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Background: Atherosclerosis is a multifactorial inflammatory disease that begins with the accumulation of lipid in arterial endothelium. Atherosclerosis is the leading cause of deaths attributable to cardiovascular disease in the United States. Epidemiological studies showing high-density lipoprotein (HDL) cholesterol is inversely correlated with atherosclerosis has made it a pharmacological target for preventing cardiovascular disease. However, outcomes from clinical trials have raised questions about HDL’s protective properties. Investigating the molecular interactions of apolipoprotein (apo)A-I, which accounts for approximately 70% of total HDL protein, can help translate HDL structure to cardioprotective function. Lecithin: cholesterol acyl transferase (LCAT) is a critical HDL-modifying protein that performs a key function in reverse cholesterol transport by using apoA-I as a cofactor to esterify cholesterol. Data from our lab and others demonstrate that apoA-I molecules dimerize into an antiparallel stacked ring-structure that encapsulates lipid in reconstituted (r)HDL. Cross-linking analysis of rHDL implies that apoA-I molecules exist in at least two distinct organizations: one with helix 5 of an apoA-I molecule adjacent to helix 5 of its antiparallel partner (5/5 helical registry), and the other in a 5/2 registry. We hypothesized that the orientation of apoA-I molecules on rHDL modulates LCAT activity. Objective: Identify the mechanism by which apoA-I activates LCAT to determine how HDL interacts with its immense proteome. Linking HDL structure and function will allow for therapeutic development that targets HDL-associated inflammatory diseases. Major Findings: 1)Antiparallel apoA-I molecules adopt a thumbwheel mechanism to generate a discontinuous epitope for LCAT activation. Site-directed cysteine mutagenesis was used to “lock” two apoA-I molecules into an antiparallel 5/5, 5/2, and 5/1 helical registry on rHDL. The 5/5 mutant demonstrated higher LCAT activity than wildtype (WT) rHDL, while the 5/2 and 5/1 mutants showed dramatically lower LCAT activity (p |
