Regulation of CETP by lipid transfer inhibitor protein

‘描述(由申请人提供)：血浆胆固醇和低密度脂蛋白(LDL)浓度是动脉粥样硬化的主要风险因素，而高密度脂蛋白(HDL)水平升高则降低风险。胆固醇酯转运蛋白(CETP)在脂蛋白之间转运脂类，从而直接影响脂蛋白代谢，改变低密度脂蛋白和高密度脂蛋白的浓度。CETP水平对饮食中的脂质和遗传因素有很高的反应。此外，CETP的活性受脂转移抑制蛋白(LTIP)的调节。我们已经证明，LTIP并不降低CETP活性，而是选择性地抑制低密度脂蛋白上的CETP活性，对HDL2上的CETP活性有较小的抑制作用，但对HDL3上的CETP活性有刺激作用。因此，通过高密度脂蛋白的胆固醇流量增加，血浆胆固醇酯合成率增加。因此，LTIP调整CETP介导的脂转移事件，导致脂蛋白谱不同于仅通过提高或降低CETP水平而实现的脂蛋白谱。 在接下来的申请中，我们将更充分地定义LTIP的功能，并表征其调节和作用机制。我们假设LTIP改变CETP活性以产生更有益或更具动脉粥样硬化保护作用的脂蛋白图谱。将讨论三个具体目标：目标1)确定LTIP抑制CETP的机制，并确定对这一活动重要的LTIP的结构特征。我们将量化LTIP与LDL、HDL2和HDL3的结合动力学，确定LTIP结合如何与CETP抑制和CETP从脂蛋白表面置换有关，并确定LTIP结合如何影响脂蛋白表面的组成和结构。突变研究将确定LTIP功能所需的区域和特定氨基酸。目的2)确定LTIP在调节脂蛋白代谢中的作用。腺病毒介导的LTIP过表达或LTIP抑制对脂蛋白组成以及对极低密度脂蛋白和高密度脂蛋白代谢的影响将在仓鼠模型中确定。LTIP表达的改变对前β-高密度脂蛋白形成和血浆促进细胞胆固醇外流的能力的影响也将被量化。重组组分的生化研究将确定LTIP影响极低密度脂蛋白和高密度脂蛋白代谢的特定方面的能力，并为解释体内观察提供机制基础。目的3)鉴定失活的LTIP复合体，明确其在调节LTIP活性中的作用。我们观察到，LTIP是由一个不活跃的复合体的隔离作用控制的。我们将分离这种复合体，并确定其蛋白质和脂肪成分。此外，由于与这种复合体的关联是动态的，我们将确定代谢过程和血脂水平的变化如何改变LTIP在活动池和非活动池之间的分布。 总体而言，这些研究将为LTIP控制CETP提供新的见解，并增加我们对血管内脂蛋白重塑事件如何影响稳态脂蛋白浓度和组成的理解。

'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.