Effect of Apolipoprotein Structural Adaptability

载脂蛋白结构适应性的影响

基本信息

批准号：
7624191
负责人：
ROBERT O'Mara RYAN
金额：
$ 37.95万
依托单位：
CHILDREN'S HOSPITAL & RES CTR AT OAKLAND
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-06-01 至 2010-06-03
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7624191
关键词：
Acidic Amino Acids Adopted Adoption Agonist Amino Acids Apolipoprotein E Apolipoproteins Arginine Binding Biological Assay Blood Vessels Cardiovascular Diseases Cell Surface Receptors Charge Cholesterol Cholesterol Homeostasis Complement Data Disease Electron Spin Resonance Spectroscopy Electrons Electrostatics Experimental Designs Family Family member Fluorescence Goals Hand Health High Density Lipoproteins Histidine Human Knowledge Label Length Ligand Binding Ligands Lipid Binding Lipids Lipoproteins Low Density Lipoprotein Receptor Lysine Maintenance Mediating Metabolic Metabolism Modification Molecular Molecular Conformation Mutagenesis N-terminal NMR Spectroscopy Nature Pathway interactions Plasma Protein Region Proteins Recombinants Regulation Research Research Personnel Resolution Role Signal Transduction Site Site-Directed Mutagenesis Spectrum Analysis Structure Surface Plasmon Resonance Testing Therapeutic Agents Time X-Ray Crystallography alpha helix base cardiovascular disorder prevention epidermal growth factor precursor improved insight lipid transport member novel particle protein protein interaction receptor receptor binding reconstitution research study three dimensional structure

项目摘要

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.

描述（由申请人提供）：我们研究的长期目标是了解脂质转运和代谢如何受脂蛋白与细胞表面受体之间的分子相互作用来调节。在设计实验策略的设计中，将对载脂蛋白E（APOE）和低密度脂蛋白受体（LDLR）的代谢作用的了解将与可用的结构信息结合使用，以剖析生产受体 - 配体相互作用的决定因素。另外，将进行研究以阐明从受体中释放配体的机理。据推测，载脂蛋白E（APOE）经历了脂质结合诱导的构象变化，从而导致螺旋4超过其无脂质螺旋束状态所确定的边界。我们假设该螺旋延伸是一个关键步骤，它允许对齐带正电的氨基酸，从而采用构象，从而促进LDLR的配体结合重复序列相互作用。在不存在和存在脂质的情况下，将进行特定标记的APOE的电子顺磁性自旋共振和NMR光谱分析，允许在蛋白质的这一区域中直接评估从随机线圈（无脂质状态）到α螺旋（脂质相关状态）的假定结构过渡。进一步假设，无脂质APOE中螺旋4的终止是由序列特异性终止信号引起的，该信号可以通过脂质关联来克服。将进行定位的诱变研究，以破坏假定的螺旋帽基序以及对APOE Helix 4终止的影响。在没有脂质的情况下，将在受体结合测定中测试螺旋帽基序的破坏将导致螺旋延伸并采用受体活动构象。在其他研究中，LDLR的可溶片段涵盖了其七个补体的配体结合，LDL-A重复序列和表皮生长因子前体同源性结构域，包括其A-螺旋桨段，用于使用APOE的配体结合和释放研究。 LDL-A重复交换实验和重复之间的间隔序列的修改将用于确定与含有重构的高密度脂蛋白配体的APOE相互作用的分子需求。将检查以下假设，即A-Propeller段与特定LDL-A重复序列之间的分子内相互作用是对配体释放的原因。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。基于以下事实：LDLR途径中的像差与心血管疾病的发作呈正相关，我们预计从这些研究中获得的新知识将提供对调节健康和疾病中血管胆固醇通量的分子机制的见解。

项目成果

期刊论文数量（54）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Domain swapping reveals that low density lipoprotein (LDL) type A repeat order affects ligand binding to the LDL receptor.

结构域交换揭示了低密度脂蛋白 (LDL) A 型重复顺序影响配体与 LDL 受体的结合。

DOI：
10.1074/jbc.m900194200
发表时间：
2009
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Yamamoto,Taichi;Ryan,RobertO
通讯作者：
Ryan,RobertO

Apolipoprotein A-V dependent modulation of plasma triacylglycerol: a puzzlement.

DOI：
10.1016/j.bbalip.2011.12.002
发表时间：
2012-05
期刊：
Biochimica et biophysica acta
影响因子：
0
作者：
Sharma V;Ryan RO;Forte TM
通讯作者：
Forte TM

Curcumin nanodisks: formulation and characterization.

DOI：
10.1016/j.nano.2010.08.002
发表时间：
2011-04
期刊：
NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE
影响因子：
5.4
作者：
Ghosh, Mistuni;Singh, Amareshwar T. K.;Xu, Wenwei;Sulchek, Todd;Gordon, Leo I.;Ryan, Robert O.
通讯作者：
Ryan, Robert O.

Complete 1H, 15N, and 13C assignments of an exchangeable apolipoprotein, Locusta migratoria apolipophorin III.

完成可交换载脂蛋白 Locusta migratoria apolipophorin III 的 1H、15N 和 13C 分配。