Biochemistry and Cell Biology of CHC22 Clathrin

CHC22 网格蛋白的生物化学和细胞生物学

• 批准号: 8459867
• 负责人: Frances M. Brodsky
• 金额: $ 34.08万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-12-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8459867
• 关键词: 3T3-L1 Cells; Address; Adipocytes; Amino Acid Sequence; Binding; Binding Proteins; Biochemical; Biochemistry; Biogenesis; Blood Circulation; Cell membrane; Cells; Cellular biology; Chromosomes; Clathrin; Clathrin Heavy Chains; Clathrin-Coated Vesicles; Computer Simulation; Crystallography; Cultured Cells; Data; Defect; Diabetes Mellitus; Electron Microscopy; Endocytosis; Endosomes; Evolution; Familiarity; Fatty acid glycerol esters; GLUT4 gene; Gene Duplication; Genes; Genetic Variation; Glucose; Glucose Transporter; Goals; Hela Cells; Human; Human Chromosomes; In Vitro; Individual; Insulin; Knowledge; Laboratories; Mediating; Membrane; Membrane Protein Traffic; Metabolic; Metabolism; Molecular; Morphology; Mus; Muscle; Muscle Cells; Myoblasts; Names; Non-Insulin-Dependent Diabetes Mellitus; Organelles; Pathway interactions; Peptide Sequence Determination; Physiological; Play; Population; Property; Protein Biochemistry; Protein Fragment; Protein Isoforms; Proteins; Pseudogenes; Publishing; Recombinants; Recruitment Activity; Regulation; Relative (related person); Research; Role; Skeletal Muscle; Sorting - Cell Movement; Structure; Symptoms; Transgenes; Transgenic Organisms; Variant; Vertebrates; base; blood glucose regulation; cell growth regulation; comparative; diabetic; diabetic patient; genetic analysis; glucose metabolism; glucose transport; human tissue; protein structure; public health relevance; receptor mediated endocytosis; research study; response; retrograde transport; self assembly; tandem mass spectrometry; trafficking; trans-Golgi Network

DESCRIPTION (provided by applicant): In humans, there are two isoforms of clathrin protein, CHC17 and CHC22, named for their encoding chromosomes. The CHC17 isoform is well studied and forms the outer layer of clathrin-coated vesicles, which sort cargo during receptor-mediated endocytosis from the plasma membrane (PM), endosomal traffic, and organelle biogenesis from the trans-Golgi network (TGN). The gene encoding CHC22 is present in humans and other vertebrates but evolved into a pseudogene in mice, so humans, but not mice, express CHC22 clathrin. The Brodsky laboratory has extensively studied CHC17 clathrin, contributing to general knowledge about its biochemistry, regulation and cell biology. Capitalizing on our familiarity with CHC17, we investigated the relative function of CHC22 and discovered that CHC22 participates uniquely in retrograde transport from endosomes to the TGN and thereby targets the GLUT4 glucose transporter to the insulin-responsive GLUT4 storage compartment (GSC) in human muscle and fat cells. Formation of the GSC and its release of GLUT4 to the PM in response to insulin is the major pathway for insulin-regulated glucose clearance. Aspects of this pathway are defective in type 2 diabetes and we showed that CHC22 associates with abnormal GLUT4- containing structures in type 2 diabetic patients. Furthermore, transgenic expression of CHC22 in mice, which use CHC17 to form their GSC, altered murine GSC properties leading to diabetic symptoms. These data indicate that CHC22-mediated membrane traffic is needed for human glucose metabolism, defining differences between human and mouse glucose homeostasis. Having demonstrated the physiological importance of CHC22, the goal of this proposal is to define the molecular basis for CHC22 action and address three hypotheses about its role in human glucose metabolism. The experiments proposed involve protein biochemistry, electron microscopy, in vitro cell biology, and in silico genetic analysis. Aim 1 will define CHC22 morphology, properties of self-assembly, CHC22 subunits and binding proteins, and structure to address the hypothesis that CHC22 has unique biochemical features that distinguish the stability and dynamics of the human GSC from its murine counterpart. Aim 2 will establish the regulation of CHC22 intracellular recruitment to membranes in HeLa cells, human myoblasts and transfected 3T3-L1 cells to address the hypothesis that CHC22 influences GLUT4 transport in muscle and fat cells because it competes with CHC17 to function preferentially in a localized step of membrane traffic. Aim 3 characterizes functional and biochemical differences between existing allelic variants of CHC22 to address the hypothesis that variant allotypes of CHC22 could contribute to metabolic differences between individuals, and exacerbate or protect against diabetes. The proposed research will characterize the molecular mechanism of CHC22 function, its cellular regulation and its relationship to membrane traffic pathways that malfunction in diabetes.

描述（由申请人提供）：在人类中，有两种甲状腺素蛋白CHC17和CHC22的同工型，以它们的编码染色体命名。对CHC17同工型进行了充分的研究，并形成了涂有网格蛋白的囊泡的外层，这些囊泡在受体介导的内吞作用期间从质膜（PM），内体流量和细胞体生物发生物中对货物进行了排序。编码CHC22的基因存在于人类和其他脊椎动物中，但在小鼠中演变成伪代，因此人类（而不是小鼠）表达CHC22克拉瑟蛋白。 Brodsky实验室已经广泛研究了CHC17甲板蛋白，促进了有关其生物化学，调节和细胞生物学的一般知识。利用我们对CHC17的熟悉程度，我们研究了CHC22的相对功能，发现CHC22独特地参与了从内体到TGN的逆行运输，因此将GLUT4葡萄糖转运蛋白靶向人类肌肉和脂肪细胞中的胰岛素 - 反应性GLUT4储存室（GSC）。 GSC的形成及其对胰岛素响应于PM的GLUT4的释放是胰岛素调节的葡萄糖清除率的主要途径。该途径的各个方面在2型糖尿病中是有缺陷的，我们表明CHC22与2型糖尿病患者中含有异常glut4的结构相关。此外，使用CHC17形成GSC的小鼠中CHC22的转基因表达改变了导致糖尿病症状的鼠GSC特性。这些数据表明，人类葡萄糖代谢需要CHC22介导的膜流量，从而定义了人与小鼠葡萄糖稳态之间的差异。该提议的目的是证明了CHC22的生理重要性，是定义CHC22作用的分子基础，并解决有关其在人类葡萄糖代谢中的作用的三个假设。提出的实验涉及蛋白质生物化学，电子显微镜，体外细胞生物学和计算机遗传分析。 AIM 1将定义CHC22形态，自组装的特性，CHC22亚基和结合蛋白，以及结构，以解决CHC22具有独特的生化特征的假设，这些特征将人类GSC的稳定性和动力学与鼠类鼠的鼠类相对应区分开。 AIM 2将建立对HeLa细胞，人成肌细胞和转染的3T3-L1细胞中CHC22对膜的调节，以解决CHC22在肌肉和脂肪细胞中影响GLUT4转运的假设，因为它会与CHC17竞争，以在杂膜流量的局部范围内竞争。 AIM 3表征了CHC22现有等位基因变体之间的功能和生化差异，以解决以下假设：CHC22的变体异型可能导致个体之间的代谢差异，并加剧或预防糖尿病。拟议的研究将表征CHC22功能的分子机制，其细胞调节及其与糖尿病故障故障的膜交通途径的关系。