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Studies on Regulation of Cellular Function by Calpastatin

钙蛋白酶抑制剂调节细胞功能的研究

基本信息

批准号：
01580157
负责人：
MAKI Masatoshi
金额：
$ 1.28万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01580157/
关键词：
Calpain Calpastatin Proteinase Proteinase Inhibitor Protein Kinase C calcium channel プロテア-ゼ

项目摘要

Calpastatin is endogenous inhibitor protein acting specifically on intracellular calcium dependent proteinase(calpain). In the present study we extended structure-function analyses of calpastatin and examined roles of this inhibitor in animal cells. The major results obtained were as follows :1. Locus of human calpastatin gene was assigned to chromosome 5ql4-22.2. Highly conserved regions among the four internally repetitive domains were separated by introns in the genomic DNA.3. The 27-residue synthetic oligopeptide corresponding to'the Exon 1B was active as calpain inhibitor, and it suppressed TPA induced down-regulation of protein kinase C when added to culture medium of BIM cells(neuroblastoma cell line).4.2-dimensional NMR studies have revaeled that the oligopeptide forms type I beta-turn structure in the highly conserved sequence.5. Monoclonal antibodies were prepared for human calpastatin, and a method of distinguishing two types(muscle and erythrocyte)of calpastatin molecules were developed.In contrast to mammalian erythrocytes, chicken erythrocytes(nucleated cells)were suggested to retain amino-terminal domains of calpastatin and to have a muscle type structure.6. MRNA and protein levels of m-calpain and calpastatin were found to be elevated in HTLV-I infected T-cell leukemia cell lines, but not in HTLV-I tax-transformed fibroblast cell lines.7. Besides calpain inhibition, calpastatin was suggested to have L-type calcium channel activating function.

钙蛋白酶抑制素是一种内源性抑制蛋白，特异性作用于细胞内钙依赖蛋白酶（calpain）。在本研究中，我们扩展了钙蛋白酶抑制剂的结构-功能分析，并检查这种抑制剂在动物细胞中的作用。主要研究结果如下：1.人钙蛋白酶抑制素基因定位于染色体5 q14 -22.2。在基因组DNA中，四个内部重复结构域之间的高度保守区域被内含子隔开。合成的27个氨基酸残基的寡肽对应于外显子1B，具有钙蛋白酶抑制剂活性，加入到BIM细胞（神经母细胞瘤细胞系）培养液中，可抑制TPA诱导的蛋白激酶C的下调。4.二维核磁共振研究表明，该寡肽在高度保守的序列中形成I型β-转角结构。制备了人钙蛋白酶抑制剂的单克隆抗体，建立了区分肌型和红细胞型钙蛋白酶抑制剂的方法，并与哺乳动物红细胞（有核细胞）进行了比较，结果表明鸡红细胞（有核细胞）保留了钙蛋白酶抑制剂的氨基端结构域，具有肌型结构.发现m-钙蛋白酶和钙蛋白酶抑制素的mRNA和蛋白水平在HTLV-I感染的T细胞白血病细胞系中升高，但在HTLV-I tax转化的成纤维细胞系中不升高。除钙蛋白酶抑制作用外，钙蛋白酶抑制蛋白还具有L-型钙通道激活作用。