Structure-Function Analysis of Triglyceride Regulators ApoC-III and ApoA-V Using Natural Variants

项目总结 冠状动脉疾病(CAD)仍然是现代世界最高的疾病负担之一。尽管 有效的治疗侧重于特定的风险因素，如低密度脂蛋白(LDL-C)、患病率和死亡率 这种疾病的发病率仍然很高。这就需要调查更多的风险因素并确定 治疗的新靶点。载脂蛋白C-III和A-V是血浆和WARE中甘油三酯的相互调节 最近通过人类遗传学被认为与发生CAD的风险显著相关。遗传 研究表明，灭活载脂蛋白C-III或增强载脂蛋白A-V可能对冠心病有保护作用。然而，我们 目前对这两种蛋白质的结构和机制以及如何 疾病相关突变发挥其调节甘油三酯水平和冠心病风险的作用。在这项提案中，我们寻求 为了阐明这两种载脂蛋白野生型(WT)的螺旋和结构动力学 通过氢-氚交换和质谱学对氨基酸的脱脂和脂结合状态- 级别分辨率。我们还将研究已识别的编码突变对蛋白质折叠、动力学和 脂类结合。我们推测，已识别的突变会影响脂结合，并导致甘油三酯代谢改变。 通过影响脂蛋白结合、LPL活性、富含甘油三酯的脂蛋白清除和肝脏甘油三酯 新陈代谢。我们将通过生化方法测试假设每个变种是如何起作用的 WT及其突变体ApoC-III/A-V在人源化小鼠体内表达的互补研究 模特们。最后，我们将评估与CAD相关的ApoC-III/A-V突变对动脉粥样硬化形成的影响 活着。拟议的实验将破译载脂蛋白C-III和载脂蛋白A-V在体内的具体作用机制 调节TG代谢和冠心病的风险，并确定对这些最关键的结构特征 功能。

PROJECT SUMMARY Coronary artery disease (CAD) remains among the highest disease burdens in the modern world. Despite effective treatments focused on specific risk factors such as LDL cholesterol (LDL-C), prevalence and mortality from this disease remains very high. This necessitates investigating additional risk factors and identifying novel targets for treatment. Apolipoproteins C-III and A-V are reciprocal regulators of TG in plasma and were recently implicated through human genetics as being significantly related to risk of developing CAD. Genetic studies suggested that inactivating ApoC-III or enhancing ApoA-V, may protect against CAD. However, we currently have very little understanding of the structure and mechanisms of these two proteins and how the disease-associated mutations exert their effects to regulate TG levels and CAD risk. In this proposal, we seek to elucidate the helical and structural dynamics of the wild-type (WT) forms of these two apolipoproteins in the lipid-free and lipid-bound states through hydrogen-deuterium exchange and mass spectrometry to amino acid- level resolution. We will also study the effects the identified coding mutations on protein folding, dynamics, and lipid binding. We posit that the identified mutations influence lipid binding and cause altered TG metabolism through impacting lipoprotein association, LPL activity, TG-rich lipoprotein clearance and hepatic TG metabolism. We will test hypotheses for how each variant is acting through biochemical approaches and complementary in vivo studies comparing expression of WT and mutant ApoC-III/A-V in humanized mouse models. Finally, we will assess the impact of the CAD-associated ApoC-III/A-V mutations on atherogenesis in vivo. The proposed experiments will decipher the specific mechanisms of action of ApoC-III and ApoA-V in regulating TG metabolism and risk of CAD, and determine the structural features most crucial to these functions.