Abstract
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.
Highlights
The post-translational modification of proteins by ubiquitin (Ub), termed ubiquitination, dynamically modulates their stability and activity
We aimed to investigate the properties of this novel chicken ubiquitin-specific proteases (USPs)-like protein, which we termed chicken USP1 homologue (chUSP1) because of its homology to human USP1 (hUSP1), by cloning the chUAF1 and chUSP1 genes
We further investigated the hydrolysis activity of chUSP1FL/ chUAF1 using a variety of ubiquitin-like substrates
Summary
The post-translational modification of proteins by ubiquitin (Ub), termed ubiquitination, dynamically modulates their stability and activity. Monoubiquitination and K63-linked polyubiquitination are widely known to regulate intracellular processes such as DNA damage repair, genomic stability, protein activity, inflammation, apoptosis, endocytic trafficking, and translation [2]. Human USP1 (hUSP1) is critical for the regulation of DNA damage repair and genomic stability [12, 13]. The hUSP1/hUAF1 dimer has been shown to deubiquitinate monoubiquitinated FANCD2 and PCNA, modulating DNA damage repair and genomic stability [12, 13], and to regulate the degradation of T cell receptors and CD4 + molecules on the surface of T lymphocytes upon viral infection, leading to their functional impairment [14, 15]
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