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 脑胆固醇在衰老和阿尔茨海默氏病中受到干扰。我们发现胆固醇可以直接 与蛋白质尼加斯特林相互作用以改变γ-分泌酶复合函数。了解这种互动，以及 通过调节复杂功能的机制，可以促进开发疗法以防止 阿尔茨海默氏病。我们假设胆固醇及其代谢物与 γ-分泌酶的尼卡斯特林亚基改变辅助蛋白的募集并改变选择性和功能 综合体。通过拟议的实验，我们将： 目标1。识别尼加斯特林上的胆固醇结合位点。尼卡斯特林有一个较大的细胞外域 已提议包含一个底物识别域，这是一个与近去的域，该结构域与之相互作用 presenilin和单个跨膜结构域。使用与质谱耦合的胆固醇探针，我们 将识别胆固醇 - 抗抗激素加合物。提议的相互作用位点将在人尼加斯特林上突变 然后将对野生型和突变的尼加斯特林进行免疫沉淀，并确认探针相互作用。 目标2。确定响应于尼卡斯特林的辅助蛋白的组成 胆固醇和胆固醇代谢产物。我们假设胆固醇与尼卡斯特林结合的形式将 将附件蛋白的募集更改为复合物，这将改变功能。我们将翻译 尼古斯特林敲除原发性神经元培养物与人尼加斯特林。然后将细胞用胆固醇处理 代谢物和复合物免疫沉淀。分离的蛋白质将通过质谱法测量。 使用DSTORM超分辨率成像，我们将确定胆固醇代谢物是否会差异影响 辅助蛋白/γ-分泌酶共定位。 AIM 3。分析γ-分泌酶复合物的底物的加工如何通过不同的不同 胆固醇代谢产物。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。这些代谢物可以改变复合物的成熟 综合体的选择和/或过程。我们将测试胆固醇代谢物的影响能力 复杂的成熟，底物应用程序的差异裂解以及应用程序的尺寸 使用Western印迹，记者分析，ELISA和质谱法产生。 这些研究将提供有关胆固醇如何通过与NICASTRIN亚基的新型相互作用的胆固醇作为γ-分泌酶的内源性调节剂的见解，并为将来提供了基础 调节特定胆固醇代谢物以预防阿尔茨海默氏病的干预措施。