Biophysical Studies of Caveolin

项目总结/摘要 小窝是瓶状的内陷微区，它在质膜上起着重要的作用。 在多种细胞过程中发挥核心作用，包括机械传感、内吞作用和信号传导。 膜蛋白小窝蛋白（三种亚型-1，-2和-3）是在细胞膜中发现的最重要的蛋白质。 小窝，并且是小窝生物发生所必需的。大量研究表明，不适当的监管 并且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。根据间接证据，提出了一个挑衅性的假设， 第四，小窝蛋白在脂质双层中采用“U形”构象，据我们所知， 没有明确地表征任何膜蛋白。此外，有证据表明，窖蛋白 具有同源寡聚化的能力，尽管我们实验室最近的证据挑战了这一概念， 使人们关注到一种令人不安的可能性，即许多用于研究窖蛋白的方法可能是 无意中促进了非生物学相关的聚集。利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 [NMR]光谱，化学交联和计算建模），我们的目标是探索 结构，地形和同源寡聚状态的小窝蛋白-1（最普遍的三种异构体）在一个 脂质双层这将通过追求以下三个具体目标来实现。1.调查推定的 膜内结构域的“U形”构象。2.研究了大豆异黄酮的二级结构 N-末端结构域。3.同源寡聚体相互作用的研究。具体目标1将确定第三个 膜内结构域的结构和膜地形，这将解决持续的怀疑， 围绕着原子水平的相互作用，这些相互作用可以使膜埋藏的多肽变成可能。 具体目标2将解决长期存在的问题：𝛂 N-末端结构域，并且随着这一目标的实现，完整的骨架NMR数据将最终可用于 小窝蛋白-1。具体目标3将评估小窝蛋白-1是否具有自身寡聚化的能力 当在体内（即高）表面密度下重构成双层时， 避免了可能使以前对低聚反应的研究脱轨并导致错误的 结论（例如，使用去污剂，标记蛋白质等）。在完成这些目标后，关键 Caveolin-1的神秘特征将显现出来，为深入了解疾病打开大门。 发病机制和最终可能的治疗干预，可以解决极其复杂的 一系列与异常小窝蛋白功能相关的疾病状态。

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.