DEFINING THE FUNCTION OF PROTEASOMAL DEUBIQUITINASES

项目摘要 26S蛋白酶体是一种庞大的、调节复杂的依赖于ATP的蛋白酶，它负责 大多数细胞蛋白质的降解。蛋白酶体不能精确调节蛋白质水平是一种 是许多癌症和神经退行性疾病的标志。将蛋白质靶向蛋白酶体需要 具有聚合物(Ub)链的共价改性。因此，令人困惑的是，蛋白酶体实际上包含了 负责去除Ub链的酶(脱泛素酶/DUBS)。虽然已经变得很明显， 固有的Dub，RPN11，通过阻止蛋白质的过早去泛素化来促进降解 与蛋白酶体结合，其他蛋白酶体Dub的作用很差，例如UCH37/UCHL5 明白了。在初步研究中，我们发现蛋白酶体结合的UCH37通过以下方式发挥链编辑作用 正在删除分支点。本申请旨在阐明UCH37如何选择分支Ub链 编辑，这一活动如何整合到蛋白酶体降解的整个过程中，以及它如何影响 细胞蛋白质的周转。在目标1中，我们将研究UCH37介导链脱支的作用 在使用不同的、纯化的人类蛋白酶体复合体和荧光降解蛋白酶体的过程中， 多泛素化底物。在目标2中，我们建议识别蛋白酶体结合的UCH37的细胞靶标 使用定量蛋白质组学、细胞内邻近标记和Ub中间向下的创新组合 质谱学。在目标3中，我们重点了解UCH37的S特异性的分子基础 分支链。最后，在目标4中，我们将开发新型的UCH37环肽抑制剂，以促进努力 来剖析它在任何生物范式中的功能。从这项研究中获得的知识将有助于 泛素蛋白酶体系统的基本方面，为新的发展铺平道路 调节蛋白酶体功能的治疗学。

Project Summary The 26S proteasome is a massive, intricately regulated ATP-dependent protease that is responsible for the degradation of most cellular proteins. Failure of the proteasome to precisely regulate protein levels is a hallmark of many cancers and neurodegenerative diseases. Targeting proteins to the proteasome requires covalent modification with a polymeric (Ub) chain. Thus, it is perplexing that the proteasome actually houses enzymes (deubiquitinases/DUBs) responsible for removing Ub chains. While it has become clear that the intrinsic DUB, RPN11, promotes degrades by preventing premature deubiquitination of proteins not yet engaged with the proteasome, the roles of other proteasomal DUBs, e.g., UCH37/UCHL5, are poorly understood. In preliminary studies, we discovered that proteasome-bound UCH37 acts as a chain editor by removing branch points. This application proposes to elucidate how UCH37 selects branched Ub chains for editing, how this activity is integrated into the entire process of proteasomal degradation, and how it impacts the turnover of cellular proteins. In Aim 1, we will investigate the role of UCH37-mediated chain debranching during proteasomal degradation using distinct, purified human proteasome complexes and fluorescent, polyubiquitinated substrates. In Aim 2, we propose to identify cellular targets of proteasome-bound UCH37 using an innovative combination of quantitative proteomics, in-cell proximity labeling, and Ub middle-down mass spectrometry. In Aim 3, we focus on understanding the molecular basis of UCH37's specificity toward branched chains. Finally, in Aim 4, we will develop novel cyclic peptide inhibitors of UCH37 to facilitate efforts to dissect its function in any biological paradigm. The knowledge gained from this research will shed light on fundamental aspects of the ubiquitin proteasome system and pave the way for the development of new therapeutics that regulate proteasome function.