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

项目摘要 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 有效的治疗集中在特定的风险因素，如低密度脂蛋白胆固醇（LDL-C），患病率和死亡率 这种疾病的发病率仍然很高。这就需要调查额外的风险因素， 新的治疗目标。载脂蛋白C-III和A-V是血浆中TG的相互调节剂， 最近通过人类遗传学暗示与发展CAD的风险显著相关。遗传 研究表明，失活ApoC-III或增强ApoA-V可以预防CAD。但我们 目前对这两种蛋白质的结构和机制以及它们是如何在细胞中形成的了解很少。 疾病相关突变发挥其调节TG水平和CAD风险的作用。在这一建议中，我们寻求 为了阐明这两种载脂蛋白的野生型（WT）形式在人结肠癌中的螺旋和结构动力学， 通过氢-氘交换和质谱分析， 水平分辨率。我们还将研究所确定的编码突变对蛋白质折叠，动力学和生物学的影响。 脂质结合我们认为，已确定的突变影响脂质结合，并导致改变TG代谢 通过影响脂蛋白结合、LPL活性、富TG脂蛋白清除和肝TG 新陈代谢.我们将通过生物化学方法测试每个变体如何起作用的假设， 比较WT和突变体ApoC-III/A-V在人源化小鼠中的表达的互补体内研究 模型最后，我们将评估CAD相关ApoC-III/A-V突变对动脉粥样硬化形成的影响， vivo.所提出的实验将解释ApoC-III和ApoA-V在人乳腺癌中的具体作用机制。 调节TG代谢和CAD风险，并确定对这些最关键的结构特征 功能协调发展的

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.