ACAT inhibition regulates ERAD of APP and Abeta production

ACAT 抑制调节 APP 和 Abeta 产生的 ERAD

基本信息

批准号：
7452364
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
项目类别：
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/7452364
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

项目摘要

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）加工和AP产生。作为减少与广告相关的淀粉样蛋白病理学的潜在策略，已经为血脂异常开发的疗法变得有吸引力。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溶剂，目前尚未销售，但在临床试验中已经积极开发，以治疗心血管疾病。我们先前已经表明，良好的ACAT抑制剂CP-113,818降低了神经元和非神经元细胞中的分泌AP水平，可极大地改善HAPP转基因小鼠的大脑中的AD样病理，并调节内源性小鼠脑应用程序的处理。我们的初步数据表明，ACAT抑制会诱导APP的ER相关降解。在ACAT抑制期间与APP共免疫沉淀的MS分析表明，ER伴侣GRP94是一种由ACAT调节的新型应用结合蛋白。 ACAT抑制不仅诱导GRP94与未成熟APP的结合，还诱导了基于细胞的实验和用ACAT抑制剂治疗的APP TG小鼠的蛋白酶/伴侣HTRA2的结合。 HTRA2在体外完全降解应用程序。另外，将APP的一部分脱位至细胞质，似乎被HTRA2和蛋白酶体降解。尽管其他机制可能有助于ACAT介导的APP处理调节，但我们的初步数据强烈表明ACAT抑制会导致APP在早期分泌途径中的降解，从而减少可用于AP生成的APP的量。因此，我们在这里建议解决ACAT抑制会导致ER保留和与ER相关的APP降解的假设。具体目标1将集中在体外和体内受ACAT抑制作用影响的应用程序结合蛋白的鉴定和表征。我们将使用LC/MS-MS在神经元细胞和用ACAT抑制剂治疗的HAPP小鼠的大脑中鉴定APP的结合伴侣。一旦表征，我们还将在APP中识别其结合域以及APP中介导ACAT调节的应用程序处理中的精确蛋白质序列。在特定目标2中，我们将确定ACAT抑制如何影响APP的ER保留和降解。为此，我们将跟进我们的初步数据，并在ACAT抑制过程中保留APP和APP贩运的ER。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ACAT抑制剂处理的细胞中的降解。最后，我们将评估由ACAT抑制引起的蛋白酶体和HTRA2在APP的降解中的作用。这些研究应进一步阐明采用ACAT抑制作用作为AD的新型治疗的前景。