Biochemistry and Cell Biology of CHC22 Clathrin

描述（由申请人提供）：在人类中，网格蛋白有两种异构体，CHC17和CHC22，以其编码染色体命名。CHC17异构体被充分研究并形成网格蛋白包被囊泡的外层，在受体介导的质膜内吞作用（PM）、内体运输和反式高尔基网络（TGN）的细胞器生物发生过程中对货物进行分选。编码CHC22的基因存在于人类和其他脊椎动物中，但在小鼠中进化为假基因，因此人类而不是小鼠表达CHC22网格蛋白。Brodsky实验室对CHC17网格蛋白进行了广泛的研究，对其生物化学，调控和细胞生物学的一般知识做出了贡献。利用我们对CHC17的熟悉程度，我们研究了CHC22的相关功能，发现CHC22独特地参与了从内体到TGN的逆行运输，从而将GLUT4葡萄糖转运蛋白靶向到人体肌肉和脂肪细胞中胰岛素反应性的GLUT4储存室（GSC）。在胰岛素的作用下，GSC的形成及其向PM释放GLUT4是胰岛素调节的葡萄糖清除的主要途径。该通路的某些方面在2型糖尿病中存在缺陷，我们发现CHC22与2型糖尿病患者中异常的含GLUT4结构相关。此外，CHC22在小鼠体内的转基因表达改变了小鼠GSC的特性，从而导致糖尿病症状。这些数据表明，chc22介导的膜运输是人体葡萄糖代谢所必需的，这定义了人类和小鼠葡萄糖稳态的差异。在证明了CHC22的生理重要性之后，本提案的目标是确定CHC22作用的分子基础，并提出关于其在人体葡萄糖代谢中的作用的三个假设。实验包括蛋白质生物化学、电子显微镜、体外细胞生物学和硅基因分析。目的1将定义CHC22的形态、自组装特性、CHC22亚基和结合蛋白以及结构，以解决CHC22具有独特生化特征的假设，这些特征将人类GSC的稳定性和动力学与小鼠GSC区分开来。Aim 2将在HeLa细胞、人成肌细胞和转染的3T3-L1细胞中建立CHC22细胞内募集到细胞膜的调控，以解决CHC22影响肌肉和脂肪细胞中GLUT4运输的假设，因为它与CHC17竞争，在膜运输的局部步骤中优先发挥作用。目的3分析现有CHC22等位基因变体之间的功能和生化差异，以解决CHC22变异异型可能导致个体之间代谢差异，并加剧或预防糖尿病的假设。本研究将探讨CHC22在糖尿病中功能的分子机制、细胞调控及其与膜交通通路功能障碍的关系。

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.