ACAT inhibition regulates ERAD of APP and Abeta production

ACAT 抑制调节 APP 和 Abeta 产生的 ERAD

• 批准号: 7800928
• 负责人: DORA M KOVACS
• 金额: $ 34.11万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-23 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7800928
• 关键词: Abeta synthesis; Acyl Coenzyme A; Address; Affect; Amino Acid Sequence; Amyloid beta-Protein Precursor; Atherosclerosis; Binding; Binding Proteins; Brain; Cardiovascular Diseases; Cell Fractionation; Cell Line; Cells; Cellular biology; Cholesterol; Cholesterol Esters; Cholesterol Homeostasis; Clinical Treatment; Clinical Trials; Cytoplasm; Cytosol; Data; Dimerization; Dyslipidemias; Epidemiology; Event; Fatty Acids; Fluorescence; GRP94; Generations; Genetic; Immunoprecipitation; In Vitro; Kinetics; Marketing; Mediating; Membrane; Metabolism; Molecular; Molecular Chaperones; Mus; N-terminal; Neuroglia; Neurons; Pathogenesis; Pathology; Pathway interactions; Peptide Hydrolases; Peptide Sequence Determination; Point Mutation; Post-Translational Protein Processing; Prevention; Process; Production; Protein Analysis; Proteins; Publishing; Regulation; Research Personnel; Role; Side; Sterol O-Acyltransferase; System; Tertiary Protein Structure; Testing; Transgenic Mice; Transgenic Organisms; Ubiquitination; amyloid pathology; amyloid precursor protein processing; base; brain cell; cholesterol control; follow-up; glycosylation; improved; in vivo; inhibitor/antagonist; insight; multicatalytic endopeptidase complex; mutant; novel; novel therapeutics; prevent; programs; protein protein interaction; research study; trafficking

DESCRIPTION (provided by applicant): Increasing evidence shows that intracellular cholesterol regulates amyloid precursor protein (APR) processing and Ap production. Therapies already developed for dyslipidemia and atherosclerosis are becoming attractive as potential strategies for reducing AD-related amyloid pathology. Acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitors, which prevent conversion of cholesterol and fatty acids into cholesteryl-esters, are not currently marketed, but have been actively developed in clinical trials for the treatment of cardiovascular disease. We have previously shown that a well-characterized ACAT inhibitor, CP-113,818, reduces secreted Ap levels in neuronal and non-neuronal cells, dramatically improves AD-like pathology in the brains of hAPP transgenic mice, and also regulates processing of endogenous mouse brain APP. Our preliminary data indicate that ACAT inhibition induces ER-associated degradation of APP. MS analysis of proteins coimmunoprecipitating with APP during ACAT inhibition revealed that the ER chaperone GRP94 is a novel APP-binding protein regulated by ACAT. ACAT inhibition not only induces binding of GRP94 to immature APP, but also that of the protease/chaperone HtrA2 in cell-based experiments and in the brains of APP tg mice treated with an ACAT inhibitor. HtrA2 fully degrades APP in vitro. In addition, a fraction of APP is dislocated to the cytosol and appears to be degraded by HtrA2 and the proteasome. While additional mechanisms may contribute to ACAT-mediated regulation of APP processing, our preliminary data strongly suggest that ACAT inhibition induces degradation of APP in the early secretory pathway, thereby reducing the amount of APP available for Ap generation. Therefore, here we propose to address the hypothesis that ACAT inhibition induces ER retention and ER-associated degradation of APP. Specific Aim 1 will focus on the identification and characterization of APP-binding proteins affected by ACAT inhibition in vitro and in vivo. We will use LC/MS-MS to identify binding partners of APP in neuronal cells and in brains of hAPP mice treated with ACAT inhibitors. Once characterized, we will also identify their binding domains in APP and the exact protein sequences in APP mediating ACAT-regulated APP processing. In Specific Aim 2, we will determine how ACAT inhibition affects ER retention and degradation of APP. For this purpose, we will follow up on our preliminary data, and characterize ER retention of APP and APP trafficking during ACAT inhibition. We will also test how N-glycosylation, ubiquitination, and dimerization of APP influence the retention of APP and its degradation in cells treated with ACAT inhibitors. Finally, we will assess the roles of the proteasome and HtrA2 in ER-associated degradation of APP, induced by ACAT inhibition. These studies should further elucidate the prospects for employing ACAT inhibition as a novel therapeutic for AD.

描述（由申请人提供）：越来越多的证据表明细胞内胆固醇调节淀粉样蛋白前体蛋白（APR）的加工和Ap的产生。已经开发的针对血脂异常和动脉粥样硬化的治疗方法正成为减少ad相关淀粉样蛋白病理的潜在策略。酰基辅酶A：胆固醇酰基转移酶（ACAT）抑制剂，可防止胆固醇和脂肪酸转化为胆固醇酯，目前尚未上市，但已积极开发用于治疗心血管疾病的临床试验。我们之前已经证明，一种表征良好的ACAT抑制剂CP-113,818可以降低神经元和非神经元细胞中分泌的Ap水平，显著改善hAPP转基因小鼠大脑中的ad样病理。我们的初步数据表明，ACAT抑制可诱导ER相关的APP降解。质谱分析显示，ACAT抑制期间与APP共免疫沉淀的蛋白发现，ER伴侣GRP94是一种受ACAT调控的新型APP结合蛋白。ACAT抑制不仅诱导GRP94与未成熟APP结合，而且在细胞实验和ACAT抑制剂处理的APP tg小鼠的大脑中也诱导了蛋白酶/伴侣HtrA2的结合。HtrA2在体外完全降解APP。此外，APP的一部分被脱位到细胞质中，似乎被HtrA2和蛋白酶体降解。虽然ACAT介导的APP加工调节可能有其他机制，但我们的初步数据强烈表明，ACAT抑制诱导APP在早期分泌途径中降解，从而减少了可用于Ap生成的APP数量。因此，在这里，我们提出解决ACAT抑制诱导内质网保留和内质网相关的APP降解的假设。Specific Aim 1将侧重于体外和体内受ACAT抑制影响的APP结合蛋白的鉴定和表征。我们将使用LC/MS-MS鉴定ACAT抑制剂处理的hAPP小鼠神经元细胞和大脑中APP的结合伙伴。一旦确定，我们还将确定它们在APP中的结合域以及APP中介导acat调节的APP加工的确切蛋白质序列。在具体目标2中，我们将确定ACAT抑制如何影响内质网保留和APP的降解。为此，我们将跟踪我们的初步数据，并表征ACAT抑制期间APP的内质网保留和APP贩运。我们还将测试APP的n -糖基化、泛素化和二聚化如何影响在ACAT抑制剂处理的细胞中APP的保留及其降解。最后，我们将评估蛋白酶体和HtrA2在ACAT抑制诱导的er相关APP降解中的作用。这些研究应该进一步阐明利用ACAT抑制作为一种新的治疗AD的前景。