DEGRADATION OF E12/E47 IN SMOOTH MUSCLE CELLS

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DESCRIPTION (Adapted from applicants' abstract) The proliferation and dedifferentiation of vascular smooth muscle cells (VSMC) is a principal component of occlusive vascular disease, which is the leading cause of death in developed countries. As a preventive cardiologist, the candidate's principle focus has been to identify and treat the root causes of atherosclerosis in patients with established cardiovascular disease. As an investigator, he seeks to further the understanding of this disease. The basic helix-loop-helix proteins E12 and E47 promote cellular differentiation and retard cellular proliferation. They are expressed in VSMC. The applicant cloned a novel ubiquitin conjugating enzyme, UBC-E2A, directed against the E12 and E47. The ubiquitin-proteasome system can dramatically alter the cellular phenotype by regulating the degradation of trans-acting factors. By targeting E12 and E47 for turnover, UBC-E2A may promote VSMC proliferation and dedifferentiation. The project herein will characterize E12 and E47 protein degradation in VSMC. First he will map the UBC-E2A binding site on the E2A proteins, then he will determine the role of E2A protein phosphorylation on their degradation. This analysis will permit the design of a peptide to compete with UBC-E2A for binding the E2A proteins and inhibit E2A protein degradation. Stable constitutive and tetracycline-inducible smooth muscle cell clones of full-length sense and antisense UBC-E2A and UBC-E2A inhibitor will be generated. He will use these clones to study the effect of UBC-E2A on the cell cycle restriction point, which regulates cellular proliferation. In addition, he will study the effects of UBC-E2A on VSMC migration, adhesion, and matrix protein production. The Cardiovascular Biology Laboratory of the Harvard School of Public Health will serve as the site for these studies. The laboratory encompasses 12,000 square feet and is equipped with instruments designed for research in cellular and molecular biology. (End of Abstract)