Investigation of Caveolin Structure, Topology, and Oligomerization

描述（由申请人提供）：小窝是位于质膜中的高度弯曲的内陷微域，在各种细胞过程中发挥核心作用。小窝蛋白（1、2和3）是小窝中发现的最重要的蛋白质，并且负责赋予小窝其不寻常的“烧瓶状”形状。最近的证据表明，小窝蛋白的不适当调节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。小窝蛋白采用一种不寻常的膜内“马蹄铁”构象，其N-和C-末端都面向细胞质，这种构象被认为是促进膜弯曲。此外，通过高阶低聚反应，小窝蛋白形成稳定膜曲率的结构骨架。使用生物物理技术，如核磁共振（NMR），荧光光谱，和分析超离心，我们的目标是表征小窝蛋白-1的基本水平。这将通过追求以下两个具体目标来实现：1.小窝蛋白-1的膜拓扑结构和三维结构研究。2.小窝蛋白-1寡聚化的研究。具体目标1将确定小窝蛋白-1的高分辨率三维溶液结构以及检查色氨酸残基的溶剂可及性以评估小窝蛋白-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“马蹄铁”构象的产生和/或稳定中发挥的作用将使用定点诱变来探测。具体目标2将表征在胆固醇存在和不存在下由小窝蛋白-1形成的寡聚体的大小和分布。此外，脯氨酸亮氨酸突变体在寡聚化过程中发挥的作用将被探测。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，这些干预可以解决与小窝蛋白功能障碍相关的疾病。

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.