Apolipoprotein E/Lipoprotein Binding Mechanism

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。它通过作为低密度脂蛋白受体（LDLr）家族蛋白的配体介导其作用，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形成高密度脂蛋白（HDL），在动脉粥样硬化的逆向胆固醇运输中发挥作用。在这篇文章中，我们将重点了解两种主要的人类apoE亚型，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，是心血管疾病（CVD）和阿尔茨海默病（AD）的危险因素。然而，无论是脂质结合的机制，还是同种异构体之间生理行为差异的分子基础都是未知的。我们将检验apoE3和apoE4构象存在同种异构体特异性变异的总体假设，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)利用荧光偏振（FP）和氢氘交换耦合质谱（HDX-MS）的组合确定重组apoE3和apoE4的展开模式；（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，并确定不同螺旋脂质结合顺序的可能的异构体特异性差异，（iii）检查脂质结合构象

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.