Investigation of role of the Hrd 1 membrane core complex in retro-translocation

描述(申请人提供)：内质网(ER)中错误折叠的蛋白质必须被有效地降解，因为异常蛋白质的积累可能会产生严重的有害影响，正如大量蛋白质错误折叠相关疾病所证明的那样。在真核细胞中，许多在内质网中错误折叠的蛋白质被运回胞浆进行降解。这个过程被称为内质网相关降解(ERAD)。除了在细胞维持中的重要作用外，该通路还与许多人类疾病有关，包括囊性纤维化、艾滋病毒和巨细胞病毒。人们对这一途径知之甚少，也许关于ERAD最紧迫的问题之一是如何使管腔底物能够被细胞质降解机制所利用。该建议旨在通过研究ERAD酵母模型中的Hrd1膜核心复合体来阐明腔底物的逆转位的机制，并确定可能的蛋白质传导通道。这种ER膜复合体包含所有关键的ER膜插入成分，对于降解错误折叠的发光ERAD底物至关重要，因此有望包含推测的回溯易位通道。通过免疫沉淀和交联法研究络合物与底物的相互作用，将确定形成通道的成分。此外，纯化的Hrdlp核心复合体的结构和通道的构象将使用电子冷冻显微镜进行研究。识别追溯易位通道将是理解ERAD的机制、该途径在人类疾病中的作用以及追溯易位机制如何被许多病毒和毒素劫持的关键进展。相关性：细胞拥有特殊的机制来分解不正确组装的蛋白质，但当这个系统发生故障时，异常蛋白质的积累可能会导致许多疾病。剖析这种组装如何在降解过程中发挥作用的分子水平的细节，对于了解系统的故障如何导致疾病非常重要，对于解释某些病毒和毒素如何利用和劫持这一重要的细胞机制也是至关重要的。

DESCRIPTION (provided by applicant): Proteins that misfold in the endoplasmic reticulum (ER) must be degraded efficiently because the accumulation of aberrant proteins can have severe deleterious effects, as evidenced by the large number of protein-misfolding associated diseases. In eukaryotic cells, many proteins that misfold in the endoplasmic reticulum are transported back to the cytosol for degradation. This process is known as ER-associated degradation (ERAD). In addition to its important role in cellular maintenance, the pathway has also been implicated in a number of human diseases, included cystic fibrosis, HIV, and cytomegalovirus. The pathway is only poorly understood, and perhaps one of the most pressing questions about ERAD is how luminal substrates are made accessible to the cytosolic degradation machinery. This proposal is aimed at elucidating the mechanisms of retro-translocation of luminal substrates and identifying the putative protein-conducting channel by investigating the Hrd1 membrane core complex from a yeast model of ERAD. This ER membrane complex contains all of the key ER-membrane inserted components central to to the degradation of luminally misfolded ERAD substrates and is therefore expected to contain the putative retro-translocation channel. By investigating the interactions of the complex with substrate through immunoprecipitation and cross-linking assays, the components that form the channel will be identified. Furthermore, the architecture of the purified Hrdlp core complex and the conformation of the channel will be investigated using electron cryomicroscopy. Identifying the retro-translocation channel will be a key development in understanding the mechanisms of ERAD, the role of the pathway in human diseases, and how retro-translocation machinery is hijacked by a number of viruses and toxins. Relevance: Cells possess special machinery to breakdown improperly assembled proteins, but when this system malfunctions, the accumulation of aberrant proteins can lead to a number of diseases. Dissecting the molecular level details of how this assembly functions in degradation will be important for understanding how breakdowns in the system can lead to disease and will be essential for explaining how certain viruses and toxins can take advantage of and hijack this important cellular machinery.