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

 描述(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)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。由于基因突变、转录/翻译过程中的错误或应激条件，内质网中的蛋白质错误折叠是常见的，并持续发生。超过70种疾病与蛋白质错误折叠或蛋白质稳态缺陷有关。内质网中错误折叠的蛋白质被错位回到胞浆中，穿过内质网细胞膜，由蛋白酶体降解。这种称为内质网相关降解(ERAD)的质量控制过程需要内质网腔、膜和胞浆中的蛋白质组成的复杂系统。控制这一过程的分子机制尚不清楚。ERAD机制被认为是围绕着嵌入E3泛素连接酶的膜而模块化的。Hrd1是一种在ERAD中与几个已知结合伙伴起作用的E3泛素连接酶。然而，到目前为止，大多数研究都是在蛋白质过度表达的背景下检查哺乳动物Hrd1复合体的组成，这可能导致人工的蛋白质-蛋白质相互作用。因此，基因组编辑技术被用来构建细胞系，从而允许对内源水平表达的Hrd1复合体进行生化分析。这项研究的总体目标是使用这种最先进的方法全面剖析Hrd1复合体的架构。本提案的具体目标#1结合生化分离和质谱分析(MS)来定义处于稳定状态的天然Hrd1复合体的成分。在第二个目标中，将使用绝对定量MS(Aqua-MS)来揭示已鉴定的Hrd1络合物中各组分的化学计量比。这些实验将对ERAD复合体的结构提供前所未有的详细程度，并对这一蛋白质质量控制系统的功能产生新的见解。具体目标#3探索这些复合体对细胞扰动的动态适应。这些研究涉及将AIMS#1和AIMS#2中开发的方法和工作流程应用于不同的细胞环境，包括应激条件和与疾病相关的ERAD底物的表达。总之，提出的研究将极大地促进我们对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.