DEFINING THE FUNCTION OF PROTEASOMAL DEUBIQUITINASES

项目摘要 26 S蛋白酶体是一种巨大的、复杂调节的ATP依赖性蛋白酶，负责 大多数细胞蛋白质的降解。蛋白酶体不能精确地调节蛋白质水平是一个 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 用聚合物（Ub）链共价修饰。因此，令人困惑的是，蛋白酶体实际上 负责去除Ub链的酶（去泛素化酶/DUB）。虽然很明显， 内源性DUB，RPN 11，通过阻止蛋白质的过早去泛素化而促进降解， 参与蛋白酶体，其他蛋白酶体DUB的作用，例如，UCH 37/UCHL 5，较差 明白在初步研究中，我们发现蛋白酶体结合的UCH 37通过以下方式充当链编辑器： 去除分支点。本申请提出阐明UCH 37如何选择分支Ub链用于 编辑，这种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UCH 37介导的链脱支的作用 在使用不同的、纯化的人蛋白酶体复合物和荧光， 多泛素化底物。在目标2中，我们提出鉴定蛋白酶体结合的UCH 37的细胞靶点 使用定量蛋白质组学、细胞内邻近标记和Ub中下 质谱分析法来在目标3中，我们专注于了解UCH 37对肿瘤特异性的分子基础。 支链最后，在目标4中，我们将开发新的UCH 37环肽抑制剂，以促进努力 来剖析它在任何生物学范式中的功能。从这项研究中获得的知识将有助于了解 泛素蛋白酶体系统的基本方面，并为新的开发铺平道路 调节蛋白酶体功能的治疗剂。

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.