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。这种疾病的定义是： 形成含有活化的巨噬细胞（MO）和功能障碍的内皮细胞的血管病变 (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中诱导的细胞过程，以及在经处理的培养细胞（MO、EC和SMC）中 已知的促动脉粥样硬化诱导剂。这项资助的主要目的是探索小说的功能 基因CHAC 1。该蛋白质由UPR激活特异性诱导，并且位于ATF 4-ATF 3-ATF 4的下游。 CHOP级联反应是UPR重要的促凋亡途径。CHAC 1是可溶性胞质肽， 当过度表达时诱导细胞凋亡，siRNA敲低证实了其促凋亡作用。 基因CHAC 1激活TNFRSF 6 B，一种FASL诱饵受体，其功能是拮抗FASL-FAS 诱导凋亡信号传导。该途径是重要的，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。该提案 本文将在分子水平上定义CHAC 1的功能，并表征其对细胞凋亡的贡献 MO、EC和SMC参与动脉粥样硬化的病变过程。CHAC 1在CHOP中的作用， UPR诱导的细胞凋亡将使用建立的过表达质粒和siRNA来定义， 敲除细胞，并测定推定的信号级联的不同组分。直接结合 CHAC 1的伴侣也将使用TAP方法进行探索，以确定其分子功能。 基因TAP筛选的候选物将使用共免疫沉淀进行验证。基于 鉴定候选物，将测定chaC结构域的pitative功能，并靶向缺失和位点 用于确定蛋白质重要区域的定向诱变。最后，使用具有 将CHAC 1基因敲除后产生的细胞系作为研究对象，探讨CHAC 1基因敲除对CHAC 1基因表达的影响。 在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.