Defining the Function of Proteasomal Deubiquitinases

定义蛋白酶体去泛素酶的功能

• 批准号: 10623537
• 负责人: ERIC Robert STRIETER
• 金额: $ 36.15万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623537
• 关键词: 26S proteasome; ATP-Dependent Proteases; Antibodies; Biochemical Pathway; Biochemistry; Catalytic Domain; Cell Cycle; Cells; DNA Damage; Deubiquitination; Development; Embryo; Foundations; Goals; Histones; Human; In Vitro; Malignant Neoplasms; Molecular; Mus; Oxidative Stress; Pharmaceutical Preparations; Prognosis; Protein Engineering; Proteins; Proteome; Research; Role; Tumor Suppressor Proteins; Ubiquitin; X-Ray Crystallography; design; human disease; insight; multicatalytic endopeptidase complex; nanobodies; nervous system disorder; new therapeutic target; novel therapeutics; overexpression; programs; protein degradation; receptor binding; recruit; response; targeted treatment; tool; ubiquitin isopeptidase

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.

26S 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。该研究计划的目标是确定如何降解 26S 蛋白酶体受去泛素酶调节。由于蛋白质降解的失调是一个标志 许多人类癌症和神经系统疾病，这些研究的结果可用于设计新的 针对 26S 蛋白酶体关键调节因子的药物。该研究计划的一个主要重点将是 去泛素酶 UCHL5/UCH37，由泛素结合受体招募到蛋白酶体 ADRM1/RPN13。 UCH37 缺陷导致小鼠胚胎死亡和 UCH37 过度表达 RPN13 与多种癌症的不良预后相关。 X射线的组合 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 UCH37 如何选择其泛素链靶标，为什么这些靶标的切割对于 促进蛋白酶体降解，以及细胞蛋白质组中的哪些蛋白质需要 UCH37 活性 有效降解。我们还将开发一套能够 干扰 UCH37 的独特活性。这些新工具将有助于定义 UCH37 在不同生化途径中的作用，例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 BAP1 与催化因子具有相似的序列 UCH37 的结构域，我们的工具和方法也可用于更好地了解其在组蛋白之外的作用 去泛素化。拟议研究产生的分子见解将为 开发针对需要过度蛋白酶体活性的癌症的新治疗剂 生存。