Effect of Apolipoprotein Structural Adaptability

描述(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是如何调节脂类的运输和代谢的。对载脂蛋白E(ApoE)和低密度脂蛋白受体(LDLR)代谢作用的了解将与现有的结构信息相结合，以设计一种实验策略，以剖析有效的受体-配体相互作用的决定因素。此外，还将进行研究，以阐明从受体释放配体的机制。据推测，载脂蛋白E(ApoE)经历了脂结合诱导的构象变化，导致螺旋4延伸到其无脂螺旋束状态的边界之外。我们假设，这种螺旋延伸是允许正电荷氨基酸比对的关键步骤，这样它们就可以采用一种构象来促进与LDLR的配体结合重复序列的相互作用。在没有和存在脂类的情况下，将对特定标记的apoE进行电子顺磁共振和核磁共振波谱分析，从而直接评估蛋白质这一区域从随机卷曲(无脂状态)到α螺旋(脂结合状态)的结构转变。进一步推测，无脂apoE中螺旋4的终止是由序列特异性终止信号引起的，该信号可以被脂质结合所克服。将进行定点突变研究，以破坏假定的螺旋帽基序，并研究其对apoE螺旋4终止的影响。在没有脂类的情况下，螺旋帽基序的破坏将导致螺旋延伸和采用受体活性构象的假设将在受体结合分析中得到验证。在其他研究中，LDLR的一个包含其七个补体样配体结合、低密度脂蛋白-A重复序列和表皮生长因子前体同源结构域(包括其α-螺旋桨片段)的可溶片段将被用于与apoE的配体结合和释放研究。低密度脂蛋白-A重复交换实验和重复序列之间间隔区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进行有效相互作用的分子要求。A-推进器片段和特定的低密度脂蛋白-A重复序列之间依赖于pH的分子内相互作用是配体释放的原因的假设将得到检验。这些实验结果将扩大我们对低密度脂蛋白受体通路、载脂蛋白介导脂蛋白代谢和血浆胆固醇动态平衡的理解。基于低密度脂蛋白受体通路的异常与心血管疾病的发生呈正相关这一事实，我们预计从这些研究中获得的新知识将为深入了解调节健康和疾病中血管胆固醇流量的分子机制提供帮助。

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.