Selective editing of cellular protein degradation by modulating ubiquitin specific protease function

通过调节泛素特异性蛋白酶功能选择性编辑细胞蛋白质降解

• 批准号: 2433747
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2433747
• 关键词: Selective; editing; cellular; protein; degradation

Manipulation of a cell's proteome by selectively increasing the longevity of beneficial proteins or removing aberrant or undesirable proteins is highly attractive in basic research, therapy and biotechnology applications. This strategy offers advantages over gene silencing and genome editing techniques in that it impacts directly on the protein levels potentially enhancing precision. In eukaryotic cells, the ubiquitin proteasome system regulates selective protein degradation. Therefore, interference with the ubiquitin system offers valuable avenues for modulating cellular protein clearance and in consequence protein levels. Ubiquitin modification of a substrate protein is reversible, whereby ubiquitin specific proteases (USPs), a class of deubiquitinating enzymes, can salvage proteins from destruction by the proteasome by removing the ubiquitin tag. These cysteine proteases are often dysregulated in cancer and also play a role in neurodegenerative disorders and the host's response to infection. The main aim of this project is the identification and evaluation of novel ubiquitin system modulators that interfere with ubiquitin-mediated protein degradation.To this end we will generate engineered fluorescent ubiquitin variants to test ubiquitin specific protease activity in vitro. We will then use the developed assay to identify novel USP inhibitors by screening an in-house compound collection. As a target we will focus on USP15, a multi-functional protease that has been shown to play a regulatory role in transforming growth factor beta (TGF-beta signalling that regulates cell growth, differentiation and apoptosis. In cancer TGF-beta signalling promotes tumour development during the advanced stages of tumorigenesis, but induces cell-cycle arrest and consequently tumour suppression in the early stages. In addition, USP15 suppresses mitophagy, which in turn is related with Parkinson disease and other dysfunctional mitochondria diseases associated with ageing. USP15 also influences the inflammatory response and promotes RIG-I-mediated antiviral signalling. We have recently solved crystal structures of the USP15 N-terminal and catalytic domains. The known crystallisation conditions for the protein will facilitate the determination of structures of USP15-inhibtor complexes to gain insight into the interactions and in combination with binding assays investigate the specificity of identified inhibitors with regards to related USPs. We will then test the top scoring inhibitors in cellular assays focusing on the viability and impact on protein levels of USP15 substrates such as the E3 ubiquitin ligase SMURF2 that targets the TGF beta receptor (T beta R) complex for ubiquitin-mediated degradation. Together, the project will offer skill development in an interdisciplinary setting including protein engineering, fluorescent assays, structural biology and cellular assays and will deliver novel insights into USP activity, structure and inhibition and create tools for the manipulation of protein degradation.

通过选择性地增加有益蛋白质的寿命或去除异常或不需要的蛋白质来操纵细胞的蛋白质组在基础研究、治疗和生物技术应用中非常有吸引力。这种策略提供了优于基因沉默和基因组编辑技术的优势，因为它直接影响蛋白质水平，从而可能提高精度。在真核细胞中，泛素蛋白酶体系统调节选择性蛋白质降解。因此，干扰泛素系统为调节细胞蛋白质清除和蛋白质水平提供了有价值的途径。底物蛋白的泛素修饰是可逆的，其中泛素特异性蛋白酶（USP），一类去泛素化酶，可以通过去除泛素标签来挽救蛋白质免受蛋白酶体的破坏。这些半胱氨酸蛋白酶通常在癌症中失调，并且在神经退行性疾病和宿主对感染的反应中也起作用。本项目的主要目的是鉴定和评价新的干扰泛素介导的蛋白质降解的泛素系统调节剂，为此，我们将产生工程化的荧光泛素变体，以测试体外泛素特异性蛋白酶活性。然后，我们将使用开发的检测方法，通过筛选内部化合物集合来鉴定新型USP抑制剂。作为一个目标，我们将集中在USP 15，一种多功能蛋白酶，已被证明在转化生长因子β（TGF-β信号，调节细胞生长，分化和凋亡发挥调节作用。在癌症中，TGF-β信号传导在肿瘤发生的晚期促进肿瘤发展，但在早期诱导细胞周期停滞并因此抑制肿瘤。此外，USP 15抑制线粒体自噬，这反过来又与帕金森病和其他与衰老相关的线粒体功能障碍性疾病有关。USP 15还影响炎症反应并促进RIG-I介导的抗病毒信号传导。我们最近解决了USP 15 N-末端和催化域的晶体结构。已知的蛋白质结晶条件将有助于测定USP 15-抑制剂复合物的结构，以深入了解相互作用，并结合结合试验研究已鉴别抑制剂对相关USP的特异性。然后，我们将在细胞试验中测试得分最高的抑制剂，重点关注USP 15底物的活力和对蛋白质水平的影响，例如靶向TGF β受体（T β R）复合物进行泛素介导降解的E3泛素连接酶SMURF 2。该项目将共同提供跨学科环境中的技能发展，包括蛋白质工程，荧光测定，结构生物学和细胞测定，并将提供对USP活性，结构和抑制的新见解，并创建用于操纵蛋白质降解的工具。