Functional Analysis of Triglyceride Regulator ApoA-V Using Natural Variants

使用天然变体进行甘油三酯调节剂 ApoA-V 的功能分析

• 批准号: 10021411
• 负责人: Sylvia Georgieva Stankov
• 金额: $ 4.55万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-16 至 2022-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10021411
• 关键词: APOA5 gene; Alanine Transaminase; Apolipoproteins; Apolipoproteins A; Apolipoproteins C; Arterial Fatty Streak; Atherosclerosis; Biochemical; Biomedical Research; Blood Vessels; Cause of Death; Cell surface; Cholesterol; Code; Coronary Arteriosclerosis; Development; Etiology; Event; Exhibits; Extrahepatic; Family; Fatty acid glycerol esters; Frequencies; Genes; Genetic study; Goals; Health; Heparin; High Density Lipoproteins; Human; Human Genetics; Hypertriglyceridemia; In Vitro; Injections; Investigation; Knock-out; Knockout Mice; LDL Cholesterol Lipoproteins; Lipase; Lipids; Lipoprotein Binding; Lipoproteins; Liver; Low Density Lipoprotein Receptor; Measures; Medicine; Molecular; Mus; Mutation; Myocardial Infarction; Nonesterified Fatty Acids; Olives - dietary; Oral; Outcome; Pathway interactions; Patients; Phenotype; Phospholipids; Plasma; Prevention; Process; Proprotein Convertases; Prospective Studies; Proteins; Recombinants; Residual state; Risk; Risk Factors; Role; Sampling; Stomach; Structure; Subtilisins; Techniques; Testing; Therapeutic; Tissues; Training; Triglyceride Metabolism; Triglycerides; Triolein; Variant; Work; absorption; base; biobank; career; case control; clinically relevant; design; disorder risk; effective therapy; exome sequencing; experience; experimental study; fast protein liquid chromatography; gain of function; genetic variant; genome wide association study; head-to-head comparison; in vitro Assay; in vivo; insight; interest; lipoprotein lipase; liver injury; loss of function; mouse model; novel; novel therapeutic intervention; overexpression; plasmid DNA; preference; segregation

Project Summary Coronary artery disease (CAD) is a leading cause of death worldwide. A major causal risk factor for CAD is elevated low-density lipoprotein cholesterol (LDL-C) levels. While reduction in LDL-C is a cornerstone of the prevention and treatment of CAD, many patients continue to experience CAD events despite highly effective LDL-C reduction, indicating important residual risk. Plasma triglycerides (TGs) are an independent predictor of CAD risk. TGs are strongly associated with CAD events even in statin-treated patients with low LDL-C levels, and are one of the strongest predictors of on-statin vascular risk. TGs are carried in TG-rich lipoproteins (TRLs). TRLs provide energy to extrahepatic tissues through the activity of cell-surface lipoprotein lipase (LPL), which hydrolyzes TGs in TRLs for the local absorption of the released free fatty acids. Recent large-scale human genetics studies have established that genetic variants associated with TG levels are also strongly associated with CAD. These genetic studies have especially pointed to the LPL pathway, and the apolipoprotein ApoA-V has been identified as a particularly interesting modulator of LPL. ApoA-V is primarily secreted from the liver and can exchange between high-density lipoproteins (HDLs) and TRLs. ApoA-V has been shown to enhance LPL activity, although the precise mechanism remains unclear. Human genetics have strongly supported ApoA-V’s role in TG metabolism: case-control, family-based sequencing studies, and an exome sequencing study have implicated several coding variants in hypertriglyceridemia (hyperTG), hyperchylomicronemia, and early myocardial infarction, respectively. Additionally, the hyperTG phenotype in apoa5 knockout mice can be suppressed by recombinant human ApoA-V or human APOA5 AAV. My work seeks to functionally characterize the effects of selected natural APOA5 variants, both in vitro and in vivo. Preliminary evidence supports 2 predicted loss-of-function (LoF) variants Q275X and Q305X and 3 ambiguous and/or potentially beneficial variants (predicted gain-of-function (GoF)) D37E, P215L, and T292I, that may be particularly informative for elucidating ApoA-V function. My first goal is to assess lipoprotein binding and LPL activity enhancement of selected APOA5 variants, as ApoA-V’s ability to bind lipoproteins is critical to localizing the protein at the TRL-LPL interface. I hypothesize that APOA5 predicted LoF variants decrease lipoprotein binding, blunt LPL activation, and increase plasma TG levels, while APOA5 predicted GoF variants increase lipoprotein binding, resulting in reciprocal phenotypes. My second goal is to determine APOA5 variant impacts on long-term TG metabolism and atherosclerosis. I hypothesize that atherosclerotic mouse models expressing APOA5 predicted LoF variants will have decreased TG clearance and augmented atherosclerotic progression while APOA5 predicted GoF variants will have reciprocal phenotypes. The proposed studies will provide substantial new insight into ApoA-V’s modulation of plasma TGs, a targetable CAD risk factor, that could guide development of rationally-designed therapeutics.

项目摘要 冠状动脉疾病(CAD)是世界范围内主要的死亡原因。一个主要的致病风险因素 冠心病是指低密度脂蛋白胆固醇(LDL-C)水平升高。虽然降低低密度脂蛋白是一个基石 在冠心病的预防和治疗中，许多患者尽管高度重视，但仍继续经历CAD事件 有效降低低密度脂蛋白胆固醇，表明存在重要的残余风险。血浆甘油三酯(TGS)是一种独立的 冠心病风险的预测指标。TGS与冠心病事件密切相关，即使在他汀类药物治疗的低血糖患者中也是如此 低密度脂蛋白水平，是他汀类药物血管风险的最强预测因子之一。Tg以富含Tg的形式携带 脂蛋白(TRL)。TRL通过细胞表面脂蛋白的活性为肝外组织提供能量 脂肪酶(LPL)，它能分解TRL中的TGS，使释放的游离脂肪酸得到局部吸收。近期 大规模的人类遗传学研究已经证实，与甘油三酯水平相关的遗传变异也是 与CAD密切相关。这些遗传学研究特别指出了LPL途径，以及 载脂蛋白ApoA-V已被确定为一种特别有趣的LPL调节剂。载脂蛋白A-V主要是 由肝脏分泌，可在高密度脂蛋白(HDL)和TRL之间交换。载脂蛋白A-V有 已被证明可以增强LPL活性，尽管确切的机制尚不清楚。人类的遗传学已经 有力支持载脂蛋白A-V在甘油三酯代谢中的作用：病例对照、基于家族的测序研究和 外显子组测序研究发现高甘油三酯血症(HyperTG)涉及几个编码变异， 分别为高乳糜血症和早期心肌梗死。此外，高甘油三酯在 载脂蛋白5基因敲除小鼠可被重组人载脂蛋白A-V或人载脂蛋白AAV抑制。 我的工作试图从功能上表征选定的天然APOA5变体的影响，这两种变体都是在体外 在活体内。初步证据支持两种预测的功能丧失(LoF)变体Q275X和Q305X和3 模棱两可和/或潜在有益的变体(预测的函数增益(GoF))D37E、P215L和T292I， 这对于阐明载脂蛋白A-V的功能可能特别有用。我的第一个目标是评估脂蛋白 由于载脂蛋白A-V与脂蛋白结合的能力是 对于将蛋白质定位在TRL-LPL界面上至关重要。我假设APOA5预测了LOF变种 减少脂蛋白结合，钝化LPL激活，升高血浆TG水平，而APOA5 预测的Gof变异体增加脂蛋白结合，导致相互的表型。我的第二个目标 目的是确定APOA5变异对长期甘油三酯代谢和动脉粥样硬化的影响。我假设 表达APOA5预测的LOF变异体的动脉粥样硬化小鼠模型将降低甘油三酯 清除和动脉粥样硬化进展加剧，而APOA5预测的GoF变体将 相互作用的表型。拟议的研究将为载脂蛋白A-V对 血浆TGS，一个有针对性的CAD危险因素，可以指导合理设计的治疗方法的发展。