The primary goal of this grant proposal is to demonstrate that self-association of apolipoprotein A-I (apoA-I) is a functional feature of the protein and that alteration of its native state of self-association is involved in mechanisms leading to disease. Self-association is an inherent property of the lipid-free forms of several exchangeable apolipoproteins, including apoA-I, the main protein component of HDL and an established antiatherogenic factor. Monomeric lipid-free apoA-I is believed to be the biologically active species. But abnormal conditions, such as specific mutations or oxidation, produce an altered state of self-association that may contribute to apoA-I dysfunction. Although the functional role of self-association in some apolipoproteins has been established, the influence of self-association on apoA-I function has not been studied before because of technical limitations that are addressed and overcome in this project. Replacement of apoA-I's Trps with Phes (W-apoA-I) leads to unusually large and stable self-associated species. At least four self-associated species of W-apoA-I can be isolated and will be used here as a model of self-association to analyze its role in determining apoA-I's structure, function, and susceptibility to mechanisms leading to dysfunction. The three overlapping areas to be investigated in this project are: 1. Define the effects of self-association at all levels of apoA-I structure, from secondary to quaternary. The nature of the inter-molecular interactions that are involved in apoA-I self-association will be established and the structural details underlying the loss of lipid-binding efficiency for increasing degrees of self- association will be determined. This structural knowledge will help to understand the mechanisms whereby alteration of the protein self-association state affects its biological function. 2. Characterize how the self-association state o lipid-free apoA-I affects its function as recipient of lipids released from cells in the biogenesisof HDL. The efficiency of different apoA-I self-associated species in activating lipid release mediated by different cell membrane transporters will be determined. Including ABCA1, which is the primary mechanism underlying the anti-atherogenic function of apoA-I. a. Demonstrate that the self-association state of lipid-free apoA-I modulate the protein susceptibility to mechanisms leading to dysfunction. The vulnerability of different self-associated species to reactions that are implicated in the pathogenesis of atherosclerosis and diabetes will be tested. The possible role of self-association in protecting apoA-I from conditions which promote amyloid fibril formation, a contributing mechanism to atherosclerosis progression, will be also evaluated. These studies are highly significant for public health because determining a new functional aspect of apoA- I, which is one of the most important known anti-atherogenic factors, bears potential for the formulation of new therapies and the development of new biomarkers for the evaluation of cardiovascular disease risk. PUBLIC HEALTH RELEVANCE: Cardiovascular related diseases are the leading cause of mortality in developed countries. This project is highly relevant for public health because it addresses a fundamental structural and functional feature of apolipoprotein A-I, the most reliable predictor of cardiovascular disease risk. Lipid-free apolipoprotein A-I most effectively removes excess cholesterol from macrophages in the arterial wall, which is the primary mechanism whereby apoA-I exerts its antiatherogenic function. Yet the influence of self-association, one central structural property of lipid-free apolipoprotein A-I, on protein function has never been studied. This project takes advantage of a novel variant of apolipoprotein A-I that enables the isolation and study of individual self- association species. This research will show how self-association is a functional aspect of lipid-free apoA-I structure and demonstrate that alteration of this native state contributes to dysfunction mechanisms implicated in disease progression. Knowledge of this unexplored functional property of such an important factor for cardiovascular health will prompt the design of new therapeutic strategies and the development of new biomarkers for the evaluation of cardiovascular disease risk.