CALPAINS IN REGULATION OF FIBROBLAST PROLIFERATION

钙蛋白酶调节成纤维细胞增殖

• 批准号: 6302211
• 负责人: RONALD L MELLGREN
• 金额: $ 15.82万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6302211
• 关键词: antisense nucleic acid; autoradiography; calpain; cardiac myocytes; cell growth regulation; cell line; cell proliferation; enzyme substrate; fibroblasts; gel electrophoresis; heart cell; heart metabolism; immunoprecipitation; laboratory rat; myocardium; protease inhibitor; protein purification; protein sequence; proteolysis; tissue /cell culture; tumor suppressor proteins

Mechanisms regulating heart cell growth have attracted much interest, since hypertrophy of cardiomyocytes and proliferative growth of cardiac fibroblasts is a hallmark of pathologic myocardial hypertrophy and associated fibrosis. It has recently become apparent that selective, regulated proteolysis of intracellular protein targets is an important mechanism in regulation of cell growth. The calcium-dependent intracellular proteases, calpains, appear to be required for cell growth; however, the mechanism is not yet known. The long range hypothesis to be tested is that calpains catalyze proteolysis of signal transducing proteins, allowing progression through critical steps in cell growth regulatory pathways. In stable cell lines and cardiac fibroblasts, growth- promoting signal transduction pathways which require calpains will be defined (specific aim 1). As probes for calpain function, cell-permeant calpain inhibitors will be utilized in conjunction with growth factors known to stimulate hyperplastic growth of fibroblasts. In other studies, established cell lines will be permanently transfected with plasmids bearing cDNAS for mu-calpain which may serve as dominant-negative mutants. These studies will be complemented by isolation of growth-related calpain substrates by affinity absorption to a calpain-gel or co- immunoprecipitation with endogenous calpains (specific aim 2). Cells will be labeled with 35S-Met, and purified proteins detected on 2D-gel electrophoresis followed by autoradiography. Known signal transduction proteins will be detected by western blots of the 2D gels. Unique major protein bands will be purified. Partial peptide sequences will be obtained for generation of antibodies, and isolation of the protein by cDNA cloning. Other experiment will focus on the calpain-catalyzed proteolysis of the p53 tumor suppressor protein prior to S-phase in serum-stimulated WI-38 fibroblasts (specific aim 3). Specific goals to be achieved by these studies will include determination of the effects of calpain or p53 phosphorylation state of p53 proteolysis, and the possibility that p53- association with nucleic acids or proteins is important for signaling its degradation.

调节心脏细胞生长的机制引起了人们的广泛兴趣， 由于心肌细胞肥大和心脏增殖性生长 成纤维细胞是病理性心肌肥厚的标志 相关纤维化。最近变得很明显，选择性， 细胞内蛋白质靶标的调节蛋白水解是一个重要的 细胞生长调节机制。钙依赖性 细胞内蛋白酶、钙蛋白酶似乎是细胞生长所必需的； 然而，其机制尚不清楚。长期假设为 测试结果是钙蛋白酶催化信号转导蛋白水解 蛋白质，允许细胞生长中关键步骤的进展 监管途径。在稳定细胞系和心脏成纤维细胞中，生长- 促进需要钙蛋白酶的信号转导途径 定义（具体目标 1）。作为钙蛋白酶功能的探针，可渗透细胞 钙蛋白酶抑制剂将与生长因子结合使用 已知可刺激成纤维细胞的增生性生长。在其他研究中， 建立的细胞系将被质粒永久转染 带有 mu-calpain 的 cDNAS，可以作为显性失活突变体。 这些研究将通过生长相关钙蛋白酶的分离得到补充 通过钙蛋白酶凝胶或共酶亲和吸收来底物 内源性钙蛋白酶免疫沉淀（具体目标 2）。细胞会 用 35S-Met 标记，并在 2D 凝胶上检测纯化的蛋白质 电泳后进行放射自显影。已知的信号转导 蛋白质将通过 2D 凝胶的蛋白质印迹进行检测。独特专业 蛋白质条带将被纯化。将获得部分肽序列 用于产生抗体并通过 cDNA 分离蛋白质 克隆。其他实验将集中于钙蛋白酶催化的蛋白水解作用 血清刺激中 S 期之前的 p53 肿瘤抑制蛋白 WI-38 成纤维细胞（具体目标 3）。 具体要达到的目标 这些研究将包括确定钙蛋白酶或 p53 的作用 p53 蛋白水解的磷酸化状态，以及 p53- 的可能性 与核酸或蛋白质的关联对于发出信号非常重要 降解。