Biophysical Studies of Caveolin

Caveolin 的生物物理学研究

• 批准号: 10577560
• 负责人: Kerney Jebrell Glover
• 金额: $ 7.39万
• 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/10577560
• 关键词: Address; Adopted; Behavior; Biochemical; Biogenesis; Biological Process; Biophysics; Caveolae; Caveolins; Cell membrane; Cell physiology; Cells; Chemicals; Circular Dichroism Spectroscopy; Complex; Computer Models; Data; Detergents; Disease; Endocytosis; Fluorescence Spectroscopy; Heart Diseases; Integral Membrane Protein; Investigation; Link; Lipid Bilayers; Lipodystrophy; Lung diseases; Malignant Neoplasms; Membrane; Membrane Proteins; Methods; Molecular Conformation; Muscular Dystrophies; N-terminal; NMR Spectroscopy; Nature; Nuclear Magnetic Resonance; Pathogenesis; Play; Protein Isoforms; Proteins; Regulation; Role; Signal Transduction; Structure; Surface; Techniques; Therapeutic Intervention; Vertebral column; base; biophysical analysis; caveolin 1; crosslink; density; experimental study; flasks; in vivo; insight; mechanotransduction; mutant; polypeptide; reconstitution

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 小凹，并且是小凹生物发生所必需的。大量研究表明，监管不当 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、 癌症、心脏病和肺病。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，caveolin 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。使用一整套生物物理和生化方法 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 脂质双层。这将通过追求以下三个具体目标来实现。 1. 假设的调查 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. 同寡聚相互作用的研究。具体目标1将确定第三级 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 卡维奥林-1。具体目标3将评估caveolin-1是否具有自身寡聚的能力 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。

项目成果

Facile production of tagless membrane scaffold protein for nanodiscs.

• DOI: 10.1016/j.ab.2021.114497
• 发表时间: 2022-02-01
• 期刊: Analytical biochemistry
• 影响因子: 2.9
• 作者: Julien JA;Mutchek SG;Fernandez MG;Glover KJ
• 通讯作者: Glover KJ

One-step site-specific S-alkylation of full-length caveolin-1: Lipidation modulates the topology of its C-terminal domain.

• DOI: 10.1002/pro.4791
• 发表时间: 2023-11
• 期刊: Protein science : a publication of the Protein Society

Rapid preparation of nanodiscs for biophysical studies.

• DOI: 10.1016/j.abb.2021.109051
• 发表时间: 2021-11-15
• 期刊: Archives of biochemistry and biophysics
• 影响因子: 3.9
• 作者: Julien JA;Fernandez MG;Brandmier KM;Del Mundo JT;Bator CM;Loftus LA;Gomez EW;Gomez ED;Glover KJ
• 通讯作者: Glover KJ