:This is a new application for an interdisciplinary program project led by a cohesive group of investigators. The central theme of the Program Project is to understand the nature and regulation of the actin noncontractile cytoskeletal dynamics in the differentiated vascular smooth muscle cell (dVSMC). Preliminary data already collected by this working group demonstrate that the actin cytoskeleton of the dVSMC is, indeed, dynamic. Additionally, key molecular insights into the function of actin binding proteins in the nonmuscle cytoskeleton, published in part by members of this group, have positioned the field to allow major advances in our understanding of this system over the next five years. This program, in essence, aims to determine the integrative protein biology of the dVSMC cytoskeleton, a goal too broad for an individual RO1, but ideally suited for the PPG mechanism. Project 1 will focus at the level of dVSM tissue and cells, using techniques including protein knockdown, decoy peptides, quantitative imaging, in-cell FRET and crosslinking to investigate the mechanisms of actin remodeling, testing the hypothesis that remodeling of the noncontractile cytoskeleton modulates VSM contractility. Project 2 will focus on physical protein chemical and cell biology approaches to study phosphorylation-induced alterations of the conformation of actin binding proteins and test the hypothesis that conformational changes explain synergistic protein-protein interactions. Project 3 aims to understand the structural and mechanical effects of actin binding proteins (ABP) on the cytoskeleton of dVSMCs using helical reconstruction and single-particle analysis and laser trap methods. Project 4 will test the hypotheses that tandem WH2 domains function in filament nucleation, and that the adjacent proline-rich regions participate in filament elongation and will determine the mechanism by which alpha-actinin and plectin interact with actin using X-ray crystallography, biophysical methods, EM and cell biology. Core B will support all projects by producing custom peptide and protein tools and providing expertise and training in the use of sophisticated biophysical equipment. ? ? Lay Summary: The interdisciplinary, integrative, approach of the program will allow individual protein sequences to be directly related to vascular function and, thus, will identify entirely novel targets for the possible development of new therapeutics for the treatment of cardiovascular disease.
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