Apolipoprotein E/Lipoprotein Binding Mechanism

载脂蛋白 E/脂蛋白结合机制

• 批准号: 8856278
• 负责人: VASANTHY NARAYANASWAMI
• 金额: $ 10.84万
• 依托单位: CALIFORNIA STATE UNIVERSITY LONG BEACH
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8856278
• 关键词: Address; Alzheimer' s Disease; Amino Acids; Antiatherogenic; Apolipoprotein E; Apolipoproteins; Atherosclerosis; Behavior; Biomedical Research; Brain; C-terminal; Cardiovascular Diseases; Carrier Proteins; Cholesterol; Cholesterol Homeostasis; Collaborations; Coupled; Deuterium; Development; Disease; Fluorescence Polarization; Fluorescence Spectroscopy; Fluorescent Probes; Goals; Health; High Density Lipoproteins; Human; Hydrogen; Individual; International; Intervention; Lead; Ligands; Lipid Binding; Lipids; Lipoprotein Binding; Lipoproteins; Low Density Lipoprotein Receptor; Mass Spectrum Analysis; Mediating; Medical; Mentorship; Molecular; Molecular Conformation; Monitor; N-terminal; Outcome; Pattern; Physiological; Plasma; Play; Positioning Attribute; Predisposition; Process; Protein Family; Protein Isoforms; Proteins; Publishing; Recombinants; Relative (related person); Research; Research Activity; Research Personnel; Research Training; Rest; Risk Factors; Role; Solvents; Specific qualifier value; Staging; Structure; Students; Testing; Time; Ursidae Family; Variant; Vertebral column; Vesicle; apolipoprotein E-3; apolipoprotein E-4; base; crosslink; dimer; disulfide bond; fluorophore; high risk; innovation; lipid transport; macrophage; meetings; particle; receptor mediated endocytosis; reconstitution; reverse cholesterol transport

DESCRIPTION (provided by applicant): Apolipoprotein E (apoE) is an exchangeable apolipoprotein that plays a critical role in lipid transport and in maintaining plasma and brain cholesterol homeostasis. It mediates its role by serving as a ligand for the low- density lipoprotein receptor (LDLr) family of proteins, which facilitate receptor-mediated endocytosis of lipoproteins, thereby eventually lowering plasma lipid levels. It also plays a role in reverse cholesterol transport in atherosclerosis, by promoting cholesterol efflux from macrophages to form high-density lipoproteins (HDL). In this proposal, we will focus on understanding the differences between the two major human apoE isoforms, apoE3 and apoE4, and their role in HDL formation. ApoE3 is associated with normal plasma lipid profiles (anti- atherogenic), while apoE4 is considered pro-atherogenic and is a risk factor for cardiovascular disease (CVD) and Alzheimer's disease (AD). However, neither the mechanism of lipid binding nor the molecular basis for the differences in the physiological behavior between the isoforms is known. We will test the overall hypothesis that there are isoform-specific variations in the conformation of apoE3 and apoE4, which lead to differences in lipid binding mechanism and in lipid (HDL)-bound conformation. To accomplish this, we will: (i) determine the unfolding pattern of recombinant apoE3 and apoE4 using a combination of fluorescence polarization (FP) and hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS); (ii) compare the lipid binding mechanism between apoE3 and apoE4 by capturing 'snapshots' of the proteins during lipid binding by FP and HDX-MS, and identifying possible isoform-specific differences in the order of lipid binding of the different helices, and, (iii) examine the lipid-bound conformation of apoE in reconstituted and macrophage-generated HDL following cholesterol efflux by fluorescence spectroscopy and cross-linking analysis. Completion of these studies will significantly advance our understanding of the isoform-specific differences in the physiological behavior of apoE and the predisposition of apoE4 to CVD and AD. By determining the mechanistic basis of lipid binding, our studies will provide timely and much-needed opportunities to identify potential intervention strategies to treat CVD and AD. The developmental objectives of the PI are to establish and sustain a highly productive and scholarly research activity at CSULB, publish research findings, offer research training opportunities and mentorship for student trainees from diverse backgrounds in state-of-the-art biomedical research, actively participate in national and international meetings, initiate and continue collaboration with established investigators outside CSULB, and expand her expertise to address questions related to AD.

描述（由申请人提供）：载脂蛋白E（APOE）是一种可兑换的载脂蛋白，在脂质转运以及维持血浆和脑胆固醇稳态方面起着至关重要的作用。它通过用作低密度脂蛋白受体（LDLR）蛋白质的配体来介导其作用，从而促进受体介导的脂蛋白内吞作用，从而最终降低血浆脂质水平。它还通过促进巨噬细胞的胆固醇外排以形成高密度脂蛋白（HDL），在动脉粥样硬化中的反向胆固醇转运中起作用。在此提案中，我们将专注于理解两个主要的人类APOE同工型APOE3和APOE4及其在HDL形成中的作用之间的差异。 APOE3与正常的血浆脂质谱（抗动脉粥样硬化）有关，而APOE4被认为是促动脉粥样硬化的，是心血管疾病（CVD）和阿尔茨海默氏病（AD）的危险因素。但是，脂质结合的机制和同工型之间生理行为差异的分子基础均不知道。我们将测试总体假设，即APOE3和APOE4的构象存在同工型特异性变化，这会导致脂质结合机理和脂质（HDL）结合构象的差异。为此，我们将：（i）使用荧光极化（FP）和氢氘交换的组合确定重组APOE3和APOE4的展开模式，该模式耦合到质谱（HDX-MS）； （ii）通过捕获FP和HDX-MS脂质结合期间蛋白质的“快照”，比较APOE3和APOE4之间的脂质结合机制，并鉴定出可能在不同螺旋的脂质结合顺序的同工型特异性差异，并检查了不同螺旋的脂质结合的顺序，并检查了（III）（III），（III），（iii），（iii）（III） 通过荧光光谱和交联分析，胆固醇外排的重建和巨噬细胞产生的HDL中的APOE。这些研究的完成将显着提高我们对APOE生理行为以及APOE4对CVD和AD的倾向的同工型特异性差异的理解。 通过确定脂质结合的机械基础，我们的研究将提供及时且急需的机会，以确定治疗CVD和AD的潜在干预策略。 PI的发展目标旨在在CSULB建立和维持一项高产和学术的研究活动，发布研究结果，为来自各种背景的学生学员提供研究培训机会和指导，积极地参与国家和国际会议，与CSULB以外的CSULB以外的既定调查员一起，并继续与CSULB之外的研究人员进行探索，并继续与她的问题相关问题，并继续与她的专业相关的问题。