酵母去泛素化酶与泛素链特异性关系的定量蛋白质组学研究

The ubiquitination signal network is reversely trimmed through deubiquitinating enzymes (DUB), which were potential drug targets. The specificity of DUBs and ubiquitin chains was challenging but meritorious, which help us deeply understand the precise regulatory mechanism of UPS enzymes and substrates. However the systematic assessment of specificity between DUBs and ubiquitin linkages in vivo are still less studied. Technically it is remaining challenge to directly display the specificity of certain DUB for their corresponding ubiquitin linkages. In this study, we employed SILAC quantitative proteomics approaches to systematically evaluate the specificity of DUBs on all seven types of ubiquitin chains of Saccharomyces cerevisiae. We present here a novel screening of DUB specificity with ubiquitin chains in vivo. We assumed that all existing DUBs maintained the ubiquitin chains homeostasis, while deletion one certain DUB will result in the accumulation of its preferred chains. First we should construct a series of 19 single DUB deletion strains. In summary, diverse DUB deletion strains differed from each other in terms of growth abnormalities and ubiquitination. We would apply swapped SILAC strategy to quantify seven ubiquitin chains in each DUB deletion strain with WT and prove that the accumulation of Ub chains in each DUB deletion strain distinguished obviously with each other. Through hierarchical clustering of linkages quantification of USP DUBs deletion mutants, which proved the what rules of DUB and ubiquitin linkage contributed to the specificity. In addition, we will prove the specificity of DUBs and 7 ubiquitin chains through in vitro reaction. Using DUB specificity, we will prove the precise regulation and functions of ubiquitin modification on substrates through DUBs. The signal “DUBs – Ub chains – substrate – function” become the basis of precise regulation mechanism of ubiquitin networks.

去泛素化酶在泛素化网络稳态调控中发挥重要作用，诸多已经成为潜在的药物设计靶点。去泛素化酶与泛素链的特异性关系是该研究领域的前沿与热点，是深刻理解泛素化网络精准调控并实现靶向调节的理论基础。然而目前去泛素化酶与泛素链特异性调控关系尚不清楚，如何开展体内特异性关系研究成为该领域的挑战。本项目以酵母为模型，首次建立体内去泛素化酶与泛素链特异性关系的研究策略。基于敲除特定去泛素化酶造成其偏好泛素链累积的假设，通过SILAC定量与泛素链靶向鉴定技术，全面比较酵母内去泛素化酶单敲除菌株中7种泛素链的变化情况，聚类分析挖掘两者特异性关系的内在规律；通过去泛素化酶与泛素链体外反应，验证体内两者的特异性关系。最后，根据去泛素化酶突变株生长表型结果，结合发现的去泛素化酶与泛素链特异性关系，开展重要去泛素化酶及其相关底物泛素链的调控机制以及功能研究，揭示“去泛素化酶-泛素链-底物-功能”的信息流精准调控模式。

去泛素化酶在泛素化网络稳态调控中发挥重要作用，诸多已经成为潜在的药物设计靶点。去泛素化酶与泛素链的特异性关系是该研究领域的前沿与热点，是深刻理解泛素化网络精准调控并实现靶向调节的理论基础。然而目前去泛素化酶与泛素链特异性调控关系尚不清楚，如何开展体内特异性关系研究成为该领域的挑战。本项目发展了高灵敏泛素化检测技术体系，基于镜像胰蛋白酶的泛素化位点鉴定策略，为泛素化修饰位点的深度覆盖与精确鉴定提供了技术支撑（Anal Chem, 2019）；基于前期发展的串联杂合泛素结合结构域（ThUBD）发展了8种泛素链无偏性固相检测技术（Anal Chem，2020），显著提升了泛素链的检测灵敏度。以酵母为模型，通过SILAC定量与高灵敏泛素化靶向鉴定技术，全面比较酵母内去泛素化酶单敲除菌株中7种泛素链的变化情况，并据此发展了“泛素链-修饰位点-底物-去泛素化酶”一站式的筛选策略DLUS（iScience,2020），为后续特定泛素链（如K63、K11等）功能研究提供重要靶点以及技术手段。首次大规模筛选得到Ubp2调控的新底物群，证明其广泛参与细胞周期、胞吞胞吐等生物学过程，发现去泛素化酶Ubp2对K63泛素链的偏好性，证明了抑免蛋白Cpr1在K151位点发生K63泛素链修饰，为进一步探究其功能提供了生化基础，证明了该K63泛素链介导了Zpr1的入核过程。基于定量蛋白质组学发现K48、K11泛素链作为信号转导的载体，实现对转录因子活性的调控（Mol Cell, 2019）。该课题结合发现的去泛素化酶与泛素链特异性关系，开展重要去泛素化酶及其相关底物泛素链的调控机制以及功能研究，揭示“去泛素化酶-泛素链-底物-功能”的信息流精准调控模式为以泛素链为载体的“泛素密码”解析提供了范例。