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，在脂质运输以及维持血浆和脑胆固醇稳态中发挥着关键作用。它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，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。它还通过促进胆固醇从巨噬细胞流出形成高密度脂蛋白（HDL），在逆转动脉粥样硬化中胆固醇转运方面发挥作用。在本提案中，我们将重点了解两种主要的人类 apoE 同工型 apoE3 和 apoE4 之间的差异，以及它们在 HDL 形成中的作用。 ApoE3 与正常血浆脂质谱相关（抗动脉粥样硬化），而 apoE4 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) 的危险因素。然而，脂质结合机制和异构体之间生理行为差异的分子基础均未知。我们将检验总体假设，即 apoE3 和 apoE4 的构象存在异构体特异性变异，这导致脂质结合机制和脂质 (HDL) 结合构象的差异。为了实现这一目标，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 apoE3 和 apoE4 的解折叠模式； (ii) 通过 FP 和 HDX-MS 捕获脂质结合过程中蛋白质的“快照”，比较 apoE3 和 apoE4 之间的脂质结合机制，并识别不同螺旋的脂质结合顺序中可能存在的异构体特异性差异，以及 (iii) 检查 通过荧光光谱和交联分析，在胆固醇流出后重建和巨噬细胞生成的 HDL 中 apoE。这些研究的完成将显着促进我们对 apoE 生理行为中亚型特异性差异以及 apoE4 对 CVD 和 AD 的易感性的理解。 通过确定脂质结合的机制基础，我们的研究将为确定治疗 CVD 和 AD 的潜在干预策略提供及时且急需的机会。 PI 的发展目标是在 CSULB 建立和维持高产的学术研究活动，发表研究成果，为来自不同背景的最先进生物医学研究的学生学员提供研究培训机会和指导，积极参加国内和国际会议，发起并继续与 CSULB 之外的知名研究人员合作，并扩展她的专业知识以解决与 AD 相关的问题。