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

描述（由申请人提供）：在美国，动脉粥样硬化是发病率和死亡率的主要原因。该疾病过程由包含活化的巨噬细胞（MO）和功能障碍的内皮细胞（EC）和平滑肌细胞（SMC）的血管病变的形成来定义。未折叠蛋白反应（UPR）已被认为是在动脉粥样硬化病变中以及在用已知促动脉粥样硬化诱导剂处理的培养细胞（MO、EC和SMC）中诱导的细胞过程。该基金的主要目的是探索一种新基因CHAC 1的功能。该蛋白质由UPR激活特异性诱导，并且是ATF 4-ATF 3-CHOP级联的下游，ATF 4-ATF 3-CHOP级联是UPR的重要促凋亡途径。CHAC 1是一种可溶性胞质肽，当过表达时诱导凋亡，siRNA敲除证实了该基因的促凋亡作用。CHAC 1激活TNFRSF 6 B，TNFRSF 6 B是一种FASL诱饵受体，其功能是拮抗FASL-FAS诱导的凋亡信号传导。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，并且这可能加剧血管疾病的进展。本文将在分子水平上定义CHAC 1的功能，并表征其在MO、EC和SMC中的凋亡和动脉粥样硬化疾病过程中的作用。CHAC 1在CHOP和UPR诱导的细胞凋亡中的作用将使用建立的过表达质粒和siRNA、敲除细胞的产生以及测定推定的信号传导级联的不同组分来确定。还将使用TAP方法探索CHAC 1的直接结合伴侣，以定义该基因的分子功能。TAP筛选的候选物将使用共免疫沉淀进行验证。基于所鉴定的候选物，将测定chaC结构域的推定功能，并使用靶向缺失和定点诱变来限定蛋白质的重要区域。最后，将产生CHAC 1基因敲除的小鼠的用途，以在小鼠疾病模型中探索CHAC 1基因消除在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