Regulation of CETP by lipid transfer inhibitor protein

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'DESCRIPTION (provided by applicant): Plasma cholesterol and low density lipoprotein (LDL) concentrations are major risk factors for atherosclerosis, whereas elevated high density lipoprotein (HDL) levels decreased risk. Cholesteryl ester transfer protein (CETP) transports lipids between lipoproteins and, consequently, directly affects lipoprotein metabolism and alters LDL and HDL concentrations. CETP levels are highly responsive to dietary lipids and genetic factors. Additionally, CETP activity is regulated by lipid transfer inhibitor protein (LTIP). We have demonstrated that LTIP does not lower CETP activity generically but selectively suppresses CETP activity on LDL, and to a lesser extent on HDL2, but stimulates CETP activity on HDL3. Consequently, cholesterol flux through HDL is enhanced and plasma cholesteryl ester synthesis rates are increased. Thus LTIP tailors CETP-mediated lipid transfer events resulting in a lipoprotein profile that is different from that achieved by raising or lowering CETP levels alone. In this continuation application we will more fully define the functions of LTIP, and characterize its regulation and mechanism of action. We hypothesize that LTIP alters CETP activities to generate a more beneficial or atheroprotective lipoprotein profile. Three specific aims will be addressed: AIM 1) Define the mechanism by which LTIP inhibits CETP and identify the structural features of LTIP important for this activity. We will quantify binding kinetics of LTIP to LDL, HDL2 and HDL3 and determine how LTIP binding relates to CETP inhibition and the displacement of CETP from the lipoprotein surface, and define how LTIP binding influences the composition and structure of the lipoprotein surface. Mutagenesis studies will define regions and specific amino acids required for LTIP function. AIM 2) Determine the role of LTIP in regulating lipoprotein metabolism. The effects of adenoviral-mediated LTIP overexpression or LTIP suppression on lipoprotein composition and on VLDL and HDL metabolism will be defined in vivo in a hamster model. The effects of altered LTIP expression on prebeta-HDL formation and the capacity of plasma to promote cholesterol efflux from cells will also be quantified. Biochemical studies with reconstituted components will define the capacity of LTIP to influence specific aspects of VLDL and HDL metabolism, and provide a mechanistic basis for interpreting in vivo observations. AIM 3) Characterize the inactive LTIP complex and define its role in regulating LTIP activity. We have observed that LTIP is controlled by sequestration into an inactive complex. We will isolate this complex and define its protein and lipid components. Further, as association with this complex is dynamic, we will determine how metabolic processes and changes in plasma lipid levels alter the distribution of LTIP between active and inactive pools. Overall, these studies will provide novel insight into the control of CETP by LTIP and add to our understanding of how intravascular lipoprotein remodeling events contribute to steady-state lipoprotein concentration and composition.