Regulation of B-arrestin2's pro-atherogenic activity by the deubiquitinase USP20

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DESCRIPTION (provided by applicant): Atherosclerosis remains a leading cause of death in the United States. Accumulating evidence suggests that aortic wall smooth muscle cell gene expression contributes substantially to atherogenesis. ß-arrestin2 (ßarr2) an endocytic and signaling adaptor for G protein-coupled receptors (GPCRs), growth factor receptors and ion- channels is also known to promote neointimal hyperplasia and atherosclerosis in mice. Reversible ßarr2 ubiquitination, as regulated by deubiquitinases (DUBs) is a critical post-translational modification that is required for ßarr2's adaptor functions in mediating cell-signaling. Our Preliminary Studies suggests that the DUB USP20 might affect ßarr2 ubiquitination as well as NFκB signaling induced by the atherogenic Toll-like receptor 4 (TLR4). To delineate the role(s) of ßarr2 ubiquitination/deubiquitination dynamics in vivo and to evaluate whether the ubiquitination status of ßarr2 could engender pro-inflammatory signaling in SMCs, we have generated transgenic mice expressing USP20 or its catalytically inactive isoform (DN-USP20) under control of the SMC-specific SM22α promoter. In this model, we expect that by de-ubiquitinating ßarr2, USP20 would reduce ßarr2 activity and thereby reduce the SMC pro-atherogenic proliferation and migration that engenders neointimal hyperplasia, whereas the DN-USP20 would have reciprocal effects. By utilizing these and additional novel reagents and in vivo methods involving diet and gene-dependent atherosclerosis and in vitro techniques employing primary vascular smooth muscle cells we will test the hypotheses that USP20 in SMCs mitigates atherosclerosis through mechanisms involving deubiquitination of ßarr2, and/or deubiquitination of TRAF6 or TRAF2 in a manner dependent upon ßarr2 scaffolding by accomplishing following specific aims: (1) To determine the atheroprotective role of SMC USP20, and whether USP20's mechanism of action requires de-ubiquitination of ßarr2 (2) To determine whether USP20 activity regulates ßarr2-dependent SMC proliferation, migration and signaling triggered by inflammatory stimuli and (3) To elucidate the mechanistic basis of USP20's effects on ßarr2-dependent signaling.