Discovering the Origin of Vascular Aging Amyloid Protein Medin

摘要 年龄是心脑血管疾病（CVD）最重要的危险因素 美国和全世界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，同时放大了后者的损害。麦丁是一个 源自其母蛋白乳脂球-EGF因子8蛋白的50个氨基酸淀粉样蛋白前体 （MFGE8）。随着年龄的增长，它会在脉管系统中积累，并有助于形成人类最常见的形式 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 失智。对于medin的产生机制知之甚少，我们也不知道 负责 MFGE8 切割的蛋白酶。该提案的主要目标是解决这一重大问题 通过使用新型基因组和生物信息学工具来弥补知识差距。在目标 1 中，我们将发展人类 内皮细胞是一个可靠的定量报告系统，用于 MFGE8 的加工活性，并使用它 作为可选表型进行全基因组 CRISPR/Cas9 敲除筛选以鉴定基因 直接或间接参与medin的生成，但特别关注蛋白酶基因。在 目标 2，我们将进行计算机分子动力学研究以了解 MFGE8 构象变化 需要暴露 medin 切割位点，并根据目标 1 中确定的基因目标进行信息，在计算机中使用 蛋白质生物信息学，以选择在结构上预测与 MFGE8 相互作用的潜在蛋白酶 适当的构象。在目标 3 中，我们将从功能上询问目标中的候选基因/蛋白质 1和2通过在报告内皮细胞和幼稚细胞中单独过度表达或敲除它们 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。 鉴定药物生成的酶、蛋白质和分子结构决定因素是药物生成的关键一步 了解和逆转医学病理生理学，这将有助于解决血管老化问题， 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.