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

确定促凋亡 UPR 基因 CHAC1 在动脉粥样硬化中的作用。

• 批准号: 7739253
• 负责人: Imran Mungrue
• 金额: $ 8.95万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7739253
• 关键词: Ablation; Americas; Apoptosis; Apoptotic; Arterial Fatty Streak; Atherosclerosis; Binding; Biological Assay; Blood Vessels; Cell physiology; Cells; Clear Cell; Cultured Cells; DNA; Development; Diet; Disease; Disease model; Generations; Genes; Genetic; Grant; Knock-out; Knockout Mice; Lesion; Mediating; Messenger RNA; Methods; Molecular; Morbidity - disease rate; Mus; Pathway interactions; Peptides; Plasmids; Precipitation; Principal Investigator; Process; Protein Region; Proteins; Role; Screening procedure; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Small Interfering RNA; Smooth Muscle Myocytes; TNFRSF6B gene; TNFSF6 gene; Vascular Diseases; base; caspase 12; gene function; in vivo; macrophage; mortality; novel; promoter; protein function; receptor; research study; response

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

描述（由申请人提供）：动脉粥样硬化是美国发病和死亡的主要原因。这种疾病过程的定义是血管病变的形成，其中包含活化的巨噬细胞（MO）以及功能失调的内皮细胞（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中以及在用已知促动脉粥样硬化诱导物处理的培养细胞(MO、EC和SMC)中诱导的细胞过程。这笔资助的主要目的是探索新基因 CHAC1 的功能。该蛋白由 UPR 激活特异性诱导，位于 ATF4-ATF3-CHOP 级联的下游，是 UPR 的重要促凋亡途径。 CHAC1 是一种可溶性胞浆肽，过度表达时会诱导细胞凋亡，而 siRNA 敲低证实了该基因的促细胞凋亡作用。 CHAC1 激活 TNFRSF6B（一种 FASL 诱饵受体），其作用是拮抗 FASL-FAS 诱导的细胞凋亡信号传导。该途径很重要，因为表达 FASL 的细胞可以诱导 MO、EC 或 SMC 凋亡，这可能会加剧血管疾病的进展。本文的提议将在分子水平上定义 CHAC1 的功能，并描述其对 MO、EC 和 SMC 中细胞凋亡和动脉粥样硬化疾病过程的贡献。 CHAC1 在 CHOP 和 UPR 诱导的细胞凋亡中的贡献将通过使用已建立的过表达质粒和 siRNA、生成敲除细胞以及分析假定的信号级联的不同成分来定义。还将使用 TAP 方法探索 CHAC1 的直接结合伴侣，以确定该基因的分子功能。 TAP 筛选的候选者将使用免疫共沉淀进行验证。根据确定的候选物，将分析 chaC 结构域的推定功能，并使用靶向删除和定点诱变来定义蛋白质的重要​​区域。最后，将产生使用CHAC1基因敲除的小鼠来探索CHAC1基因敲除对小鼠疾病模型中动脉粥样硬化发展的影响