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.
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