Defining the Function of Proteasomal Deubiquitinases

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。这项研究计划的目标是确定如何降解， 26 S蛋白酶体受去泛素化酶调节。因为蛋白质降解的失调是 许多人类癌症和神经系统疾病，这些研究的结果可以用来设计新的 针对26 S蛋白酶体关键调节因子的药物。这项研究计划的一个主要重点将是 去泛素化酶UCHL 5/UCH 37，其被泛素结合受体募集到蛋白酶体 ADRM 1/RPN 13。UCH 37缺陷导致小鼠胚胎死亡和UCH 37过表达 与RPN 13一起沿着的是几种癌症中的不良预后。X射线和 晶体学、核磁共振、体外生物化学、蛋白质工程和基于细胞的方法将用于 了解UCH 37如何选择其泛素链靶点，为什么这些靶点的切割是必要的， 促进蛋白酶体降解，并且细胞蛋白质组中的哪些蛋白质需要UCH 37活性用于 高效降解。我们还将开发一套单链抗体（称为纳米抗体）， 干扰UCH 37的不同活动。这些新的工具将有助于确定 UCH 37在不同的生化途径，如细胞周期和细胞对氧化应激的反应 和DNA损伤考虑到肿瘤抑制因子BAP 1与催化肿瘤的转录因子BAP 1具有相似的序列， UCH 37的结构域，我们的工具和方法也可以用来更好地了解它在组蛋白之外的作用 去泛素化从拟议的研究中获得的分子见解将为以下方面提供基础： 开发靶向需要过度蛋白酶体活性的癌症的新治疗剂， 生存

The 26S proteasome is a massive, intricately regulated ATP-dependent protease responsible for the degradation of most cellular proteins. The goal of this research program is to determine how degradation by the 26S proteasome is regulated by deubiquitinases. Since misregulation of protein degradation is a hallmark of many human cancers and neurological disorders, the results of these studies can be used to design new drugs that target key regulators of the 26S proteasome. A major focus of this research program will be on the deubiquitinase UCHL5/UCH37, which is recruited to the proteasome by the ubiquitin-binding receptor ADRM1/RPN13. Deficiencies in UCH37 leads to embryonic lethality in mice and overexpression of UCH37 along with RPN13 is associated with poor prognoses in several cancers. A combination of x-ray crystallography, NMR, in vitro biochemistry, protein engineering, and cell-based approaches will be used to understand how UCH37 selects its ubiquitin chain targets, why the cleavage of those targets is necessary for promoting proteasomal degradation, and which proteins in a cellular proteome require UCH37 activity for efficient degradation. We will also develop a set of single-chain antibodies (referred to as nanobodies) capable of interfering with distinct activities of UCH37. These new tools will be instrumental in defining the function of UCH37 in different biochemical pathways such as the cell cycle and the cellular response to oxidative stress and DNA damage. Considering the tumor suppressor BAP1 shares a similar sequence with the catalytic domain of UCH37, our tools and approaches can also be used to better understand its role outside of histone deubiquitination. The molecular insights that will result from the proposed studies will provide a foundation for the development of new therapeutic agents that target cancers requiring excessive proteasome activity for survival.