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

描述（由申请人提供）：动脉粥样硬化仍然是美国的主要死亡原因。越来越多的证据表明，主动脉壁平滑肌细胞基因表达在动脉粥样硬化发生中起着重要作用。ß-arrestin2 （ßarr2）是一种G蛋白偶联受体（gpcr）、生长因子受体和离子通道的内吞和信号适配器，已知也可促进小鼠新生内膜增生和动脉粥样硬化。可逆的ßarr2泛素化，由去泛素酶（deubiquitinases, DUBs）调控，是一个关键的翻译后修饰，是ßarr2在介导细胞信号传导中的适配器功能所必需的。我们的初步研究表明，DUB USP20可能影响ßarr2泛素化以及由致动脉粥样硬化toll样受体4 （TLR4）诱导的NFκB信号传导。为了描述体内ßarr2泛素化/去泛素化动力学的作用，并评估ßarr2的泛素化状态是否会在SMCs中产生促炎信号，我们在SMCs特异性SM22α启动子的控制下产生了表达USP20或其催化失活亚型（DN-USP20）的转基因小鼠。在该模型中，我们预计通过去泛素化ßarr2， USP20会降低ßarr2活性，从而减少SMC促动脉粥样硬化增殖和迁移，从而导致新生内膜增生，而DN-USP20则会产生相互作用。通过利用这些和其他新试剂以及涉及饮食和基因依赖性动脉粥样硬化的体内方法，以及采用原代血管平滑肌细胞的体外技术，我们将测试SMCs中的USP20通过依赖于ßarr2支架的ßarr2去泛素化和/或TRAF6或TRAF2去泛素化的机制来减轻动脉粥样硬化的假设，实现以下具体目标：(1)确定SMC USP20的动脉粥样硬化保护作用，以及USP20的作用机制是否需要ßarr2的去泛素化(2)确定USP20活性是否调节炎症刺激引发的ßarr2依赖性SMC增殖、迁移和信号传导(3)阐明USP20对ßarr2依赖性信号传导作用的机制基础。

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.