NMR Studies of ApoE and Its Interactions with Receptors

载脂蛋白E （apoE）是一种可交换的载脂蛋白，在脂质/脂蛋白代谢和心血管疾病中起重要作用。最近的证据表明，apoE在其他几个重要的生物学过程中也至关重要，包括阿尔茨海默病、认知功能、免疫调节、细胞信号传导和传染病。ApoE是一种多态蛋白，主要有apoE2、apoE3和apoE4三种亚型。载脂蛋白e异构体彼此之间只有一个氨基酸取代，但它们在细胞和分子水平上都具有深远的功能影响。虽然在1991年就解决了apoE n端结构域的x射线晶体结构，但apoE的寡聚化性质阻碍了对全长apoE和apoE c端结构域的结构研究。c端结构域引起apoE聚集已被证实。最近，我们实验室生成了一个具有生物活性的单体apoE c端结构域，解决了apoE结构研究中的主要技术难题。这一具有生物活性的单体apoE c端结构域的核磁共振波谱已被完全确定，其核磁共振结构将很快得到解析。这一进展使我们能够很好地提出全长apoE的核磁共振结构测定。本研究计划重点利用核磁共振（NMR）和分子生物学技术解决无脂状态下人类apoE全长的核磁共振结构。此外，我们还建立了LDL受体配体结合域重复序列（LDLR-LBDR）和LRP配体结合域2重复序列（LRP- lbd2r）的高水平表达和重折叠系统，使我们能够表征apoE与受体相互作用时结合残基的结构变化。最后，鉴定参与apoE结构域-结构域相互作用的关键残基也在本提案中提出。由于apoE和apoE/受体相互作用在包括动脉粥样硬化和阿尔茨海默病在内的几种主要人类疾病中的重要性，这一提议的意义是非常合理的。值得注意的是，该应用程序的一个独特之处在于，为每个特定目标提出了几个独立的方法，确保一种方法的任何成功都将实现该目标的总体目标。

DESCRIPTION (provided by applicant): Apolipoprotein E (apoE) is an exchangeable apolipoprotein that plays an important role in lipid/lipoprotein metabolism and cardiovascular diseases. Recent evidence indicates that apoE is also critical in several other important biological processes, including Alzheimer's disease, cognitive function, immunoregulation, cell signaling, and infectious diseases. ApoE is a polymorphic protein with three major isoforms, apoE2, apoE3 and apoE4. The apoE isoforms differ from one another only by a single amino acid substitution, yet they have profound functional consequences at both the cellular and molecular levels. Although the X-ray crystal structure of the apoE N-terminal domain was solved in 1991, the structural studies of full-length apoE and the apoE C-terminal domain is hindered by apoE's oligomerization property. It is well established that the C-terminal domain causes apoE aggregation. A monomeric, biologically active apoE C-terminal domain has been generated in our laboratory recently, which has solved the major technical problem in the apoE structural study. The NMR spectra of this monomeric, biologically active apoE C-terminal domain has been completely assigned and its NMR structure will be solved soon. This progress places us in a very good position to propose a NMR structural determination of full-length apoE. This research proposal focuses on solving the NMR structure of full-length human apoE in the lipid-free state using nuclear magnetic resonance (NMR) and molecular biology techniques. In addition, a high-level expression and refolding system has been established for the LDL receptor ligand-binding domain repeats (LDLR-LBDR) and LRP ligand binding domain 2 repeats (LRP-LBD2R), allowing us to propose to characterize the structural changes of the binding residues in apoE upon interaction with receptors. Finally, identification of the critical residues that are involved in the apoE domain-domain interactions has also been proposed in this proposal. Due to the importance of apoE and the apoE/receptor interactions in several major human diseases, including atherosclerosis and Alzheimer's disease, the significance of this proposal is very well justified. It is worth noting that one unique feature of this application is that several independent approaches have been proposed for each specific objective, ensuring that any success in one approach will achieve the overall goal of this objective.