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

在基础研究、治疗和生物技术应用中，通过选择性地延长有益蛋白质的寿命或去除异常或不受欢迎的蛋白质来操纵细胞的蛋白质组是非常有吸引力的。这一策略比基因沉默和基因组编辑技术更具优势，因为它直接影响蛋白质水平，潜在地提高了精确度。在真核细胞中，泛素蛋白酶体系统调节选择性的蛋白质降解。因此，对泛素系统的干扰为调节细胞蛋白质清除，从而调节蛋白质水平提供了有价值的途径。泛素对底物蛋白质的修饰是可逆的，泛素特异性蛋白水解酶(USPS)是一类脱泛素化酶，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调节失调，也在神经退行性疾病和宿主对感染的反应中发挥作用。这个项目的主要目的是鉴定和评价干扰泛素介导的蛋白质降解的新型泛素系统调节剂。为此，我们将产生工程化的荧光泛素变异体来测试体外泛素特异性蛋白酶的活性。然后，我们将使用开发的方法通过筛选内部化合物集合来鉴定新的USP抑制剂。作为目标，我们将重点关注USP15，这是一种多功能蛋白酶，已被证明在调节细胞生长、分化和凋亡的转化生长因子-β信号转导中发挥调节作用。在癌症中，转化生长因子-β信号在肿瘤形成的晚期阶段促进肿瘤的发展，但在早期阶段诱导细胞周期停滞，从而抑制肿瘤。此外，USP15抑制有丝分裂，这反过来又与帕金森病和其他与衰老相关的功能障碍的线粒体疾病有关。USP15还影响炎症反应，并促进RIG-I介导的抗病毒信号。我们最近已经解决了USP15N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确定USP15-抑制物复合体的结构，以深入了解相互作用，并结合结合分析，研究已确定的抑制剂相对于相关USP的特异性。然后，我们将在细胞测试中测试得分最高的抑制剂，重点关注USP15底物的活性和对蛋白质水平的影响，例如E3泛素连接酶SMURF2，它以转化生长因子β受体(TβR)复合体为靶点，进行泛素介导的降解。总而言之，该项目将在包括蛋白质工程、荧光分析、结构生物学和细胞分析在内的跨学科背景下提供技能发展，并将提供对USP活性、结构和抑制的新见解，并为操纵蛋白质降解创造工具。

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.