Biophysical Studies of Caveolin

Caveolin 的生物物理学研究

• 批准号: 10198303
• 负责人: Kerney Jebrell Glover
• 金额: $ 47.11万
• 依托单位: LEHIGH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10198303
• 关键词: Address; Adopted; Behavior; Biochemical; Biogenesis; Biological; Biological Process; Biophysics; Caveolae; Caveolins; Cell membrane; Cell physiology; Cells; Cellular Membrane; Chemicals; Circular Dichroism Spectroscopy; Complex; Computer Models; Data; Detergents; Disease; Endocytosis; Environment; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Glare; Hand; Heart Diseases; Hybrids; Integral Membrane Protein; Investigation; Knowledge; Length; Link; Lipid Bilayers; Lipids; Lipodystrophy; Lung diseases; Malignant Neoplasms; Membrane; Membrane Proteins; Methods; Modality; Modeling; Molecular; Molecular Conformation; Morphologic artifacts; Muscular Dystrophies; N-terminal; NMR Spectroscopy; Nature; Nuclear Magnetic Resonance; Nuclear Protein; Pathogenesis; Phase; Physiological; Play; Precipitation; Process; Protein Isoforms; Proteins; Regulation; Role; Shapes; Signal Transduction; Structure; Surface; Techniques; Testing; Therapeutic Intervention; Uncertainty; Vertebral column; Work; base; biophysical analysis; caveolin 1; cell behavior; crosslink; density; experimental study; flasks; in silico; in vivo; innovation; insight; intermolecular interaction; mechanotransduction; molecular dynamics; mutant; novel; novel strategies; polypeptide; preference; preservation; reconstitution; restraint

Project Summary/Abstract Caveolae are flask-shaped invaginated microdomains that punctuate the plasma membrane, and play a central role in a variety of cellular processes including mechanosensing, endocytosis, and signal transduction. The integral membrane protein caveolin (three isoforms -1, -2, and -3) is the most important protein found in caveolae, and is required for caveolae biogenesis. A plethora of studies have shown that improper regulation and mutant forms of caveolin can result in a variety of diseases including lipodystrophy, muscular dystrophy, cancer, and heart and lung disease. Based on indirect evidence, the provocative postulation has been put forth that caveolin adopts a ‘U-shaped’ conformation in the lipid bilayer, a disposition that to our knowledge has not been definitively characterized for any membrane protein. Furthermore, evidence suggests that caveolin has the ability to homooligomerize, although recent evidence from our lab has challenged that notion and brought into focus the uncomfortable possibility that many of the methods used to study caveolin may be inadvertently promoting non-biologically relevant aggregation. Using a panoply of biophysical and biochemical techniques (i.e. fluorescence spectroscopy, circular dichroism spectroscopy, nuclear magnetic resonance [NMR] spectroscopy, chemical cross-linking, and computational modeling) our objective is to probe the structure, topography and homooligomeric state of caveolin-1 (the most ubiquitous of the three isoforms) in a lipid bilayer. This will be achieved by pursuing the following three specific aims. 1. Investigation of the putative ‘U-shaped’ conformation of the intramembrane domain. 2. Investigation of the secondary structure of the N-terminal domain. 3. Investigation of homooligomeric interactions. Specific aim 1 will determine the tertiary structure and membrane topography of the intramembrane domain which will address the persistent skepticism surrounding the atomic-level interactions that could make a membrane-buried polypeptide turn possible. Specific aim 2 will address the long standing question of whether any 𝛂-helices or β-strands are present in the N-terminal domain, and with the fulfillment of this aim, complete backbone NMR data will finally be available for caveolin-1. Specific aim 3 will evaluate whether caveolin-1 possesses the ability to oligomerize on its own when reconstituted into the bilayer at in vivo (i.e. high) surface densities by devising an experiment that will circumvent the pitfalls that may have derailed previous investigations into oligomerization and led to false conclusions (e.g. use of detergents, tagging of the protein, etc.). Upon completion of these aims, the key enigmatic features of caveolin-1 will be manifest, opening the door to deeper insights into disease pathogenesis and ultimately possible therapeutic interventions that could address the immensely complex array of disease states linked to aberrant caveolin function.

项目摘要/摘要 小窝是烧瓶状的凹陷微域，它点缀在质膜上，发挥着 在各种细胞过程中发挥中心作用，包括机械传感、内吞作用和信号转导。 完整的膜蛋白小窝蛋白(三种异构体-1、-2和-3)是在 Caveolae，这是小窝生物发生所必需的。大量研究表明，不适当的监管 小窝蛋白的突变形式会导致各种疾病，包括脂肪营养不良、肌肉营养不良、 癌症、心脏病和肺病。基于间接证据，这一挑衅性的假设已经被提出 第四，小窝蛋白在脂双层中采用‘U形’构象，据我们所知，这种倾向具有 对于任何膜蛋白都没有明确的特征。此外，有证据表明，洞穴 具有同源齐聚的能力，尽管我们实验室的最新证据挑战了这一概念 让人感到不安的是，许多用于研究洞穴的方法可能是 无意中促进了与生物无关的聚集。使用一整套生物物理和生物化学 技术(即荧光光谱、圆二色光谱、核磁共振 [核磁共振]光谱学、化学交联和计算模型)我们的目标是探索 三种异构体中最普遍的小窝蛋白-1的结构、形貌和同源低聚状态 脂质双分子层。这将通过追求以下三个具体目标来实现。1.推定人的调查 膜内结构域呈“U”形构象。2.研究了黄曲霉的二级结构 N端域。3.同源低聚物相互作用的研究。具体目标1将决定第三 膜内结构域的结构和膜形貌将解决持续存在的怀疑 围绕着原子水平的相互作用，这可能使膜埋藏的多肽成为可能。 特定目标2将解决长期存在的问题，即是否有𝛂-螺旋或β-链存在于 N-端域，随着这一目标的实现，完整的主干核磁共振数据最终将可用于 洞穴1号。特殊目标3将评估小窝蛋白-1是否具有自身寡聚的能力 当在体内(即高)表面密度下重组为双层时，通过设计一种实验来 绕过可能使先前对寡聚的调查脱轨并导致错误的陷阱 结论(例如，洗涤剂的使用、蛋白质的标记等)。在实现这些目标后，关键是 洞穴-1的神秘特征将会显现，为更深入地了解疾病打开了大门 发病机制和最终可能的治疗干预措施，可以解决极其复杂的 一系列与小窝功能异常有关的疾病状态。