The objective of the proposed research is to determine fundamental factors that regulate cholesterol distribution within the cell. Cholesterol may accumulate in specific cell membranes during aging and atheroschlerosis. Specifically we wish to focus on the role of sterol carrier protein (SCP) in conferring specificity to the intracellular movement of cholesterol that is not accounted for by spontaneous exchange or transfer. Using fluorescent cholesterol analogues, we demonstrated for the first time that SCP binds sterols in vivo and in vitro. The SCP can also exchange the fluorescent sterols into and between membranes.
The specific aims of this proposal are as follows: 1) Isolate plasma membranes, phagosomes, microsomes and mitochondria from cultured LM fibroblasts. These subcellular membranes differ over a ten-fold range in cholesterol content. The membranes will be used to examine the role of SCP in conferring specificity to cholesterol movement between membranes. 2) Intact membranes, lipids extracted from the membranes, and model membranes will be used to determine if lipids or another membrane constiutent (e.g. proteins) are involved as """"""""targets"""""""" or SCP """"""""binding sites"""""""" that confer specificity to SCP mediated sterol movement. 3) The mechanism(s) whereby SCP may mediate cholesterol transfer between membranes (carrier mediated, collisional transfer, fusion, or physical state alterations) will be examined. 4) The properties of sterols in membranes, sterol-phospholipid interactions in membranes, and sterol-SCP interactions will be explored using fluorescent sterol analogues. The significance of this research is to elucidate a potential role for SCP, a ubiquitous cytosolic protein (2-14% cytosolic protein), in enriching and/or transferring cholesterol to specific membranes. Understanding of how membrane sterol content is regulated may be important to a variety of diseases in which cholesterol may accumulate in membranes as in aging, cancer, cholestasis, and intracellular cholesterol accumulation. These results may ultimately lead to a better understanding of one potential biological mechanism involved in cardiovascular disease.
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