Discovering the Origin of Vascular Aging Amyloid Protein Medin

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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.