泛素化和去泛素化是存在于生物体内的可逆动态过程，其紊乱可引发多种疾病包括肿瘤。去泛素化酶DUB的活性结构域是优良的药物靶标。传统的DUB活性筛选方法局限于体外，使得在生理及病理条件下高效捕获去泛素化酶的研究滞足不前。为此，我们结合生物和化学方法构建了新型去泛素化酶探针，利用非天然氨基酸Ffact标记泛素后与活化的DUB活性中心半胱氨酸反应形成共价键的特性，开发了新型DUB探针和体内DUB活性筛选的生物正交反应方法。用此方法在高转移性的乳腺癌细胞中捕获了促进肿瘤转移的关键DUB并建立了体外筛选小分子DUB抑制剂的体系。本项目旨在发展新DUB探针，发展DUB调控蛋白组泛素修饰的机制的新研究方法，为临床治疗和药物筛选提供理论基础。

Ubiquitination and de-ubiquitination are reversible processes involved in nearly all cellular processes. Aberrations in this system give rise to numerous diseases, including cancer. Deubiquitin enzymes (DUB), responsible for protein de-ubiquitination and holding special catalytic domain, is considering a promising target. Traditional DUB activity capturing or screening assay is technically limited only to in vitro assay which could not enable the discovery of real DUB activity in the physiological and pathological conditions. Here, we genetically introduce a new type of covalent bond into the ubiquitin protein by enabling an unnatural amino acid to react with a proximal enzymatic cysteine of the endogenous active DUBs. Design of this novel probe for endogenous DUB combined biological and chemical expertise. By using this probe, we developed a new bio-orthogonal reaction by expressing this probe in live cells. We have demonstrated the utility of this probe and method for capturing endogenous ubiquitin and its covalently associated DUB in the high metastatic breast cancer cells, such as UCHL1, USP8 and USP4. Moreover, we also developed high throughput screening system by reading DUB activity towards Ub-AMC substrate in vitro. Our preliminary work have identified potent lead compound that could suppress UCHL1 activity. By developing novel DUB probe and inhibitor, we aim to identify the mechanism of which DUB regulates ubiquitination status of proteome and figure out whether chemical targeting of DUB could limit or even reverse certain DUB-drived biological events such as cancer metastasis. We also aim to further improve the drug screen model that target the DUBs probed by our new probe. Upon successful conclusion of this work, we will have provided not only new probe for DUB in vivo, but also proposed new methods for understanding DUB’s working mechanism and efficient DUB-related drug discovery.