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: 8532027
• 负责人: Imran Mungrue
• 金额: $ 23.54万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 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 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8532027
• 关键词: Ablation; Americas; Apoptosis; Apoptotic; Arterial Fatty Streak; Atherosclerosis; Binding; Biological Assay; Biology; Blood Vessels; Cell physiology; Cells; Clear Cell; Cultured Cells; DNA; Development; Diet; Disease; Disease model; Generations; Genes; Genetic; Grant; Heart Arrest; Knock-out; Knockout Mice; Lesion; Mediating; Messenger RNA; Methods; Molecular; Morbidity - disease rate; Mus; Pathway interactions; Peptides; Plasmids; Precipitation; Process; Protein Region; Proteins; Role; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Small Interfering RNA; Smooth Muscle Myocytes; Stroke; Systems Biology; TNFRSF6B gene; TNFSF6 gene; Vascular Diseases; base; caspase 12; in vivo; macrophage; mortality; novel; promoter; receptor; research study; response; screening

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.

动脉粥样硬化是美国发病和死亡的主要原因。该疾病过程定义为 形成含有活化巨噬细胞 (MO) 和功能失调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 在动脉粥样硬化病变以及经过处理的培养细胞（MO、EC 和 SMC）中诱导的细胞过程 与已知的促动脉粥样硬化诱导剂。这笔赠款的主要目的是探索小说的功能 基因，CHAC1。该蛋白是由 UPR 激活特异性诱导的，位于 ATF4-ATF3- 的下游 CHOP级联，UPR的重要促凋亡途径。 CHAC1是一种可溶性胞质肽， 过度表达时会诱导细胞凋亡，而 siRNA 敲低证实了其促凋亡作用 基因。 CHAC1 激活 FASL 诱饵受体 TNFRSF6B，其作用是拮抗 FASL-FAS 诱导细胞凋亡信号传导。该途径很重要，因为 FASL 表达细胞可以诱导 MO、EC 或 SMC 的凋亡，这可能会加剧血管疾病的进展。提案 本文将在分子水平上定义CHAC1的功能，并表征其对细胞凋亡的贡献 以及 MO、EC 和 SMC 中的动脉粥样硬化疾病过程。 CHAC1 在 CHOP 中的贡献 UPR 诱导的细胞凋亡将使用已建立的过表达质粒和 siRNA 进行定义，生成 敲除细胞，并分析假定的信号级联的不同成分。直接绑定 CHAC1的合作伙伴也将使用TAP方法进行探索，以定义其分子功能 基因。 TAP 筛选的候选者将使用免疫共沉淀进行验证。基于 确定候选者后，将分析 chaC 结构域的 pijt 功能，并确定目标删除和位点 定向诱变用于定义蛋白质的重要​​区域。最后，使用具有 将生成敲除的CHAC1基因，以探讨CHAC1基因敲除对人体的影响 小鼠疾病模型中动脉粥样硬化的发展。