The long-term objectives of this research proposal are two-fold: 1) to investigate the potential role of cytosolic, calcium-dependent endoproteases in the degradation of selected vascular smooth muscle cytoskeletal proteins and 2) to explore the possible role of these enxymes in disassembly of the smooth muscle cytoskeleton during the early stages of cell transformation and proliferation. We exntertain the hypothesis that calcium-dependent proteolysis is an important early step in cell proliferation and that this proteolysis is fundamental to remodeling on the vascular smooth muscle cell cytoskeleon.
The specific aims that will be pursued in order to attain the long-term objectives are: 1) to purify vascular calcium-dependents proteases and characterize certain physical and structural properties, 2) to explore several avenues of potential regulation, including intramolecular and exogenous proteolysis, the presence and effect of endogenous activators and inhibitors and various forms of covalent and non-covalent protein modification, 3) to examine several purified vascular smooth muscle proteins as substrates in vitro and to determine the kinds of fragments produced, the functional activity of these fragments and what factors specifically promote or protect the selected proteins from calcium-dependent proteolysis, 4) to determine the effects of calcium-dependent proteases on contractile activity and ultrastructures of chemically-skinned vascular smooth muscle, and 5) to investigate the localization and potential function of the calcium-dependent proteases in cultured vascular smooth muscle cells treated with agents that may activate calcuim-dependent proteolysis, transform the cells or stimulate proliferation. A variety of methods will be employed including protein purification, analysis of protein structure, radioimmunoassay, measurements of muscle mechanical properties, electron microscopy and cell culture. We believe this research is important because of the link between mitogenesis and specific calcium-dependent proteases. Moreover, we believe this research pertains directly vascular smooth muscle cell proliferation which has been implicated as an early step in the disease, atherosclerosis.
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