The Discovery of Molecules and Mechanisms in ERAD Retrotranslocation Pathways

项目摘要/摘要 通过内质网相关蛋白降解(ERAD)消除错误折叠的蛋白质确保蛋白质进入 分泌途径被正确折叠，内质网应激保持在可接受的低水平。所有ERAD 途径包括被称为逆转位的蛋白质转位过程，在该过程中泛素化的ERAD 在胞质26S蛋白酶体降解之前，从内质网中选择性地提取底物。尽管 它在ERAD中的共性，许多逆转易位的特征仍然是个谜。我们最近做了 在理解回溯易位方面取得了重大突破。通过使用全基因组酵母阵列，我们 发现了菱形家族蛋白Dfm1对膜底物的去除、打开 这是深入理解逆转位机制和生物学的大门。具体来说，我们 WILL： 1)确定Dfm1介导的逆转录易位的机制和机制。 2)研究了在缺乏Dfm1的情况下诱导的一种新的逆转录易位途径。 3)探索在缺乏Dfm1的情况下可能出现的新的应激途径。 我们将使用包括生物化学、细胞生物学、遗传学、功能基因组学和 蛋白质组学来解决ERAD和膜生物学中的这些中心问题。我们将利用我们独特的 体内和体外分析-并继续设计新的-剖析菱形介导的基本机制 逆转移位及其在细胞和生物生物学中的地位。对逆转位的一种机械理解 而与它的缺失相关的压力将在揭开面纱的同时建立基础的生物学见解 多种关键途径的治疗靶点，包括蛋白质错误折叠、蛋白质质量控制、内质网应激、 以及宿主与病原体的相互作用。

Project Summary/Abstract Elimination of misfolded proteins by ER-associated protein degradation (ERAD) ensures that proteins entering the secretory pathway are correctly folded and that ER stress is maintained at acceptably low levels. All ERAD pathways include a protein translocation process termed retrotranslocation, in which ubiquitinated ERAD substrates are selectively extracted from the ER before degradation by the cytosolic 26S proteasome. Despite its commonality in ERAD, many features of retrotranslocation have remained mysterious. We have recently made a major breakthrough in understanding retrotranslocation. By employing whole-genome yeast arrays, we have discovered the rhomboid family protein Dfm1 to be critical for the removal of membrane substrates, opening the door to a deep mechanistic understanding of retrotranslocation mechanisms and biology. Specifically, we w ill: 1) Determine the machinery and mechanisms involved in Dfm1-mediated retrotranslocation. 2) Characterize a novel retrotranslocation pathway induced in the absence of Dfm1. 3) Explore the new stress pathway that can arise in the absence of Dfm1. We will use a multifaceted approach including biochemistry, cell biology, genetics, functional genomics and proteomics to address these central questions in ERAD and membrane biology. We will leverage our unique in vivo and in vitro assays-and continue to devise new ones-to dissect the basic mechanisms of rhomboid-mediated retrotranslocation and its place in cell and organismal biology. A mechanistic understanding of retrotranslocation and the stress associated with its absence will establish foundational biological insights while unveiling therapeutic targets for a variety of critical pathways including protein misfolding, protein quality control, ER stress, and host-pathogen interactions.