ACAT inhibition regulates ERAD of APP and Abeta production

ACAT 抑制调节 APP 和 Abeta 产生的 ERAD

• 批准号: 7316133
• 负责人: DORA M KOVACS
• 金额: $ 34.45万
• 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/7316133
• 关键词: Abeta synthesis; Acyl Coenzyme A; Address; Affect; Amino Acid Sequence; Amyloid beta-Protein Precursor; Atherosclerosis; Binding; Binding Proteins; Brain; CP 113818; Cardiovascular Diseases; Cell Fractionation; Cell Line; Cells; Cellular biology; Cholesterol; Cholesterol Esters; Cholesterol Homeostasis; Clinical Treatment; Clinical Trials; Cytoplasm; Cytosol; Data; Dimerization; Dyslipidemias; Endopeptidases; 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; Purpose; 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（由申请方提供）：越来越多的证据表明，细胞内胆固醇调节淀粉样前体蛋白（APR）加工和Ap产生。已经开发的用于血脂异常和动脉粥样硬化的疗法作为减少AD相关淀粉样病变的潜在策略正变得越来越有吸引力。酰基辅酶A：胆固醇酰基转移酶（ACAT）抑制剂防止胆固醇和脂肪酸转化为胆固醇酯，目前尚未上市，但已在临床试验中积极开发用于治疗心血管疾病。我们先前已经证明，一种充分表征的ACAT抑制剂CP-113，818降低了神经元和非神经元细胞中分泌的Ap水平，显著改善了hAPP转基因小鼠脑中的AD样病理学，我们的初步数据表明，ACAT抑制诱导ER-1表达，在ACAT抑制期间与APP共免疫沉淀的蛋白质的MS分析揭示ER伴侣GRP 94是由ACAT调节的新型APP结合蛋白。ACAT抑制不仅诱导GRP 94与未成熟APP的结合，而且在基于细胞的实验中以及在用ACAT抑制剂处理的APP tg小鼠的脑中诱导蛋白酶/伴侣HtrA 2的结合。HtrA 2在体外完全降解APP。此外，APP的一部分被移位到胞质溶胶，似乎是由HtrA 2和蛋白酶体降解。虽然其他机制可能有助于ACAT介导的APP加工调节，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，从而减少可用于Ap生成的APP量。因此，在这里，我们建议解决的假设，ACAT抑制诱导ER滞留和ER相关的APP降解。具体目标1将集中在ACAT抑制在体外和体内的APP结合蛋白的鉴定和表征。我们将使用LC/MS-MS鉴定神经元细胞和用ACAT抑制剂处理的hAPP小鼠脑中APP的结合伴侣。一旦表征，我们还将确定它们在APP中的结合结构域和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ER保留和APP降解。为此，我们将跟进我们的初步数据，并描述ACAT抑制期间APP的ER保留和APP运输的特征。我们还将测试APP的N-糖基化、泛素化和二聚化如何影响APP的保留及其在用ACAT抑制剂处理的细胞中的降解。最后，我们将评估蛋白酶体和HtrA 2在ACAT抑制诱导的ER相关APP降解中的作用。这些研究将进一步阐明采用ACAT抑制剂作为AD的新型治疗剂的前景。