NMR Studies of ApoE and Its Interactions with Receptors

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/脂蛋白代谢和心血管疾病中发挥重要作用。最近的证据表明，apoE在其他几个重要的生物学过程中也是至关重要的，包括阿尔茨海默病、认知功能、免疫调节、细胞信号转导和传染病。载脂蛋白E是一种多态蛋白质，有三种主要亚型，即载脂蛋白2、载脂蛋白E3和载脂蛋白E4。载脂蛋白E亚型之间的差异只有一个氨基酸替代，但它们在细胞和分子水平上都有深刻的功能后果。虽然1991年已经解决了apoE N-末端结构域的X射线晶体结构问题，但apoE的低聚特性阻碍了对apoE全长结构和apoE C-末端结构的研究。众所周知，C-末端结构域导致apoE聚集。本实验室最近合成了一个具有生物活性的单体apoE C-末端结构域，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-末端结构域的核磁共振谱已被完全指认，其核磁共振结构将很快得到解决。这一进展使我们处于非常有利的地位，可以提出全长载脂蛋白E的核磁共振结构测定。本研究计划利用核磁共振技术和分子生物学技术解决脱脂状态下全长人载脂蛋白E的核磁共振结构。此外，还建立了低密度脂蛋白受体配体结合结构域重复序列(LDLR-LBdr)和LRP配体结合结构域2重复序列(LRP-LBD2R)的高水平表达和复性体系，使我们能够表征载脂蛋白E与受体相互作用时结合残基的结构变化。最后，在该提案中还提出了apoE结构域-结构域相互作用中关键残基的识别。由于载脂蛋白E和载脂蛋白E/受体的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，这一建议的意义是非常充分的。值得注意的是，这一应用程序的一个独特特点是，为每个具体目标提出了几种独立的方法，确保在一种方法中取得任何成功都将实现这一目标的总体目标。

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.