Elucidating the architecture of native multiprotein complexes involved in ER-associated degradation

 描述（由申请人提供）：大约30%的哺乳动物蛋白质组被合成到内质网（ER）中并进入分泌途径。由于内质网的拥挤环境，这些蛋白质的折叠是一个复杂的过程。ER中的蛋白质错误折叠是常见的，并且由于基因突变、转录/翻译过程中的错误或应激条件而持续发生。超过70种疾病与蛋白质错误折叠或蛋白质稳态缺陷有关。内质网内错误折叠的蛋白质穿过内质网膜移位回胞质溶胶中，由蛋白酶体降解。这种质量控制过程称为ER相关降解（ERAD），需要ER腔、膜和胞质溶胶中的复杂蛋白质系统。控制这一过程的分子机制尚不清楚。ERAD机制被假设为围绕膜包埋的E3泛素连接酶的模块化。Hrd 1是一种E3泛素连接酶，在ERAD中与几种已知的结合伴侣起作用。然而，迄今为止，大多数研究已经检查了哺乳动物Hrd 1复合物在蛋白质过表达的背景下的组成，这可能导致人为的蛋白质-蛋白质相互作用。因此，利用基因组编辑技术来构建细胞系，从而允许对以内源水平表达的Hrd 1复合物进行生化分析。本研究的总体目标是使用这种最先进的方法全面剖析Hrd 1复合物的结构。该提案的具体目标#1结合了生物化学分馏和质谱（MS），以确定稳态下天然Hrd 1复合物的组分。在第二个目标中，绝对定量MS（AQUA-MS）将用于揭示已识别的Hrd 1复合物内组分的化学计量。这些实验将提供ERAD复合物结构的前所未有的细节水平，并对该蛋白质质量控制系统的功能产生新的见解。具体目标#3探索这些复合物对细胞扰动的动态适应。这些研究涉及在不同的细胞环境中应用目标#1和#2中开发的方法和工作流程，包括应激条件和疾病相关ERAD底物的表达。总之，所提出的研究将大大推进我们对ERAD所涉及的功能网络的理解。

 DESCRIPTION (provided by applicant): Approximately 30% of the mammalian proteome is synthesized into the endoplasmic reticulum (ER) and enters the secretory pathway. The folding of these proteins is a complicated process due to the crowded environment of the ER. Protein misfolding in the ER is common and occurs continually because of genetic mutations, errors during transcription/translation, or conditions of stress. Over 70 diseases are related to protein misfolding or defects in protein homeostasis. Misfolded proteins within the ER are dislocated back into the cytosol across the ER membrane for degradation by the proteasome. This quality control process called ER-associated degradation (ERAD) requires a complex system of proteins in the ER lumen, membrane, and cytosol. The molecular mechanisms governing this process are unclear. ERAD machinery is hypothesized to be modular around membrane embedded E3 ubiquitin ligases. Hrd1 is one such E3 ubiquitin ligase functioning in ERAD with several known binding partners. However, most studies to date have examined the composition of mammalian Hrd1 complexes in the context of protein overexpression, which can result in artifactual protein- protein interactions. Therefore, genome editing techniques were utilized to construct cell lines permitting the biochemical analysis of Hrd1 complexes expressed at endogenous levels. The overall goal of this research is to comprehensively dissect the architecture of Hrd1 complexes using this state-of-the-art approach. Specific aim #1 of this proposal combines biochemical fractionation and mass spectrometry (MS) to define components of native Hrd1 complexes at steady-state. In the second aim, absolute quantitation MS (AQUA-MS) will be used to reveal the stoichiometry of components within identified Hrd1 complexes. These experiments will provide an unprecedented level of detail in the architecture of ERAD complexes and yield new insight into the function of this protein quality control system. Specific aim #3 explores the dynamic adaption of these complexes to cellular perturbations. These studies involve applying the approaches and workflow developed in aims #1 and #2 in distinct cellular contexts, including stress conditions and the expression of disease-related ERAD substrates. Together, the research proposed will significantly advance our understanding of the functional networks involved in ERAD.