Intracellular cholesterol sorting and transport pathways govern the physiologic utilization of lipoprotein-derived cholesterol, and are central to pathophysiology of inherited disorders such as Niemann-Pick type C (NPC) and of acquired atherosclerotic disease. The NPC1 protein, which functions in these pathways, bears sequence similarity to Patched, HMG- CoA reductase, and SCAP. Mutations in NPC1 result in accumulation of unesterified lysosomal cholesterol, but the specific function of NPC1 in cellular cholesterol metabolism is not well understood. The present proposal is designed to test the hypothesis that NPC1 is a key participant in the post-plasma membrane (PM) trafficking of low density lipoprotein (LDL) cholesterol. We propose that NPC1 resides within an endocytic organelle, whose function is to sort and recycle internalized PM lipids. NPC1 may perform an essential function in the endocytic pathway by facilitating vesicular trafficking of the lipid cargo back to the PM. Moreover, NPC1 may play an important role in cellular cholesterol homeostasis, serving to maintain normal levels of PM cholesterol, as well as to shuttle excess unesterified cholesterol to the endoplasmic reticulum (ER) for esterification. These hypotheses will be tested by the following specific aims: (1) Examination of the role of NPC1 in intracellular trafficking of LDL cholesterol, (2) Use of a functional genetic screen to identify genes required for LDL cholesterol trafficking, (3) Characterization of NPC1- interacting proteins identified by a yeast two-hybrid screen and their roles in cholesterol trafficking, and (4) Characterization of the function of NPC1 and proteins identified by our genetic screens in cell culture models of atherogenesis. These studies will contribute to our understanding of the molecular basis of intracellular cholesterol trafficking under normal conditions and in atherogenesis. The work proposed herein may identify novel targets for pharmacologic therapy within the sterol transport pathway for treatment of coronary and cerebral vascular disease and for inherited metabolic disorders.
