Biochemistry and Cell Biology of CHC22 Clathrin

描述（由申请方提供）：在人体中，网格蛋白有两种亚型，CHC 17和CHC 22，以其编码染色体命名。CHC 17同种型被充分研究，并形成网格蛋白包被的囊泡的外层，其在受体介导的胞吞作用期间从质膜（PM）、内体运输和来自trans-Golgi网络（TGN）的细胞器生物发生中分选货物。编码CHC 22的基因存在于人类和其他脊椎动物中，但在小鼠中进化成假基因，因此人类而不是小鼠表达CHC 22网格蛋白。Brodsky实验室广泛研究了CHC 17网格蛋白，有助于了解其生物化学，调控和细胞生物学。利用我们对CHC 17的熟悉，我们研究了CHC 22的相关功能，发现CHC 22独特地参与了从内体到TGN的逆行转运，从而将GLUT 4葡萄糖转运蛋白靶向人类肌肉和脂肪细胞中的胰岛素响应性GLUT 4储存室（GSC）。GSC的形成及其响应于胰岛素向PM释放GLUT 4是胰岛素调节的葡萄糖清除的主要途径。该途径的某些方面在2型糖尿病中存在缺陷，我们发现CHC 22与2型糖尿病患者中含有GLUT 4的异常结构相关。此外，在使用CHC 17形成其GSC的小鼠中CHC 22的转基因表达改变了导致糖尿病症状的鼠GSC性质。这些数据表明，CHC 22介导的膜运输是人类葡萄糖代谢所必需的，定义了人类和小鼠葡萄糖稳态之间的差异。已经证明了CHC 22的生理重要性，本提案的目标是定义CHC 22作用的分子基础，并解决关于其在人体葡萄糖代谢中作用的三个假设。建议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。目的1将定义CHC 22的形态，自组装，CHC 22亚基和结合蛋白的性质，和结构，以解决CHC 22具有独特的生化特征，区分稳定性和动力学的人GSC从其小鼠对应的假设。目的2将在HeLa细胞、人成肌细胞和转染的3 T3-L1细胞中建立CHC 22细胞内募集到膜的调节，以解决CHC 22影响肌肉和脂肪细胞中GLUT 4转运的假设，因为它与CHC 17竞争优先在膜运输的局部步骤中发挥作用。目的3表征CHC 22的现有等位基因变体之间的功能和生化差异，以解决CHC 22的变体同种异型可能导致个体之间的代谢差异，并加重或预防糖尿病的假设。拟议的研究将表征CHC 22功能的分子机制，其细胞调节及其与糖尿病中发生故障的膜交通途径的关系。

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.