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主要的死亡原因。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形成中起重要作用。？ARR2是G蛋白偶联受体(GPCRs)、生长因子受体和离子通道的内吞和信号转接子，也被认为可以促进小鼠的新生内膜增生和动脉粥样硬化。受脱泛素酶(DUBS)调控的可逆性的arr2泛素化是一种关键的翻译后修饰，在介导细胞信号转导过程中起着非常重要的作用。我们的初步研究表明，Dub USP20可能会影响由致动脉粥样硬化的Toll样受体4(TLR4)诱导的泛素化和NFκB信号转导。为了阐明在体内泛素化/去泛素化动力学过程中的作用(S)，并评价其泛素化状态是否可以在SMC中产生促炎信号，我们在SMC特异的Sm22α启动子控制下获得了表达USP20或其催化失活亚型(dN-USP20)的转基因小鼠。在这个模型中，我们预计通过去泛素化？arr2，USP20将降低？arr2的活性，从而减少导致新生内膜增生的SMC促动脉粥样硬化的增殖和迁移，而dN-USP20将具有相互作用。通过利用这些和其他新的试剂和体内涉及饮食和基因依赖性动脉粥样硬化的方法以及使用原代血管平滑肌细胞的体外技术，我们将检验这样的假设，即SMC中的USP20通过涉及çarr2去泛素化和/或TRAF6或TRAF2去泛素化的机制来缓解动脉粥样硬化，这种机制依赖于？arr2支架，从而实现以下特定目的：(1)确定SMC USP20的动脉粥样硬化保护作用，以及USP20的S作用机制是否需要去泛素化？arr2(2)以确定USP20的活性是否调节？arr2依赖的SMC的增殖、增殖、迁移和炎症刺激触发的信号转导；(3)阐明USP20‘S作用于B arr2依赖信号转导的机制基础。

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.