Cholesterol Metabolites as Regulators of Nicastrin

胆固醇代谢物作为尼卡斯特林的调节剂

• 批准号: 10680096
• 负责人: Heather Ferris
• 金额: $ 53.96万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10680096
• 关键词: 25-hydroxycholesterol; 27-hydroxycholesterol; 7-ketocholesterol; Aging; Alzheimer disease prevention; Alzheimer' s Disease; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Binding; Binding Proteins; Binding Sites; Biological Assay; Brain; Cells; Cholesterol; Clinical Trials; Complex; Coupled; Data; Disease; Enzyme-Linked Immunosorbent Assay; Excision; Extracellular Domain; Future; Generations; Goals; Human; Hydroxycholesterols; Image; Impairment; Inflammatory; Intervention; Knock-out; Lead; Mass Spectrum Analysis; Measures; Mutate; Neurons; Patients; Play; Prevention; Protein Fragment; Proteins; Proteolysis; Reporter; Resolution; Role; Senile Plaques; Site; Substrate Specificity; Testing; Therapeutic; Toxic effect; Transmembrane Domain; Western Blotting; adduct; crosslink; drug development; experimental study; gamma secretase; insight; liquid chromatography mass spectrometry; nicastrin protein; notch protein; novel; presenilin; presenilin-1; presenilin-2; prevent; protein complex; recruit; response; side effect; success; therapy development

Abstract Brain cholesterol is disrupted in aging and Alzheimer’s disease. We have found that cholesterol can directly interact with the protein Nicastrin to alter gamma-secretase complex function. Understanding this interaction, and the mechanisms by which it regulates complex function, could facilitate efforts to develop therapies to prevent Alzheimer’s disease. We hypothesize that cholesterol and its metabolites differentially interact with the Nicastrin subunit of gamma-secretase to change accessory protein recruitment and alter selectivity and function of the complex. Through the proposed experiments we will: Aim 1. Identify the cholesterol binding site on Nicastrin. Nicastrin has a large extracellular domain which has been proposed to contain a substrate recognition domain, a juxtamembrane domain which interacts with Presenilin and a single transmembrane domain. Using the cholesterol probe coupled to mass spectrometry, we will identify the cholesterol-Nicastrin adducts. Proposed sites of interaction will be mutated on human Nicastrin and then wildtype and mutated Nicastrin will be immunoprecipitated and probe interaction confirmed. Aim 2. Determine the composition of accessory proteins recruited to Nicastrin in response to cholesterol and cholesterol metabolites. We hypothesize that the form of cholesterol bound to Nicastrin will change the recruitment of accessory proteins to the complex, which will alter function. We will transduce Nicastrin knockout primary neuron cultures with human Nicastrin. Cells will then be treated with cholesterol metabolites and the complexes immunoprecipitated. Isolated proteins will be measured by mass spectrometry. Using dSTORM super-resolution imaging we will determine if cholesterol metabolites differentially influence accessory protein/gamma-secretase co-localization. Aim 3. Analyze how processing of substrates of the gamma-secretase complex is altered by different cholesterol metabolites. Our preliminary data suggest that cholesterol metabolites are able to act as endogenous gamma-secretase modulators. These metabolites could alter maturation of the complex, substrate selection and/or processivity of the complex. We will test cholesterol metabolites for their ability to influence complex maturation, differential cleavage of the substrates APP and Notch and size of APP products generated, using western blot, reporter assays, ELISA and mass spectrometry. These studies will provide insights into how cholesterol acts as an endogenous modulator of gamma-secretase through a novel interaction with the Nicastrin subunit and provide the basis for future interventions to modulate specific cholesterol metabolites for the prevention of Alzheimer’s disease.

抽象的 大脑胆固醇在衰老和阿尔茨海默病中受到破坏。我们发现胆固醇可以直接 与 Nicastrin 蛋白相互作用，改变 γ-分泌酶复合物的功能。了解这种相互作用，以及 它调节复杂功能的机制，可以促进开发预防疗法的努力 阿尔茨海默病。我们假设胆固醇及其代谢物与 γ-分泌酶的烟碱亚基改变辅助蛋白的募集并改变选择性和功能 复杂的。通过建议的实验，我们将： 目标 1. 识别 Nicastrin 上的胆固醇结合位点。尼卡斯特林有一个大的细胞外结构域 已被提议包含一个底物识别域，一个与 早老素和单个跨膜结构域。使用胆固醇探针与质谱联用，我们 将鉴定胆固醇-尼卡斯特林加合物。拟定的相互作用位点将在人类尼卡斯特林上发生突变 然后对野生型和突变型 Nicastrin 进行免疫沉淀并确认探针相互作用。 目标 2. 确定 Nicastrin 响应的辅助蛋白的组成 胆固醇和胆固醇代谢物。我们假设与 Nicastrin 结合的胆固醇形式 改变辅助蛋白向复合物的募集，这将改变功能。我们将转译 尼卡斯特林用人尼卡斯特林敲除原代神经元培养物。然后用胆固醇处理细胞 代谢物和复合物免疫沉淀。分离的蛋白质将通过质谱法进行测量。 使用 dSTORM 超分辨率成像，我们将确定胆固醇代谢是否会产生不同的影响 辅助蛋白/γ-分泌酶共定位。 目标 3. 分析 γ-分泌酶复合物底物的加工如何被不同的因素改变 胆固醇代谢物。我们的初步数据表明胆固醇代谢物能够充当 内源性γ-分泌酶调节剂。这些代谢物可能会改变复合物、底物的成熟 复合物的选择和/或持续合成能力。我们将测试胆固醇代谢物的影响能力 复杂的成熟、底物 APP 和 Notch 的差异裂解以及 APP 产品的大小 使用蛋白质印迹、报告分析、ELISA 和质谱法生成。 这些研究将深入了解胆固醇如何通过与 Nicastrin 亚基的新型相互作用作为 γ-分泌酶的内源性调节剂，并为未来提供基础 调节特定胆固醇代谢物以预防阿尔茨海默病的干预措施。