Discovering the Origin of Vascular Aging Amyloid Protein Medin

摘要 年龄是心血管和脑血管疾病（CVD）最重要的危险因素， 是美国和世界范围内死亡和发病的主要原因。衰老导致血管损伤 独立于传统的心脏代谢危险因素，同时放大后者的损害。Medin是一个 50-来源于其母体蛋白乳脂肪球-EGF因子8蛋白的氨基酸淀粉样前体 （MFGE8）。随着年龄的增长，它在血管系统中积累，并导致人类最常见的形式 淀粉样变性Medin与血管老化、主动脉疾病、阿尔茨海默病和血管疾病有关。 痴呆关于medin产生的机制知之甚少，我们也不知道medin是什么。 负责MFGE 8切割的蛋白酶。该提案的主要目标是解决这一重大问题， 通过使用新的基因组学和生物信息学工具缩小知识差距。在目标1中，我们将在人类中开发 内皮细胞的MFGE 8加工活性的可靠和定量报告系统，并使用它 作为可选择的表型，进行全基因组CRISPR/Cas9敲除筛选， 直接或间接参与medin生成，但特别关注蛋白酶的基因。在 目的2，我们将进行计算机分子动力学研究，以了解MFGE 8构象变化 需要暴露medin切割位点，并根据目标1中确定的基因靶点，通过计算机模拟使用 蛋白质生物信息学来选择在结构上预测与MFGE 8相互作用的潜在蛋白酶， 合适的构造。在目标3中，我们将功能性地询问来自目标的候选基因/蛋白质 1和2通过在报告内皮细胞和幼稚内皮细胞中过表达或单独敲除它们来表达。 内皮细胞，以评估medin生成的预测变化并确认其生物学相关性。 确定酶，蛋白质和分子结构决定因素的medin产生是一个关键步骤， 了解和逆转medin的病理生理学，这将有助于解决血管老化， 血管性痴呆、AD和主动脉疾病。

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.