JP4232423B2 - Novel ubiquitin-specific protease - Google Patents

Description

【図面の簡単な説明】
【図１】 ｂｆ０４２７４の脱ユビキチン化活性が、ｂｆ０４２７４と人工基質（Ｕｂ−Ｒ−ＧＳＴ、Ｕｂ−Ｍ−ＧＳＴ、Ｕｂ−Ｉ−ＧＳＴ、またはＵｂ−Ｐ−ＧＳＴ）とを共に大腸菌で発現させた共発現系で認められたことを示す図である。図中、ＵＳＰ１５およびルシフェラーゼは、それぞれ脱ユビキチン化活性の陽性コントロールおよび陰性コントロールである。レーン左側の数値は分子量を示す。
【図２】 ｂｆ０４２７４の推定酵素活性部位である、Ｎ末端から第６３４番目のシステイン（Ｃ）残基をセリン（Ｓ）残基に置換すると（ｂｆ０４２７４^{Ｃ６３４Ｓ}）、その脱ユビキチン化活性が消失したことを示す図である。図中、ＵＳＰ１５およびルシフェラーゼは、それぞれ脱ユビキチン化活性の陽性コントロールおよび陰性コントロールである。レーン左側の数値は分子量を示す。
【図３】 ｂｆ０４２７４のＮ末端から５２１アミノ酸残基を欠失させたＫＩＡＡ１０５７−１の脱ユビキチン化活性が、ＫＩＡＡ１０５７−１と人工基質（Ｕｂ−Ｒ−ＧＳＴ、Ｕｂ−Ｍ−ＧＳＴ、Ｕｂ−Ｉ−ＧＳＴ、またはＵｂ−Ｐ−ＧＳＴ）とを共に大腸菌で発現させた共発現系で認められたことを示す図である。図中、ＵＳＰ１５およびルシフェラーゼは、それぞれ脱ユビキチン化活性の陽性コントロールおよび陰性コントロールである。レーン左側の数値は分子量を示す。[0001]
[Industrial application fields]
The present invention relates to a novel protease having deubiquitination activity (hereinafter sometimes abbreviated as USP) and a gene encoding the same. More specifically, a polypeptide or peptide having all or part of the amino acid sequence of a novel USP, a polynucleotide encoding the polypeptide or a polynucleotide encoding the peptide, or a complementary strand thereof, and a recombinant vector containing the polynucleotide , A transformant containing the recombinant vector, the polypeptide or an antibody against the peptide, a compound interacting with the polypeptide or the polynucleotide, an antagonist of the polypeptide, a pharmaceutical composition comprising one or more of these Product, method for producing the polypeptide or the peptide, method for identifying the polypeptide or the compound having interaction with the peptide or the polynucleotide, method for measuring the polypeptide or the peptide or the polynucleotide, and the method for identifying the same Or the measurement method A reagent kit for use.
[0002]
[Prior art]
Ubiquitin (hereinafter sometimes abbreviated as Ub) is a peptide chain consisting of 76 amino acid residues, and its amino acid sequence is highly conserved from yeast to human. Ub has various roles in vivo, such as carcinogenesis (Non-patent documents 1 to 4), cell cycle (non-patent documents 5 to 7), viral infection (non-patent document 8), and neurodegenerative diseases (non-patent document). It is involved in many biological reactions such as 9-11).
[0003]
The most important function of Ub is to act as a signal in proteolysis in the 26S proteasome. By a series of ubiquitinases such as ubiquitin activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3), Ub is isopeptide-bonded to the target protein to form a polyubiquitin chain. The polyubiquitin chain is recognized by the proteasome as a degradation signal, whereby the ubiquitinated protein is degraded.
[0004]
On the other hand, the presence of a deubiquitinase (DUB) that catalyzes a deubiquitination reaction in which Ub dissociates from a ubiquitinated protein has been reported. DUBs are roughly classified into two families based on their structures (Non-Patent Documents 12 to 14). One is called ubiquitin C-terminal hydrolase (UCH), which has many molecular weights of 20 kDa to 30 kDa, and preserves the primary structure between different species. UCH dissociates Ub mainly when a small molecule is bound to the C-terminus of Ub. The other is called a ubiquitin specific protease (USP, UBP, or UCH_type II), and its molecular weight varies from 40 kDa to 150 kDa, and there is little common amino acid sequence between different species. USP has a cysteine (Cys) domain (Cys box), a histidine (His) domain (His box), and an aspartic acid (Asp) domain as its active domain, and a cysteine residue present in the Cys domain as an active site It is a protease. There is also a report (Non-patent Document 15) that the USP N-terminal sequence is involved in substrate recognition. USP dissociates Ub when a polymer is bound to the C-terminus of Ub.
[0005]
The functions of USP in vivo can be roughly divided into three. The first is a function of generating Ub from a precursor Ub such as a ribosomal protein-fused ubiquitin or a peptide-bonded polyubiquitin chain. This involves Ub-CEP52, which is one of the USPs. Part 2 is a function of dissociating Ub from an isopeptide-bonded ubiquitinated protein, and suppresses protein degradation by suppressing ubiquitination of the protein. The third is a function of disassembling an isopeptide-linked polyubiquitin chain after being degraded by the proteasome. For example, isopeptidase T known as USP5 has this function (Non-patent Document 16). .
[0006]
One of the functions of the ubiquitin system composed of Ub, USP, and the like is removal of abnormal proteins generated in vivo, and quantitative adjustment by degradation of transcription factors, signal transduction factors, etc. (Non-patent Document 17) USP dysfunction causes this system to malfunction. It has been suggested that USP abnormalities are associated with carcinogenesis and neurodegenerative diseases (Non-Patent Documents 17 to 19). For example, it has been reported that many protein aggregates observed in Alzheimer's disease and Parkinson's disease react with anti-ubiquitin antibodies (Non-patent Document 17). USP is also involved in maintaining the chromosomal structure, and it is known that deubiquitination of ubiquitinated histones is important for chromosome aggregation (Non-patent Document 20), and one of the USP families. USP16 is reported to deubiquitinate H2A (Non-patent Document 21). Furthermore, it has been reported that the ubiquitin pathway is associated with muscle atrophy (Non-patent Document 22).
[0007]
[Non-Patent Document 1]
"FEBS Letters", 1997, vol. 420, p. 25-
[Non-Patent Document 2]
“Oncogene”, 1993, Vol. 8, p. 2307-
[Non-Patent Document 3]
“Oncogene”, 1995, Vol. 10, p. 2179-
[Non-Patent Document 4]
“Nature”, 1993, 366, p. 313
[Non-Patent Document 5]
“Annual Review of Biochemistry”, 1998, Vol. 67, p. 425-
[Non-Patent Document 6]
“Proceedings of the National Academy of the United States of America”, 1996, Vol. 93, “Proceedings of the National Academy of Sciences of the United States of America”. 3275-
[Non-Patent Document 7]
“Journal of Biological Chemistry”, 1997, Vol. 272, p. 51-
[Non-Patent Document 8]
“EMBO Journal”, 1997, Vol. 16, p. 1519-
[Non-patent document 9]
“Trends In Neurosciences”, 1998, Vol. 21, p. 516-
[Non-Patent Document 10]
“Nature”, 1998, Vol. 395, p. 451-452
[Non-Patent Document 11]
“Nature Genetics”, 1998, Vol. 23, p. 47-
[Non-Patent Document 12]
“Biochemical And Biophysical Research Communications”, 1999, Vol. 266, p. 633
[Non-Patent Document 13]
“FASEB Journal”, 1997, Vol. 11, p. 1245-
[Non-Patent Document 14]
“Critical Reviews in Biochemistry and Molecular Biology”, 1998, Vol. 33, p. 337-
[Non-Patent Document 15]
“Journal of Biological Chemistry”, 2001, Vol. 276, p. 20357-20363
[Non-Patent Document 16]
“Biochemistry”, 1995, Vol. 34, p. 14535
[Non-Patent Document 17]
Toshiaki Suzuki, Hideki Shimura, Nobutaka Hattori, “Ubiquitin and Neurodegenerative Diseases”, “Experimental Medicine”, 2000, Vol. 18, p. 1478-1482
[Non-Patent Document 18]
Toshiaki Suzuki, “Various Actions of Deubiquitinating Enzymes”, “Experimental Medicine”, 2001, Vol. 19, p. 193-
[Non-Patent Document 19]
Anan Tadashi, Nakao Mitsuyoshi, “Molecular Mechanisms of Ubiquitin Disease”, “Proteins / Nucleic Acids / Enzymes”, 1999, Vol. 44, p. 776-
[Non-Patent Document 20]
“BioEssays”, 1992, Vol. 14, p. 9-
[Non-patent document 21]
“Proceedings of the National Academy of Sciences of United States of America,” 1996, Vol. 96, “Proceedings of the National Academy of Sciences of the United States of America”. 2828-
[Non-Patent Document 22]
“Current Opinion In Clinical Nutrition And Metabolic Care”, 2001, Vol. 4, p. 183-190
[Non-Patent Document 23]
“Kazusa DNA Research Institute DNA Sequence Analysis Analysis Information Database, HUGE”, Internet <http: // www. kazusa. or. jp / huge / gfpage>
[0008]
[Problems to be solved by the invention]
Many USPs exist in the living body, and it is considered that each has different substrate specificity and physiological function. Therefore, in order to elucidate diseases caused by abnormal USPs, such as carcinogenesis and neurodegenerative diseases, and to prevent, treat and diagnose them, it is necessary to discover and use many new USPs. .
[0009]
One of the problems to be solved by the present invention is to find a new USP and to control the USP in vivo. More specifically, it is to provide a USP having a novel property, and a polypeptide or peptide derived from the novel USP that is useful in connection therewith, a polynucleotide encoding the same, and an antibody against the polypeptide or the peptide Is to provide. Furthermore, it is to identify an inhibitor, antagonist or promoter of the expression and / or physiological activity of a novel USP, and to provide the identified compound. Moreover, it is providing the pharmaceutical composition using the said polypeptide or the said peptide, the said polynucleotide, the said antibody, and the said compound, and the measuring method of the said polypeptide, the said peptide, or the said polynucleotide. Still another object of the present invention is to provide a novel USP-derived polypeptide or peptide production method by genetic engineering techniques using the above polynucleotide.
[0010]
[Means for Solving the Problems]
In order to solve the above problems, the present inventors have made intensive efforts and succeeded in obtaining a USP gene and its protein having novel characteristics. More specifically, a cDNA clone was extracted as a novel protease candidate gene from the Kazusa DNA Laboratory human long-chain cDNA analysis information database, and expressed in a gene expression system using Escherichia coli to obtain a protein encoded by the gene. . Furthermore, the obtained protein exhibits deubiquitination activity, and the protein lacking 521 consecutive amino acid residues from the N-terminal first to 521-th sequence does not exhibit deubiquitination activity The present invention was completed.
[0011]
That is, the present invention
(1) a polypeptide selected from the following group;
(1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing;
(2) a polypeptide containing a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing;
(3) A polypeptide having at least about 70% homology on the amino acid sequence with a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and having deubiquitination activity,
and
(4) The polypeptide according to any one of (1) to (3) has a mutation such as deletion, substitution, addition or insertion of one to several amino acids in the amino acid sequence, and deubiquitination activity A polypeptide having
(2) A polypeptide selected from the following group, having a deubiquitination activity;
(1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing;
(2) a polypeptide containing a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing;
(3) A polypeptide having at least about 70% homology on the amino acid sequence with a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing,
and
(4) A polypeptide having a mutation such as deletion, substitution, addition, or insertion of one to several amino acids in the amino acid sequence in the polypeptide of any one of (1) to (3) above,
(3) a peptide having at least about 5 consecutive amino acid sequences of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing;
(4) a polypeptide selected from the following group;
(A) a polypeptide comprising 521 consecutive amino acid residues from the N-terminal first amino acid residue to the 521st amino acid residue of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing ,
(B) A polypeptide comprising 521 consecutive amino acid residues from the N-terminal first amino acid residue to the 521st amino acid residue of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing And a polypeptide that has at least about 70% amino acid sequence homology and inhibits the deubiquitination activity of the polypeptide of (1) or (2) above,
and
(C) The polypeptide of (a) or (b) has a mutation such as deletion, substitution, addition or insertion of one to several amino acids in the amino acid sequence, and (1) or A polypeptide that inhibits the deubiquitination activity of the polypeptide of (2),
(5) A polypeptide selected from the following group, which inhibits the deubiquitination activity of the polypeptide of (1) or (2);
(A) a polypeptide comprising 521 consecutive amino acid residues from the N-terminal first amino acid residue to the 521st amino acid residue of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing ,
(B) A polypeptide comprising 521 consecutive amino acid residues from the N-terminal first amino acid residue to the 521st amino acid residue of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing A polypeptide having at least about 70% amino acid sequence homology with
and
(C) a polypeptide having a mutation such as deletion, substitution, addition or insertion of one to several amino acids in the amino acid sequence in the polypeptide of (a) or (b),
(6) A polypeptide comprising 521 consecutive amino acid residues from the N-terminal first amino acid residue to the 521st amino acid residue of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing A peptide having an amino acid sequence of at least about 5 consecutive amino acid sequences and inhibiting the deubiquitination activity of the polypeptide of (1) or (2) above,
(7) The polypeptide of (1) or (2), or the polynucleotide encoding the peptide of (3) or a complementary strand thereof,
(8) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 2 in the sequence listing or a complementary strand thereof,
(9) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2 in the sequence listing or a polynucleotide comprising at least about 15 consecutive nucleotide sequences of its complementary strand, (10) the above (7) to (9) A polynucleotide that hybridizes with any polynucleotide or its complementary strand under stringent conditions;
(11) The polypeptide of (4) or (5), or the polynucleotide encoding the peptide of (6) or a complementary strand thereof,
(12) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 4 in the sequence listing or a complementary strand thereof,
(13) A recombinant vector containing the polynucleotide according to any one of (7) to (12),
(14) The recombinant vector according to (13), wherein the recombinant vector is an expression recombinant vector,
(15) A transformant into which the recombinant vector of (13) or (14) has been introduced,
(16) A method for producing the polypeptide of (1), (2), (4) or (5) above, or the peptide of (3) or (6) above, A method comprising culturing a transformant into which a recombinant vector has been introduced, or a cell-free protein synthesis means using the recombinant vector of (13) or (14),
(17) An antibody that immunologically recognizes the polypeptide of (1), (2), (4) or (5), or the peptide of (3) or (6),
(18) The antibody according to (17), which inhibits deubiquitination activity,
(19) A compound that interacts with the polypeptide of (1) or (2) to inhibit or enhance its physiological activity, and / or interacts with the polynucleotide of (7) or (8). A method for identifying a compound that inhibits or promotes the expression of the polypeptide of (1), (2), (4) or (5), (3) or (6) A peptide, the polynucleotide of any one of (7) to (12), the recombinant vector of (13) or (14), the transformant of (15), and the (17) or (18) A method comprising using at least one of the antibodies
(20) interacts with the polypeptide of (1) or (2) to inhibit or enhance its physiological activity and / or interacts with the polynucleotide of (7) or (8) A method of identifying a compound that inhibits or promotes its expression, wherein the polypeptide or the polynucleotide and the compound are contacted under conditions that allow the compound to interact with the polypeptide or the polynucleotide. The compound interacts with the polypeptide or polynucleotide by detecting the presence or absence or change of a signal resulting from the interaction of the compound with the polypeptide or the polynucleotide, Whether to inhibit or promote the physiological activity of the polynucleotide or the expression of the polynucleotide How to constant,
(21) The compound identified by the method of (19) or (20),
(22) interacting with the polypeptide of (1) or (2) to inhibit or enhance deubiquitination activity, or interacting with the polynucleotide of (7) or (8) A compound that inhibits or promotes its expression,
(23) The antagonist of the polypeptide of (1) or (2), comprising the polypeptide of (4) or (5) and / or the peptide of (6),
(24) The polypeptide of (1), (2), (4) or (5), the peptide of (3) or (6), any of (7) to (12) The polynucleotide of (13) or (14), the transformant of (15), the antibody of (17) or (18), the compound of (21) or (22) And a pharmaceutical composition comprising at least one of the antagonists of (23) above,
(25) The polypeptide of (1), (2), (4) or (5), the peptide of (3) or (6), any of (7) to (12) The polynucleotide of (13) or (14), the transformant of (15), the antibody of (17) or (18), the compound of (21) or (22) And an agent for preventing and / or treating a neurodegenerative disease, comprising at least one of the antagonists of (23),
(26) The preventive and / or therapeutic agent for neurodegenerative disease according to (25), wherein the neurodegenerative disease is Alzheimer's disease and / or Parkinson's disease,
(27) The polypeptide of (1), (2), (4) or (5), the peptide of (3) or (6), any of (7) to (12) The polynucleotide of (13) or (14), the transformant of (15), the antibody of (17) or (18), the compound of (21) or (22) And an agent for preventing and / or treating muscular atrophy, comprising at least one of the antagonists of (23),
(28) The polypeptide of (1), (2), (4) or (5) above, or the polynucleotide of (7), (8), (11) or (12) above A method of measuring quantitatively or qualitatively,
(29) The polypeptide of (1), (2), (4) or (5), the peptide of (3) or (6), any of (7) to (12) The recombinant vector of (13) or (14), the transformant of (15), the antibody of (17) or (18), and the antagonist of (23) A reagent kit comprising at least one of them.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
(New USP)
Human USP provided in the present invention is a protein encoded by cDNA clone bf04274 selected as a gene having a protease motif from a long-chain cDNA library derived from human brain. The USP was obtained by expressing in E. coli into which an expression plasmid incorporating the above gene was introduced. The USP is a novel USP that has almost no homology except that it has a highly homologous Cys domain and His domain in the amino acid sequence of known USP. The USP gene consists of 7744 bases (SEQ ID NO: 2 in the sequence listing), its open reading frame (ORF) full length is 4671 bases, and the gene product of the gene consists of 1556 amino acid residues (SEQ ID NO: 1). ). Hereinafter, this gene is referred to as a new USP gene, and the gene product of the gene is referred to as a new USP. The C-terminal 5618 bases of the novel USP gene and the C-terminal 977 amino acid residues of the novel USP are common to the gene of KIAA1057 (GenBank accession number: AB026980) and the gene product thereof, respectively.
[0013]
When the novel USP gene was expressed together with an artificial substrate in a gene co-expression system, it acted on the artificial substrate and showed deubiquitination activity. On the other hand, when 521 amino acid residues deleted from the N-terminus of novel USP (KIAA1057-1) and an artificial substrate were reacted in vitro or in a gene co-expression system, no deubiquitination activity was observed. KIAA1057-1 includes KIAA1057. KIAA1057 has already been disclosed to have a base sequence, an encoded amino acid sequence, and a Cys domain and His domain that are characteristic of USP (Non-patent Document 23). However, KIAA1057-1 containing the amino acid sequence disclosed as KIAA1057 did not show deubiquitination activity. That is, by isolating and identifying a novel USP in the present invention, it was confirmed for the first time that the novel USP had deubiquitination activity, and a protein having enzyme activity could be obtained. Furthermore, it has been clarified that the novel USP has substrate selectivity and, in the study using an artificial substrate, selectively acts on Ub bound to the protein via arginine. None of the known USPs are known to act selectively on such Ub. In addition, since it has been disclosed that KIAA1057 cDNA is strongly expressed in brain, skeletal muscle, heart, etc., particularly in skeletal muscle, it is considered that a novel USP gene is also expressed in brain, skeletal muscle, etc. It is done.
[0014]
(Polypeptide or peptide)
The polypeptide according to the present invention is a gene product of a novel USP gene, and is a polypeptide obtained by expressing the gene in cells such as Escherichia coli. Here, the polypeptide means a long-chain peptide such as a protein among arbitrary peptides containing two or more amino acids bonded to each other by peptide bonds or modified peptide bonds, Short chain peptides, also called oligomers, are simply called peptides. In the present specification, amino acids may be represented by 3 letters or 1 letter.
[0015]
One embodiment of the polypeptide according to the present invention is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing. Another embodiment is a polypeptide containing a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1.
[0016]
Another embodiment is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing, and about 70% or more, preferably about 80% or more, more preferably about 90% or more, more preferably on the amino acid sequence. Is a polypeptide having about 95% or more homology. More preferably, it is a polypeptide having an activity equivalent to that of the polypeptide shown in SEQ ID NO: 1 in the sequence listing, for example, deubiquitination activity. For example, as shown in the Examples described later, the deubiquitination activity is obtained by binding a glutathione_S-transferase (GST) to the C terminus of human ubiquitin via arginine, isoleucine, methionine, or proline as a substrate. Whether or not ubiquitin can be dissociated from the substrate can be measured by detecting GST after ubiquitin dissociation by a known method such as an immunoblotting method using an anti-GST antibody. More preferably, the polypeptide exhibits a deubiquitination activity by selectively acting on a substrate to which Ub is bound via arginine. Techniques for determining amino acid sequence homology are known per se. For example, a method for directly determining the amino acid sequence, a method for determining the base sequence of cDNA and estimating the amino acid sequence encoded by this can be used. Naturally, homologous gene products of animal species other than humans are also included in the scope of the present invention.
[0017]
Furthermore, by using deubiquitination activity as an index based on the polypeptide thus identified, one or more, for example, 1 to 100, preferably 1 to 30, more preferably 1 A polypeptide comprising an amino acid sequence having mutations such as deletion, substitution, addition or insertion of 1 to 20, more preferably 1 to 10, particularly preferably 1 to several amino acids is also provided. The peptide or polypeptide having a mutation may be naturally occurring or may have a mutation introduced therein. Means for introducing mutations such as deletion, substitution, addition and insertion are known per se. For example, site-specific mutagenesis, gene homologous recombination, primer extension, or polymerase chain amplification (PCR) alone Or as appropriate, for example, Sambrook et al., “Molecular Cloning Laboratory Manual”, 2nd edition, Cold_Spring_Harbor_Laboratory_Press, 1989, edited by Masaki Matsumura, “Lab Manual Genetic Engineering”, Maruzen Co., Ltd. 1988, Ehrlich, “PC Technology”, “Principle and Application of DNA Amplification”, Stockton Press, 1989, etc. For example, Ulmer technology ("Science ( cience) ", 1983, No. 219 Volume, p.666-) can be utilized.
[0018]
From the viewpoint of not changing the basic properties (physical properties, physiological activity, immunological activity, etc.) of the polypeptide in the introduction of mutations as described above, for example, homologous amino acids (polar amino acids, nonpolar amino acids, Mutual substitution between hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) is easily envisaged. Furthermore, these peptides can be altered to the extent that they do not undergo significant changes in function, such as modification of their constituent amino groups or carboxyl groups.
[0019]
Thus, a polypeptide having an activity equivalent to the physiological activity possessed by the novel USP, for example, an equivalent deubiquitination activity can be provided in the present invention. In addition, polypeptides with altered activity intensity or substrate specificity can be provided.
[0020]
Furthermore, a polypeptide or peptide having a partial sequence of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing is also included in the scope of the present invention. The polypeptide or peptide having the partial sequence consists of 5 or more amino acids, preferably 8 or more amino acids, more preferably 12 or more, still more preferably 15 or more consecutive amino acids as the minimum unit. is there. For example, a polypeptide or peptide consisting of the minimally active unit (region or domain) of the biological activity possessed by the novel USP is also provided in the present invention. The polypeptide or peptide having the partial sequence is a novel USP or a substance that modulates the activity of the polypeptide having a physiological activity equivalent to that of the novel USP, for example, a deubiquitination activity, or an identification of a substance that modulates the physiological activity It is useful as a reagent used for the above.
[0021]
Specifically, for example, the polypeptide or peptide having the partial sequence can be used as an antagonist of the above polypeptide having a novel USP or a physiological activity equivalent to that of the novel USP, for example, a deubiquitination activity. For example, a polypeptide consisting of 521 amino acid residues from the N-terminal first amino acid residue to the 521st amino acid residue of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing (sequence listing In SEQ ID NO: 3), when this site is deleted, the deubiquitination activity of the polypeptide consisting of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing disappears, and the ubiquitin-specific protease (UBP) N-terminal side Since there is a report that the sequence is involved in substrate recognition (Non-patent Document 15), it is considered that the sequence is involved in substrate recognition of a novel USP. Such a polypeptide having a substrate recognition site but no enzyme active site can be used as an antagonist of the enzyme from which it is derived. Therefore, the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing is a novel USP or an antagonist of the above polypeptide having a physiological activity equivalent to the novel USP, and has a physiological activity such as deubiquitination activity. Can be used for inhibition.
[0022]
The polypeptide that inhibits the physiological activity of the above-mentioned polypeptide having a physiological activity equivalent to that of the new USP or the novel USP, for example, the deubiquitination activity is not limited to the polypeptide described in SEQ ID NO: 3 in the Sequence Listing. The polypeptide may be any polypeptide that can inhibit the physiological activity of, for example, deubiquitination activity, for example, about 70% or more, preferably about 80% or more in terms of the amino acid sequence with the polypeptide of SEQ ID NO: 3 in the Sequence Listing. Preferably, the polypeptide having the homology of about 90% or more, more preferably about 95% or more, and the polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or the physiological activity of the polypeptide having the same physiological activity as the polypeptide Polypeptides that can inhibit the activity, for example, deubiquitination activity. Further, for example, based on the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing, by using the ability to inhibit deubiquitination activity as an index, one or more, for example, 1 to 100, preferably Amino acid sequence having mutations such as deletion, substitution, addition or insertion of 1 to 30, more preferably 1 to 20, more preferably 1 to 10, particularly preferably 1 to several amino acids A polypeptide consisting of can also be provided. The same means as described above can be used for deletion, substitution, addition, or insertion.
[0023]
Furthermore, it is a partial peptide of a polypeptide that inhibits the physiological activity of the above-mentioned polypeptide having a physiological activity equivalent to that of the novel USP or the novel USP, for example, the deubiquitination activity, and inhibits the physiological activity, for example, the deubiquitination activity Such peptides are also included in the scope of the present invention.
[0024]
A novel USP or a polypeptide or peptide that inhibits the physiological activity of the above-mentioned polypeptide having a physiological activity equivalent to that of the novel USP, for example, a polypeptide or peptide that inhibits the deubiquitination activity in an experimental system for measuring the deubiquitination activity. Can be obtained by examining the above. As the experimental system, for example, as shown in the Examples described later, a substrate in which glutathione_S-transferase (GST) is bound to the C-terminus of human ubiquitin via arginine, isoleucine, methionine, or proline is used as a substrate. An experimental system for detecting whether ubiquitin can be dissociated from the substrate by a known method such as an immunoblotting method using an anti-GST antibody after the ubiquitin dissociation can be used.
[0025]
Further, among the polypeptides or peptides having the above partial sequence, the peptide that can be immunologically recognized is, for example, an epitope peptide, as an antigen for producing an antibody specific for a novel USP as described later. Alternatively, it can be used in combination with a carrier (for example, keyhole limpet hemocyanin or ovalbumin).
[0026]
The scope of the present invention also includes a polypeptide or peptide according to the present invention bound to another type of protein or substance such as a carrier. For example, in order to facilitate the detection or purification of the polypeptide or the like according to the present invention or to add another function, another type of protein or peptide such as glutathione_S-transferase is added to the N-terminal side or C-terminal side thereof. (GST), luciferase, GFP, β-galactosidase, immunoglobulin Fc fragment such as IgG, His-tag, Myc-tag, Flag-tag etc. directly or indirectly via linker peptide It may be added by using a technique or the like.
[0027]
(Polynucleotide)
The present invention provides the above polypeptide or a polynucleotide encoding the above peptide and its complementary strand. For example, the polynucleotide according to the present invention is a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing or a complementary strand thereof. Preferably, it is a polynucleotide consisting of the base sequence set forth in SEQ ID NO: 2 in the sequence listing or its complementary strand. Furthermore, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 of the sequence listing according to the present invention or a complementary strand thereof, preferably a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4 of the sequence listing, or a complementary strand thereof. Included in the range.
[0028]
Furthermore, the present invention provides a polynucleotide that hybridizes under stringent conditions to the above-mentioned polynucleotide or a complementary strand thereof, preferably a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2 in the sequence listing or a corresponding region of the complementary strand thereof. I will provide a. The hybridization conditions can be in accordance with, for example, Sambrook et al., “Molecular Cloning Laboratory Manual”, 2nd edition, Cold Spring_Harbor_Laboratory_Press, 1989, and the like. These polynucleotides are not necessarily complementary sequences as long as they hybridize to the target polynucleotide, preferably the polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2 in the Sequence Listing or its complementary strand.
[0029]
The polynucleotide according to the present invention is a polynucleotide or oligonucleotide comprising a sequence of 10 or more nucleotides corresponding to the designated base sequence region of the polynucleotide, preferably 15 or more, more preferably 20 or more. Includes nucleotides or their complementary strands.
[0030]
These polynucleotides or oligonucleotides provide gene information useful for the production of the polypeptide according to the present invention, or can be used as a reagent or standard for nucleic acids. For example, it can be used as a probe or primer for detecting a nucleic acid encoding a novel USP, for example, its gene or mRNA, or as an antisense oligonucleotide for regulating gene expression. In that sense, the polynucleotides and oligonucleotides according to the present invention include not only translated regions but also those corresponding to untranslated regions. Here, for selection of a polynucleotide encoding a novel USP or the above-mentioned polypeptide having physiological activity equivalent to that of the USP, for example, a known protein expression system is used to confirm the expressed protein, and its physiological activity, for example, deactivation. It can be performed using ubiquitination activity as an index. As a known protein expression system, for example, a cell-free protein expression system utilizing a ribosome technology derived from embryos or rabbit reticulocytes (“Nature, 1957, 179, pp. 160-161). ).
[0031]
(Recombinant vector)
A recombinant vector can be obtained by incorporating the polynucleotide into an appropriate vector DNA. The vector DNA to be used is appropriately selected depending on the type of host and intended use. The vector DNA may be one in which a part of the DNA other than the part necessary for growth is missing in addition to the one extracted from naturally occurring ones. For example, vectors derived from chromosomes, episomes and viruses such as bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosome elements, such as baculovirus, papovavirus, SV40, vaccinia virus, adenovirus, Examples include vectors derived from viruses such as fowlpox virus, pseudorabies virus and retrovirus, and vectors in combination thereof, such as vectors derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. Moreover, an expression vector, a cloning vector, etc. can be used depending on the purpose.
[0032]
Recombinant vectors are composed of the gene sequence of interest and a gene sequence carrying information on replication and control, such as a promoter, a ribosome binding site, a terminator, a signal sequence, an enhancer, etc., and these are combined by a method known per se. Produced. As a method for incorporating the polynucleotide according to the present invention into the vector DNA, a method known per se can be applied. For example, a method is used in which an appropriate restriction enzyme is selected and treated to cleave DNA at a specific site, and then mixed with DNA used as a similarly treated vector and religated by ligase. Alternatively, the desired recombinant vector can also be obtained by ligating a suitable linker to the polynucleotide of interest and inserting it into a multicloning site of a vector suitable for the purpose.
[0033]
(Transformant)
A transformant can be obtained by introducing a vector DNA incorporating the polynucleotide into a host known per se by a method known per se. Examples of the host include Escherichia coli, yeast, Bacillus subtilis, insect cells, and animal cells. In the case of introducing a gene, a more preferable system is an integration method into a chromosome if the stability of the gene is taken into account, but an autonomous replication system using an extranuclear gene can be used conveniently. The introduction of vector DNA into host cells can be performed according to the standard methods described in, for example, Sambrook et al., “Molecular Cloning Laboratory Manual”, Second Edition, Cold Spring Spring Harbor Laboratory Press, 1989, etc. It can be done by a method. Specifically, calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, and infection Etc.
[0034]
Moreover, if an expression vector is used as the vector DNA to be introduced into the transformant, the polypeptide or peptide according to the present invention can be provided. The transformant introduced with the expression vector DNA into which the polynucleotide is incorporated is cultured under the optimum conditions for the culture conditions of each host. The culture is performed by the action of the polypeptide or peptide of the present invention expressed by the transformant, for example, at least the deubiquitination activity or the like, or the polypeptide or the peptide produced in the host or produced outside the host. Although the amount may be used as an index, subculture or batch culture may be performed using the amount of transformant in the medium as an index.
[0035]
(Manufacture of polypeptide or peptide)
The polypeptide or peptide according to the present invention can be produced by genetic engineering techniques as described above using the above vector or transformant. It can also be produced by methods known in normal peptide chemistry. For example, methods described in “Peptide Synthesis”, Maruzen Co., Ltd., 1975, “Peptide Synthesis”, Interscience, New York, 1996 can be exemplified, but of course known methods Is widely available.
[0036]
Purification or recovery of the polypeptide or peptide according to the present invention can be performed by combining molecular sieve, ion column chromatography, affinity chromatography, etc., using its physiological activity, for example, at least deubiquitination activity as an index, It can be purified and recovered by fractionation means such as alcohol. Preferably, based on the information of the polypeptide to be recovered or the amino acid sequence of the peptide, a polyclonal antibody or monoclonal antibody specific to these is prepared, and a method of specifically adsorbing and recovering using the antibody is used. .
[0037]
(antibody)
The antibody is produced using the polypeptide or the peptide as an antigen. The antigen may be the above polypeptide or the above peptide, or a fragment thereof, and is composed of at least 8, preferably at least 10, more preferably at least 12, further preferably 15 or more amino acids. In order to produce an antibody specific for a new USP, it is preferable to use a region consisting of an amino acid sequence unique to the new USP other than a conserved region between USP families. The amino acid sequence of a polypeptide or peptide used as an antigen is not necessarily homologous to a polypeptide or peptide, for example, the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing or a partial sequence consisting of a continuous amino acid sequence in the sequence. It is not necessary to have an exposed site on the three-dimensional structure of the protein, and even if the amino acid sequence of the exposed site is discontinuous on the primary structure, it may be a continuous amino acid sequence for the exposed site. The antibody is not particularly limited as long as it binds or recognizes a peptide or polypeptide comprising an immunologically novel USP or a derivative thereof. The presence or absence of this binding or recognition is determined by a known antigen-antibody binding reaction.
[0038]
In order to produce an antibody, an antibody production method known per se can be used. For example, the polypeptide or peptide of the present invention is administered to an animal alone or in combination with a carrier in the presence or absence of an adjuvant to induce immunity such as a humoral response and / or a cellular response. Can be obtained. The carrier is not particularly limited as long as it does not itself have a harmful effect on the host and enhances antigenicity, and examples thereof include cellulose, polymerized amino acid, albumin, keyhole limpet hemocyanin (KLH) and the like. Is done. Adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Ribi (MPL), Ribi (TDM), Ribi (MPL + TDM), pertussis vaccine (Bordetella pertussis vaccine), muramyl dipeptide (MDP), aluminum Adjuvants (ALUM) and combinations thereof are exemplified. As the animal to be immunized, a mouse, rat, rabbit, goat, horse or the like is preferably used.
[0039]
Polyclonal antibodies are obtained from the sera of animals that have been subjected to the above-mentioned immunization means by a known antibody recovery method. A preferable means is obtained by immunoaffinity chromatography.
[0040]
In order to produce a monoclonal antibody, antibody-producing cells (for example, spleen or lymph node-derived lymphocytes) are collected from an animal that has been subjected to the above-described immunization means, and known per se proliferative cells (for example, It is carried out by introducing a means for transformation into a myeloma strain such as the P3-X63-Ag8 strain. For example, an antibody-producing cell and a permanently proliferating cell are fused by a method known per se to prepare a hybridoma, which is then cloned, and a hybridoma that produces an antibody that specifically recognizes the polypeptide or the peptide is selected. The antibody is recovered from the culture medium of the hybridoma.
[0041]
The thus-obtained polypeptide or polyclonal antibody or monoclonal antibody that recognizes and binds to the peptide can be used as an antibody for purification, a reagent, or a marker marker for the polypeptide or the peptide. For example, among polyclonal antibodies or monoclonal antibodies that recognize and bind to the polypeptide or the peptide, an antibody that directly binds to the novel USP according to the present invention and inhibits its deubiquitination activity results from an abnormality in the USP. It is useful for elucidating, preventing and / or treating various diseases.
[0042]
(screening)
A polypeptide or peptide according to the present invention, a polynucleotide according to the present invention or a complementary strand thereof, a vector incorporating the polynucleotide or the complementary strand thereof, a transformant obtained by introducing the vector, a protein expression system using these, In addition, the polypeptide or an antibody that immunologically recognizes the peptide can be used alone or in combination to identify a novel USP or an activity inhibitor or activity enhancer of the above polypeptide having physiological activity equivalent to that of the USP. To provide an effective method. These also provide an effective method for identifying an expression inhibitor or expression promoter for a polynucleotide encoding a novel USP or a polypeptide having the same physiological activity as the USP. This method can be constructed using a pharmaceutical screening system known per se. According to the identification method of the present invention, for example, selection of an antagonist by drug design based on the three-dimensional structure of a novel USP or the above-mentioned polypeptide having physiological activity equivalent to the USP, expression at a gene level using a protein synthesis system It is possible to select regulators, antibody recognition substances using antibodies, and the like.
[0043]
For example, using the polypeptide or peptide according to the present invention, conditions that allow interaction between the test compound and these polypeptides or peptides are selected, and under these conditions, these polypeptides or peptides and the compounds are selected. And the presence or absence or change of a signal caused by the interaction is detected, whereby the physiological activity of the novel USP or the above polypeptide having physiological activity equivalent to that of the USP, for example, deubiquitination activity is increased. Compounds that enhance or inhibit can be identified.
[0044]
In addition, a condition that allows interaction between a test compound and a polynucleotide encoding a novel USP or the above polypeptide having a physiological activity equivalent to that of the USP is selected, and the polynucleotide and the A compound that binds to the polynucleotide can be identified by contacting the compound and detecting the presence or absence or change of a signal resulting from the interaction.
[0045]
Furthermore, using the transformant according to the present invention, the test compound or the above-identified compound is contacted under appropriate conditions, and the novel USP according to the present invention or the above-mentioned physiological activity equivalent to the USP is obtained. By detecting the presence or absence or change of polypeptide expression, it is possible to identify compounds that inhibit or promote the expression of these polypeptides. The presence / absence or change of expression of these polypeptides can be conveniently detected using as an index the physiological activity of the expressed polypeptide, for example, deubiquitination activity. The deubiquitination activity can be measured, for example, by measuring GST generated by degradation of the artificial substrate Ub-R-GST. In such an identification method, a compound that inhibits or enhances the physiological activity of these polypeptides, for example, deubiquitination activity, can also be identified. Alternatively, in order to detect the presence or absence or change of expression of a novel USP, a system known per se using a signal or marker for detection may be introduced to detect the presence or absence or change of this signal or marker. Good. Here, the signal refers to a signal that can be directly detected by its physical or chemical properties, and the marker refers to a signal that can be indirectly detected by using the physical or biological properties of the signal as an index. Signals include luciferase and green fluorescent protein (GFP), and markers include reporter genes such as chloramphenicol acetyltransferase (CAT) gene, or detection tags such as 6 × His tags. Available. A vector incorporating these signals or markers may be prepared, and the vector may be introduced into a host cell to produce a transformant. Methods for detecting these signals or markers are well known to those skilled in the art.
[0046]
Specifically, for example, according to the examples described later, a test compound is added thereto in an experimental system in which a novel USP and a substrate are co-expressed in, for example, Escherichia coli and the deubiquitination activity of the USP is measured. Thus, compounds that inhibit, promote or enhance the expression or physiological activity of the USP can be identified. This experimental system explains one of the identification methods, and the identification method of the compound according to the present invention is not limited to this.
[0047]
(Compound)
The compound identified by the above method can be used as a candidate compound for an inhibitor, antagonist, or enhancer of a novel USP or an activity of the above polypeptide having a physiological activity equivalent to the USP, for example, deubiquitination activity . It can also be used as a candidate compound for an inhibitor or promoter related to the expression of a novel USP at the gene level or the above-mentioned polypeptide having a physiological activity equivalent to the USP. These candidate compounds are effective in preventing various pathological symptoms caused by expression or physiological activity of the novel USP or the above-mentioned polypeptide having the same physiological activity as the USP, for example, increase, decrease or deletion of deubiquitination activity. And / or a therapeutic effect can be expected. As will be described later, since USP has been reported to be associated with neurodegenerative diseases and muscle atrophy (Non-Patent Document 22), it can be used as a preventive and / or therapeutic agent for these diseases.
[0048]
(Pharmaceutical composition)
The candidate compound thus selected can be prepared as a pharmaceutical composition by further selecting in consideration of the balance between biological usefulness and toxicity. In addition, a polypeptide or peptide comprising the novel USP according to the present invention and a derivative thereof, a polynucleotide according to the present invention or a complementary strand thereof, a vector comprising the polynucleotide or the complementary strand thereof, and a polypeptide comprising the novel USP and a derivative thereof A peptide or an antibody that immunologically recognizes a peptide itself is a novel USP or the expression or physiological activity of the above-mentioned polypeptide having a physiological activity equivalent to the USP, such as an increase, decrease or deletion of deubiquitination activity, etc. It can be used for prevention and / or treatment of various pathological symptoms caused by. That is, this invention provides the pharmaceutical composition containing at least 1 of these by using these individually or in combination of multiple. In formulating, formulation means suitable for each target such as a polypeptide, peptide, protein, polynucleotide, antibody, etc., known per se may be introduced.
[0049]
As a method for treating abnormal symptoms caused by the expression of the novel USP according to the present invention or the above polypeptide having a physiological activity equivalent to the USP and / or the decrease or deletion of the physiological activity, the USP is used as one method. A compound that enhances the physiological activity of the polypeptide itself or a physiological activity equivalent to that of the USP (enhancing agent) and / or a therapeutically effective amount of a compound (accelerating agent) that promotes the expression of the gene encoding the USP And a method characterized in that it is administered together with a pharmaceutically acceptable carrier, thereby improving abnormal symptoms. Alternatively, gene therapy may be used to generate the activity of a novel USP or a polypeptide having a physiological activity equivalent to that of the USP in cells in a subject. For gene therapy using the above-mentioned polynucleotide, known methods can be used. For example, a replication-deficient retrovirus vector incorporating the polynucleotide according to the present invention may be prepared and used for gene therapy as described above. Also, for example, cells derived from a subject can be treated ex vivo with, for example, a retroviral plasmid vector using DNA or RNA encoding the protein, and then the cells can be introduced into the subject. it can.
[0050]
When the expression of the novel USP according to the present invention or the above-mentioned polypeptide having physiological activity equivalent to the USP and / or its physiological activity is excessive, an effective amount of the inhibitor compound is administered to the subject together with a pharmaceutically acceptable carrier. Thus, the physiological activity of the novel USP or the above-mentioned polypeptide having a physiological activity equivalent to that of the USP can be inhibited, whereby abnormal symptoms can be improved. For example, a partial polypeptide or peptide of a novel USP that inhibits the physiological activity of the novel USP, such as deubiquitination activity, can be used as an antagonist of the novel USP. Specifically, for example, the physiological activity of the novel USP can be inhibited by administering the polypeptide described in SEQ ID NO: 3 in the sequence listing to a subject together with a pharmaceutically acceptable carrier. Alternatively, a partial polypeptide or peptide of a novel USP that inhibits the physiological activity of the novel USP, such as the deubiquitination activity, such as the polypeptide of SEQ ID NO: 3, and a polynucleotide encoding the polypeptide Then, it may be generated in cells in the subject as described above by gene therapy. Thereby, it is possible to prevent and / or treat a disease caused by the expression of the above-mentioned polypeptide having a physiological activity equivalent to that of the novel USP or the novel USP and / or excessive physiological activity thereof. The antagonist can also be used as a component of the pharmaceutical composition.
[0051]
Furthermore, expression of a gene encoding the above-mentioned polypeptide having an endogenous new USP or a physiological activity equivalent to that of USP may be inhibited using an expression blocking method. An antisense sequence of the gene generated intracellularly or administered separately can be used to inhibit the expression of the gene. These oligonucleotides can be designed and synthesized based on the polynucleotide according to the present invention. The oligonucleotide can be administered per se or the relevant oligomer can be expressed in vivo.
[0052]
The administration form may be systemic administration or local administration. One preferred embodiment of systemic administration includes injection, for example intravenous injection. Other injection routes such as subcutaneous, intramuscular or intraperitoneal can also be used. Another mode of administration can be oral if the enteric or capsule formulation is well formulated. In addition, transmucosal or transdermal administration using penetrants such as bile salts, fusidic acid, or other surfactants can be used. For topical administration, forms such as salves, pasta and gels can be used.
[0053]
The required dose range is a polypeptide or peptide comprising a novel USP and its derivatives, a polynucleotide encoding these or a complementary strand thereof, a vector comprising the polynucleotide or its complementary strand, a polypeptide comprising a novel USP and its derivative. It depends on the effectiveness of the peptide or the antibody immunologically recognizing the peptide, the compound, the antagonist, and the pharmaceutical composition, the route of administration, the nature of the formulation, the nature of the subject's symptoms, and the judgment of the attending physician. Specifically, a suitable dose is, for example, in the range of 0.1 to 100 μg / kg of the subject's body weight. However, these dose modifications can be made using general routine experimentation for optimization well known in the art.
[0054]
In formulating, for example, known formulation means corresponding to the physical properties of each target such as peptides, proteins, oligonucleotides, polynucleotides, antibodies, and compounds may be introduced. Specifically, for example, methods for formulating powders, pills, tablets, capsule preparations, aqueous solution preparations, ethanol solution preparations, liposome preparations, fat emulsions, inclusion bodies such as cyclodextrins and the like can be used.
[0055]
Powders, pills, capsules and tablets are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxypropyl It can be produced using a binder such as cellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin. For the production of tablets and capsules, solid pharmaceutical carriers are used.
[0056]
The suspension can be produced using water, sugars such as sucrose, sorbitol, and fructose, glycols such as PEG, and oils.
[0057]
Injectable solutions can be prepared using a carrier consisting of a salt solution, a glucose solution, or a mixture of saline and glucose solution.
[0058]
Liposomeization is performed, for example, by adding a solution in which the substance is dissolved in a solvent (ethanol, etc.) to a solution in which phospholipid is dissolved in an organic solvent (chloroform, etc.), then distilling off the solvent and adding a phosphate buffer solution to the solution. Additionally, after shaking, sonication and centrifugation, the supernatant can be filtered and collected.
[0059]
For fat emulsification, for example, the substance, oil components (vegetable oils such as soybean oil, sesame oil, olive oil, etc., MCT, etc.), emulsifiers (phospholipids, etc.), etc. are mixed and heated to a solution, and then the required amount of water is added. , Emulsification and homogenization can be performed using an emulsifier (homogenizer, such as a high-pressure jet type or an ultrasonic type). It can also be lyophilized. When emulsifying fat, an emulsification aid may be added. Examples of the emulsification aid include glycerin and saccharides (eg, glucose, sorbitol, fructose, etc.).
[0060]
For cyclodextrin inclusion, for example, a solution in which cyclodextrin is heated and dissolved in water or the like is added to a solution in which the substance is dissolved in a solvent (such as ethanol), and then cooled and the deposited precipitate is filtered and sterilized and dried. It can be done by doing. At this time, the cyclodextrin used may be appropriately selected from cyclodextrins (α, β, γ types) having different pore diameters according to the size of the substance.
[0061]
A dysfunction such as USP expression or increase, decrease, or deletion of physiological activity causes abnormalities in the ubiquitin system associated with USP, which in turn causes pathological symptoms. For example, it has been suggested that USP abnormalities are associated with carcinogenesis and neurodegenerative diseases (Non-Patent Documents 17 to 19). USP is also involved in maintaining the chromosomal structure, and it is known that deubiquitination of ubiquitinated histones is important for chromosome aggregation (Non-patent Document 20) and is one of the USP families. It has been reported that USP16 deubiquitinates H2A (Non-patent Document 21). In addition, many protein aggregates observed in Alzheimer's disease and Parkinson's disease react with anti-ubiquitin antibodies (Non-patent Document 17), suggesting a relationship between neurodegenerative diseases and USP dysfunction. It has also been reported that the ubiquitin pathway is associated with muscle atrophy (Non-patent Document 22). The USP according to the present invention has a relatively high expression in brain and skeletal muscle, particularly skeletal muscle, and it is highly likely that the USP is related to neurodegenerative diseases and muscle atrophy. Therefore, the present invention is intended to elucidate biological functions involved in USP, e.g., elucidation of carcinogenic processes, elucidation of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, elucidation of muscular atrophy, and their inhibitors and / or It enables the development of therapeutic agents and the development of measurement methods used as diagnostic means for them, and is very useful.
[0062]
(Measurement methods and reagents for diagnosis)
A polypeptide or peptide comprising the novel USP and its derivative according to the present invention, a polynucleotide according to the present invention and its complementary strand, and an antibody that immunologically recognizes the polypeptide or peptide comprising the USP and its derivative As a diagnostic marker, a reagent, etc., it can be used in a method for quantitatively or qualitatively measuring the novel USP according to the present invention and a polypeptide derived therefrom or a polynucleotide encoding them. The present invention also provides a reagent kit comprising one or more containers filled with one or more of these. The reagent kit can be used for the identification method and the measurement method. In formulating, a formulation means corresponding to each known polypeptide or peptide, protein, polynucleotide, antibody or the like may be introduced. According to the measurement method described above, it is possible to diagnose various pathological symptoms related to the expression, activity increase, decrease or deletion of new USP.
[0063]
The diagnostic means for a disease caused by abnormal expression or physiological activity of a peptide or polypeptide comprising the novel USP and its derivative according to the present invention utilizes, for example, interaction and reactivity with a nucleic acid encoding the USP. Determining the abundance of the corresponding nucleic acid and / or determining the biodistribution in the individual for the USP and / or determining the presence of the USP in the sample from the individual Is done by. Specifically, the new USP is tested as a diagnostic marker. Measurement methods that can be used to detect the USP in a sample or to determine its abundance are well known to those skilled in the art. Such measurement methods include radioimmunoassay, competitive binding assay, Western blot analysis, and ELISA assay. In addition, as a method for detecting and quantifying a polynucleotide encoding the novel USP according to the present invention, measurement is performed at the RNA level using, for example, amplification, PCR, RTPCR, RNase protection, Northern blotting, and other hybridization methods. be able to.
[0064]
Examples of the sample to be measured include cells derived from an individual, such as blood, urine, saliva, spinal fluid, tissue biopsy or autopsy material. Moreover, the nucleic acid to be measured is obtained from each of the above samples by a known nucleic acid preparation method. Nucleic acids may be used directly for detection of genomic DNA or may be amplified enzymatically by using PCR or other amplification methods prior to analysis. RNA or cDNA may be used as well. In comparison with the normal genotype, deletions and insertions can be detected by a change in size of the amplified product. Point mutations can be identified by hybridizing amplified DNA to labeled DNA encoding the USP.
[0065]
By detecting the mutation, decrease, or increase of the USP according to the present invention and the DNA encoding the USP by the above measurement, diseases caused by abnormalities in the USP, such as cancer or neurodegenerative diseases such as Alzheimer's disease, Diagnosis such as Parkinson's disease becomes possible.
[0066]
【Example】
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated more concretely, this invention is not limited to the following Example.
[Example 1]
(Isolation and identification of new USP gene)
The USP gene according to the present invention was extracted from the human long-chain cDNA analysis information database of Kazusa DNA Laboratory as a novel protease candidate gene by bioinformatics. The base sequence of the 3 ′ end region of this gene was a sequence common to cDNA clone KIAA1057 (GenBank accession number: AB026980). The cDNA clone KIAA1057 is a clone containing 5618 bp containing a region encoding 977 amino acids, and is a Cys domain (Cys-box) (G- [LIVFMFY] -x (1,3 )-[AGC]-[NASM] -xC- [FYW]-[LIVMFC]-[NST]-[SACV] -x- [LIVMS] -Q) and His domain (His-box) (Yx) -L-x- [SAG]-[LIVMFT] -x (2) -Hx-Gx (4,5) -GHY) and aspartic acid (Asp) domains. The gene according to the present invention has one amino acid residue that does not match the known domain sequence in the Cys domain.
[0067]
Since the cDNA clone KIAA1057 was presumed to have been cloned by deleting the 5 'terminal region in the ORF, the above database was further searched based on the base sequence of KIAA1057. As a result, a cDNA clone bf04274 containing the base sequence of KIAA1057 was found.
[0068]
bf04274 is a cDNA consisting of 7744 bp. The first ATG present at positions 389 to 391 of the base sequence was predicted to be a translation initiation codon because it had a Kozak consensus sequence immediately before that. That is, bf04274 includes a translation region (CDS) encoding a polypeptide consisting of 1556 amino acid residues at positions 389 to 5059 of the base sequence. The base sequence of bf04274 is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence encoded by bf04274 is shown in SEQ ID NO: 1. KIAA1057 corresponds to the nucleotide sequence from position 2124 to position 7741 of the base sequence of bf04274, and the deduced amino acid sequence of KIAA1057 corresponds to the amino acid sequence from position 580 to 1556 of the amino acid sequence encoded by bf04274. . Cys-box and His-box are present in the 626th to 641st and 890th to 907th amino acid sequences of the amino acid sequence encoded by bf04274.
[0069]
[Example 2]
(Expression of new USP)
The cDNA clone bf04274 expression plasmid encoding the novel USP isolated and identified in Example 1 was used as a gateway. ^TM _Cloning_Technology (Life Technologies) was used. First, bf04274 clone (pBC SK ⁺ Using the advantage-HF2_PCR kit (Clontech) or Expand_high-fidelity_PCR_system (Roche) as a template, and the putative CDS region (positions 389 to 5059 of the base sequence described in SEQ ID NO: 2). Was amplified by two-step PCR, and then inserted into an entry vector (pDONR201) by a recombination reaction using BP clonase enzyme to prepare pDONR-bf04274. The primers were bf04274-AttB (SEQ ID NO: 5) and PrDONR1057 (-) (SEQ ID NO: 6) for the first stage PCR, and the AttB1 adapter primer (SEQ ID NO: 7) for the second stage PCR and the above. Pr-DONR1057 (-) was used. Next, using pDONR-bf04274 and N-terminal His-tagged protein expression vector pDEST17, expression of His-tagged bf04274 by recombination reaction with LR clonase enzyme. A plasmid (pHis-04274) was prepared. The expression plasmid is an E. coli that can be induced for expression by NaCl. E. coli BL21-SI (Life Technologies) was introduced. It was confirmed by sequencing that the CDS base sequence was correctly inserted. The sequence reaction was carried out using BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (PE biosystems) using Big Dye_Terminator_Cycle_Sequencing_FS_Ready_Reaction_Kit.
[0070]
Using the expression plasmid prepared above, expression of bf04274 was induced in E. coli. LBON / Amp medium (LB medium containing 50 mg / ml ampicillin but not NaCl) is inoculated with 1/10 amount of E. coli preculture and OD at 37 ° C. ₆₀₀ After culturing until about 1.0, NaCl was added to a final concentration of 0.3M. Further, after culturing for about 4 to 5 hours, the cells were collected. The cells were suspended in phosphate buffered saline (PBS) and then subjected to sonication, and the supernatant was collected by centrifugation to obtain an extract. The extract was separated by 10% SDS-PAGE and anti-His-tag antibody (Penta-His ^TM Immunoblotting was performed using an antibody (QIAGEN). As a result, a protein (186.8 kDa) having the same molecular weight as that predicted from the deduced amino acid sequence was detected. Most of the expressed protein was found in the insoluble fraction and the distribution to the soluble fraction was small. For detection, an ECL western blotting detection kit (Amersham pharmacia biotech) was used.
[0071]
[Example 3]
(Deubiquitination activity of bf04274)
The protein encoded by bf04274 was examined for its deubiquitination activity in a co-expression system with a substrate. In this experimental system, an enzyme and a substrate are co-expressed in the same cell by using a substrate expression plasmid having p15A ori that is compatible with the pBR322 ori of the enzyme expression plasmid. ("Archives Of Biochemistry And Biophysics", 2000, 379, p. 198-).
[0072]
First, various substrate expression plasmids for co-expression were constructed. As the substrate, an artificial substrate Ub-GST in which glutathione_S-transferase (GST) was bound to the C terminus of human ubiquitin was used. After amplifying the Ub-GST coding region by PCR using pTV118N / Ub-GST as a template, the gateway ^TM_ Using cloning technology, it was inserted into the vector for expression of E. coli, pDEST14 to prepare pDEST14Ub-GST. Next, a region containing the T7 promoter and the Ub-GST coding region was excised from pDEST14Ub-GST by treatment with SphI and HindIII, and incorporated into SphI-HindIII of pACYC184 (Nippon Gene) having ori derived from p15A, and pACUb-M-GST Was made. This was used as a Ub-M-GST expression plasmid for co-expression. Next, pACUb-M-GST was treated with SalI and HindIII, and the region containing the T7 promoter and Ub-GST coding region was incorporated between pBluescriptII SK (-) (Stratagene) SalI-HindIII to prepare pBSUbGST. Further, using pBSUbGST as a template, a codon corresponding to the N-terminal amino acid of GST is obtained from ATG (methionine: M) using a QuickChange Site-Directed Mutagenesis Kit (Stratagene). PBSUb-P-GST, pBSUb-I-GST, or pBSUb-R-GST converted to CCG (proline: P), ATC (isoleucine: I), or CGT (arginine: R). Codon conversion was confirmed by sequencing. Hereinafter, Ub-M-GST, Ub-R-GST, Ub-P-GST, and Ub-I-GST are collectively referred to as Ub-X-GST. Next, each pBSUb-X-GST was treated with HindIII and SphI, and a region containing the T7 promoter and Ub-X-GST coding region was integrated between HindIII-SphI of pACYC184, and Ub-X-GST expression for each co-expression. A plasmid, pACUb-X-GST, was prepared. The four types of pACUb-X-GST produced were E. coli. E. coli BL21-SI was made into competent cells by the calcium chloride method.
[0073]
E. holding each pACUb-X-GST. The plasmid pHis-04274 prepared in Example 2 was introduced into E. coli BL21-SI competent cells. At this time, in some competent cells, instead of pHis-04274, a known ubiquitin specific protease 15 (hereinafter abbreviated as USP15) cDNA (KIAA0529 clone) as a template was used as a template, and the method described in Example 2 was used. The His-tagged USP15 expression plasmid (pHis-USP15) prepared in step 1 or a His-tagged luciferase (Luc) expression plasmid (pHis-Luc) was introduced as a negative control. The gene-introduced competent cells were cultured on LBON (LB medium without NaCl) plate containing 50 mg / ml ampicillin and 34 mg / ml chloramphenicol, and various co-expression strains were selected and obtained. . In the preparation of pHis-USP15, KIAA0529 was deleted from the N-terminal 3 amino acid residues (MAE) of USP15, and therefore, according to the method of Baker et al. (“Genomics”, 1999, Vol. 59, p. 264-), a codon corresponding to these 3 amino acid residues was designed in the primer to form a complete CDS. PHis-Luc is a gateway similar to Example 2 using pGL3-Luc vector (Promega) as a template. ^TM _Cloning_ technology was used to make.
[0074]
The various co-expression strains were OD in LBON medium containing 50 mg / ml ampicillin and 34 mg / ml chloramphenicol. ₆₀₀ After culturing at 37 ° C. until about 0.5 to 1.2, NaCl was added to a final concentration of 0.3M. After further cultivation for about 4 or 5 hours, the cells were collected. The cells were suspended in 1/10 volume of 50 mM Tris-HCl, pH 7.6 / 5 mM ethylenediaminetetraacetic acid (EDTA) / 1 mM dithiothreitol (DTT), and then disrupted by sonication and centrifuged. Thus, the supernatant was recovered to prepare an E. coli extract. The extract was separated by 15% SDS-PAGE, and anti-GST antibody (Amersham pharmacia biotech) was used as the primary antibody, and horseradish_peroxidase (HRP) -labeled anti-goat antibody (Alpha diagnostic international) was used as the secondary antibody. And immunoblotting was performed to detect Ub-X-GST and released GST. For detection, an ECL Western blotting detection kit was used.
[0075]
As a result, as shown in FIG. 1, in the extract prepared from Escherichia coli co-expressed with plasmid pHis-04274 and Ub-R-GST, Ub from Ub-R-GST other than Ub-R-GST GST produced by hydrolysis was detected. However, GST was not detected in the co-expression system with Ub-M-GST, Ub-P-GST, or Ub-I-GST. On the other hand, when USP15, a positive control, was co-expressed with a substrate instead of bf04274, deubiquitination activity was observed for any of the above four types of substrates, but not with luciferase, a negative control. It was.
[0076]
Thus, the protein expressed by plasmid pHis-04274 showed substrate selectivity against an artificial substrate in which Ub was bound to the protein via an arginine (R) residue. Substrate selectivity was similarly examined for known USPs, but only the new USP according to the present invention showed substrate selectivity for Ub-R-GST. Ub-GST is considered a precursor Ub model because Ub is peptide-bonded to protein (GST). Since the protein encoded by bf-04274 dissociates Ub using Ub-GST as a substrate, it may be involved in Ub generation from precursor Ub in vivo.
[0077]
[Example 4]
(Identification of enzyme active site of protein encoded by bf-04274)
USP is one of cysteine proteases, and cysteine residues present in the Cys-box are considered to be active sites. Therefore, a mutant (bf04274) in which the 634th cysteine (C), which is a putative active residue of the protein encoded by bf04274, is substituted with serine (S). ^C634S ) Was produced. First, the 634th cysteine was replaced with serine using a quick change_site-directed_mutagenesis_kit. The primer used was designed so that TGT which is the codon of 634th cysteine was replaced with TCT which was the codon of serine (SEQ ID NO: 8). Mutation introduction was confirmed by sequencing, and His-tagged bf04274. ^C634S An expression plasmid, pHis-04274Mut, was obtained. Sequencing reaction was performed using Cy5_thermo-sequenase_die_therminator_kit (ThermoSequenase Dye Thermistor Kit), and electrophoresis and analysis were performed using Long Read Tower (both performed by Amersham Pharmacia Biotech) .
[0078]
pHis-04274Mut, pHis-04274, pHis-USP15, or pHis-Luc prepared in Example 2 or Example 3 were co-expressed in Escherichia coli together with pACUb-R-GST in the same manner as in Example 3. As a result, as shown in FIG. 2, the deubiquitination activity for Ub-R-GST observed when bf04274 was expressed was ^C634S It was found to disappear. That is, it was confirmed that the protein encoded by bf04274 is a cysteine protease having the 634th cysteine residue as an active site.
[0079]
[Example 5]
(Examination of deubiquitination activity of gene product encoded by KIAA1057-1) Example of plasmid containing KIAA1057-1 encoding a polypeptide in which the N-terminal side of bf04274 is deleted by 521 amino acid residues (SEQ ID NO: 3) 2 and co-expressed with E. coli together with the substrate in the same manner as in Example 3 to examine its deubiquitination activity. As a result, KIAA1057-1 possesses Cys-Box and His-Box, which are features of the USP family, but did not show deubiquitination activity in the co-expression system with each of the four types of artificial substrates ( FIG. 3).
From the above results, it is considered that a site involved in substrate recognition is present on the N-terminal side of the protein encoded by bf04274, as in USP described in the literature (Non-patent Document 15).
[0080]
【The invention's effect】
A ubiquitin-specific protease that extracts bf04274 as a novel protease candidate gene from the long-term cDNA analysis information database of Kazusa DNA Research Institute by bioinformatics, and the gene product of this gene deubiquitinates the ubiquitinated protein I found it to be one of (USP). Furthermore, the USP according to the present invention has found that the deubiquitination activity disappears when 521 amino acid residues are deleted from the N-terminus.
The present invention enables elucidation of biological functions involved in USP, for example, elucidation of carcinogenic processes, muscle atrophy, and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and prevention, treatment and diagnosis thereof. Is very useful.
[0081]
[Sequence Listing Free Text]
SEQ ID NO: 5: oligonucleotide designed for use as a primer.
SEQ ID NO: 6: oligonucleotide designed for use as a primer.
SEQ ID NO: 7: oligonucleotide designed for use as a primer.
SEQ ID NO: 8: oligonucleotide designed for use as a primer.
[0082]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows that the deubiquitination activity of bf04274 was expressed in E. coli together with bf04274 and an artificial substrate (Ub-R-GST, Ub-M-GST, Ub-I-GST, or Ub-P-GST). It is a figure which shows having been recognized by the co-expression system. In the figure, USP15 and luciferase are a positive control and a negative control for deubiquitination activity, respectively. The numerical value on the left side of the lane indicates the molecular weight.
[Fig. 2] Substitution of the 634th cysteine (C) residue from the N-terminus, which is a putative enzyme active site of bf04274, with a serine (S) residue (bf04274). ^C634S ), The deubiquitination activity disappeared. In the figure, USP15 and luciferase are a positive control and a negative control for deubiquitination activity, respectively. The numerical value on the left side of the lane indicates the molecular weight.
FIG. 3 shows that the deubiquitination activity of KIAA1057-1 in which 521 amino acid residues are deleted from the N-terminus of bf04274 shows that KIAA1057-1 and artificial substrates (Ub-R-GST, Ub-M-GST, Ub-I) -GST or Ub-P-GST) is a figure showing that it was observed in a co-expression system in which E. coli was expressed together. In the figure, USP15 and luciferase are a positive control and a negative control for deubiquitination activity, respectively. The numerical value on the left side of the lane indicates the molecular weight.

Claims (15)

A polypeptide selected from the group consisting of the following (1) to (3) :
(1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing ;
(2) a polypeptide comprising a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and having deubiquitination activity ;
and
(3) A polypeptide having a deletion, substitution, addition, or insertion of one to several amino acids in the amino acid sequence and having deubiquitination activity in the polypeptide of (1) or (2) .   A polynucleotide encoding the polypeptide of claim 1 or a complementary strand thereof.   A polynucleotide comprising the base sequence set forth in SEQ ID NO: 2 in the sequence listing or a complementary strand thereof. A polynucleotide that hybridizes with one polynucleotide selected from the group consisting of the following (1) to (3) or a complementary strand thereof under a stringent condition and encodes a polypeptide having deubiquitination activity :
(1) a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing;
(2) a polynucleotide comprising a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and encoding a polypeptide having deubiquitination activity;
and
(3) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 2 in the sequence listing .   A recombinant vector containing the polynucleotide according to any one of claims 2 to 4.   The recombinant vector according to claim 5, wherein the recombinant vector is an expression recombinant vector.   A transformant into which the recombinant vector according to claim 5 or 6 has been introduced.   A method for producing the polypeptide according to claim 1, wherein a step of culturing a transformant into which the recombinant vector according to claim 6 has been introduced, or the recombination according to claim 5 or 6 is provided. A method comprising cell-free protein synthesis means using a vector.   An antibody that immunologically recognizes the polypeptide of claim 1. The identification method of the compound which inhibits or enhances the deubiquitination activity of the polypeptide of Claim 1 which comprises the following process (i) or (ii) .
(I) a step of adding a test compound to a system for co-gene expression of the polypeptide of claim 1 and its substrate; and a step of measuring deubiquitination activity.
(Ii) a step of bringing a transformant into which the recombinant vector containing the polynucleotide according to any one of claims 2 to 4 has been introduced into contact with a test compound; and measuring deubiquitination activity Process. A method for identifying a compound that inhibits or enhances the deubiquitination activity of a polypeptide according to claim 1, wherein the polypeptide is allowed to interact with the polypeptide under conditions that allow interaction between the test compound and the polypeptide. contacting a test compound, followed by detecting the presence or absence or change in signal caused by the interaction of the test compound and the polypeptide, the test compound to de-ubiquitination activity of the polypeptide wherein the determining whether to inhibit or promote. The method according to claim 11, comprising the following step (i) or (ii).
(I) a step of adding a test compound to a system for co-gene expression of the polypeptide of claim 1 and its substrate; and a step of measuring deubiquitination activity.
(Ii) a recombinant vector containing the polynucleotide according to any one of claims 2 to 4; A step of bringing a transformant into which a test substance has been introduced into contact with a test compound; and a step of measuring deubiquitination activity. A method for quantitatively or qualitatively measuring the polypeptide according to claim 1 or the polynucleotide according to any one of claims 2 to 4 , comprising the following step (1) or (2): Method.
(1) A step of measuring the deubiquitination action of the polypeptide by detecting dissociation of ubiquitin from the substrate using the polypeptide and its substrate.
(2) Using a gene co-expression system in which the polynucleotide and its substrate are co-expressed, the deubiquitination of the polypeptide encoded by the polynucleotide is detected by the dissociation of ubiquitin from the substrate by the polypeptide. The process to measure by. A method for quantitatively or qualitatively measuring the polypeptide according to claim 1 or the polynucleotide according to any one of claims 2 to 4, wherein the polypeptide or Methods comprising measuring polynucleotides: radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays, as well as PCR, RTPCR, RNase protection, Northern blotting, and hybridization methods.   A polypeptide according to claim 1, a polynucleotide according to any one of claims 2 to 4, a recombinant vector according to claim 5 or 6, a transformant according to claim 7, and A reagent kit comprising at least one of the antibodies according to claim 9.

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