Investigation of Caveolin Structure, Topology, and Oligomerization

Caveolin 结构、拓扑和寡聚化的研究

基本信息

批准号：
8975780
负责人：
Kerney Jebrell Glover
金额：
$ 39.08万
依托单位：
LEHIGH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-01-01 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8975780
关键词：
Address Adopted Alzheimer&apos s Disease Behavior Biological Biology Caveolae Caveolins Cell membrane Cell physiology Cells Cholesterol Cytoplasm Data Disease Event Face Fluorescence Fluorescence Spectroscopy Gel Chromatography Goals Hand Health Heart Diseases Imagery Individual Integral Membrane Protein Investigation Knowledge Leucine Link Lipid Bilayers Liposomes Malignant Neoplasms Membrane Membrane Proteins Modeling Molecular Conformation Monitor Muscular Dystrophies Mutate Mutation Nature Nuclear Magnetic Resonance Play Process Proline Proteins Regulation Resolution Role Sequence Analysis Shapes Signal Transduction Site-Directed Mutagenesis Solvents Stretching Structure Testing Therapeutic Intervention Transmembrane Domain Tryptophan Vertebral column Work analytical ultracentrifugation base biophysical techniques caveolin 1 design flasks in vivo innovation membrane model mutant polypeptide protein structure reconstitution research study scaffold three dimensional structure

项目摘要

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.

描述（由申请人提供）：小凹是位于质膜中的高度弯曲的内陷微域，在多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，它们使小窝具有不寻常的“瓶状”形状。最近的证据表明，小窝蛋白的不当调节和突变形式可能导致多种疾病，包括阿尔茨海默病、肌肉萎缩症、癌症和心脏病。 Caveolin 采用一种不寻常的膜内“马蹄形”构象，其 N 端和 C 端都面向细胞质，这种构象被认为可以促进膜弯曲。此外，通过高阶低聚，caveolin 形成稳定膜曲率的结构主链。使用核磁共振 (NMR)、荧光光谱和分析超速离心等生物物理技术，我们的目标是在基本水平上表征 Caveolin-1。这将通过追求以下两个具体目标来实现： 1. 研究 Caveolin-1 的膜拓扑和三维结构。 2. Caveolin-1 寡聚化的研究。具体目标 1 将确定 Caveolin-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 Caveolin-1 在双层中的拓扑结构。接下来，将使用定点诱变来探讨两个保守的脯氨酸残基在膜内“马蹄”构象的创建和/或稳定中所起的作用。具体目标 2 将表征在存在和不存在胆固醇的情况下由 Caveolin-1 形成的寡聚物的大小和分布。此外，还将探讨脯氨酸到亮氨酸突变体在寡聚过程中所起的作用。对 Caveolin-1 结构和寡聚化的基本了解无疑将为可能的治疗干预措施打开大门，从而解决与 Caveolin 功能障碍相关的疾病。