Biophysical Studies of Caveolin

Caveolin 的生物物理学研究

基本信息

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
项目类别：
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

项目摘要

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/摘要小窝是烧瓶形的无知微区，可打断质膜，并玩在各种细胞过程中的中心作用，包括机理，内吞作用和信号转导。整体膜蛋白可爱素（三种同工型-1，-2和-3）是发现中最重要的蛋白小窝，是小窝生物发生所必需的。大量的研究表明，调节不当小窝蛋白的突变形式会导致多种疾病，包括脂肪营养不良，肌肉营养不良，癌症，心脏和肺部疾病。基于间接证据，提出了挑衅性的假设 Caveolin在脂质双层中采用“ U形”会议，据我们所知没有针对任何膜蛋白的明确特征。此外，有证据表明卡韦洛林尽管我们实验室的最新证据挑战了这一概念和将重点放在焦点中，许多用于研究小窝蛋白的方法可能是不舒服的可能性无意中促进非生物学相关的聚集。使用一系列生物物理和生化技术（即荧光光谱，圆形二色性光谱，核磁共振 [NMR]光谱，化学交联和计算建模）我们的目标是探测 Caveolin-1的结构，地形和家族性状态（三种同工型中最普遍）脂质双层。这将通过追求以下三个特定目标来实现。 1。对推定的调查膜内域的“ U形”构象。 2。调查二级结构 N末端结构域。 3。调查家族相互作用。具体目标1将确定第三纪膜内结构域的结构和膜地形，这将解决持续的怀疑论周围的原子级相互作用可以使膜燃烧的多肽变成可能。具体目标2将解决一个长期存在的问题，即在 N末端域，并且随着此目标的实现，完整的骨干NMR数据最终将用于 caveolin-1。特定目标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）表面密度的双层重构时，通过设计实验将避免了可能已经使以前对低聚的调查脱轨的陷阱，并导致了错误结论（例如使用确定剂，蛋白质标记等）。完成这些目标后，关键 Caveolin-1的神秘特征将显示出来，为疾病的深入了解打开了大门发病机理以及最终可能解决极其复杂的治疗干预措施与异常的小窝蛋白功能相关的疾病状态阵列。