Discovering the Origin of Vascular Aging Amyloid Protein Medin

发现血管老化淀粉样蛋白的起源

• 批准号: 10351895
• 负责人: Ming Li
• 金额: $ 34.74万
• 依托单位: ARIZONA VETERANS RESEARCH AND EDUCATION FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10351895
• 关键词: Address; Age; Aging; Alzheimer' s Disease; Amino Acids; Amyloid; Amyloid Proteins; Amyloid beta-Protein; Amyloidosis; Aortic Aneurysm; Aortic Diseases; Automobile Driving; Biochemistry; Bioinformatics; Biological; Biological Process; Biology; Blood Vessels; CRISPR/Cas technology; Candidate Disease Gene; Cardiovascular Diseases; Cell surface; Cells; Cerebrovascular Disorders; Clustered Regularly Interspaced Short Palindromic Repeats; Dementia; Diabetes Mellitus; Disease; EGF gene; Endothelial Cells; Endothelium; Enzymes; Epitopes; Exposure to; Functional disorder; Gene Proteins; Gene Targeting; Generations; Genes; Genomics; Goals; Human; Hyperlipidemia; Hypertension; Impairment; Individual; Inflammatory; Investigation; Knock-out; Knowledge; Lead; Measures; Metabolism; Milk Proteins; Molecular; Molecular Biology; Molecular Conformation; Morbidity - disease rate; Mus; Myocardial Infarction; Outcome Study; Parents; Pathology; Peptide Hydrolases; Peptides; Phenotype; Process; Production; Protein Conformation; Proteins; Proteolysis; Reporter; Research; Risk; Risk Factors; Site; Smoking; Stroke; Structural Models; System; Vascular Dementia; age related; aged; amyloid formation; bioinformatics tool; candidate validation; cardiometabolic risk; cardiovascular risk factor; cerebrovascular; endothelial dysfunction; functional genomics; genome-wide; genomic tools; human disease; in silico; knock-down; medin; milk fat globule; milk production; molecular dynamics; mortality; novel; overexpression; preservation; screening

ABSTRACT Age is the most important risk factor for cardiovascular and cerebrovascular diseases (CVD), the leading causes of mortality and morbidity in the US and worldwide. Aging causes vascular impairment independent of the traditional cardiometabolic risk factors, while magnifying the latter’s damage. Medin is a 50-amino acid amyloid precursor derived from its parent protein milk fat globule-EGF factor 8 protein (MFGE8). It accumulates in the vasculature with aging and contributes to the most common form of human amyloidosis. Medin is implicated in vascular aging, aortic disease, Alzheimer’s disease and vascular dementia. Little is known as to the mechanism by which medin is generated and we do not know the protease(s) responsible for MFGE8 cleavage. The main goal of the proposal is to address this major knowledge gap through use of novel genomic and bioinformatics tools. In Aim 1, we will develop in human endothelial cells a reliable and quantitative reporter system for the processing activity for MFGE8, and use it as a selectable phenotype to conduct genome wide CRISPR/Cas9 knockout screening to identify genes involved in medin generation either directly or indirectly, but with particular focus on genes for protease/s. In Aim 2, we will conduct in silico Molecular Dynamics study to understand MFGE8 conformation changes required to expose the medin cleavage sites and, informed by gene targets identified in Aim 1, use in silico protein bioinformatics to select potential protease/s that are structurally predicted to interact with MFGE8 in appropriate conformation. In Aim 3, we will functionally interrogate the candidate genes/proteins from Aims 1 and 2 by over-expressing or knocking them out individually in reporter endothelial cells and in naïve endothelial cells, to assess predicted changes in medin generation and confirm their biologic relevance. Identifying enzymes, proteins and molecular structural determinants of medin generation is a critical step in understanding and reversing medin pathophysiology that would be useful in addressing vascular aging, vascular dementia, AD and aortic disease.

摘要 年龄是心脑血管疾病最重要的危险因素， 在美国和世界范围内导致死亡和发病的主要原因。衰老导致血管损伤 与传统的心脏代谢风险因素无关，同时放大了后者的损害。梅敏是一位 50个氨基酸的淀粉样前体来源于其亲本蛋白乳脂球-EGF因子8蛋白 (MFGE8)。随着年龄的增长，它在血管系统中积累，并对人类最常见的形式做出贡献 淀粉样变性。梅丁与血管老化、主动脉疾病、阿尔茨海默病和血管疾病有关 痴呆症。关于Medin的产生机制我们知之甚少，我们也不知道 负责裂解MFGE8的蛋白酶(S)。该提案的主要目标是解决这一主要问题 通过使用新的基因组和生物信息学工具实现知识差距。在目标1中，我们将在人类中发展 内皮细胞MFGE8加工活性的可靠定量报告系统及其应用 作为一种可选择的表型进行全基因组CRISPR/Cas9基因敲除筛选以识别基因 直接或间接地参与了麦德林的生成，但特别关注蛋白酶/S的基因。 目的2，我们将在分子动力学研究中了解MFGE8的构象变化 需要暴露Medin裂解位点，并由Aim 1中确定的基因靶标告知，在硅胶中使用 蛋白质生物信息学筛选结构上预测与MFGE8相互作用的潜在蛋白酶/S 适当的构象。在目标3中，我们将从AIMS中对候选基因/蛋白质进行功能查询 1和2通过在报告内皮细胞和幼稚中单独过度表达或敲除它们 评估Medin生成的预期变化，并确认它们的生物学相关性。 确定酶、蛋白质和分子结构决定因素的生成是关键的一步 了解和逆转Medin的病理生理学将有助于解决血管老化问题， 血管性痴呆、阿尔茨海默病和主动脉疾病。