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

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          Abstract

          (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.

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          Most cited references43

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          Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes.

          Deubiquitinating enzymes (DUBs) are proteases that process ubiquitin or ubiquitin-like gene products, reverse the modification of proteins by a single ubiquitin(-like) protein, and remodel polyubiquitin(-like) chains on target proteins. The human genome encodes nearly 100 DUBs with specificity for ubiquitin in five gene families. Most DUB activity is cryptic, and conformational rearrangements often occur during the binding of ubiquitin and/or scaffold proteins. DUBs with specificity for ubiquitin contain insertions and extensions modulating DUB substrate specificity, protein-protein interactions, and cellular localization. Binding partners and multiprotein complexes with which DUBs associate modulate DUB activity and substrate specificity. Quantitative studies of activity and protein-protein interactions, together with genetic studies and the advent of RNAi, have led to new insights into the function of yeast and human DUBs. This review discusses ubiquitin-specific DUBs, some of the generalizations emerging from recent studies of the regulation of DUB activity, and their roles in various cellular processes.
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            Enhanced protein degradation by branched ubiquitin chains.

            Posttranslational modification of cell-cycle regulators with ubiquitin chains is essential for eukaryotic cell division. Such chains can be connected through seven lysine residues or the amino terminus of ubiquitin, thereby allowing the assembly of eight homogenous and multiple mixed or branched conjugates. Although functions of homogenous chain types have been described, physiological roles of branched structures are unknown. Here, we report that the anaphase-promoting complex (APC/C) efficiently synthesizes branched conjugates that contain multiple blocks of K11-linked chains. Compared to homogenous chains, the branched conjugates assembled by the APC/C strongly enhance substrate recognition by the proteasome, thereby driving degradation of cell-cycle regulators during early mitosis. Our work, therefore, identifies an enzyme and substrates for modification with branched ubiquitin chains and points to an important role of these conjugates in providing an improved signal for proteasomal degradation. Copyright © 2014 Elsevier Inc. All rights reserved.
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              Ubiquitin-like proteins.

              The eukaryotic ubiquitin family encompasses nearly 20 proteins that are involved in the posttranslational modification of various macromolecules. The ubiquitin-like proteins (UBLs) that are part of this family adopt the β-grasp fold that is characteristic of its founding member ubiquitin (Ub). Although structurally related, UBLs regulate a strikingly diverse set of cellular processes, including nuclear transport, proteolysis, translation, autophagy, and antiviral pathways. New UBL substrates continue to be identified and further expand the functional diversity of UBL pathways in cellular homeostasis and physiology. Here, we review recent findings on such novel substrates, mechanisms, and functions of UBLs.
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                Author and article information

                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                05 March 2020
                March 2020
                : 21
                : 5
                : 1783
                Affiliations
                [1 ]College of Animal Science, Jilin University, 5333 Xi An Road, Changchun 130062, Jilin, China; linjunyan919@ 123456163.com (J.L.); aiyx@ 123456jlu.edu.cn (Y.A.); hongdazhou521@ 123456163.com (H.Z.); lvyanty@ 123456jlu.edu.cn (Y.L.); menghanwang927@ 123456163.com (M.W.); jcxu@ 123456jlu.edu.cn (J.X.)
                [2 ]Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Institute of Zoonosis, Jilin University, 5333 Xi An Road, Changchun 130062, Jilin, China
                [3 ]State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Avenue, Changchun 130022, Jilin, China; congyu@ 123456ciac.ac.cn
                [4 ]Avian Disease and Oncology Laboratory, Agriculture Research Service, United States Department of Agriculture, 4279 East Mount Hope Road East Lansing, MI 48823, USA
                Author notes
                [* ]Correspondence: huanmin.zhang@ 123456usda.gov (H.Z.); mengyun_wang1124@ 123456163.com (M.W.); Tel.: +1-517-3376835 (H.Z.); +86-18744027109 (M.W.)
                [†]

                These authors contributed equally to this work.

                Author information
                https://orcid.org/0000-0002-6082-1818
                https://orcid.org/0000-0001-5212-3609
                https://orcid.org/0000-0003-4896-2391
                Article
                ijms-21-01783
                10.3390/ijms21051783
                7084888
                32150874
                b39acb9a-8974-4201-898a-55eea1cb29dd
                © 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/).

                History
                : 07 February 2020
                : 02 March 2020
                Categories
                Article

                Molecular biology
                ul36,deubiquitinase,mdv,catalytic activity,substrate specificity
                Molecular biology
                ul36, deubiquitinase, mdv, catalytic activity, substrate specificity

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