Plasma membrane cholesterol-rich microdomains, lipid rafts and caveolae, mediate HDL reverse cholesterol transport, signaling, antibody recognition, and serve as entry portals for potential bioterror toxins, viruses, and parasites. While cholesterol is essential for lipid raft/caveolae function, almost nothing is known regarding distribution, structure, and regulation of cholesterol in lipid rafts or caveolae. Preliminary Data show sterol carrier protein-2 (SCP-2) binds caveolin-1 in vitro and in vivo, alters lipid raft/caveolae cholesterol domains, and inhibits HDL-mediated cholesterol efflux from cells rich in caveolin-1 (L-cell fibroblasts). In contrast, cultured primary hepatocytes (from wild-type, SCP-2 overexpressing, and SCP-2 gene targeted mice) provide a model for addressing cholesterol membrane dynamics in cells essentially deficient in caveolin-1, but rich in proteins that mediate cholesterol transport between plasma membrane and HDL (SRB1, P-gp, ABCA1). We now propose four specific aims.
Aim 1. Determine which proteins (SRB1, ABCA1, P-gp) mediating HDL-cholesterol efflux/uptake are present in plasma membrane lipid rafts versus caveolae, interactions between them, and interactions with caveolin-1.
Aim 2. Examine how SCP-2 interacts with caveolin-1, and whether SCP-2 binds lipid raft or caveolae proteins mediating HDL-cholesterol uptake/efflux (SRB 1, ABCA1, P-gp).
Aim 3. Resolve if SCP-2 expression alters cholesterol distribution, cholesterol dynamics, and structure in purified rafts and caveolae. SCP-2 level will be modified with SCP-2 overexpressing L-cell fibroblasts and primary cultured hepatocytes from SCP-2 overexpressed and gene-ablated mice.
Aim 4. In living cells, determine if SCP-2 expression alters cholesterol distribution, structure, and dynamics in lipid rafts or caveolae. Results will provide fundamental, mechanistic details of cholesterol organization and dynamics in lipid rafts, regulation by SCP-2, differential roles of rafts versus caveolae in mediating HDL-cholesterol uptake/efflux, and insights on potential intervention in metabolic disease, pathogen entry, and bioterror agents. ? ?
Elevated blood cholesterol predisposes to atherosclerosis. Cholesterol is derived from bothdiet and made in the body. To maintain optimum cholesterol levels; the human liverexcretes nearly 2 grams of cholesterol per day into bile. Decreased cholesterol disposalresults in increased blood cholesterol; while excessive secretion or disproportionate biliaryconstituents may lead to gallstones and gallbladder inflammation. Currently; mosteffective cholesterol-lowering drugs interfere with cholesterol synthesis (statins); blockintestinal cholesterol absorption (ezetimibe); or enhance fecal excretion (bile acidsequestrants). Resolving mechanisms and candidate proteins involved in rapid uptakeand transhepatocyte transport of cholesterol will identify new therapeutic targets forlowering elevated cholesterol and therefore treatment of cardiovascular disease;gallstones; and obesity.
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