Mengyun Wang

(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)^H234A and UL36(480)^D232A, 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.

Interleukin-2 (IL-2) is a pleiotropic cytokine regulating the immune and nervous systems. Mammalian and bird IL-2s have different protein sequences, but perform similar functions. In the current study, two bands were detected by immunoblotting using an antibody against freshly purified chicken IL-2 (chIL-2). The molecular weight of the larger band was approximately twice as much of the chIL-2 monomer, although a chIL-2 complex or homodimer has never been reported. To explain this intriguing result, several dissociation reagents were used to examine the intermolecular forces between components of the proposed chIL-2 complex. It was found that intermolecular disulphide bond promotes homodimerization of chIL-2. Subsequently, mutation of Cys residues of chIL-2 revealed that mutation of all four Cys residues disrupted homodimerization, but a single, dual, or triple Cys mutation failed to disrupt homodimerization, suggesting that all four Cys residues on chIL-2 contribute to this dimerization. Functional analysis showed that both monomeric and dimeric chIL-2 consisting of either wild type or mutant chIL-2 were able to stimulate the expansion of CD4⁺ T cell in vivo or in vitro, and effectively bind to chIL-2 receptor. Overall, this study revealed that the recombinant chIL-2 purified from either Escherichia coli (E. coli) or Spodoptera frugiperda (Sf9) cells could homodimerize in vitro, with all four Cys residues on each chIL-2 protein contributing to this homodimerization, and dimerization and Cys mutation not impacting chIL-2 induced stimulation of chicken CD4⁺ T cells.

Ubiquitination and deubiquitination of cellular proteins are reciprocal reactions catalyzed by ubiquitination-related enzymes and deubiquitinase (DUB) which regulate almost all cellular processes. Marek’s disease virus (MDV) encodes a viral DUB that plays an important role in the MDV pathogenicity. Chicken CD4⁺ T-cell lymphoma induced by MDV is a key contributor to multiple visceral tumors and immunosuppression of chickens with Marek’s disease (MD). However, alterations in the ubiquitylome of MDV-induced T lymphoma cells are still unclear. In this study, a specific antibody against K-ε-GG was used to isolate ubiquitinated peptides from CD4⁺ T cells and MD T lymphoma cells. Mass spectrometry was used to compare and analyze alterations in the ubiquitylome. Our results showed that the ubiquitination of 717 and 778 proteins was significantly up- and downregulated, respectively, in T lymphoma cells. MDV up- and downregulated ubiquitination of a similar percentage of proteins. The ubiquitination of transferases, especially serine/threonine kinases, was the main regulatory target of MDV. Compared with CD4⁺ T cells of the control group, MDV mainly altered the ubiquitylome associated with the signal transduction, immune system, cancer, and infectious disease pathways in T lymphoma cells. In these pathways, the ubiquitination of CDK1, IL-18, PRKCB, ETV6, and EST1 proteins was significantly up- or downregulated as shown by immunoblotting. The current study revealed that the MDV infection could exert a significant influence on the ubiquitylome of CD4⁺ T cells.