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(申请人提供)：载脂蛋白E(ApoE)是一种可交换的载脂蛋白，在脂质运输和维持血浆和脑内胆固醇稳态方面发挥关键作用。它作为低密度脂蛋白受体(LDLR)家族的配体，促进受体介导的脂蛋白内吞作用，从而最终降低血脂水平，从而发挥其作用。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)，在动脉粥样硬化中发挥反向胆固醇运输的作用。在这个提案中，我们将重点了解人类两种主要的载脂蛋白E亚型，apoE3和apoE4之间的差异，以及它们在高密度脂蛋白形成中的作用。载脂蛋白3与正常的血脂谱(抗动脉粥样硬化)有关，而载脂蛋白E4被认为是促动脉粥样硬化的，是心血管疾病(CVD)和阿尔茨海默病(AD)的危险因素。然而，脂质结合的机制和不同亚型之间生理行为差异的分子基础都不清楚。我们将检验这一总体假设，即apoE3和apoE4的构象存在异构体特异性变化，从而导致脂结合机制和脂(HDL)结合构象的不同。为此，我们将：(I)使用荧光偏振(FP)和氢重离子交换与质谱联用(HDX-MS)相结合的方法来确定重组apoE3和apoE4的展开模式；(Ii)通过捕捉FP和HDX-MS在脂结合过程中蛋白质的快照来比较apoE3和apoE4之间的脂结合机制，并确定不同螺旋在脂结合顺序上可能存在的异构体特异性差异，以及(Iii)检查apoE3和apoE4的脂结合构象 用荧光光谱和交联法分析胆固醇外流后重组和巨噬细胞产生的高密度脂蛋白中的载脂蛋白E。这些研究的完成将极大地促进我们对apoE生理行为的异构体特异性差异以及apoE4对CVD和AD易感性的理解。通过确定脂质结合的机制基础，我们的研究将提供及时和急需的机会，以确定治疗心血管疾病和AD的潜在干预策略。该协会的发展目标是在CSULB建立和维持一个高效和学术的研究活动，发表研究成果，为来自最先进生物医学研究的不同背景的学生学员提供研究培训机会和指导，积极参与国内和国际会议，启动并继续与CSULB以外的知名研究人员合作，并扩大她的专业知识，以解决与AD相关的问题。