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.

摘要 脑胆固醇在衰老和阿尔茨海默病中被破坏。我们发现胆固醇可以直接 与蛋白质Nicastrin相互作用以改变γ-分泌酶复合物功能。理解这种相互作用， 它调节复杂功能的机制，可以促进开发治疗方法， 老年痴呆症我们假设胆固醇及其代谢产物与胆固醇的相互作用是不同的。 γ-分泌酶的Nicastrin亚基改变辅助蛋白募集并改变细胞的选择性和功能 复杂的。通过拟议的实验，我们将： 目标1.确定Nicastrin上的胆固醇结合位点。Nicastrin具有大的胞外结构域， 已经提出含有底物识别结构域，即与 早老素和单个跨膜结构域。使用胆固醇探针与质谱联用， 将识别胆固醇-尼卡斯特罗林加合物。拟定的相互作用位点将在人Nicastrin上突变 然后将野生型和突变的Nicastrin免疫沉淀并确认探针相互作用。 目标2.确定响应于以下而募集至Nicastrin的辅助蛋白的组成： 胆固醇和胆固醇代谢物。我们假设，与Nicastrin结合的胆固醇形式将 改变辅助蛋白对复合物的募集，这将改变功能。我们将 用人Nicastrin敲除Nicastrin的原代神经元培养物。然后用胆固醇处理细胞 代谢物和复合物免疫沉淀。将通过质谱法测量分离的蛋白质。 使用dSTORM超分辨率成像，我们将确定胆固醇代谢物是否差异性地影响 辅助蛋白/γ-分泌酶共定位。 目标3.分析γ-分泌酶复合物的底物加工如何被不同的 胆固醇代谢物。我们的初步数据表明，胆固醇代谢物能够作为 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， 选择性和/或合成能力。我们将测试胆固醇代谢物影响 复合物成熟、底物APP和Notch的差异切割以及APP产物的大小 使用蛋白质印迹、报告基因测定、ELISA和质谱法产生。 这些研究将为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提供见解，并为未来的研究提供基础。 因此，我们需要采取干预措施来调节特定的胆固醇代谢物，以预防阿尔茨海默病。