Atherosclerosis is a chronic inflammatory disease that is promoted by the consumption of dietary fat andcholesterol. The extent to which atherosclerosis progresses to cause coronary occlusion and/or death ismediated by the presence of plasma apoA-l, the main protein constituent of high density lipoproteins (HDL).The role of HDL apoA-l in whole body cholesterol homeostasis has been extensively investigated and its roleis to direct cholesterol from the periphery to the liver for excretion. Despite intensive investigations, majorquestions remain regarding the mechanism by which apoA-l regulates cellular cholesterol levels and how theapoprotien's unique structural features facilitate cholesterol transport. To more completely understand thestructural basis for apoA-l's role in cholesterol transport, we will carry out three specific aims that will clarifythe structural reorganization necessary for the formation of nascent HDL via ABCA1.
In Aim 1 we propose toaddress the role of apoA-l in cholesterol homeostasis by determining the conformation of apoA-l on fourdifferent sized subclasses of ABCA1 generated nascent HDL. Also as part of Aim 1, we will examine the'unfolding' steps' through which lipid-free apoA-l acquires lipid from ABCA1. To do this we will construct aseries of 'tethered' disulfide apoA-l mutants that will prevent key intermediate unfolding steps through whichthe 4-helix bundle must transition as it organizes and binds phospholipid and cholesterol. In the Aim 2, wewill investigate the mechanistic basis for the dominant negative repression of wild-type apoA-l HDL, asobserved in humans who carry this mutation, by investigating the lipidation of the helix 6 mutant, L159RapoA-l by ABCA1. Our preliminary studies suggest that this single amino acid substitution mutant, L159RapoA-l, competes with wild-type apoA-l for phospholipid and cholesterol, resulting in a reduction in theoverall lipidation of wild-type apoA-l by ABCA1 in a model cell culture system.
In Aim 3, we plan to determinewhether the dominant negative phenotype associated with the L159R apoA-l mutant is pro- or antiatherogenicin hyperlipidemic mice. Using transgenic mice that express wild-type or L159R apoA-l, we willdetermine the extent to which the mutant apoA-l protects against the development of atherosclerosis, as wellas investigating the in vivo basis for the low concentration of L159R apoA-l in plasma as it relates to thedominant negative phenotype.
Showing the most recent 10 out of 242 publications
