Assembly Chaperone Complex for Membrane Proteins in the Endoplasmic Reticulum

项目说明 在理解多亚单位膜蛋白是如何形成的过程中，有一个显著的知识缺口。 在内质网(ER)的膜上插入、折叠和组装，内质网是一种细胞 在分子伴侣的帮助下进行蛋白质质量控制的细胞器。我们使用γ- A型氨基丁酸(GABAA)受体回答这个问题。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 五聚体蛋白质复合体。尽管对血浆中GABAA受体的生理学进行了广泛的研究 膜及其在神经回路中控制抑制-兴奋平衡的作用，几乎没有努力 被用来研究蛋白质稳态(蛋白质稳态)网络是如何调节折叠和 GABAA蛋白在内质网中的组装。这给遗传病的治疗带来了巨大的障碍。 癫痫是因为GABAA受体亚单位的大量癫痫相关突变导致的 亚基蛋白质在内质网中的错误折叠和/或破坏五聚体复合体的组装，导致 细胞表面受体复合体的局部化程度降低，神经回路失衡。这里, 具体地说，我们基于亲和纯化-质谱仪的蛋白质组学分析确定了一种潜在的 与内源性GABAA相互作用的膜蛋白组装伴侣复合体(MPACC) 受体，包括内质网管腔中的热休克蛋白47(Hsp47)和内质网膜蛋白 内质网中的复合体(EMC)。在目标1中，我们将测试我们的假设，即Hsp47是肯定的 调节内源性GABAA受体在内质网及其功能表面的组装 表情。在目标2中，我们将检验我们的假设，即EMC正向调节 GABAA受体位于内质网细胞膜，因此其功能表面表达。在《目标3》中，我们将 操纵内质网中的组装伴侣复合体以纠正致病GABAA的功能 感受器。

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.