Function and Modulation of USP11 in Cancer

通过泛素化修饰底物蛋白质是调控真核生物蛋白质活性和水平的重要机制。因此，对泛素系统的干扰为调节细胞蛋白质功能和降解对蛋白质水平的影响提供了有价值的途径，在基础研究、药物开发和生物技术应用中具有应用价值。本项目的主要目的是鉴定和评估改变泛素介导的蛋白质降解途径的泛素特异性蛋白水解酶调节剂。泛素特异性蛋白水解酶(USPS)可以逆转泛素化，使蛋白质免于被蛋白酶体破坏。USP11与同源重组蛋白相关，是DNA损伤修复的关键调节因子，逆转了几种对细胞功能至关重要的蛋白质的泛素化，如p21、组蛋白、感冒素和p53。此外，USP11还调节自噬，促进蛋白毒性、先天免疫反应和炎症。耗尽或沉默USP11已被证明有效地靶向DNA损伤反应(DDR)途径缺陷的肿瘤，杀死临床相关的铂耐药癌细胞，并能抑制胰腺癌细胞的生存。在以前的研究中，我们发现了一种新的USP11多肽结合体，它与N-末端区域的一个新位点相互作用，并对一组细胞系的存活产生不同的影响。在这个项目中，我们将进一步探索USP11中这个新的结合位点的功能，并进行荧光偏振分析，以从内部化合物集合中识别新的抑制剂。结构生物学技术，包括X射线结晶学或冷冻电子显微镜，将表征USP11-抑制剂复合体的结构，以深入了解相互作用，并与结合分析一起研究已识别的抑制剂的特异性。评分最高的抑制剂将在细胞分析中进行评估，以胰腺癌细胞为模型系统，探索对USP11底物蛋白水平和细胞活力的影响。同时，这一跨学科项目提供了蛋白质生物化学、荧光分析、结构生物学和细胞生物学方面的技能发展，并将增进我们对USP11功能、结构和抑制的理解，并为操纵泛素化最终靶向癌细胞创造新的工具。

The modification of substrate proteins by ubiquitination is a crucial mechanism for regulating the activity and levels of proteins in eukaryotes. Therefore, interference with the ubiquitin system offers valuable avenues for modulating cellular protein function and degradation impacting on protein levels with applications in basic research, drug development and biotechnological applications.The main aim of this project is the identification and evaluation of ubiquitin specific protease modulators that alter ubiquitin-mediated protein degradation pathways. Ubiquitin specific proteases (USPs) can salvage proteins from destruction by the proteasome by reversing ubiquitination. USP11 associates with homologous recombination proteins and is a key regulator of DNA damage repair reversing ubiquitination of several proteins critical to cellular function such as p21, histones, senataxin and p53. In addition, USP11 also regulates autophagy and contributes to proteotoxicity, the innate immune response and inflammation. Depletion or silencing of USP11 has been shown to be effective in targeting DNA damage response (DDR) pathway deficient tumours, killing clinically relevant platinum-resistant cancer cells and can inhibit pancreatic cancer cell survival. In previous studies we identified a novel peptide binder of USP11 that interacted with a novel site in the N-terminal region and differentially affected survival of a panel of cell lines. In this project, we will further probe the function of this novel binding site in USP11 and conduct fluorescence polarization assays to identify novel inhibitors from an in-house compound collection. Structural biology techniques including X-ray crystallography or cryo-electron microscopy will characterise structures of USP11-inhibitor complexes to gain insight into the interactions and together with binding assays investigate the specificity of identified inhibitors. The top scoring inhibitors will be evaluated in cellular assays probing the impact on protein levels of USP11 substrates and cell viability using pancreatic cancer cells as model system.Together, this interdisciplinary project offers skill development in protein biochemistry, fluorescence assays, structural biology and cell biology and will advance our understanding of USP11 function, structure and inhibition and create novel tools for the manipulation ubiquitination to ultimately target cancer cells.