Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2000-10-06
2003-12-30
Chan, Christina (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Reexamination Certificate
active
06670450
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel protein involved in myocyte differentiation and DNA encoding the protein.
BACKGROUND OF THE INVENTION
Genes, such as muscle creatine kinase, troponin, caveolin 3, &agr;-actin, and myosin, are reported to be expressed predominantly in the skeletal muscles. A family of transcription factors specifically expressed in the muscles, including myoD, myogenin, myf-5, and MRF-4/herculin/myf-6, have been cloned. These factors are phosphorylated nuclear proteins containing a helix-loop-helix (bHLH) motif, as required for both dimerization and DNA binding, and are believed to be determinants of the cell-specific differentiation program (Olson and Klein (1994), Genes & Dev. 8:1-8). When one of these factors is introduced into non-myogenic cells, differentiation into mature muscle cells is initiated (Weintraub et al. (1991), Science 251:761-766). The myoD family, a group of transcription factors, has been found to direct muscle formation, inhibit proliferation, activate differentiation and induce a contractile phenotype. While myoD and myf-5 are expressed within the proliferating myoblasts, myogenin and MRF-4 are not expressed until the myoblasts withdraw from the cell cycle in response to mitogen withdrawal. Based on these findings, it was demonstrated that myogenin and MRF-4 activate and maintain the expression of muscle-specific genes (Emerson (1993), Curr. Opin. Genet. Dev. 3:265-274), while myoD and myf-5 are thought to play a role in the proliferation of myoblasts. Other cell-cycle regulatory proteins, such as RB (Shiio et al. (1996), Oncogene 12:1837-1845, Wang et al. (1997), Cancer Research 57:351-354), p21 (Guo et al. (1995), Mol. Cell Biol. 15:3823-3829), cyclin D, cdk2, cdk4 (Kiess et al. (1995), Oncogene 10:159-166) and tumor suppressor gene p53 (Soddu et al. (1996), J. Cell Biol. 134:193-204) are involved in the muscle cell differentiation program. Recently, caveolin 3 (Song et al. (1996), J. Cell Biol. 271:15160-15165), &agr;-dystroglycan (Kostrominova and Tanzer (1995), J. Cell Biochem. 58:527-534) and DNA methyltransferases (Takagi et al. (1995), Eur. J. Biochem. 231:282-291) have been shown to play positive roles in myogenic differentiation.
SUMMARY OF THE INVENTION
An objective of the present invention is to provide a novel protein and gene involved in myocyte differentiation, and the production and use thereof.
The inventors carried out an antibody screening, using an antibody raised against a protein specific to immortalized cells, to isolate genes specifically expressed in the immortalized cells. Unexpectedly, a novel gene was isolated, which was not an initial objective gene. By analyzing the isolated gene, the inventors found that this gene is a novel gene showing no significant homology with any known genes deposited in the database, and is strongly expressed in skeletal muscle and undifferentiated cells. The inventors also analyzed the protein encoded by the gene, and found that the protein has an inhibitory effect on the differentiation of myoblasts into myotubes. The inventors also found that the protein interacts with p53, a transcription factor involved in tumor suppression, to inhibit the p53 transactivation function.
The present invention relates to a novel protein having an inhibitory effect on the differentiation of myoblasts into myotubes, and the gene encoding the protein, and the production and the use thereof. More specifically the present invention relates to:
(1) a protein comprising the amino acid sequence of SEQ ID NO:1, or a protein comprising said amino acid sequence in which one or more amino acids are substituted, deleted or added and exhibiting an inhibitory effect on the differentiation of myoblasts into myotubes;
(2) a protein encoded by DNA that hybridizes with the DNA comprising the nucleotide sequence of SEQ ID NO:2, wherein said protein exhibits an inhibitory effect on the differentiation of myoblasts into myotubes;
(3) a DNA encoding the protein according to (1);
(4) a DNA hybridizing with the DNA comprising the nucleotide sequence of SEQ ID NO:2, wherein said DNA encodes a protein exhibiting an inhibitory effect on the differentiation of myoblasts into myotubes;
(5) a vector containing the DNA according to (3);
(6) a transformant retaining the DNA according to (3) in an expressible manner;
(7) a method for producing the protein according to (1) or (2), said method comprising culturing the transformant according to (6);
(8) an antibody binding to the protein according to (1) or (2);
(9) a method of screening for a compound that binds to the protein according to (1) or (2), said method comprising the steps of:
a) contacting a test sample with said protein or a partial peptide thereof;
b) detecting the binding activity of the test sample to said protein or a partial peptide thereof; and
c) selecting a compound binding to said protein or a partial peptide thereof;
(10) a compound, binding to the protein according to (1) or (2), wherein said compound can be isolated using the method according to (9);
(11) a method of screening for a compound that promotes or inhibits the activity of the protein according to (1) or (2), the method comprising the steps of:
a) contacting myoblasts with said protein in the presence of a test sample;
b) detecting the differentiation of the cells into myotubes; and
c) selecting a compound which can increase or decrease the inhibitory activity of the protein, compared with its inhibitory activity as detected in the absence of said test sample;
(12) a method of screening for a compound that promotes or inhibits the activity of the protein according to (1) or (2), said method comprising the steps of:
a) providing p53-deficient cells with a vector expressing said protein, a vector expressing p53, and a vector expressing a reporter gene in response to p53;
b) contacting a test sample with said cells;
c) detecting the reporter activity in said cells; and
d) selecting a compound that can reduce or increase the reporter activity compared with the activity in the cells without contact with said test sample (control);
(13) a compound that promotes or inhibits the activity of the protein according to (1) or (2), wherein said compound can be isolated using the method according to (11) or (12); and
(14) a DNA comprising at least 15 nucleotides in length and specifically hybridizing with the DNA comprising the nucleotide sequence of SEQ ID NO:2.
The present invention relates to a novel protein, “striamin,” that inhibits the differentiation of myoblasts into myotubes. (The inventors initially designated the protein “striatin” in the original application (Japanese Patent Application No. Hei 10-115975), but another protein was later found to have the same name; hence the renaming to “striamin”). The nucleotide sequence of striamin cDNA derived from mouse DNA is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO:2. As shown in SEQ ID NO:1, mouse striamin cDNA has an ORF encoding a protein of 149 amino acids. As determined by immunoprecipitation of the striamin protein translated in vitro (FIG.
2
A), and by Western blotting of the recombinant striamin protein (FIG.
2
B), the mouse-derived striamin protein has a molecular weight of about 18 kDa. Northern blot analysis showed that the striamin gene is expressed in the undifferentiated cells, and that the expression of this gene is inhibited during myoblast differentiation into myotubes (FIG.
4
C). Overexpression of the gene actually blocked the differentiation of myoblasts into myotubes (FIG.
5
). These facts suggest that the striamin protein is involved in the duration of the undifferentiated state of the cells.
The expression of the striamin protein also inhibited expression of the p53 transactivation function. The expression of this transcription factor is known to be upregulated during muscle differentiation (FIG.
7
and FIG.
8
). The striamin protein was further shown to interact with p53 both in vivo and in vitro (FIG.
9
and FIG.
10
). It is reported that p53
Kaul Sunil C.
Reddel Roger R.
Wadhwa Renu
Belyavskyi Michail
Chan Christina
Chugai Seiyaku Kabushiki Kaisha
Fish & Richardson P.C.
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