Assembly Chaperone Complex for Membrane Proteins in the Endoplasmic Reticulum

项目描述 在理解多亚基膜蛋白是如何 插入，折叠，并组装在内质网（ER）的膜，细胞 在分子伴侣的协助下进行蛋白质质量控制的细胞器。我们用γ- 氨基丁酸A型（GABAA）受体来回答这个问题。GABAA受体，主要的 哺乳动物中枢神经系统中的抑制性神经递质门控离子通道，是一种 五聚体蛋白复合物。尽管对血浆中GABAA受体生理学进行了广泛的研究， 膜及其在控制神经回路中抑制-兴奋平衡中的作用，很少有努力 研究了蛋白质稳态（proteostasis）网络如何调节折叠， GABAA蛋白在ER中的组装。这为遗传性疾病的治疗带来了重大障碍。 癫痫，因为GABAA受体亚单位中的许多癫痫相关突变导致 亚基蛋白在ER中错误折叠和/或破坏五聚体复合物的组装，导致 受体复合物的细胞表面定位减少和神经回路不平衡。在这里， 具体来说，我们的亲和纯化-质谱为基础的蛋白质组学分析确定了一个潜在的 与内源性GABAA相互作用的膜蛋白组装伴侣复合物（MPACC） 受体，由ER腔中的热休克蛋白47（Hsp 47）和ER膜蛋白组成 复合物（EMC）在ER膜。在目标1中，我们将测试我们的假设，即Hsp 47阳性 调节内源性GABAA受体在ER及其功能表面的组装 表情在目标2中，我们将测试我们的假设，即EMC积极调节 GABAA受体在ER膜上的表达及其功能性表面表达。在目标3中，我们 操纵ER中的组装伴侣复合物以纠正致病性GABAA的功能 受体。

Project Description There is a significant knowledge gap in understanding how multi-subunit membrane proteins are inserted, folded, and assembled in the membrane of the endoplasmic reticulum (ER), a cellular organelle for protein quality control with the assistance of molecular chaperones. We use γ- aminobutyric acid type A (GABAA) receptors to answer this question. The GABAA receptors, the primary inhibitory neurotransmitter-gated ion channels in the mammalian central nervous systems, are a pentameric protein complex. Despite extensive research on GABAA receptors physiology on the plasma membrane and their role in controlling the inhibition-excitation balance in neural circuits, little effort has been made to investigate how the protein homeostasis (proteostasis) network regulates the folding and assembly of GABAA proteins in the ER. This brings a significant barrier for the treatment of genetic epilepsies because numerous epilepsy-associated mutations in GABAA receptor subunits cause subunit protein misfolding in the ER and/or disrupt assembly of the pentameric complex, leading to decreased cell surface localization of the receptor complex and imbalanced neural circuits. Here, specifically, our affinity purification-mass spectrometry-based proteomics analysis identified a potential Membrane Protein Assembly Chaperone Complex (MPACC) that interacts with endogenous GABAA receptors, consisting of heat shock protein 47 (Hsp47) in the ER lumen and the ER membrane protein complex (EMC) in the ER membrane. In Aim 1, we will test our hypothesis that Hsp47 positively regulates the assembly of endogenous GABAA receptors in the ER and their functional surface expression. In Aim 2, we will test our hypothesis that the EMC positively regulates the assembly of GABAA receptors in the ER membrane and thus their functional surface expression. In Aim 3, we will manipulate the assembly chaperone complex in the ER to correct the function of pathogenic GABAA receptors.