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：
8847846
负责人：
Kerney Jebrell Glover
金额：
$ 6.53万
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 2016-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8847846
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 Solutions 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）：Caveolae是位于质膜中高度弯曲的无知的微型域，在各种细胞过程中起着中心作用。小窝蛋白（1、2和3）是在小窝中发现的最重要的蛋白质，并负责使Caveolae具有不寻常的“烧瓶状”形状。最近的证据表明，不当调节小窝蛋白的调节和突变形式会导致各种疾病，包括阿尔茨海默氏症，肌肉营养不良，癌症和心脏病。小窝蛋白采用异常的膜内“马蹄形”构象，其N-和C-termini都面对细胞质，并且这种构型被认为可以促进膜曲率。另外，通过高阶寡聚化，小窝蛋白形成了稳定膜曲率的结构主链。使用生物物理技术，例如核磁共振（NMR），荧光光谱和分析性超速离心，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特定目的来实现：1。对Caveolin-1的膜拓扑结构和三维结构进行研究。 2。对小窝蛋白-1低聚的研究。具体的目标1将确定小窝1的高分辨率三维溶液结构，并检查色氨酸残基的溶剂可访问性，以评估双层中的小窝蛋白-1的拓扑结构。接下来，将使用位置定向的诱变探测两个保守的脯氨酸残基在创建和/或稳定中的作用。特定的目标2将表征在存在和不存在胆固醇的情况下由Caveolin-1形成的低聚物的大小和分布。另外，将探测脯氨酸到亮氨酸突变体在寡聚过程中发挥作用的作用。对Caveolin-1结构和低聚的基本理解无疑将为可能的治疗干预措施打开大门，这些干预措施可能解决与Caveolin障碍有关的疾病。