Investigation of Caveolin Structure, Topology, and Oligomerization

描述（由申请人提供）：小泡是位于质膜上的高度弯曲的内陷微结构域，在各种细胞过程中起核心作用。小泡蛋白（1、2和3）是在小泡中发现的最重要的蛋白质，并且负责赋予小泡不寻常的“瓶状”形状。最近的证据表明，小窝蛋白的调节不当和突变形式可导致多种疾病，包括阿尔茨海默氏症、肌肉萎缩症、癌症和心脏病。小窝蛋白在膜内呈不寻常的“马蹄形”构象，其N端和c端都面向细胞质，这种构象被认为促进了膜的弯曲。此外，通过高阶寡聚化，小洞蛋白形成稳定膜曲率的结构骨架。利用生物物理技术，如核磁共振（NMR）、荧光光谱和分析性超离心，我们的目标是在基本水平上表征小窝蛋白-1。这将通过追求以下两个具体目标来实现：小洞蛋白-1的膜拓扑和三维结构研究。2. 小洞蛋白-1寡聚的研究。具体目标1将确定小洞蛋白-1的高分辨率三维溶液结构，并检查色氨酸残基的溶剂可及性，以评估小洞蛋白-1在双层中的拓扑结构。接下来，两个保守的脯氨酸残基在膜内“马蹄形”构象的形成和/或稳定中所起的作用将通过定点诱变来探索。特异性目标2将描述在存在和不存在胆固醇的情况下由小窝蛋白-1形成的低聚物的大小和分布。此外，脯氨酸到亮氨酸突变体在寡聚化过程中所起的作用也将被探讨。对小窝蛋白-1结构和寡聚化的基本理解无疑将为可能的治疗干预打开大门，从而解决与小窝蛋白功能失调有关的疾病。

DESCRIPTION (provided by applicant): Caveolae are highly-curved invaginated micro-domains located in the plasma membrane that play a central role in a variety of cellular processes. Caveolins (1, 2, and 3) are the most important proteins found in caveolae, and are responsible for giving caveolae their unusual "flask-like" shape. Recent evidence has shown that improper regulation and mutant forms of caveolin can result in a variety of diseases including Alzheimer's, muscular dystrophy, cancer, and heart disease. Caveolin adopts an unusual intra-membrane "horseshoe" conformation where both its N- and C-termini face the cytoplasm, and this conformation is thought to promote membrane curvature. In addition, via high-order oligomerization, caveolin forms a structural backbone which stabilizes the membrane curvature. Using biophysical techniques such as nuclear magnetic resonance (NMR), fluorescence spectroscopy, and analytical ultracentrifugation, our objective is to characterize caveolin-1 on a fundamental level. This will be achieved by pursuing the following two specific aims: 1. Investigation of the membrane topology and three-dimensional structure of caveolin-1. 2. Investigation of caveolin-1 oligomerization. Specific aim 1 will determine the high-resolution three-dimensional solution structure of caveolin-1 as well as examine the solvent accessibility of tryptophan residues to assess the topology of caveolin-1 in a bilayer. Next, the role that two conserved proline residues play in the creation and/or stabilization of the intra-membrane "horseshoe" conformation will be probed using site-directed mutagenesis. Specific aim 2 will characterize both the size and distribution of oligomers formed by caveolin-1 in the presence and absence of cholesterol. Additionally, the role that a proline to leucine mutant plays in the oligomerization process will be probed. A fundamental understanding of caveolin-1 structure and oligomerization will undoubtedly open the door to possible therapeutic interventions that could address diseases linked to caveolin misfunction.