Apolipoprotein E/Lipoprotein Binding Mechanism

载脂蛋白 E/脂蛋白结合机制

• 批准号: 8666778
• 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 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8666778
• 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; 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; public health relevance; 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）是一种可交换的载脂蛋白，在脂质转运和维持血浆和脑胆固醇稳态中起关键作用。其通过充当低密度脂蛋白受体（LDLr）蛋白家族的配体来介导其作用，所述蛋白家族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。它还通过促进胆固醇从巨噬细胞流出形成高密度脂蛋白（HDL），在动脉粥样硬化中的胆固醇逆向转运中发挥作用。在这个建议中，我们将重点了解两个主要的人类apoE亚型，apoE 3和apoE 4之间的差异，以及它们在HDL形成中的作用。ApoE 3与正常血浆脂质谱（抗动脉粥样硬化）相关，而apoE 4被认为是促动脉粥样硬化的，并且是心血管疾病（CVD）和阿尔茨海默病（AD）的风险因素。然而，脂质结合的机制和异构体之间生理行为差异的分子基础都是未知的。我们将测试的整体假设，有异构体特异性的apoE 3和apoE 4的构象的变化，这导致在脂质结合机制和脂质（HDL）结合构象的差异。为了实现这一点，我们将：（i）使用荧光偏振（FP）和氢氘交换耦合质谱（HDX-MS）的组合来确定重组apoE 3和apoE 4的解折叠模式;（ii）通过FP和HDX-MS在脂质结合期间捕获蛋白质的“快照”，比较apoE 3和apoE 4之间的脂质结合机制，并鉴定不同螺旋的脂质结合顺序中可能的同种型特异性差异，以及（iii）检查 通过荧光光谱和交联分析，在胆固醇流出后，重组和巨噬细胞产生的HDL中的apoE。这些研究的完成将大大推进我们对apoE的生理行为和apoE 4对CVD和AD的易感性的异构体特异性差异的理解。 通过确定脂质结合的机制基础，我们的研究将提供及时和急需的机会，以确定治疗CVD和AD的潜在干预策略。PI的发展目标是在CSULB建立和维持一个高效的学术研究活动，发表研究成果，为来自不同背景的学生学员提供研究培训机会和指导，积极参加国家和国际会议，启动并继续与CSULB以外的既定研究人员合作，并扩展她的专业知识，以解决与AD相关的问题。