Legionella pneumophila regulates the activity of UBE 2N by deamidase‐mediated deubiquitination
2019; Springer Nature; Volume: 39; Issue: 4 Linguagem: Inglês
10.15252/embj.2019102806
ISSN1460-2075
AutoresNinghai Gan, Hongxin Guan, Yini Huang, Ting Yu, Jiaqi Fu, Ernesto Nakayasu, Kedar Puvar, Chittaranjan Das, Dongmei Wang, Songying Ouyang, Zhao‐Qing Luo,
Tópico(s)Autophagy in Disease and Therapy
ResumoArticle11 December 2019free access Source DataTransparent process Legionella pneumophila regulates the activity of UBE2N by deamidase-mediated deubiquitination Ninghai Gan orcid.org/0000-0001-7238-4056 Purdue Institute for Inflammation, Immunology and Infectious Disease and Department of Biological Sciences, Purdue University, West Lafayette, IN, USA Search for more papers by this author Hongxin Guan Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, The Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China Search for more papers by this author Yini Huang Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, The Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China Search for more papers by this author Ting Yu Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, The Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China Search for more papers by this author Jiaqi Fu Purdue Institute for Inflammation, Immunology and Infectious Disease and Department of Biological Sciences, Purdue University, West Lafayette, IN, USA Search for more papers by this author Ernesto S Nakayasu orcid.org/0000-0002-4056-2695 Biological Science Division, Pacific Northwest National Laboratory, Richland, WA, USA Search for more papers by this author Kedar Puvar orcid.org/0000-0003-1788-2604 Department of Chemistry, Purdue University, West Lafayette, IN, USA Search for more papers by this author Chittaranjan Das Department of Chemistry, Purdue University, West Lafayette, IN, USA Search for more papers by this author Dongmei Wang Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, The Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China Search for more papers by this author Songying Ouyang Corresponding Author [email protected] orcid.org/0000-0002-1120-1524 Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, The Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China Search for more papers by this author Zhao-Qing Luo Corresponding Author [email protected] orcid.org/0000-0001-8890-6621 Purdue Institute for Inflammation, Immunology and Infectious Disease and Department of Biological Sciences, Purdue University, West Lafayette, IN, USA Search for more papers by this author Ninghai Gan orcid.org/0000-0001-7238-4056 Purdue Institute for Inflammation, Immunology and Infectious Disease and Department of Biological Sciences, Purdue University, West Lafayette, IN, USA Search for more papers by this author Hongxin Guan Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, The Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China Search for more papers by this author Yini Huang Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, The Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China Search for more papers by this author Ting Yu Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, The Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China Search for more papers by this author Jiaqi Fu Purdue Institute for Inflammation, Immunology and Infectious Disease and Department of Biological Sciences, Purdue University, West Lafayette, IN, USA Search for more papers by this author Ernesto S Nakayasu orcid.org/0000-0002-4056-2695 Biological Science Division, Pacific Northwest National Laboratory, Richland, WA, USA Search for more papers by this author Kedar Puvar orcid.org/0000-0003-1788-2604 Department of Chemistry, Purdue University, West Lafayette, IN, USA Search for more papers by this author Chittaranjan Das Department of Chemistry, Purdue University, West Lafayette, IN, USA Search for more papers by this author Dongmei Wang Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, The Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China Search for more papers by this author Songying Ouyang Corresponding Author [email protected] orcid.org/0000-0002-1120-1524 Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, The Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China Search for more papers by this author Zhao-Qing Luo Corresponding Author [email protected] orcid.org/0000-0001-8890-6621 Purdue Institute for Inflammation, Immunology and Infectious Disease and Department of Biological Sciences, Purdue University, West Lafayette, IN, USA Search for more papers by this author Author Information Ninghai Gan1,‡, Hongxin Guan2,3,‡, Yini Huang2,3, Ting Yu2,3, Jiaqi Fu1, Ernesto S Nakayasu4, Kedar Puvar5, Chittaranjan Das5, Dongmei Wang2,3, Songying Ouyang *,2,3 and Zhao-Qing Luo *,1 1Purdue Institute for Inflammation, Immunology and Infectious Disease and Department of Biological Sciences, Purdue University, West Lafayette, IN, USA 2Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, The Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China 3Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China 4Biological Science Division, Pacific Northwest National Laboratory, Richland, WA, USA 5Department of Chemistry, Purdue University, West Lafayette, IN, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +86 591 22868199; E-mail: [email protected] *Corresponding author. Tel: +17654966697; E-mail: [email protected] EMBO J (2020)39:e102806https://doi.org/10.15252/embj.2019102806 See also: F Yan et al (February 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The Legionella pneumophila effector MavC induces ubiquitination of the E2 ubiquitin-conjugating enzyme UBE2N by transglutamination, thereby abolishing its function in the synthesis of K63-type polyubiquitin chains. The inhibition of UBE2N activity creates a conundrum because this E2 enzyme is important in multiple signaling pathways, including some that are important for intracellular L. pneumophila replication. Here, we show that prolonged inhibition of UBE2N activity by MavC restricts intracellular bacterial replication and that the activity of UBE2N is restored by MvcA, an ortholog of MavC (50% identity) with ubiquitin deamidase activity. MvcA functions to deubiquitinate UBE2N-Ub using the same catalytic triad required for its deamidase activity. Structural analysis of the MvcA-UBE2N-Ub complex reveals a crucial role of the insertion domain in MvcA in substrate recognition. Our study establishes a deubiquitination mechanism catalyzed by a deamidase, which, together with MavC, imposes temporal regulation of the activity of UBE2N during L. pneumophila infection. Synopsis Transglutaminase MavC of the bacterial pathogen Legionella pneumophila catalyzes atypical ubiquitination of the host cell E2 ubiquitin conjugation enzyme UBE2N. Here, the L. pneumophila ubiquitin deamidase MvcA is found to reverse MavC-induced UBE2N ubiquitination and to restore its activity to promote host cell survival at later stages of L. pneumophila infection. Expression of MvcA reduces MavC-induced UBE2N ubiquitination. MvcA promotes synthesis of K63-type polyubiquitin chains by UBE2N during L. pneumophila infection. MvcA is a UBE2N-specific deubiquitinase that cleaves the isopeptide bond between Gln40 of ubiquitin and Lys92 of UBE2N. Structure of the MvcA-UBE2N-Ub complex reveals involvement of the MvcA “insertion domain” in substrate recognition. MvcA counteracts MavC activity at the late stage of infection to promote intracellular replication of L. pneumophila. Introduction Immune cells detect the presence of pathogens by sensing PAMPs such as flagellin, nucleic acids, and lipopolysaccharide or DAMP signals resulting from alternations or damages caused by virulence factors (Vance et al, 2009; Diacovich & Gorvel, 2010; Kieser & Kagan, 2017). The engagement of these ligands or perception of the activity of virulence factors leads to the activation of distinct signal transduction pathways and the induction of genes involved in the production of cytokines and antimicrobial agents (Vance et al, 2009; Diacovich & Gorvel, 2010; Kieser & Kagan, 2017). NFκB is a major immune transcriptional factor that becomes activated in response to diverse stimuli including bacterial infection (Li & Verma, 2002). NFκB activation is often achieved by the ubiquitin–proteasome system-mediated degradation of IκBα, a labile protein that sequesters its components, such as RelA and RelB, in the cytoplasm by forming stable protein complexes (Li & Verma, 2002). Signals that cause IκBα destruction are transduced by a number of proteins with distinct enzymatic activities, including protein kinases and enzymes involved in the formation of specific types of polyubiquitin chains. Among these, the E2 ubiquitin-conjugating enzyme, UBE2N, along with its heterodimeric partner UBE2V1 or UBE2V2 functions together with E3 enzymes, such as members of the TRAF (TNF receptor-associated factor) family, to catalyze the formation of K63-type polyubiquitin chains (Deng et al, 2000), which activate the TAK1/TAB1 kinases, leading to a signaling relay that eventually causes IκBα phosphorylation and its subsequent ubiquitination and degradation (Deng et al, 2000). UBE2N has emerged as an important checkpoint for activation of this pathway and is subjected to precise regulation by diverse mechanisms such as post-translational modifications, including several imposed by infectious agents (Song & Luo, 2019). In order to establish an intracellular niche permissive for multiplication, the bacterial pathogen Legionella pneumophila extensively manipulates host signaling with a large cohort of effector proteins (Burstein et al, 2016; Qiu & Luo, 2017b). In line with the essential roles played by the ubiquitin network in immune signaling (Komander & Rape, 2012), more than 10 L. pneumophila effectors have been found to modulate the host ubiquitin machinery as E3 ubiquitin ligases that coordinate with E1 and E2 enzymes from the host (Qiu & Luo, 2017a) or as deubiquitinases that remove Gly76-linked ubiquitin from ubiquitinated substrates (Sheedlo et al, 2015; Kubori et al, 2018). In addition, members of the SidE family catalyze ubiquitination by a tightly regulated (Bhogaraju et al, 2019; Black et al, 2019; Gan et al, 2019b) mechanism that is unrelated to the classical three-enzyme cascade of the eukaryotes (Qiu & Luo, 2017b; Song & Luo, 2019). In these reactions, ubiquitin is first activated by ADP-ribosylation via a mono-ADP-ribosyltransferase activity and is then utilized by a phosphodiesterase activity that attaches phosphoribosyl ubiquitin to serine residues of the substrate proteins (Bhogaraju et al, 2016; Qiu et al, 2016; Kotewicz et al, 2017). More recently, the L. pneumophila effector MavC (Lpg2147) was found to deamidate ubiquitin; it also interacts with the ubiquitin E2 conjugation enzyme, UBE2N, which may contribute to the inhibition of NFκB signaling during L. pneumophila infection (Valleau et al, 2018). Further studies demonstrate that MavC is a transglutaminase that induces UBE2N monoubiquitination by catalyzing the formation of an isopeptide bond between Gln40 of ubiquitin and Lys92, and, to a lesser extent, Lys94 of the E2 enzyme, leading to inhibition of NFκB activation (Gan et al, 2019a). Signaling mediated by UBE2N is pivotal for multiple important cellular processes (Ye & Rape, 2009; Hodge et al, 2016), including cell survival, which is important for productive intracellular replication of L. pneumophila (Losick & Isberg, 2006). After entry, robust bacterial replication requires the activity of genes whose expression is dependent upon NFκB (Losick & Isberg, 2006), which may mandate active UBE2N during later phases of the intracellular life cycle of L. pneumophila. Thus, the bacterium may temporally control the activity of this E2 enzyme by reversing MavC-induced monoubiquitination. In this study, we find that the activity of MavC is regulated by MvcA (Lpg2148), a protein coded by a gene adjacent to MavC. MvcA is a close homolog of MavC, both at sequence and structural levels; similar to MavC, MvcA exhibits deamidase activity toward ubiquitin (Valleau et al, 2018). Unexpectedly, our results show that MvcA functions to remove ubiquitin from the MavC-catalyzed transglutamination product, UBE2N-Ub, at later phases of L. pneumophila infection. To gain structural insights into this unprecedented deubiquitination, we crystallized and solved the structure of a complex of a catalytically inactive MvcA mutant in association with the UBE2N-Ub substrate. The structure provides an atomic-level explanation of this unique example of deubiquitination and deamidation executed by the same catalytic center while revealing a crucial role of the insertion domain of MvcA in its engagement of the substrate UBE2N-Ub, especially in the recognition of UBE2N. Thus, the presence of the insertion domain distinguishes MvcA from canonical ubiquitin deamidases. Results MvcA interferes with the modification of UBE2N induced by MavC Functional redundancy is common among L. pneumophila effectors, and in some cases, such redundancy is achieved by the ability to code for multiple members with high-level similarity to form protein families (O'Connor et al, 2012; Ghosh & O'Connor, 2017). For example, phenotypes associated with mutants lacking the sidE effector family can be complemented by any of its four members (Luo & Isberg, 2004; Bardill et al, 2005). In the Philadelphia 1 strain, MavC has a homolog (50% identity and 65% similarity) encoded by its neighboring gene called mvcA (lpg2148) (Valleau et al, 2018; Zhu et al, 2011; Fig EV1A and B). Because of the high-level similarity between these two proteins, in both primary sequence and structure, and their shared deamidation activity toward ubiquitin (Valleau et al, 2018), we considered the possibility that MvcA also ubiquitinates UBE2N or one or more other E2 enzymes. When recombinant MvcA active as a ubiquitin deamidase was added to a series of reactions containing one of a panel of several E2s that are similar to UBE2N, particularly in the region modified by MavC (Gan et al, 2019a), modification of none of these E2 enzymes could be detected (Fig EV1C and D). To test whether MvcA attacks UBE2N under more physiologically relevant conditions, we examined its ability to ubiquitinate UBE2N in cells infected by L. pneumophila using the ∆mvcA and the ∆mavC∆mvcA mutants. Consistent with earlier results (Gan et al, 2019a), L. pneumophila infection caused UBE2N modification by ubiquitin in a MavC-dependent manner. Deletion of mvcA did not affect the bacterium's ability to induce such modification (Fig 1A, 4th lane). Our antibodies for MvcA were about fivefold less sensitive compared to those against MavC (Gan et al, 2019a), and the antibodies for these two proteins did not cross-react (Appendix Fig S1). While MvcA antibodies were unable to detect endogenous protein expressed in wild-type bacteria (Fig 1A, 2nd lane), expression of MvcA from a multicopy plasmid allowed its detection in the ∆mavC∆mvcA mutant and in infected cells; yet, UBE2N modification was still not detectable in host cells even though these cells contained readily detectable levels of MvcA (Fig 1A, 8th lane). Unexpectedly, in cells infected with the ΔmvcA mutant overexpressing MvcA, UBE2N modification was no longer apparent. Importantly, the potential inhibition of UBE2N modification by overexpressed MvcA in strain ΔmvcA required the catalytic residue responsible for the ubiquitin deamidase activity (Valleau et al, 2018), as overexpression of the inactive MvcAC83A mutant failed to impose such suppression (Fig 1A, 5th and 6th lanes). In the ∆mvcA(pMvcA) strain, MavC was properly expressed and translocated into host cells (Fig 1A, 5th and 6th lanes in the middle and lower panels); therefore, the lack of UBE2N modification in cells infected by this strain is clearly not caused by disruption of MavC expression or its translocation into host cells. We further analyzed the potential interference of MvcA on MavC-induced UBE2N modification in mammalian cells by transfection. Expression of MavC alone led to almost complete modification of endogenous UBE2N. In contrast, coexpression of MvcA, but not the MvcAC83A mutant defective in ubiquitin deamidation (Valleau et al, 2018), with MavC resulted in considerable accumulation of unmodified UBE2N (Fig 1B), further supporting the notion that MvcA interferes with the activity of MavC in a manner dependent on its catalytic cysteine used in the ubiquitin deamidase activity (Valleau et al, 2018). As expected, the canonical ubiquitin deamidase Cif from Yersinia pseudotuberculosis (Cui et al, 2010) did not interfere with MavC-induced UBE2N modification (Fig 1B, 6th lane). Consistent with results from infection and transfection experiments, addition of MvcA to reactions measuring the transglutaminase activity of MavC at a 1:1 molar ratio led to complete abrogation of UBE2N ubiquitination, and such interference required its catalytic cysteine (Fig 1C, 3rd, 7th, and 8th lanes). Under our experimental conditions, a substantially higher molar ratio between MavC and MvcA, close to 10:1, was needed to overcome the inhibitory effects of MvcA (Fig 1D). Click here to expand this figure. Figure EV1. Genetic organization of mavC, mvcA, and lpg2149 in the genome of Legionella pneumophila strain Philadelphia 1 and the activity of MvcA against ubiquitin and several E2 enzymes The organization of mavC, mvcA, and lpg2149 in the genome of L. pneumophila strain Philadelphia 1. Note that the 76-base pair intergenic space between mavC and mvcA may allow them to be expressed by independent promoters. Alignment of the primary sequences of MavC and MvcA by Needleman-Wunsch Global Align. Stars indicate identical residues, and colons indicate conserved amino acids. The position of the catalytic Cys83 was colored in blue and indicated by a red arrow, whereas residues important for interacting with UBE2N-Ub were colored in red. Deamidase activity of MvcA toward ubiquitin. 10 μg of ubiquitin was incubated with 1 μg MvcA and the inactive MvcAC83A mutant for 1 h at 37°C, and the reaction products were separated in native PAGE and were detected by CBB staining. The modification of several E2 enzymes by MvcA. Reactions containing His6-tagged UBE2N or the indicated E2 enzymes were incubated with or without MvcA. Ubiquitination was assessed by molecular weight shift after Coomassie Brilliant Blue staining. None of the tested E2 enzymes were detectably modified by MvcA. Reactions that contain UBE2N and MavC were included as controls (the last sample in the gel on the right). Note that the extra band above UBE2N-Ub is an unknown protein in the MavC protein sample. Download figure Download PowerPoint Figure 1. MvcA interferes with UBE2N modification induced by MavC Infection by a Legionella pneumophila strain overexpressing MvcA but not the inactive mutant MvcAC83A interferes with UBE2N modification. Raw264.7 cells were infected with the indicated bacterial strains at an MOI of 5 for 2 h. Saponin-soluble proteins resolved by SDS–PAGE were probed with a UBE2N-specific antibody (top panel). The delivery of MavC and MvcA into infected cells was probed with antibodies specific to each of these two proteins. Note that MavC but not MvcA is detectable in cells infected with wild-type bacteria (middle panels labeled as “translocation”). Tubulin was probed as a loading control. Protein levels of MavC and MvcA associated with the bacteria were similarly probed in the saponin-insoluble fraction (lower panels labeled as “expression”). The bacterial metabolism enzyme isocitrate dehydrogenase (ICDH) was probed as a loading control. Note that endogenous MvcA was not detectable in bacteria grown in bacteriological medium (the middle panel of the lower portion). Coexpression of MvcA with MavC causes accumulation of unmodified UBE2N in mammalian cells. HEK293T cells were transfected with combinations of empty vector and plasmid directing the expression of MavC or MvcA. 16 h after transfection, modification of endogenous UBE2N was detected by immunoblotting (upper level). The canonical ubiquitin deamidase CifYpt from Yersinia pseudotuberculosis (Ypt), which cannot modify UBE2N, was included as a control. Note that coexpression of MvcA led to accumulation of unmodified UBE2N (4th lane) in samples that expressed MavC. The expression of MavC, MvcA, and Cif was detected using a Flag-specific antibody (lower panel). Tubulin was probed as a loading control (lower panel). MvcA interferes with MavC-induced UBE2N ubiquitination. Reactions containing the indicated combinations of proteins were allowed to proceed for 30 min, and samples resolved by SDS–PAGE were detected by Coomassie Brilliant Blue (CBB) staining. Note that MvcA (inclusion of MvcA with MavC, 7th lane. Compare to MavC alone in 3rd lane) but not the MvcAC83A mutant (8th lane) abolished MavC-induced UBE2N modification. Dose-dependent modification of UBE2N by MavC and MvcA. A series of reactions containing MavC and MvcA at the indicated molar ratios were established, and the reactions were allowed to proceed for 30 min. Samples resolved by SDS–PAGE were detected by CBB staining. Source data are available online for this figure. Source Data for Figure 1 [embj2019102806-sup-0005-SDataFig1.pdf] Download figure Download PowerPoint MvcA deubiquitinates the UBE2N-Ub product from MavC transglutaminase activity The inhibitory effects of MvcA can be achieved either by interference with the ubiquitin transglutaminase activity of MavC or by reversal of the modification on UBE2N. To distinguish between these two possibilities, we incubated purified UBE2N-Ub with MvcA or its catalytically inactive mutant. In reactions receiving MvcA, two distinct proteins with molecular weights corresponding to UBE2N and Ub were detected as cleaved products. This cleavage of UBE2N-Ub by MvcA required Cys83, the same catalytic Cys required for its ubiquitin deamidase activity (Valleau et al, 2018; Fig 2A). We explored the mechanism of cleavage by analyzing the two protein products by mass spectrometry, which revealed that UBE2N had been restored to its original form. In contrast, the ubiquitin product was in a modified form with its original Gln40 having been converted into a glutamate residue (Fig 2A). Thus, MvcA functions as an isopeptidase that attacks the isopeptide bond between Lys92 of UBE2N and Gln40 of ubiquitin to generate UBE2N and UbQ40E (Fig 2B), which happens to be the same product that would result from the ubiquitin deamidation reaction. We further examined the activity of MvcA by testing its ability to hydrolyze UBE2N-Ub in reactions containing these two proteins at different molar ratios. Under our experimental conditions, MvcA activity was detectable in 30 min in reactions in which the molar ratio between substrate and enzyme was nearly 2,000:1 (Fig 2C). At a ratio of only ~ 100:1, cleavage was readily detectable immediately after adding MvcA, and the reaction proceeded to almost completion in 10 min (Fig 2D). In agreement with the restoration of Lys92 to its original form, UBE2N produced by MvcA from UBE2N-Ub exhibited robust E2 enzymatic activity in catalyzing polyubiquitin chain synthesis (Fig 2E). We observed that a fraction of UBE2N-Ub was not hydrolyzed even after long duration of reaction with high amounts of MvcA (Fig 2A, C and D); mass spectrometric analysis of the uncleaved UBE2N-Ub protein revealed that in this molecule, ubiquitin is linked to UBE2N at Lys94, as a minor site of modification induced by MavC (Gan et al, 2019a; Fig EV2A). Figure 2. MvcA cleaves the isopeptide cross-link between UBE2N and ubiquitin in UBE2N-Ub The hydrolytic cleavage of UBE2N-Ub by MvcA into UBE2N and ubiquitin (Q40E variant). Reactions containing the indicated proteins were allowed to proceed for 30 min, and samples resolved by SDS–PAGE were detected by CBB staining (left). Mass spectrometric analysis of the protein band corresponding to ubiquitin produced by MvcA from UBE2N-Ub (right). The ubiquitin product is the Glu40 variant of ubiquitin. Proposed catalytic mechanism for isopeptide bond cleavage catalyzed by MvcA. Nucleophile attack of the isopeptide bond by the thiol from the catalytic center of MvcA leads to the release of UBE2N and formation of a thioester-linked MvcA-Ub intermediate, which is further attacked by a nucleophilic water to produce UbQ40E and to regenerate the active enzyme. Dose-dependent cleavage of UBE2N-Ub by MvcA. A series of reactions containing UBE2N-Ub and MvcA at the indicated molar ratios were established, and the reactions were allowed to proceed for 30 min before SDS–PAGE and CBB staining. Note that the activity was detectable in a reaction starting at the molar ratio of between UBE2N-Ub and MvcA is 2,048:1. Time-dependent cleavage of UBE2N-Ub by MvcA. UBE2N-Ub was incubated with MvcA at the molar ratio of 128. Samples taken at the indicated time points were resolved by SDS–PAGE and detected by CBB staining. UBE2N produced from UBE2N-Ub via MvcA-catalyzed deubiquitination is active in catalyzing the formation of polyubiquitin chains. UBE2N-Ub was pre-incubated with MvcA or its inactive mutant for 30 min. Then, a cocktail containing E1, UBE2V2, 3xHA-ubiquitin, the E3 enzyme TRAF6, and ATP was added into reactions. Ubiquitination was allowed to proceed for 30 min. Proteins in samples resolved by SDS–PAGE were transferred onto nitrocellulose membranes and blotted with the HA-specific antibody. Note the robust formation of polyubiquitin chains in the reaction receiving MvcA (middle lane). Source data are available online for this figure. Source Data for Figure 2
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