Determining roles of the pro-apoptotic UPR gene CHAC1 in atherosclerosis.

描述(申请人提供)：动脉粥样硬化是美国发病率和死亡率的主要原因。这一疾病过程的定义是血管病变的形成，其中包含激活的巨噬细胞(MO)、功能障碍的内皮细胞(EC)和平滑肌细胞(SMC)。未折叠蛋白反应(UPR)被认为是在动脉粥样硬化病变和L等已知促动脉粥样硬化诱导剂处理的培养细胞(MO、EC和SMC)中诱导的一种细胞过程。这笔赠款的主要目的将是探索新基因CHAC1的功能。这一蛋白是由UPR激活特异性诱导的，位于ATF4-ATF3-CHOP级联的下游，ATF4-ATF3-CHOP级联是UPR重要的促凋亡途径。CHAC1是一种可溶性胞浆多肽，在过度表达时可诱导细胞凋亡，siRNA敲除证实了该基因的促凋亡作用。CHAC1激活FASL诱骗受体TNFRSF6B，拮抗FASL-Fas诱导的细胞凋亡信号。这一途径具有重要意义，因为表达FASL的细胞可以诱导MO、EC或SMC的凋亡，这可能会加剧血管疾病的进展。本文将在分子水平上定义CHAC1的功能，并表征其在MO、EC和SMC的细胞凋亡和动脉粥样硬化性疾病过程中的作用。CHAC1在CHOP和UPR诱导的细胞凋亡中的作用将通过建立过表达的质粒和siRNAs，基因敲除细胞的产生，以及分析可能的信号级联的不同成分来确定。还将使用TAP方法探索CHAC1的直接结合伙伴，以确定该基因的分子功能。来自TAP筛查的候选人将使用共同免疫沉淀进行验证。根据确定的候选基因，将分析Chac结构域的假定功能，并使用定向缺失和定点突变来定义蛋白质的重要区域。最后，将利用CHAC1基因敲除的小鼠来探索CHAC1基因消融在小鼠疾病模型动脉粥样硬化发展中的作用

DESCRIPTION (provided by applicant): Atherosclerosis is the major cause of morbidity and mortality in America. This disease process is defined by the formation of vascular lesions that contain activated macrophages (MO) and dysfunctional endothelial (EC) and smooth muscle cells (SMC). The unfolded protein response (UPR) has been implicated as a cellular process that is induced in atherosclerotic lesions, and in cultured cells (MO, EC and SMC) treated with l<known pro-atherogenic inducers. The primary aim of this grant will be to explore the function of a novel gene, CHAC1. This protein is specifically induced by UPR activation and is downstream of the ATF4-ATF3-CHOP cascade, an important pro-apoptotic pathway of the UPR. CHAC1 is a soluble cytosolic peptide, which induces apoptosis when over-expressed, and siRNA knockdown confirms a pro-apoptotic role for this gene. CHAC1 activates TNFRSF6B, a FASL decoy receptor, which functions to antagonize FASL-FAS induced apoptosis signaling. This pathway is of importance because FASL expressing cells can induce apoptosis of MO, EC or SMC, and this may exacerbate the progression of vascular disease. The proposal herein will define the function of CHAC1 at the molecular level, and characterize its contribution to apoptosis and the atherosclerotic disease process in MO, EC and SMC. The contribution of CHAC1 in CHOP and UPR induced apoptosis will be defined using established over-expression plasmids and siRNAs, generation of knockout cells, and assaying different components of putative signaling cascades. Direct binding partners of CHAC1 will also be explored using the TAP method, to define the molecular function of this gene. Candidates from TAP screening will be validated using co-immuno-precipitation. Based on candidates identified, putative functions of the chaC domain will be assayed, and targeted deletion and site directed mutagenesis used to define important regions of the protein. Finally, the use of mice that have the CHAC1 gene knocked out will be generated to explore the effects of CHAC1 genetic ablation in the development of atherosclerosis in a mouse disease model