Discovering the Origin of Vascular Aging Amyloid Protein Medin

发现血管老化淀粉样蛋白的起源

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

抽象的年龄是心血管和脑血管疾病（CVD）的最重要危险因素，美国和全球的死亡率和发病率的主要原因。衰老会导致血管障碍独立于传统的心脏代谢危险因素，同时放大了后者的损害。梅丁是一个 50-氨基酸淀粉样蛋白前体来自其母蛋白牛奶脂肪球-EGF因子8蛋白（MFGE8）。它随着衰老而积聚在脉管系统中，并有助于人类的最常见形式淀粉样变性。 Medin在血管衰老，主动脉疾病，阿尔茨海默氏病和血管中暗示失智。几乎不知道产生Medin的机制，我们不知道负责MFGE8裂解的蛋白酶。该提案的主要目标是解决这一专业通过使用新型基因组和生物信息学工具，知识差距。在AIM 1中，我们将在人类中发展内皮细胞一种用于MFGE8处理活动的可靠和定量报告基因，并使用它作为进行基因组宽CRISPR/CAS9敲除筛查以识别基因的可选表型直接或间接地参与MEDIN生成，但特别关注蛋白酶的基因。 AIM 2，我们将进行计算机分子动力学研究以了解MFGE8构象变化需要揭露Medin裂解位点，并由AIM 1中确定的基因靶标的告知，用于用于计算机蛋白质生物信息学以选择潜在的蛋白酶，这些蛋白酶在结构上预测与MFGE8相互作用适当的构象。在AIM 3中，我们将在功能上从AIM询问候选基因/蛋白质 1和2通过在记者的内皮细胞中分别表达或将它们单独击倒，并且在幼稚中内皮细胞，以评估Medin产生的预测变化并确认其生物学相关性。鉴定麦金生成的酶，蛋白质和分子结构决定剂是关键的一步理解和逆转Medin病理生理学，这对于解决血管衰老有用，血管性痴呆，AD和主动脉疾病。