Zheng Hainan, Wang Mengyun, Zhao Chengcheng, Wu Shanli, Yu Peifeng, Lü Yan, Wang Tiedong, Ai Yongxing
College of Animal Science, Jilin University, Changchun, China.
Institute of Translational Medicine, Jilin University, Changchun, China.
PLoS One. 2017 Nov 1;12(11):e0186535. doi: 10.1371/journal.pone.0186535. eCollection 2017.
Deubiquitinases (DUBs) are essential regulators of intracellular processes involving ubiquitin (Ub) modification. The human DUB ubiquitin-specific protease 1 (hUSP1) interacts with human USP-associated factor 1 (hUAF1), and helps to regulate processes such as DNA damage repair. Previously, we identified a chicken USP1 homologue (chUSP1) during an investigation into the properties of Marek's disease virus (MDV). However, chUSP1's deubiquitination activity, interaction with chUAF1, and substrate specificity remained unknown. In the present study, we expressed and purified both chUAF1 and chUSP1 with or without putative catalytic core mutations using the Bac-to-Bac system, before investigating their deubiquitination activity and kinetics using various substrates. chUSP1 was shown to interact with chUAF1 both in cellular assays in which the two proteins were co-expressed, and in in vitro assays using purified proteins. Heterodimerization with chUAF1 increased the deubiquitination activity of chUSP1 up to 54-fold compared with chUSP1 alone. The chUSP1 mutants C91S, H603A, and D758A reduced the deubiquitination activity of the chUSP1/chUAF1 complex by 10-, 7-, and 33-fold, respectively, while the C91A and H594A chUSP1 mutants eliminated deubiquitination activity of the chUSP1/chUAF1 complex completely. This suggests that C91 and H594, but not D758, are essential for chUSP1 deubiquitination activity, and that a nucleophilic group at position 91 is needed for the deubiquitination reaction. The chUSP1/chUAF1 complex was found to have distinct substrate preferences; efficient hydrolysis of Ub dimers with K11-, K48-, and K63-linkages was seen, with weaker hydrolysis observed with K6-, K27-, and K33-linkages and no hydrolysis seen with a K29-linkage. Furthermore, other Ub-like substrates were disfavored by the complex. No activity was seen with SUMO1-GST, SUMO2- and SUMO3-dimers, ISG15-Rho, FAT10-Rho, or Ufm1-Rho, and only weak activity was observed with NEDD8-Rho. Overall, the data presented here characterize the activity and substrate preferences of chUSP1, and thus may facilitate future studies on its in vivo role.
去泛素化酶（DUBs）是涉及泛素（Ub）修饰的细胞内过程的重要调节因子。人类去泛素化酶泛素特异性蛋白酶1（hUSP1）与人类USP相关因子1（hUAF1）相互作用，并有助于调节DNA损伤修复等过程。此前，我们在对马立克氏病病毒（MDV）特性的研究中鉴定出一种鸡USP1同源物（chUSP1）。然而，chUSP1的去泛素化活性、与chUAF1的相互作用以及底物特异性仍不清楚。在本研究中，我们使用杆状病毒表达系统（Bac-to-Bac system）表达并纯化了带有或不带有假定催化核心突变的chUAF1和chUSP1，然后使用各种底物研究它们的去泛素化活性和动力学。结果表明，chUSP1在共表达这两种蛋白质的细胞试验以及使用纯化蛋白质的体外试验中均能与chUAF1相互作用。与单独的chUSP1相比，与chUAF1异源二聚化使chUSP1的去泛素化活性提高了54倍。chUSP1突变体C91S、H603A和D758A分别使chUSP1/chUAF1复合物的去泛素化活性降低了10倍、7倍和33倍，而C91A和H594A chUSP1突变体则完全消除了chUSP1/chUAF1复合物的去泛素化活性。这表明C91和H594对chUSP1的去泛素化活性至关重要，而91位上的亲核基团是去泛素化反应所必需的。发现chUSP1/chUAF1复合物具有不同的底物偏好；能有效水解具有K11-、K48-和K63-连接的Ub二聚体，对具有K6-、K27-和K33-连接的水解较弱，对K29-连接则无水解作用。此外，该复合物不倾向于其他类泛素底物。对SUMO1-GST、SUMO2-和SUMO3-二聚体、ISG15-Rho、FAT10-Rho或Ufm1-Rho均无活性，对NEDD8-Rho仅观察到微弱活性。总体而言，本文提供的数据表征了chUSP1的活性和底物偏好，因此可能有助于未来对其体内作用的研究。
