Characterization of the deubiquitination activity and substrate specificity of the chicken ubiquitin-specific protease 1/USP associated factor 1 complex

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.

UL36 Encoded by Marek’s Disease Virus Exhibits Linkage-Specific Deubiquitinase Activity

“…Some animal or pathogen DUBs have dual deubiquitinating/de-NEDDylating activity [30][31][32][33]. Although the key residues of UL36-DUB are highly conserved compared with other USP type DUBs, UL36-DUB shares no homology on amino acid sequence with these DUBs.…”

“…For the direct binding test, 5 pmol of the Strep-Ub(75)-Br or Strep-Nedd8(75)-Br probe was incubated with 4 pmol of UL36(480) for 1 h at 37 • C. The reaction products were separated with SDS-PAGE and identified by Western blotting using standard protocols [30] with primary antibody rabbit anti-Strep (Cat#: ab76949, Abcam, Cambridge, MA, USA) or anti-UL36(323) [51] antibody at a dilution of 1:4000 and HRP-conjugated goat anti-rabbit IgG secondary antibody at a dilution of 1:5000 (OriGene Technologies).…”

“…Herein, the methods for preparing active UL36-DUB, the analysis of its deubiquitinating enzyme kinetics, and substrate specificity are included in this report. Some animal or pathogen DUBs, such as chicken USP1 [30], human USP21 [31], Legionella pneumophila SdeA [32], Plasmodium falciparum PfUCH54 [33], Epstein-Barr virus BPLF1 [34], reportedly exhibit a de-NEDDylating activity and can de-conjugate NEDD8-modified protein. However, MDV-encoded UL36-DUB shares no amino acid sequence homology with these DUBs.…”

Viral deubiquitinases and innate antiviral immune response in livestock and poultry

“…Furthermore, it has been found that when combined with human USP associated factor 1 (hUAF1), the deubiquitination activity of hUSP1 can be increased by 35 times [13]. Zheng et al (2017) identified a chicken USP1 homologue (chUSP1) from MDV [81]. They expressed and purified both chUAF1 and chUSP1 with or without putative catalytic core mutations using a Bac-to-Bac system before investigating their deubiquitination activity and kinetics using various substrates.…”

Identification of active deubiquitinases in the chicken tissues

“…DUBs are universally involved in health and disease, including malignancies, neurodegenerative disorders, and pathogenicity associated with microbial infections [4][5][6][7][8]. Although more than 90 DUBs have been identified in humans and a similar number of DUBs are expected in chicken, only a few of these enzymes have been identified as catalytically active in chicken, including UBP41 [9], UBP46, UBP52, UBP66 [10], UCH6 [11,12], UCH8 [13], UCH-L5 [14], and USP1 [15][16][17]. DUBs are likely important in poultry disease and could be useful in disease prognostics, drug target, and biomarker discovery, apart from their role in basic science research.…”