College of Animal Science, Jilin University, 5333 Xi An Road, Changchun 130062, Jilin, China.
Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Institute of Zoonosis, Jilin University, 5333 Xi An Road, Changchun 130062, Jilin, China.
Int J Mol Sci. 2020 Mar 5;21(5):1783. doi: 10.3390/ijms21051783.
(1) Background: Deubiquitinase (DUB) regulates various important cellular processes via reversing the protein ubiquitination. The N-terminal fragment of a giant tegument protein, UL36, encoded by the Marek's disease (MD) virus (MDV), encompasses a putative DUB (UL36-DUB) and shares no homology with any known DUBs. The N-terminus 75 kDa fragment of UL36 exists in MD T lymphoma cells at a high level and participates in MDV pathogenicity. (2) Methods: To characterize deubiquitinating activity and substrate specificity of UL36-DUB, the UL36 N-terminal fragments, UL36(323), UL36(480), and mutants were prepared using the Bac-to-Bac system. The deubiquitinating activity and substrate specificity of these recombinant UL36-DUBs were analyzed using various ubiquitin (Ub) or ubiquitin-like (UbL) substrates and activity-based deubiquitinating enzyme probes. (3) Results: The results indicated that wild type UL36-DUBs show a different hydrolysis ability against varied types of ubiquitin chains. These wild type UL36-DUBs presented the highest activity to K11, K48, and K63 linkage Ub chains, weak activity to K6, K29, and K33 Ub chains, and no activity to K27 linkage Ub chain. UL36 has higher cleavage efficiency for K48 and K63 poly-ubiquitin than linear ubiquitin chain (M1-Ub4), but no activity on various ubiquitin-like modifiers. The mutation of C98 and H234 residues eliminated the deubiquitinating activity of UL36-DUB. D232A mutation impacted, but did not eliminated UL36(480) activity. The Ub-Br probe can bind to wild type UL36-DUB and mutants UL36(480) and UL36(480), but not C98 mutants. These in vitro results suggested that the C98 and H234 are essential catalytic residues of UL36-DUB. UL36-DUB exhibited a strict substrate specificity. Inhibition assay revealed that UL36-DUB exhibits resistance to the Roche protease inhibitor cocktail and serine protease inhibitor, but not to the Solarbio protease inhibitor cocktail. (4) Conclusions: UL36-DUB exhibited a strict substrate preference, and the protocol developed in the current study for obtaining active UL36-DUB protein should promote the high-throughput screening of UL36 inhibitors and the study on the function of MDV-encoded UL36.
(1) 背景：去泛素化酶（DUB）通过逆转蛋白质泛素化来调节各种重要的细胞过程。马立克氏病（MD）病毒（MDV）编码的巨衣壳蛋白 UL36 的 N 端片段包含一个假定的 DUB（UL36-DUB），与任何已知的 DUB 均无同源性。UL36 的 N 端 75 kDa 片段在 MD T 淋巴瘤细胞中高水平存在，并参与 MDV 致病。(2) 方法：为了表征 UL36-DUB 的去泛素化活性和底物特异性，使用 Bac-to-Bac 系统制备了 UL36 N 端片段 UL36(323)、UL36(480)和突变体。使用各种泛素（Ub）或泛素样（UbL）底物和基于活性的去泛素化酶探针分析这些重组 UL36-DUB 的去泛素化活性和底物特异性。(3) 结果：结果表明，野生型 UL36-DUB 对不同类型的泛素链显示出不同的水解能力。这些野生型 UL36-DUB 对 K11、K48 和 K63 连接的 Ub 链表现出最高的活性，对 K6、K29 和 K33 Ub 链表现出较弱的活性，对 K27 连接的 Ub 链没有活性。UL36 对 K48 和 K63 聚泛素的切割效率高于线性泛素链（M1-Ub4），但对各种泛素样修饰物没有活性。C98 和 H234 残基的突变消除了 UL36-DUB 的去泛素化活性。D232A 突变影响但没有消除 UL36(480)的活性。Ub-Br 探针可以结合野生型 UL36-DUB 和突变体 UL36(480)和 UL36(480)，但不能结合 C98 突变体。这些体外结果表明，C98 和 H234 是 UL36-DUB 的必需催化残基。UL36-DUB 表现出严格的底物特异性。抑制实验表明，UL36-DUB 对罗氏蛋白酶抑制剂混合物和丝氨酸蛋白酶抑制剂具有抗性，但对 Solarbio 蛋白酶抑制剂混合物没有抗性。(4) 结论：UL36-DUB 表现出严格的底物偏好，本研究中开发的获得活性 UL36-DUB 蛋白的方案应促进 UL36 抑制剂的高通量筛选和 MDV 编码 UL36 功能的研究。
