Effect of Apolipoprotein Structural Adaptability

描述（由申请人提供）：我们研究的长期目标是了解脂蛋白和细胞表面受体之间的分子相互作用如何调节脂质转运和代谢。载脂蛋白E （apoE）和低密度脂蛋白受体（LDLR）的代谢作用的知识将与可用的结构信息相结合，设计一个实验策略来剖析生产受体-配体相互作用的决定因素。此外，还将进一步研究该受体释放配体的机制。假设载脂蛋白E （apoE）经历了脂质结合诱导的构象变化，导致螺旋4的延伸超出了在无脂螺旋束状态下确定的边界。我们假设这种螺旋延伸是允许带正电的氨基酸排列的关键步骤，使它们采用促进与LDLR配体结合重复序列相互作用的构象。电子顺磁自旋共振和核磁共振波谱分析将在没有和存在脂质的情况下进行，允许直接评估蛋白质该区域从随机线圈（无脂状态）到α螺旋（脂相关状态）的假设结构转变。进一步假设，无脂apoE中螺旋4的终止是由一个序列特异性终止信号引起的，该信号可以通过脂质结合来克服。将进行位点定向诱变研究，以破坏假定的螺旋帽基序，并研究apoE螺旋4终止的影响。假设螺旋帽基序的破坏将导致螺旋延伸，并在缺乏脂质的情况下采用受体活性构象，将在受体结合试验中进行测试。在其他研究中，LDLR的可溶性片段包括其7个补体样配体结合，LDL-A重复序列和表皮生长因子前体同源结构域，包括其a-螺旋桨片段，将用于与apoE结合和释放配体的研究。LDL-A重复序列交换实验和重复序列之间间隔序列的修饰将用于确定与含有重组高密度脂蛋白配体的载脂蛋白e有效相互作用的分子要求。关于a-螺旋桨片段和特定LDL-A重复序列之间的ph依赖的分子内相互作用负责配体释放的假设将被检验。这些实验的结果将扩展我们对LDLR途径、载脂蛋白介导的脂蛋白代谢和血浆胆固醇稳态的理解。基于LDLR通路的畸变与心血管疾病的发病正相关这一事实，我们预计从这些研究中获得的新知识将为健康和疾病中调节血管胆固醇通量的分子机制提供深入的见解。

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand how lipid transport and metabolism are regulated by molecular interactions between lipoproteins and cell surface receptors. Knowledge of the metabolic roles of apolipoprotein E (apoE) and the low density lipoprotein receptor (LDLR) will be combined with available structural information in the design of an experimental strategy to dissect the determinants of a productive receptor-ligand interaction. In addition studies will be pursued to elucidate the mechanism of ligand release from the receptor. It is postulated that apolipoprotein E (apoE) undergoes a lipid binding induced conformational change that results in extension of helix 4 beyond the boundary identified in its lipid-free helix bundle state. We hypothesize that this helix extension is a critical step that permits alignment of positively charged amino acids such that they adopt a conformation that promotes interaction with ligand binding repeats of the LDLR. Electron paramagnetic spin resonance and NMR spectroscopy analysis of specifically labeled apoE will be performed in the absence and presence of lipid, permitting direct assessment of a postulated structural transition from random coil (lipid-free state) to alpha helix (lipid associated state) in this region of the protein. It is further postulated that termination of helix 4 in lipid-free apoE is caused by a sequence specific termination signal that can be overcome by lipid association. Site directed mutagenesis studies will be performed to disrupt a putative helix cap motif and the effect on apoE helix 4 termination investigated. The hypothesis that disruption of the helix cap motif will result in helix extension and adoption of a receptor active conformation in the absence of lipid will be tested in receptor binding assays. In other studies a soluble fragment of the LDLR encompassing its seven complement-like ligand binding, LDL-A repeats and the epidermal growth factor precursor homology domain, including its a-propeller segment, will be employed in ligand binding and release studies with apoE. LDL-A repeat swapping experiments and modification of the spacer sequences between repeats will be used to determine the molecular requirements for a productive interaction with apoE containing reconstituted high density lipoprotein ligands. The hypothesis that a pH-dependent intra-molecular interaction between the a-propeller segment and specific LDL-A repeats is responsible for ligand release will be examined. The results of these experiments will extend our understanding of the LDLR pathway, apolipoprotein-mediated lipoprotein metabolism and plasma cholesterol homeostasis. Based on the fact that aberrations in the LDLR pathway are positively correlated to onset of cardiovascular disease, we anticipate that new knowledge gained from these studies will provide insight into molecular mechanisms that regulate vascular cholesterol flux in health and disease.