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: 10245120
• 负责人: Sylvia Georgieva Stankov
• 金额: $ 4.6万
• 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/10245120
• 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）是全球范围内的主要死亡原因。一个主要的因果风险因素， CAD是低密度脂蛋白胆固醇（LDL-C）水平升高。虽然降低LDL-C是一个基石 在CAD的预防和治疗中，许多患者继续经历CAD事件， 有效降低LDL-C，表明存在重要的剩余风险。血浆甘油三酯（TG）是一种独立的 CAD风险的预测因素。TG与CAD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。 LDL-C水平，并且是他汀类药物血管风险的最强预测因子之一。TG在TG丰富的 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），其水解TRL中的TG用于释放的游离脂肪酸的局部吸收。最近 大规模的人类遗传学研究已经确定，与TG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， 载脂蛋白ApoA-V已被鉴定为LPL的特别令人感兴趣的调节剂。ApoA-V主要是 从肝脏分泌，并可以在高密度脂蛋白（HDL）和TRL之间交换。ApoA-V具有 已经显示出增强LPL活性，尽管确切的机制仍不清楚。人类遗传学 强有力地支持ApoA-V在TG代谢中的作用：病例对照，基于家族的测序研究，以及一项 外显子组测序研究表明高甘油三酯血症（hyperTG）中有几种编码变体， 高乳糜微粒血症和早期心肌梗死。此外，hyperTG表型在 apoa 5敲除小鼠可以被重组人ApoA-V或人APOA 5 AAV抑制。 我的工作旨在从功能上描述所选天然APOA 5变体的作用，无论是在体外还是在体外， 和体内。初步证据支持2种预测的功能丧失（LoF）变体Q275 X和Q305 X以及3种 不明确和/或潜在有益的变体（预测的功能获得性（GoF））D37 E、P215 L和T292 I， 这对于阐明ApoA-V功能可能是特别有用的。我的首要目标是评估脂蛋白 选择的APOA 5变体的结合和LPL活性增强，因为ApoA-V结合脂蛋白的能力是 这对于将蛋白质定位在TRL-LPL界面至关重要。我假设APOA 5预测LoF变异 降低脂蛋白结合，钝化LPL激活，并增加血浆TG水平，而APOA 5 预测的GoF变体增加脂蛋白结合，导致相互表型。我的第二个目标 是为了确定APOA 5变异体对长期TG代谢和动脉粥样硬化的影响。我假设 表达APOA 5预测LoF变体的动脉粥样硬化小鼠模型将具有降低的TG 清除和增强动脉粥样硬化进展，而APOA 5预测的GoF变体将具有 相互表型这些研究将为ApoA-V对肿瘤细胞凋亡的调节提供新的见解。 血浆TG是一种可靶向的CAD风险因素，可以指导合理设计的治疗方法的开发。