去泛素化修饰酶新靶标的体内捕获、功能解析和化学干预手段的研发

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.

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。

泛素化修饰是生物大分子上发生的共价修饰。发生在关键蛋白上的泛素链连接可以改变蛋白的稳态、蛋白亚细胞定位，改变蛋白质的相互作用网络体系，改变膜蛋白接受配体信号的能力，改变转录因子调控细胞命运决定等。泛素化过程是可逆的，其逆转由去泛素化酶 DUB 催化实现。DUB 在生物大分子的泛素化修饰及生理功能的调控中发挥重要作用，从而影响抗病毒先天免疫信号通路；以及导致促癌基因的表达与稳定，增强促肿瘤信号的传导，从而影响肿瘤细胞的增殖，干性以及转移等。.申请人专注于去泛素化酶介导的生物大分子动态修饰及生理功能的调控。本课题旨在利用 DUB 分子探针等方法，在体内外筛选鉴定病理条件下的关键 DUB新靶标，并深入解析其作用机理以及在抗病毒先天免疫、肿瘤转移等过程中发挥的作用。具体研究内容包括： ①利用体外 DUB 分子探针和体内 DUB cDNA 文库筛选得到病理条件下的关键 DUB 新靶标。②研究筛选得到的 DUB 新靶标对抗病毒免疫、肿瘤转移等过程中的主要作用底物和动态修饰调节方式，阐明其发挥的功效和作用机理。③建立以 DUB 活性为读出（Readout）的小分子抑制剂筛选体系，通过高通量筛选初步获取靶向该去泛素化酶活性的小分子化学抑制剂。. 我们发现OTUB2可以去除YAP/TAZ的多聚泛素化链从而稳定YAP/TAZ，且OTUB2对YAP/TAZ的稳定作用不依赖于上游Hippo的失活。另外，OTUB2对YAP/TAZ的稳定作用依赖于OTUB2的Lys233位多聚SUMO化修饰，而EGF（表皮生长因子）和下游的KRAS 激活会增强OTUB2的SUMO修饰，稳定YAP/TAZ的蛋白水平，从而促进肿瘤转移。我们还发现在抗病毒免疫中，OTUD3通过抑制MAVS拮抗RLR信号通路，且OTUD3对MAVS的抑制是通过水解其Lys63泛素链并抑制其聚集实现的。另一方面，OTUD3的去泛素化酶活性依赖于其Lys129的乙酰化，而病毒感染发生时，SIRT1能够去除OTUD3-Lys129的乙酰化，从而解除 OTUD3对 MAVS的抑制。