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

动脉粥样硬化是美国发病率和死亡率的主要原因。此疾病过程由以下定义 含有激活的巨噬细胞(MO)和功能障碍的内皮细胞的血管病变的形成 (EC)和平滑肌细胞(SMC)。未折叠蛋白反应(UPR)被认为是一种 在动脉粥样硬化病变和培养细胞(MO、EC和SMC)处理后诱导的细胞过程 已知的促动脉粥样硬化诱导剂。这笔赠款的主要目的将是探索一部小说的功能 基恩，CHAC1。这种蛋白是由UPR激活特异性诱导的，位于ATF4-ATF3下游- CHOP级联，是UPR重要的促细胞凋亡途径。CHAC1是一种可溶性胞浆多肽， 它在过度表达时诱导细胞凋亡，siRNA敲除证实了这一点的促凋亡作用 吉恩。CHAC1激活FASL诱骗受体TNFRSF6B，拮抗FASL-Fas 诱导细胞凋亡信号。这一途径很重要，因为表达FASL的细胞可以诱导 MO、EC或SMC的细胞凋亡，可能会加剧血管疾病的进展。这项建议 本文将在分子水平上定义CHAC1的功能，并表征其在细胞凋亡中的作用 以及MO、EC和SMC的动脉粥样硬化性疾病过程。CHAC1在CHOP和CHOP中的作用 UPR诱导的细胞凋亡将使用已建立的过表达质粒和siRNAs来定义，生成 基因敲除细胞，并分析推测的信号级联的不同成分。直接绑定 CHAC1的伙伴也将使用TAP方法进行探索，以确定其分子功能 吉恩。来自TAP筛查的候选人将使用共同免疫沉淀进行验证。基于 确定候选，将分析Chac结构域的潜在功能，并有针对性地删除和定位 定向突变用于确定蛋白质的重要区域。最后，使用具有 将产生CHAC1基因敲除，以探索CHAC1基因消融在大鼠体内的作用 小鼠疾病模型中动脉粥样硬化的发展。

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 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 ofthe ATF4-ATF3- CHOP cascade, an important pro-apoptotic pathway ofthe 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 my 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 ofthis gene. Candidates from TAP screening will be validated using co-immuno-precipitation. Based on candidates identified, pijtative functions ofthe 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.