The Discovery of Molecules and Mechanisms in ERAD Retrotranslocation Pathways

项目概要/摘要 通过ER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且ER应激被维持在可接受低水平。所有ERAD 途径包括蛋白质易位过程，称为逆易位，其中泛素化ERAD 底物在被胞质26 S蛋白酶体降解之前被选择性地从ER中提取。尽管 由于它在ERAD中的共同性，许多逆易位的特征仍然是神秘的。我们最近 在理解逆易位方面取得了重大突破通过采用全基因组酵母阵列，我们 已经发现菱形家族蛋白Dfm 1对于膜基质的去除至关重要， 这是一扇深入理解逆转位机制和生物学的大门。我们特别 威尔： 1)确定Dfm 1介导的逆转录易位中涉及的机制和机制。 2)表征在不存在Dfm 1的情况下诱导的新的逆易位途径。 3)探索在缺乏Dfm 1的情况下可能出现的新的应激途径。 我们将使用多方面的方法，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核心问题。我们将利用我们独特的 体内和体外试验-并继续设计新的-解剖菱形介导的基本机制， 逆易位及其在细胞和生物学中的地位。对逆易位的机理认识 而与之相关的压力将建立基本的生物学见解， 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，ER应激， 和宿主-病原体相互作用。

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.