present invention
The present invention relates to a protein having any one of the following amino acid sequences and a gene encoding the same.
(A) the amino acid sequence set forth in SEQ ID NO: 1;
(B) an amino acid sequence obtained by deleting, substituting and / or inserting one to several amino acids in the amino acid sequence set forth in SEQ ID NO: 1 and having binding activity to TRAF3; or
(C) an amino acid sequence having 60% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and having an activity of binding to TRAF3:
[0013]
Specific examples of the gene of the present invention include genes having any of the following base sequences.
(A) the nucleotide sequence set forth in SEQ ID NO: 2;
(B) a base sequence in which 1 to several bases are deleted, added or substituted in the base sequence shown in SEQ ID NO: 2 and encoding a protein having a binding activity to TRAF3; or
(C) a base sequence that hybridizes with the base sequence described in SEQ ID NO: 2 under stringent conditions and encodes a protein having binding activity to TRAF3:
[0014]
The range of “1 to several” in the above-mentioned “amino acid sequence in which 1 to several amino acids are deleted, substituted and / or inserted in the amino acid sequence described in SEQ ID NO: 1” is not particularly limited. To 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, particularly preferably about 1 to 3.
[0015]
The above-mentioned “amino acid sequence having 60% or more homology with the amino acid sequence described in SEQ ID NO: 1” means that the homology between the amino acid sequence and the amino acid sequence described in SEQ ID NO: 1 is at least 60% or more, It means preferably 70% or more, more preferably 80% or more, further preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more.
[0016]
The range of “1 to several” in the above-mentioned “base sequence in which 1 to several bases are deleted, added or substituted in the base sequence described in SEQ ID NO: 2” is not particularly limited. To 60, preferably 1 to 30, more preferably 1 to 20, even more preferably 1 to 10, particularly preferably about 1 to 5.
[0017]
The above-mentioned “base sequence that hybridizes under stringent conditions” means DNA obtained by using a DNA as a probe and using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. For example, after hybridization at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques or a fragment of the DNA is immobilized, is used. Examples include DNA that can be identified by washing a filter at 65 ° C. using a 0.1 to 2 × SSC solution (1 × SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). Hybridization is performed by Molecular Cloning: A laboratory Mannual, 2 ^nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989. (hereinafter abbreviated as "Molecular Cloning Second Edition") and the like.
[0018]
Examples of DNA that hybridizes under stringent conditions include DNA having a certain degree of homology with the base sequence of DNA used as a probe, for example, 70% or more, preferably 80% or more, more preferably 90%. More preferably, DNA having homology of 93% or more, particularly preferably 95% or more, and most preferably 98% or more is mentioned.
[0019]
The above-mentioned “having the binding activity to TRAF3” means that it can bind to TRAF3 with the same or higher affinity as that of the protein having the amino acid sequence shown in SEQ ID NO: 1, and thereby intracellularly mediated by TRAF3 Says that it can participate in signal transduction.
[0020]
The method for obtaining the gene of the present invention is not particularly limited. Appropriate probes and primers are prepared based on the nucleotide sequence and amino acid sequence information described in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing in this specification, and cDNA libraries such as mice are screened using them. Thus, the gene of the present invention can be isolated.
The mouse-derived cDNA library is preferably prepared from cells, organs or tissues expressing the gene of the present invention. Specific examples of cells expressing the gene include WEHI231 cells, but are not limited thereto.
[0021]
DNA having the base sequence described in SEQ ID NO: 2 can also be obtained by PCR. PCR is performed using a mouse chromosomal DNA or cDNA library as a template and a pair of primers designed to amplify the nucleotide sequence set forth in SEQ ID NO: 2.
PCR reaction conditions can be set as appropriate. For example, one cycle of a reaction step consisting of 94 ° C. for 30 seconds (denaturation), 55 ° C. for 30 seconds to 1 minute (annealing), and 72 ° C. for 2 minutes (extension) For example, after 30 cycles, a condition of reacting at 72 ° C. for 7 minutes can be exemplified. The amplified DNA fragment can then be cloned into a suitable vector that can be amplified in a host such as E. coli.
[0022]
Procedures such as probe or primer preparation, cDNA library construction, cDNA library screening, and gene cloning described above are known to those skilled in the art. For example, Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, Supplement 1 to 38, John Wiley & Sons (1987-1997) (hereinafter abbreviated as Current Protocols in Molecular Biology) and the like.
[0023]
(B) a base sequence in which one to several bases are deleted, added or substituted in the base sequence described in SEQ ID NO: 2, and encodes a protein having binding activity to TRAF3. A gene (mutant gene) having a nucleotide sequence to be prepared can be prepared by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques, or mutagenesis. Specifically, a mutant DNA can be obtained by using a DNA having the base sequence described in SEQ ID NO: 2 and introducing a mutation into the DNA.
[0024]
For example, the DNA having the base sequence shown in SEQ ID NO: 2 can be contacted with a drug that becomes a mutagen, a method of irradiating with ultraviolet rays, a genetic engineering method, or the like. Site-directed mutagenesis, which is one of the genetic engineering methods, is useful because it can introduce a specific mutation at a specific position. Molecular cloning 2nd edition, Current Protocols in Molecular. It can be performed according to the method described in biology and the like.
[0025]
The base sequence that hybridizes under stringent conditions with the base sequence described in (c) SEQ ID NO: 2 described above in the present specification and that encodes a protein having a binding activity to TRAF3 is the above-described base sequence. As described above, it can be obtained by performing a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like under certain hybridization conditions.
[0026]
Next, the method for obtaining and producing the protein of the present invention will be described. The method for obtaining and producing the protein of the present invention is not particularly limited, and may be a naturally derived protein, a chemically synthesized protein, or a recombinant protein produced by a gene recombination technique. Recombinant proteins are preferred in that they can be produced in large quantities with relatively easy operations.
[0027]
When a naturally derived protein is obtained, it can be isolated from cells or tissues expressing the protein by appropriately combining methods for protein isolation and purification. When obtaining a chemically synthesized protein, for example, the protein of the present invention can be synthesized according to a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method). . In addition, various commercially available peptide synthesizers (for example, Kuwawa Trading (manufactured by Advanced Chem Tech, USA), Perkin Elmer Jabang (manufactured by Perkin-Elmer, USA), Pharmacia Biotech (manufactured by Pharmacia Biotech, Sweden), Aloka (USA) Protein Technology Instrument), Kurabo (US Synthecell-Vega), Nihon Perceptive Limited (US PerSeptive), Shimadzu Corporation, etc.) can be used to synthesize the protein of the present invention.
[0028]
In order to produce the protein of the present invention as a recombinant protein, a DNA having a base sequence encoding the protein (for example, the base sequence described in SEQ ID NO: 2) or a variant or homologue thereof is obtained and used as a suitable protein. The protein of the present invention can be produced by introducing it into an expression system. Production of expression vectors and transformants and production of recombinant proteins using them will be described later in this specification.
[0029]
A protein having an amino acid sequence in which one to several amino acids are deleted, substituted or inserted in the amino acid sequence shown in SEQ ID NO: 1, or an amino acid having 60% or more homology with the amino acid sequence shown in SEQ ID NO: 1 A protein having a sequence can be appropriately produced or obtained by those skilled in the art based on the information of the base sequence described in SEQ ID NO: 2 showing an example of the DNA sequence encoding the amino acid sequence described in SEQ ID NO: 1.
[0030]
For example, it can be isolated by screening a DNA homolog of the DNA from a mouse or an organism other than a mouse as a DNA probe having the nucleotide sequence set forth in SEQ ID NO: 2 or a part thereof under appropriate conditions. Alternatively, a corresponding cDNA sequence can be obtained from a homology search of a DNA sequence database and prepared by PCR or the like. A protein encoded by the homologous DNA can be produced by cloning the full-length DNA of this homologous DNA, then incorporating it into an expression vector and expressing it in a suitable host.
[0031]
(2) Vector having the gene of the present invention
The gene of the present invention can be incorporated into an appropriate vector and used as a recombinant vector. The type of vector may be an expression vector or a non-expression vector, and can be selected according to the purpose.
The cloning vector is preferably one that can autonomously replicate in Escherichia coli K12 strain, and any phage vector, plasmid vector, etc. can be used. Specifically, ZAP Express (Stratagies, 5, 58 (1992)) PBluescript II SK (+) (Nuclelc Acids Research, 17, 9494 (1989)), Lambda ZAP II (Stratagene), λgt10, λgt11 (DNA Cloning, A Practical Approach, 1, 49 (1985)), λTriplEx (Clontech), λExCell (Pharmacia), pT7T318U (Pharmacia), pcD2 (Mo1. Cen. Bio1, 3, 280 (1983)), pMW218 (Wako Pure Chemical Industries), pUC118 (Takara Shuzo) , PEG400 [J. Bac., 172, 2392 (1990)], pQE-30 (manufactured by QIAGEN), and the like.
[0032]
As the expression vector, a vector that can replicate autonomously in the host cell or can be integrated into the host cell chromosome is preferably used. As the expression vector, a vector containing a promoter at a position where the gene of the present invention can be expressed is used.
[0033]
When a bacterium is used as a host cell, the expression vector for expressing the gene of the present invention is capable of autonomous replication in the bacterium, and at the same time, a set comprising a promoter, a ribosome binding sequence, the above DNA and a transcription termination sequence. A replacement vector is preferred. A gene that controls the promoter may also be included.
[0034]
Examples of expression vectors for bacteria include pBTrP2, pBTac1, pBTac2 (all available from Belinger Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega) ), PQE-8 (QIAGEN), pQE-30 (QIAGEN), pKYP10 (JP 58-110600), pKYP200 (Agrc. Biol. Chem., 48, 669 (1984)), PLSA1 (Agrc) Blo1. Chem., 53, 277 (1989)], pGEL1 (Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)), pBluescrlptII SK +, pBluescriptII SK (-) (Stratagene), pTrS30 ( FERMBP-5407), pTrS32 (FERM BP-5408), pGEX (Pharmacia), pET-3 (Novagen), pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pUC18 (Gene, 33, 103 (1985)], pUC19 (Gene, 33, 103 (1985)), pSTV28 (Takara Shuzo), pSTV29 (Takara Shuzo), pUC118 (Takara Shuzo), pQE-30 (QIAGEN), etc. Is mentioned. Examples of promoters for bacteria include trp promoter (P trp), lac promoter (P lac), P _L Promoter, P _R Promoter, P _SE Examples include promoters derived from Escherichia coli and phages, SP01 promoter, SP02 promoter, penP promoter, and the like.
[0035]
Examples of expression vectors for yeast include YEp13 (ATCC37115), YEp24 (ATCC37051), Ycp5O (ATCC37419), pHS19, pHS15 and the like. Examples of the promoter for yeast include promoters such as PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, and CUP1 promoter.
[0036]
Examples of expression vectors for animal cells include, for example, pcDNAI, pcDM8 (commercially available from Funakoshi), pAGE107 (JP 3-22979; Cytotechnology, 3, 133, (1990)), pAS3-3 (JP 2-27075), Examples include pCDM8 (Nature, 329, 840, (1987)), pcDNAI / AmP (Invitrogen), pREP4 (Invitrogen), pAGE103 (J. Blochem., 101, 1307 (1987)), pAGE210, etc. Can do. Examples of promoters for animal cells include cytomegalovirus (human CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, SRα promoter, etc. Can do.
[0037]
As an expression vector for plant cells, for example, pIG121-Hm [Plant Cell Report, 15 , 809-814 (1995)], pBI121 (EMBO J. 6 , 3901-3907 (1987)] and the like. As a promoter for plant cells, for example, cauliflower mosaic virus 35S promoter [Mol. Gen. Genet (1990) 220 , 389-392], Rubros bisphosphate carboxylase small subunit promoter and the like.
[0038]
(3) Transformant having the gene of the present invention
A transformant having a gene encoding a protein having a binding activity to TRAF3 of the present invention can be prepared by introducing the above-described recombinant vector (preferably an expression vector) into a host.
Specific examples of bacterial host cells include Escherichia, Corynebacterium, Brevibacterium, Bacillus, Microbacterium, Serratia, Pseudomonas, Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter, Chromatium Examples include genus, Erwinia genus, Methylobacterium genus, Phormidium genus, Rhodobacter genus, Rhodopseudomonas genus, Rhodospiri11um genus, Scenedesmun genus, Streptomyces genus, Synnecoccus genus, Zymomonas genus and the like. Examples of the method for introducing a recombinant vector into a bacterial host include a method using calcium ions and a protoplast method.
[0039]
Specific examples of yeast hosts include Saccharomyces cerevisae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pu11uls, Trichosporon pu11uls, Trichosporon pu11uls Schwanniomyces a11uvius). Examples of the method for introducing a recombinant vector into a yeast host include an electroporation method, a spheroblast method, and a lithium acetate method.
[0040]
Examples of animal cell hosts include Namalva cells, COS1 cells, COS7 cells, CHO cells and the like. As a method for introducing a recombinant vector into animal cells, for example, an electroporation method, a calcium phosphate method, a lipofection method, or the like can be used.
[0041]
When insect cells are used as the host, the recombinant gene transfer vector and baculovirus are co-introduced into the insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected with the insect cells. And protein can be expressed (for example, as described in Baculovirus Expression Vectors, A Laboratory Manua1; and Current Protocols in Molecular Biology, Bio / Technology, 6, 47 (1988), etc.).
[0042]
As the baculovirus, for example, Autographa californica nuclear polyhedrosis virus, which is a virus that infects Coleoptera insects, can be used.
Insect cells include Sf9, Sf21 (Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company), New York, which is an ovarian cell of Spodoptera frugiperda (1992)], HiFive (manufactured by Invitrogen), which is an ovarian cell of Trichoplusia ni, can be used.
Examples of the method for co-introducing a recombinant gene introduction vector into insect cells and the baculovirus for preparing a recombinant virus include a calcium phosphate method and a lipofection method.
[0043]
(4) Production of recombinant protein using the transformant of the present invention
In the present invention, the transformant having the gene of the present invention produced as described above is cultured, the protein of the present invention is produced and accumulated in the culture, and the protein of the present invention is collected from the culture. The recombinant protein can be isolated by
When the transformant of the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the medium for culturing these microorganisms contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the microorganism. As long as the medium can efficiently culture the transformant, either a natural medium or a synthetic medium may be used. The culture is preferably performed under aerobic conditions such as shaking culture or deep aeration and agitation culture, the culture temperature is usually 15 to 40 ° C., and the culture time is usually 16 hours to 7 days. During the cultivation, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like. Moreover, you may add antibiotics, such as an ampicillin and a tetracycline, to a culture medium as needed during culture | cultivation.
[0044]
As a medium for culturing a transformant obtained using an animal cell as a host cell, a commonly used RPM11640 medium (The Journal of the American Medical Association, 199, 519 (1967)), Eagle's MEM medium (Science, 122, 501 (1952)], DMEM medium [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] or a medium obtained by adding fetal calf serum or the like to these mediums Etc. are used. Culture is usually pH 6-8, 30-40 ° C, 5% C0 ₂ Perform for 1-7 days under conditions such as presence. Moreover, you may add antibiotics, such as kanamycin and penicillin, to a culture medium as needed during culture | cultivation.
[0045]
As a medium for culturing a transformant obtained using plant cells as host cells, a medium usually used according to the plant species, such as an MS medium or an R2P medium, is used. The culture is usually performed for 1 to 21 days under conditions of pH 6 to 8, 15 to 35 ° C, and the like. Moreover, you may add antibiotics, such as kanamycin and a hygromycin, to a culture medium as needed during culture | cultivation.
[0046]
In order to isolate and purify the protein of the present invention from the culture of the transformant, ordinary protein isolation and purification methods may be used.
For example, when the protein of the present invention is expressed in a dissolved state in a cell, the cell is recovered by centrifugation after culturing, suspended in an aqueous buffer solution, an ultrasonic crusher, a French press, a Manton Gaurin homogenizer. Then, the cells are disrupted with dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary protein isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using resin such as diethylaminoethyl (DEAE) Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), cation exchange chromatography using resin such as S-Sepharose FF (Pharmacia) Hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, electrophoresis methods such as isoelectric focusing etc. Alternatively, it can be used in combination to obtain a purified preparation.
[0047]
In addition, when the protein is expressed by forming an insoluble substance in the cell, the protein is recovered by a conventional method from the precipitate fraction obtained by crushing the cell and then performing centrifugation in the same manner. Thereafter, the insoluble material of the protein is solubilized with a protein denaturant. The solubilized solution is diluted or dialyzed into a solution that does not contain a protein denaturant or the protein denaturant has such a concentration that the protein is not denatured. A purified sample can be obtained by the same isolation and purification method.
[0048]
(5) An antibody that recognizes the protein of the present invention
By identifying a TRAF3-binding protein according to the present invention, an antibody that recognizes the protein can be prepared. Such antibody production is useful in studies such as elucidation of the dynamics of the protein or TRAF3. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, and can be produced by a conventional method.
[0049]
For example, a polyclonal antibody that recognizes the protein of the present invention is obtained by immunizing a mammal with the protein of the present invention as an antigen, collecting blood from the mammal, and separating and purifying the antibody from the collected blood. be able to. For example, mammals such as mice, hamsters, guinea pigs, chickens, rats, rabbits, dogs, goats, sheep and cows can be immunized. Methods of immunization are known to those skilled in the art and can be performed, for example, by administering the antigen one or more times. Antigen administration may be performed 2-3 times at intervals of 7 to 30 days, for example. The dose can be about 0.05 to 2 mg of antigen per dose, for example. The administration route is not particularly limited, and subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, and the like can be selected as appropriate, but by intravenous, intraperitoneal or subcutaneous injection. Administration is preferred. The antigen can be used by dissolving in an appropriate buffer, for example, a complete buffer containing a commonly used adjuvant such as Freund's complete adjuvant or aluminum hydroxide. It may not be used.
[0050]
After the immunized mammal is bred for a certain period, the serum of the mammal is sampled and the antibody titer is measured. When the antibody titer increases, booster immunization is performed using, for example, 100 μg to 1000 μg of antigen. Blood is collected from a mammal immunized 1-2 months after the last dose and the blood is collected, for example, by centrifugation, precipitation with ammonium sulfate or polyethylene glycol, gel filtration chromatography, ion exchange chromatography, affinity A polyclonal antibody that recognizes the protein of the present invention can be obtained as a polyclonal antiserum by separation and purification by a conventional method such as chromatography. Serum may inactivate the complement system by, for example, treatment at 56 ° C. for 30 minutes.
[0051]
The globulin type of the monoclonal antibody that recognizes the protein of the present invention is not particularly limited, and examples thereof include IgG, IgM, IgA, IgE, and IgD. The cell line that produces the monoclonal antibody of the present invention is not particularly limited. For example, it can be obtained as a hybridoma by cell fusion between an antibody-producing cell and a myeloma cell line. The hybridoma producing the monoclonal antibody of the present invention can be obtained by the following cell fusion method.
[0052]
As antibody-producing cells, spleen cells, lymph node cells, B lymphocytes and the like from immunized animals are used. As the antigen, the protein of the present invention or a partial peptide thereof is used. Mice, rats, and the like can be used as immunized animals, and antigens are administered to these animals by conventional methods. For example, by administering a suspension or emulsion of an adjuvant such as complete Freund's adjuvant or incomplete Freund's adjuvant and the protein of the present invention as an antigen several times into the animal's vein, subcutaneous, intradermal, intraperitoneal, etc. Immunize. For example, spleen cells are obtained as antibody-producing cells from the immunized animal and fused with myeloma cells by a known method (G. Kohler et al., Nature, 256 495 (1975)) to produce a hybridoma. Can do.
[0053]
Examples of myeloma cell strains used for cell fusion include P3X63Ag8, P3U1 strain, Sp2 / 0 strain and the like in mice. When cell fusion is performed, a fusion promoter such as polyethylene glycol or Sendai virus is used, and hypoxanthine / aminopterin / thymidine (HAT) medium is used in accordance with a conventional method for selection of hybridomas after cell fusion. Hybridomas obtained by cell fusion are cloned by limiting dilution or the like. Furthermore, if necessary, a cell line producing a monoclonal antibody that specifically recognizes the protein of the present invention can be obtained by screening by an enzyme immunoassay using the protein of the present invention.
[0054]
In order to produce the target monoclonal antibody from the hybridoma thus obtained, the hybridoma is cultured by a normal cell culture method or ascites formation method, and the monoclonal antibody is purified from the culture supernatant or ascites. . Purification of the monoclonal antibody from the culture supernatant or ascites can be performed by a conventional method. For example, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography and the like can be used in appropriate combination.
[0055]
Examples of the method for immunoassay of the TRAF3-binding protein of the present invention using the monoclonal antibody of the present invention include enzyme immunoassay, radioimmunoassay, fluorescent immunoassay, luminescence immunoassay and the like.
[0056]
The above-mentioned antibody fragments are also within the scope of the present invention. Examples of the antibody fragment include F (ab ′) 2 fragment, Fab ′ fragment and the like.
Furthermore, labeled antibodies of the above-mentioned antibodies are also within the scope of the present invention. That is, the antibody of the present invention produced as described above can be used after being labeled. The types of antibody labeling and labeling methods are known to those skilled in the art. For example, enzyme labels such as horseradish peroxidase or alkaline phosphatase, fluorescent labels such as FITC (fluorescein isothiocyanate) or TRITC (tetramethylrhodamine B isothiocyanate), labels with colorants such as colloidal metals and colored latex, biotin and the like Affinity label, or ¹²⁵ An isotope label such as I can be mentioned. Analysis such as an enzyme antibody method, an immunohistochemical staining method, an immunoblot method, a direct fluorescent antibody method or an indirect fluorescent antibody method using the labeled antibody of the present invention can be performed by methods well known to those skilled in the art.
[0057]
(6) Screening method using the protein of the present invention
The present invention further relates to a method for screening a substance that promotes or suppresses the function of the protein or antibody using the TRAF3-binding protein of the present invention or an antibody thereof. Preferably, a substance that promotes or suppresses intracellular signal transduction via TRAF3 is selected by the screening method. Such a screening system can be constructed as follows, for example.
[0058]
An expression vector having the gene of the present invention is constructed, and this expression vector is introduced into a suitable host for expressing TRAF3. In the resulting transformant, both TRAF3 and the protein of the present invention are expressed, and intracellular signal transduction proceeds in the presence of an appropriate ligand through the interaction between the two.
A test substance can be added to the screening system constructed as described above. If progression of intracellular signal transduction is inhibited when a test substance is added, the test substance is a substance that inhibits the interaction between the protein of the present invention and TRAF3 (ie, the function of the TRAF3-binding protein of the present invention). Can be selected as a candidate substance. On the other hand, if intracellular signaling is activated when a test substance is added, the test substance becomes a substance that activates the interaction between the protein of the present invention and TRAF3 (of the TRAF3-binding protein of the present invention). It can be selected as a candidate substance for a substance that promotes functions).
[0059]
Expression vectors and host cells that can be used in the above screening system can be appropriately selected by those skilled in the art, and those described above in this specification may be used.
Any substance can be used as the test substance, and the type thereof is not particularly limited. Specific examples of the test substance may be a low molecular weight synthetic compound, a natural product extract, a compound library, a phage display library, or a combinatorial library. The construction of a compound library is known to those skilled in the art, and a commercially available compound library can also be used.
[0060]
(7) Medicine of the present invention
The TNF ligand family is known as one of the most pleiotropic cytokines that induce numerous cellular responses including cytotoxicity, antiviral activity, immunomodulatory activity and transcriptional control of several genes. Cellular responses to TNF ligands are not only related to normal physiological responses but also to enhanced apoptosis or inhibition of apoptosis. Abnormalities in apoptotic regulation are numerous etiologies. Diseases associated with cell growth or inhibition of apoptosis include cancer, autoimmune disease, viral infection, inflammation, graft-versus-host disease, acute graft rejection, and chronic graft rejection. Diseases associated with augmentation include AIDS, neurodegenerative diseases, myelodysplastic syndromes, ischemic damage, toxin-induced liver disease, septic shock, cachexia and anorexia.
[0061]
Although the TRAF3-binding protein of the present invention does not belong to the TNF receptor superfamily, it binds to TRAF3, so that chronic and acute inflammation, arthritis, sepsis, autoimmune diseases involving TNF receptor / ligand superfamily (for example, Systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, psoriasis), transplant rejection, graft-versus-host disease, infection, stroke, ischemia, acute respiratory disease syndrome, restenosis, brain injury, AIDS, bone It is believed to be involved in disease, cancer (eg, lymphoproliferative disease), atherosclerosis and Alzheimer's disease and other diseases.
[0062]
Therefore, the substance that promotes or suppresses the function of the TRAF3-binding protein of the present invention obtained by the screening method described above is useful as a medicament for the treatment and / or prevention of such diseases.
That is, the present invention also relates to a medicament containing, as an active ingredient, a substance that promotes or suppresses the function of the TRAF3-binding protein of the present invention obtained by the screening method described above. The medicament of the present invention can be used, for example, for the prevention or treatment of diseases associated with abnormal intracellular signal transduction via TRAF3.
[0063]
The pharmaceutical composition of the present invention generally contains a substance that promotes or suppresses the function of the TRAF3-binding protein of the present invention as an active ingredient, and further contains a pharmaceutical additive (eg, carrier, excipient, etc.). Provided in the form of a thing.
The route of administration of the medicament of the present invention is not particularly limited, and oral administration or parenteral administration (for example, intramuscular administration, intravenous administration, subcutaneous administration, intraperitoneal administration, mucosal administration to the nasal cavity, inhalation administration, etc.) Either may be used.
[0064]
The pharmaceutical form of the present invention is not particularly limited, and examples of the preparation for oral administration include tablets, capsules, fine granules, powders, granules, liquids, syrups and the like. Examples of the preparation include injections, drops, suppositories, inhalants, transmucosal absorbents, transdermal absorbents, nasal drops, ear drops and the like.
A person skilled in the art can appropriately select the form of the medicament of the present invention, the additive for the preparation to be used, the production method of the preparation, etc.
The dosage of the medicament of the present invention can be appropriately selected in consideration of the patient's sex, age or weight, severity of symptoms, administration purpose such as prevention or treatment, or the presence or absence of other complications. . The dose is generally 0.001 μg / kg body weight / day to 1000 μg / kg body weight / day, preferably 0.001 μg / kg body weight / day to 100 μg / kg body weight / day.
[0065]
(8) A method for diagnosing a disease by measuring the level of the protein of the present invention
The present invention further relates to a method for diagnosing a disease associated with abnormality in intracellular signal transduction via TRAF3, comprising measuring the level of the protein of the present invention in a biological component.
As described above, the protein of the present invention may be involved in various diseases related to apoptosis enhancement or apoptosis inhibition. Therefore, by measuring the activity or amount of the protein of the present invention in a living body, it becomes possible to diagnose a disease associated with abnormal intracellular signal transduction via TRAF3.
[0066]
The level of the protein of the present invention can be measured according to a conventional method known to those skilled in the art. For example, the level of the protein of the present invention may be determined by an immunological method using an antibody that recognizes the protein of the present invention. It may be performed by measuring the amount of mRNA encoding.
The present invention will be described more specifically with reference to the following examples, but the present invention is not particularly limited by the following examples.
[0067]
【Example】
(A) Materials and methods
A-1. Screening for protein-protein interactions using yeast
DNA encoding full-length TRAF3 was used as a bait in the CytoTrap® two-hybrid system (Stratagene, La Jolla. CA). Yeast cdc25H strain (temperature-sensitive mutant of cdc25 gene) used in this system has the following genotype: MATα ura3, lys2, leu2, trp1, his200, ade101, cdc25-2, GAL +. A library of hybrid proteins of myristylation signal sequence and cDNA fragments from WEHI231 cells was constructed in plasmid pMry. Screening was performed without modification as recommended by the manufacturer.
[0068]
Briefly, the yeast strain was grown at 25 ° C. in YPAD medium or Burkholder's minimal medium (BMM) supplemented with appropriate additives [Sato, T., et al. (1995) FEBS Lett. 358, 113. -118], and transformed by the lithium acetate method. The yeast transformants were grown on BMM / galactose plates at 25 ° C. for 2 days, followed by growth at 37 ° C. Colonies that grew at 37 ° C. that were thought to express myristylated proteins interacting with TRAF3 were picked and tested for growth on BMM / glucose and BMM / galactose plates at 37 ° C. Library plasmids (pMry with integrated cDNA) were isolated from clones showing galactose-dependent growth at 37 ° C. (expression of cDNA incorporated into pMyr is dependent on galactose) and plasmid pSos / TRAF3 or pSos / Collagenase was used to retransform into cdc25H cells. The sequence of the plasmid that suppresses the cdc25H phenotype only in the presence of pSos / TRAF3 was determined. DNA sequencing was performed with an Applied Biosystems automated DNA sequencer, ABI PRISM 310 gene analyzer (Foster, CA).
[0069]
A-2. Northern blot hybridization
10 μg of total RNA prepared from mouse tissue (C3H / Hen Crj) by the acidic guanidine phenol chloroform method was separated on a 1% agarose gel containing formaldehyde and Hybond®-N + (Amersham Pharmacia, Uppsala, Sweden) and immobilized by UV crosslinking (Stratagene). The hybridization probe is [α- ³² It was prepared from a gel-purified DNA fragment radiolabeled by the random prime method with P] dCTP (Amersham Pharmacia). Blots were hybridized at 42 ° C. in a solution containing 5 × SSC, 5 × Denhardt's solution, 0.1% SDS, 50% deionized formamide, and 100 μg / ml salmon sperm DNA. After hybridization, the blot was washed with 2 × SSC and 0.1% SDS at room temperature followed by 0.1 × SSC and 0.1% SDS at 65 ° C. The blot was dried and analyzed using BAS 2500 (Fuji Ltd., Tokyo. Japan).
[0070]
A-3. Expression plasmid construction and transfection
To generate a T3BP expression construct, the appropriate region of mouse T3BP was amplified by PCR and subcloned into pcDNA 3.1 (−) myc his version B (Invitrogen, Carlsbad, Calif.) Using EcoRI and XhoI restriction sites. PcDNA / T3BP was obtained. T3BP was expressed as a MYC fusion protein labeled at the C-terminus.
For construction of an EGFP-tagged T3BP expression plasmid, the full-length coding region was amplified by PCR and ligated to pEGFP-N2 (Clontech, California, Calif.) Using EcoRI and XhoI restriction sites to obtain pEGFP / T3BP. .
Mammalian expression vectors encoding FLAG-tagged mice TRAF2 and TRAF3 were generated by a PCR-based method.
All expression constructs were sequenced using an ABI PRISM 310 gene analyzer.
[0071]
A-4. Cell culture and transfection
Human fetal kidney-derived 293 cells are in Eagle's minimal essential medium containing 10% calf serum (HyClone, Logan, UT), 100 U / ml penicillin and 100 μg / ml streptomycin (GIBCO-BRL, Grand Island, NY). Maintained. WEHI231 cells are 10% calf serum (HyClone), 5.5 × 10 ^Five Maintained in RPMI-1640 medium containing M 2-mercaptoethanol, 100 U / ml penicillin and 100 μg / ml streptomycin (GIBCO-BRL). 293 cells in a 10 cm culture dish ⁶ Inoculated at a concentration of pcs / dish and cultured for 2-3 days. The cells were subsequently transfected using a standard calcium phosphate coprecipitation method using a commercial solution (Eppendorf-5 Prime, Inc., Boulder, CO).
[0072]
A-5. GST pull-down assay
For GST pull-down assay, GST fusion protein was expressed in E. coli (BLR) using pGEX vector (Amersham Pharmacia) after IPTG induction and purified on glutathione beads (Amersham Pharmacia). FLAG-tagged TRAF2, TRAF3, TRAF5, TRAF6 were expressed in 293 cells. Cell lysates were prepared from transfected cells by using E1A buffer (50 mM HEPES [pH 7.6], 250 mM NaCl, 0.1% NP-40 and 5 mM EDTA) as a lysing agent. The cell lysate was cleared and incubated with 2 μg of GST or GST fusion protein immobilized on glutathione beads. The glutathione beads were thoroughly washed with E1A buffer. Samples were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane. After blocking, the membrane was treated with anti-FLAG M2 antibody and immunized with horseradish peroxidase-conjugated secondary antibody (Bio-Rad) diluted 5,000-fold according to enhanced chemiluminescence protocol (ECL, Amersham Pharmacia). Reactive protein was detected. TRAF3 in vitro transcription / translation and 35-S methionine labeling were performed according to the T7 link transcription / translation system (Amersham Pharmacia), and detection was performed by fluorography.
[0073]
A-6. Immunoprecipitation assay
The cells were detached from the plate in PBS containing 5 mM EDTA and washed 3 times with the same buffer. The cells were lysed in 1 ml E1A buffer at 4 ° C. for 10 minutes. The cell lysate was centrifuged and the supernatant was used as a cell extract. The cell extract was incubated with anti-FLAG M2 antibody, anti-MYC 9E10 antibody or control antibody for 2 hours at 4 ° C., followed by treatment with protein G Sepharose FF (Amersham Pharmacia). The sepharose beads were thoroughly washed with E1A buffer. Samples were subjected to 10% SDS-polyacrylamide gel electrophoresis, and immunoreactive protein was detected using anti-FLAG M2 antibody or anti-MYC 9E10 antibody as described above.
[0074]
A-7. Fluorescence microscopy
HEK293 cells cultured on cover slips were transfected with various constructs. After 24 hours, cells were fixed for 10 minutes at room temperature in PBS containing 3.7% formalin. Cells were washed 3 times with PBS and treated with 0.2% Triton X-100 in PBS for 5 minutes at room temperature. After blocking with skim milk, cells were incubated with various primary antibodies such as anti-FLAG M2 monoclonal antibody or anti-MYC 9E10 antibody (Sigma) followed by Cy5-conjugated secondary antibody (Jackson ImmunoReseach Laboratories, Inc.). . Cells were washed 3 times with PBS and placed on a glass slide.
Fluorescence was visualized using a Carl Zeiss LSM510 confocal laser scanning microscope (Oberkochen, Germany).
[0075]
A-8. Quantification of F-actin
F-actin content was measured as previously reported [Mizuno., Et al. (1998) J. Pharm. Pharmacol. 50, 645-648]. Briefly, 293 cells cultured on 12-well plates were transiently transfected with the indicated amount of T3BP expression plasmid by the calcium phosphate method. Transfected total DNA (2 μg) was kept constant by loading onto an empty vector. After 24 hours, the transfected cells were fixed with 3.7% formalin in PBS for 10 minutes at 37 ° C. After permeation with 0.2% Triton X-100 in PBS for 10 minutes at room temperature, cells were stained with 10 U / ml rhodamine-phalloidin (MolecurProbe) in PBS for 1 hour at room temperature. After extensive washing with PBS, the bound FITC-phalloidin was extracted for 1 hour on ice with methanol. The fluorescence intensity was measured using a spectrometer F-3010 (Hitachi Ltd., Tokyo, Japan) at an excitation wavelength of 488 nm and an emission wavelength of 510 nm. The results were expressed as relative fluorescence intensity calculated from the ratio of fluorescence intensity of control transfected cells.
[0076]
(B) Result
B-1. Isolation of cDNA clones encoding T3BP
To identify proteins that bind directly to TRAF3, screening based on protein-protein interactions in yeast was performed. The S. cerevisiae cdc25H strain contains a temperature-sensitive point mutation in the cdc25 gene, so that host growth is inhibited at 37 ° C., but is normal at the permissive temperature (25 ° C.). The cdc25 gene encodes a guanyl nucleotide conversion factor that leads to cell growth by binding and activating RAS. The system used in this example utilizes the ability of human-derived Sos to complement the cdc25 defect and activate the yeast RAS signaling pathway; an hSos fusion protein (hSos-TRAF3) is expressed and When localized to the plasma membrane through protein-protein interactions, the cdc25H strain also grows at 37 ° C.
[0077]
Yeast containing plasmid pSos / TRAF3 was transformed with a WEHI231 cell cDNA expression library that fused to the myristylation signal and localized to the plasma membrane. About 2 × 10 ^Five Transformants were grown on BMM / galactose plates at 25 ° C. for 2 days and then transferred to a 37 ° C. incubator. Colonies that grew at 37 ° C were picked and tested for growth on BMM / glucose and BMM / galactose plates at 37 ° C. Library plasmids were isolated from clones showing galactose-dependent growth at 37 ° C. and retransformed into cdc25H cells using pSos / TRAF3 or pSos / collagenase as a negative control, and cdc25H expression only in the presence of pSos / TRAF3 Nine cDNAs that suppress the type were obtained. One of these, clone 3, encoded a portion of the novel protein.
[0078]
In order to isolate the full-length cDNA inserted in clone 3, a pMry cDNA library derived from WEHI231 cells was screened using a partial cDNA as a probe to obtain a positive clone. DNA sequencing of positive clones revealed the presence of an open reading frame predicted to encode a 175 amino acid protein with an estimated molecular weight of 18,846 daltons (FIG. 1A, SEQ ID NO: 1 and SEQ ID NO: 2) ). This new protein is referred to as T3BP.
[0079]
FIG. 1A shows the amino acid sequence deduced from the nucleotide sequence of full-length T3BP cDNA. The sequence encoded by clone 3 is underlined. Shaded boxes indicate putative transmembrane domains.
[0080]
A database search utilizing the BLAST and FASTA programs revealed that the T3BP cDNA contains the sequence reported as a proviral flanking sequence acquired by somatic cells in mouse leukemia and lymphoma (AF193161). In addition, human B cell maturation factor (BCMA) had significant sequence similarity to T3BP, 28% identity and 42% similarity to T3BP. Hydropathy plots generated using the Tmpred program suggested that T3BP has a putative transmembrane region (amino acids 77-97). PROSITE analysis identified an N-glycosylation site (amino acids 23-26) and an N-myristoylation site (amino acids 81-86). By PSORT analysis, it was speculated that this protein has a type I membrane protein tertiary structure and is located in the plasma membrane.
[0081]
The expression pattern of T3BP was examined by Northern blot hybridization using 6 tissues and WEHI231 cells. The results are shown in FIG. 1B. In FIG. 1B, the blot was hybridized with a 200b T3BP cDNA probe. The position of ribosomal RNA is shown on the left.
As shown in FIG. 1B, the T3BP gene showed about 2 kb of mRNA in WEHI231 cells. However, T3BP mRNA was not detected in other mouse tissues tested.
[0082]
B-2. T3BP interacts specifically with TRAF3
To confirm the interaction between TRAF3 and T3BP observed in the yeast assay, clone 3 and clone 10 were expressed as GST fusion proteins and binding to TRAF2 or TRAF3 was tested in a GST pull-down assay. Cell lysates of 293 cells overexpressing FLAG-tagged TRAF2 or TRAF3 are incubated with GST-clone 3, GST-clone 10 or GST alone, and TRAF bound to the GST fusion protein is precipitated with glutathione-sepharose. It was. The TRAF was separated by SDS-PAGE and detected by immunoblotting using an anti-FLAG M2 antibody. The expression level of FLAG-tagged TRAF in the cell lysate was also monitored by immunoblotting using anti-FLAG M2 antibody. The result is shown in FIG. 2A.
[0083]
As shown in FIG. 2A, GST-clone 3 binds preferentially to TRAF3 over TRAF2. On the other hand, GST-clone 10 interacts similarly to TRAF2 and TRAF3. Sequence analysis of clone 10 revealed that this clone contained a portion of the mnb protein kinase homolog mp86 (Dyrk; MMU58497, amino acids 284-763) containing the consensus sequence for the TRAF binding domain (PXQXT / S, amino acids 583-587). It turns out to code. However, clone 3 does not contain this sequence.
Further, in order to compare the binding specificity between clone 3 and other TRAF families (TRAF5, 6), a GST pull-down assay was performed in the same manner. As shown in FIG. 2B, GST-T3BP (clone 3) Compared to TRAF, it was most strongly bound to TRAF3.
[0084]
The interaction between TRAF3 and T3BP was analyzed by a co-immunoprecipitation assay in mammalian cells. Results are shown in FIG. 2C. First, 293 cells were transiently transfected with expression vectors encoding MYC-tagged T3BP (full length T3BP containing a C-terminal MYC epitope tag) and FLAG-tagged TRAF3. Cell extracts were prepared and immunoprecipitated (IP) using anti-MYC 9E10 antibody, anti-FLAG M2 antibody (anti-FLAG monoclonal antibody), or control mouse IgG. Co-precipitated FLAG-TRAF3 or MYC-T3BP was detected by immunoblotting analysis using anti-FLAG M2 antibody (upper part of FIG. 2C) or anti-MYC 9E10 antibody (lower part of FIG. 2C), respectively. The expression levels of T3BP and TRAF3 in the cell lysate were also measured by immunoblotting using anti-MYC 9E10 antibody or anti-FLAG M2 antibody, respectively. FIG. 2C shows the positions of T3BP and TRAF3. The positions of Ig H and L chains are also shown.
[0085]
As shown in the lower figure of FIG. 2C, the anti-MYC antibody specifically recognized multiple bands (35-50 kDa) larger than those deduced from the amino acid sequence in transfection cell lysates with MYC-tagged T3BP expression plasmid, It was not recognized in the case of the control antibody. T3BP precipitated by FLAG-labeled TRAF3 was detected at a position of about 40 kDa (lower figure), and conversely, TRAF3 precipitated by MYC-labeled T3BP was detected at a position of about 64 kDa (upper figure).
[0086]
B-3. Mapping of T3BP binding domain in TRAF3
In order to determine the region of TRAF3 involved in binding to T3BP, various truncation mutants of FLAG-tagged TRAF3 were expressed in 293 cells and GST pull-down assays were performed. First, 293 cells were transiently transfected with a FLAG-tagged TRAF3 truncation mutant. Cell extracts were prepared and incubated with GST-clone 3 or GST alone. TRAF bound to the GST fusion protein was precipitated with glutathione-Sepharose, separated by SDS-PAGE, and detected by immunoblotting using an anti-FLAG M2 antibody. The expression level of the FLAG-tagged TRAF3 truncation mutant in the cell lysate was monitored by immunoblotting using anti-FLAG M2 antibody. The results are shown in FIG. 3A. As can be seen from FIG. 3A, it was found that the TRAF domain of TRAF3 is required for interaction with T3BP.
To confirm this, TRAF3 truncation mutants fused to hSos were generated and tested for interaction with T3BP in a protein-protein interaction assay in yeast. Specifically, yeast cells cdc25H were transfected with the indicated construct and assayed for proliferation in the same manner as described above. Colonies that grow above the permissible temperature are positive, and colonies that cannot grow above the permissive temperature are negative (FIG. 3B). This assay suggested that the TRAFC domain is important for T3BP binding. FIG. 3C shows an outline of the primary structure of mouse TRAF3.
[0087]
B-4. Mapping of TRAF3 binding domain in T3BP
To determine the region of T3BP involved in binding to TRAF3, various truncation mutants of T3BP were expressed in E. coli as GST fusion proteins. , In vitro transcription / translation and incubation with 35S-methionine labeled TRAF3. T3BP bound to the GST fusion protein was precipitated with glutathione sepharose, separated by SDS-PAGE, and detected by fluorography. As shown in FIG. 4A, it was found that the C-terminal portion containing the 151st and subsequent amino acids of T3BP is necessary for the interaction with TRAF3. Further, when FLAG-tagged TRAF3 was expressed in 293 cells and GST pull-down assay of 293 cell lysate with GST and GST-fused T3BP truncation mutant was performed, binding detected with anti-FRAG M2 antibody as shown in FIG. 4B The pattern was completely consistent with the binding test with the in vitro transcription / translation product of FIG. 4A. FIG. 4C shows the primary structure of T3BP and its TRAF3 binding region (TM is the transmembrane region).
[0088]
B-5. Cell distribution of T3BP
To examine the function of T3BP, its organelle localization was examined by fluorescence microscopy. 293 cells cultured on coverslips were transiently transfected with pEGFP-N2 (control plasmid; left side) or pEGFP / T3BP (EGFP-tagged T3BP expression plasmid; right side). After 24 hours the cells were fixed in 3.7% formalin. The EGFP fluorescence was then imaged using a confocal laser scanning microscope. The result is shown in FIG. 5A.
[0089]
As shown in FIG. 5A, morphological changes were induced by the expression of EGFP-tagged T3BP. In EGFP-labeled T3BP expressing cells, the cell shape became adhesive and many spikes developed from the cell surface. Most fluorescence of EGFP fusion T3BP was observed on the cell surface and spikes. This observation is consistent with the prediction by PSORT.
[0090]
Next, the effect of T3BP expression on TRAF3 cell distribution was tested. 293 cells cultured on cover slips were transformed into pEGFP-N2 and pCR / FLAG-TRAF3 (FIG. 5B-a, b, c), or pEGFP / T3BP and pCR / FLAG-TRAF3 (FIG. 5B-d, e, f). Was transfected with. Two days later, cells were fixed in 3.7% formalin and treated with 0.2% Triton X-100. After blocking, indirect immunofluorescence analysis was performed. Anti-FLAG M2 antibody was used as the primary antibody followed by Cy5-conjugated secondary antibody treatment. Fluorescence from EGFP and Cy5 was imaged using a confocal laser scanning microscope. Top; fluorescent image of EGFP, middle; fluorescent image of Cy5-conjugated secondary antibody, bottom; merged image. The bar is 50 μm.
[0091]
As can be seen from FIG. 5B-b, in cells co-expressing EGFP and FLAG-tagged TRAF3, FLAG-tagged TRAF3 was visualized around the nucleus (FIG. 5B-b). In cells co-expressing EGFP-tagged T3BP and FLAG-tagged TRAF3, FLAG-tagged TRAF3 was observed on the cell surface and spikes where EGFP-tagged T3BP had been localized (FIG. 5B-e).
[0092]
B-6. Effect of T3BP on cellular F-actin content
To examine the effect of T3BP on cellular F-actin, the cellular content of F-actin was measured in cells transfected with pcDNA / T3BP. 293 cells cultured on 12-well plates were transfected with the indicated amount of pcDNA / T3BP. After 24 hours, Factin levels were measured using FITC-phalloidin in the same manner as described above.
The results are shown in FIG. 6A. As can be seen from FIG. 6A, the cellular F-actin content increased in a concentration-dependent manner by T3BP.
[0093]
In order to confirm that the cellular F-actin content is increased by T3BP, the cell distribution of F-actin in cells expressing MYC-labeled T3BP was observed (FIG. 6B). Specifically, 293 cells cultured on coverslips were transfected with pEGFP-N2 and pcDNA (FIG. 6B-a), or pEGFP-N2 and pcDNA / MYC-T3BP (FIG. 6B-b). After 24 hours, cells were fixed in 3.7% formalin and treated with 0.2% Triton X-100. After blocking, the cells were stained with rhodamine-phalloidin. The fluorescence from EGFP (green) and the fluorescence from rhodamine (red) were imaged using a confocal laser scanning microscope. The bar is 50 μm.
As can be seen from FIGS. 6B-b, MYC-tagged T3BP expression caused a change in cell shape, and spikes induced by T3BP were stained with rhodamine-phalloidin.
[0094]
(C) Conclusion
In this example, a novel TRAF3-binding protein T3BP was identified by screening based on protein-protein interactions in yeast. The binding between T3BP and TRAF3 was confirmed by GST pull-down assay and co-immunoprecipitation assay (FIG. 2C). Furthermore, T3BP and TRAF3 were co-localized in cells that co-express both proteins (FIG. 5B). T3BP interacted preferentially with TRAF3 within the TRAF family. A recent report revealed a TRAF binding protein that interacts specifically with the only member of the TRAF family [Gamper, C., et al. (2000) Mol. Immunol. 37, 73-84; Ling L., et al. (2000) J. Biol. Chem. 275, 23852-23860; and Ling, L., et al. (2000) Proc. Natl. Acad. Sci. USA 97, 9567-9572]. MIP-T3 has been identified as a TRAF3-specific TRAF binding protein and TRAF3 binds to microtubules by binding MIP-T3 to microtubules and collecting TRAF3 into the microtubules (Ling, L., et al. (2000 ) J. Biol. Chem. 275, 23852-23860). Nucleoporins have also been identified as TRAF3-specific TRAF binding proteins (Gamper, C., et al. (2000) Mol. Immunol. 37, 73-84). Since TRAF appears to act excessively or specifically in the signal transduction cascade, the identification and analysis of TRAF binding proteins specific for a single factor of the TRAF family is in view of the diversity of roles of individual TRAF proteins. is important.
[0095]
T3BP has significant sequence similarity to human BCMA belonging to the TNFR superfamily as the 17th factor [Madry C., et al. (2000) Int. Immunol. 10, 1693-1702]. Interestingly, T3BP expression is restricted to specific stages of the B cell lineage as in BCMA [Laabi, Y., et al. (1992) EMBO J. 11, 3897-3904; and Laabi, Y. , Et al. (1994) Nucleic Acid Res. 22, 1147-1154], may be distributed on the cell surface (FIGS. 1B and 5B). In addition, it has been reported that the T3BP gene is disrupted by insertion of murine leukemia retrovirus in murine leukemia and lymphoma [Hansen, GM, et al. (2000) Genome Res. 10, 237-43]. From the above, it is considered that T3BP plays an important role as a cell surface receptor in B cell differentiation. However, T3BP does not belong to the tumor necrosis factor receptor superfamily because it does not have a cysteine-rich region in the putative extracellular region.
The novel TRAF3-binding protein revealed by the present invention is a specific receptor [Thompson, JS] of the recently reported BAFF [B cell activation factor from the TNF family (BAFF)]. , Et al. (2001) Science 293, 2108-2111]. BAFF is related to B cell proliferation [Schneider, P., et al. (1999) J. Exp. Med. 189, 1747-1756], and mice with high expression of BAFF are hyperplastic and systemic in mature B cells. Symptoms of systemic lupus erythematosus have been suggested to be related to autoimmune diseases by proliferation of autoreactive B cells [Mackay, F., et al. (1999) J. Exp. Med. 190 , 1697-1710, Batten, M., et al., J. Exp. Med. (2000) 192, 1453-1466]. Therefore, it is presumed that TRAF3 has an important function for the intracellular signal transduction system of B cell proliferation via BAFF. If only the interaction between BAFF and TRAF3-binding B cell specific receptor is regulated, First, the development of therapeutic agents for autoimmune diseases is expected by regulating the interaction between TRAF3-binding B cell specific receptor and TRAF3.
[0096]
【The invention's effect】
According to the present invention, a novel signaling molecule that binds to TRAF3 has been identified. In addition, according to the present invention, a gene encoding a signaling molecule that binds to TRAF3 has been cloned. By using the novel protein identified in the present invention, it becomes possible to develop a new medicine or provide a method for diagnosing a disease.
[0097]
[Sequence Listing]

[0099]

[Brief description of the drawings]
FIG. 1A shows the deduced amino acid sequence of T3BP, and FIG. 1B shows the result of Northern blot analysis of T3BP mRNA.
FIG. 2A shows the results of GST pulldown assay analysis of the binding characteristics of GST fusion proteins (GST-clone 3, GST-clone 10) to TRAF2 and TRAF3.
FIG. 2B shows the results of GST pull-down assay analysis of the binding characteristics of GST-T3BP to TRAF2, TRAF3, TRAF5, and TRAF6.
FIG. 2C shows the result of analysis by immunoprecipitation assay of the interaction between TRAF3 and T3BP.
FIGS. 3A and 3B show the results of mapping of the T3BP binding region in TRAF3.
FIG. 3C shows an outline of the primary structure of mouse TRAF3.
FIG. 4A shows the results of in vitro assay analysis of the binding properties of GST-fused T3BP truncation mutants to TRAF3.
FIG. 4B shows the results of GST pull-down assay analysis of the binding properties of GST-fused T3BP truncation mutants to TRAF3.
FIG. 4C shows the primary structure of T3BP and its TRAF3 binding region.
FIG. 5A shows changes in cell morphology due to T3BP expression.
FIG. 5B shows the effect of T3BP expression on the cell distribution of TRAF3.
FIG. 6A shows the increase in cellular F-actin content by T3BP.
FIG. 6B shows the results of observing the cell distribution of F-actin in cells expressing T3BP.

Claims (3)

A polypeptide comprising an amino acid sequence from the 95th to the 175th amino acid sequence of SEQ ID NO: 1 having binding activity to TRAF3. A polypeptide comprising the amino acid sequence from the 133rd to the 175th amino acid sequence of SEQ ID NO: 1 having binding activity to TRAF3. A polypeptide comprising the amino acid sequence of positions 151 to 175 of the amino acid sequence set forth in SEQ ID NO: 1 having binding activity to TRAF3.

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