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.

项目概要/摘要小凹是烧瓶状的内陷微区，刺穿质膜，并发挥在多种细胞过程中发挥核心作用，包括机械传感、内吞作用和信号转导。整合膜蛋白 Caveolin（三种亚型 -1、-2 和 -3）是在大量研究表明，不当的调节是小凹生物发生所必需的。小窝蛋白的突变形式可导致多种疾病，包括脂肪营养不良、肌肉营养不良、基于间接证据，提出了挑衅性的假设。指出小窝蛋白在脂质双层中采用“U 形”构象，据我们所知，这种配置尚未明确表征任何膜蛋白。此外，有证据表明小窝蛋白。具有同源齐聚的能力，尽管我们实验室的最新证据对这一概念提出了挑战，并且引起人们关注的一个令人不安的可能性是，许多用于研究小窝蛋白的方法可能是无意中促进了非生物学相关的聚集。技术（即荧光光谱、圆二色光谱、核磁共振 [NMR] 光谱、化学交联和计算模型）我们的目标是探索 Caveolin-1（三种同工型中最普遍的一种）的结构、形貌和同源寡聚状态这将通过追求以下三个具体目标来实现： 1. 假定的研究。膜内结构域的“U形”构象2.二级结构的研究。 N 末端结构域。 3. 研究同源寡聚体相互作用。具体目标 1 将确定三级。膜内域的结构和膜形貌将解决持续的怀疑围绕原子级相互作用，使膜埋多肽转变成为可能。具体目标 2 将解决长期存在的问题，即是否存在任何 𝛂-螺旋或 β-链 N端域，随着这一目标的实现，完整的主干NMR数据将最终可用于具体目标3将评估caveolin-1是否具有自身寡聚化的能力当通过设计实验以体内（即高）表面密度重组为双层时，规避可能使之前的低聚研究脱轨并导致错误结果的陷阱结论（例如去垢剂的使用、蛋白质的标记等）。完成这些目标后，关键是。 Caveolin-1的神秘特征将会显现出来，为更深入了解疾病打开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问题一系列与小窝蛋白功能异常相关的疾病状态。