Cargando…

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

(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...

Descripción completa

Detalles Bibliográficos
Autores principales: Lin, Junyan, Ai, Yongxing, Zhou, Hongda, Lv, Yan, Wang, Menghan, Xu, Jiacui, Yu, Cong, Zhang, Huanmin, Wang, Mengyun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084888/
https://www.ncbi.nlm.nih.gov/pubmed/32150874
http://dx.doi.org/10.3390/ijms21051783
_version_ 1783508826658439168
author Lin, Junyan
Ai, Yongxing
Zhou, Hongda
Lv, Yan
Wang, Menghan
Xu, Jiacui
Yu, Cong
Zhang, Huanmin
Wang, Mengyun
author_facet Lin, Junyan
Ai, Yongxing
Zhou, Hongda
Lv, Yan
Wang, Menghan
Xu, Jiacui
Yu, Cong
Zhang, Huanmin
Wang, Mengyun
author_sort Lin, Junyan
collection PubMed
description (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.
format Online
Article
Text
id pubmed-7084888
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-70848882020-03-23 UL36 Encoded by Marek’s Disease Virus Exhibits Linkage-Specific Deubiquitinase Activity Lin, Junyan Ai, Yongxing Zhou, Hongda Lv, Yan Wang, Menghan Xu, Jiacui Yu, Cong Zhang, Huanmin Wang, Mengyun Int J Mol Sci Article (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. MDPI 2020-03-05 /pmc/articles/PMC7084888/ /pubmed/32150874 http://dx.doi.org/10.3390/ijms21051783 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Lin, Junyan
Ai, Yongxing
Zhou, Hongda
Lv, Yan
Wang, Menghan
Xu, Jiacui
Yu, Cong
Zhang, Huanmin
Wang, Mengyun
UL36 Encoded by Marek’s Disease Virus Exhibits Linkage-Specific Deubiquitinase Activity
title UL36 Encoded by Marek’s Disease Virus Exhibits Linkage-Specific Deubiquitinase Activity
title_full UL36 Encoded by Marek’s Disease Virus Exhibits Linkage-Specific Deubiquitinase Activity
title_fullStr UL36 Encoded by Marek’s Disease Virus Exhibits Linkage-Specific Deubiquitinase Activity
title_full_unstemmed UL36 Encoded by Marek’s Disease Virus Exhibits Linkage-Specific Deubiquitinase Activity
title_short UL36 Encoded by Marek’s Disease Virus Exhibits Linkage-Specific Deubiquitinase Activity
title_sort ul36 encoded by marek’s disease virus exhibits linkage-specific deubiquitinase activity
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084888/
https://www.ncbi.nlm.nih.gov/pubmed/32150874
http://dx.doi.org/10.3390/ijms21051783
work_keys_str_mv AT linjunyan ul36encodedbymareksdiseasevirusexhibitslinkagespecificdeubiquitinaseactivity
AT aiyongxing ul36encodedbymareksdiseasevirusexhibitslinkagespecificdeubiquitinaseactivity
AT zhouhongda ul36encodedbymareksdiseasevirusexhibitslinkagespecificdeubiquitinaseactivity
AT lvyan ul36encodedbymareksdiseasevirusexhibitslinkagespecificdeubiquitinaseactivity
AT wangmenghan ul36encodedbymareksdiseasevirusexhibitslinkagespecificdeubiquitinaseactivity
AT xujiacui ul36encodedbymareksdiseasevirusexhibitslinkagespecificdeubiquitinaseactivity
AT yucong ul36encodedbymareksdiseasevirusexhibitslinkagespecificdeubiquitinaseactivity
AT zhanghuanmin ul36encodedbymareksdiseasevirusexhibitslinkagespecificdeubiquitinaseactivity
AT wangmengyun ul36encodedbymareksdiseasevirusexhibitslinkagespecificdeubiquitinaseactivity