Background: Nrf2 (nuclear factor erythroid 2-related factor 2) enables cells to mount a cytoprotective response to oxidative stress. Results: Nrf2 localizes, in part, to promyelocytic leukemia-nuclear bodies (PML-NBs), can undergo sumoylation, and can be degraded in PML-NBs. Conclusion: Polysumoylated Nrf2 is polyubiquitylated by RING finger protein 4 (RNF4) and subsequently degraded by the proteasome in PML-NB domains. Significance: Nuclear degradation of Nrf2 involves PML-NBs.
Background/Aims: The transcription factor Nrf2 is a master regulator of the antioxidant defense system, protecting cells from oxidative damage. We previously reported that the SUMO-targeted E3 ubiquitin ligase (STUbL), RING finger protein 4 (RNF4) accelerated the degradation rate of Nrf2 in promyelocytic leukemia-nuclear body (PML-NB)-enriched fractions and decreased Nrf2-mediated gene transcription. The mechanisms that regulate Nrf2 nuclear levels are poorly understood. In this study, we aim to explore the role of the second mammalian STUbL, Arkadia/RNF111 on Nrf2. Methods: Arkadia mediated ubiquitination was detected using co-immunoprecipitation assays in which whole cell lysates were immunoprecipated with anti-Nrf2 antibody and Western blotted with anti-hemagglutinin (HA) antibody or anti-Lys-48 ubiquitin-specific antibody. The half-life of Nrf2 was detected in whole cell lysates and promyelocytic leukemia-nuclear body enriched fractions by cycloheximide-chase. Reporter gene assays were performed using the antioxidant response element (ARE)-containing promoter Heme oxygenase-1 (HO-1). Results: We show that Arkadia/RNF111 is able to ubiquitinate Nrf2 resulting in the stabilization of Nrf2. This stabilization was mediated through Lys-48 ubiquitin chains, contrary to traditionally degradative role of Lys-48 ubiquitination, suggesting that Lys-48 ubiquitination of Nrf2 protects Nrf2 from degradation thereby allowing Nrf2-dependent gene transcription. Conclusion: Collectively, these findings highlight a novel mechanism to positively regulate nuclear Nrf2 levels in response to oxidative stress through Arkadia-mediated K48-linked ubiquitination of Nrf2.
BACKGROUND/AIMS: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element(s) (ARE) in target gene promoters, enabling oxidatively stressed cells to respond in order to restore redox homeostasis. Post-translational modifications (PTMs) that mediate activation of Nrf2, in the cytosol and its release from Keap1, have been extensively studied but PTMs that impact its biology after activation are beginning to emerge. In this regard, PTMs like acetylation, phosphorylation, ubiquitination and sumoylation contribute towards the Nrf2 subcellular localization, and its transactivation function. We previously demonstrated that Nrf2 traffics to the promyelocytic leukemia-nuclear bodies (PML-NB), where it is a target for modification by small ubiquitin-like modifier (SUMO) proteins (sumoylation), but the site(s) for SUMO conjugation have not been determined. In this study, we aim to identify SUMO-2 conjugation site(s) and explore the impact, sumoylation of the site(s) have on Nrf2 stability, nuclear localization and transcriptional activation of its target gene expression upon oxidative stress. METHODS: The putative SUMO-binding sites in Nrf2 for human isoform1 (NP_006155.2) and mouse homolog (NP_035032.1) were identified using a computer-based SUMO-predictive software (SUMOplot™). Site-directed mutagenesis, immunoblot analysis, and ARE-mediated reporter gene assays were used to assess the impact of sumoylation on these site(s) in vitro. Effect of mutation of these sumoylation sites of Nrf2 on expression of Heme Oxygenase1 (HO-1) was determined in HEK293T cell. RESULTS: Eight putative sumoylation sites were identified by SUMOplot™ analysis. Out of the eight predicted sites only one 532LKDE535 of human (h) and its homologous 524LKDE527 of mouse (m) Nrf2, exactly matches the SUMO-binding consensus motif. The other high probability SUMO-acceptor site identified was residue K110, in the motifs 109PKSD112 and 109PKQD112 of human and mouse Nrf2, respectively. Mutational analysis of putative sumoylation sites (human (h)/mouse (m) K110, hK533 andmK525)showed that these residues are needed for SUMO-2 conjugation, nuclear localization and ARE driven transcription of reporter genes and the endogenous HO-1 expression by Nrf2. These residues also stabilized Nrf2, as evident from shorter half-lives of the mutant protein compared to wild-type Nrf2. CONCLUSION: Our findings indicate that SUMO-2 mediated sumoylation of K110 and K533 in human Nrf2 regulates in part its transcriptional activity by enhancing its stabilization and nuclear localization.
The transcription factor Nrf2 is a master regulator of the antioxidant defense system that serves to protect cells from oxidative damage. We previously reported that the SUMO‐targeted E3 ubiquitin ligase (STUbL), RING finger protein 4 (RNF4) accelerated the degradation rate of Nrf2 in promyelocytic leukemia‐nuclear body (PML‐NB)‐enriched fractions and decreased Nrf2‐mediated gene transcription. STUbLs contain SUMO‐interaction motifs that enable it to target substrate proteins that have been modified by SUMO resulting in ubiquitylation. These ubiquitylation events help in regulating eukaryotic cellular activity. In this study, we establish the second mammalian STUbL, Arkadia/RNF111 ubiquitylates polysumolyated Nrf2 to stabilize it, but has no effect on Nrf2‐mediated gene transcription. Interestingly, we discovered that the Arkadia‐mediated stabilization of Nrf2 occurs through Ub‐K48 linkages rather than the predicted Ub‐K63 linkages. These results suggest Arkadia‐mediated ubiquitylation of Nrf2 protects it from degradation, thereby allowing Nrf2‐dependent gene transcription. Collectively, these findings highlight a novel mechanism to positively regulate nuclear Nrf2 levels in response to oxidative stress.Support or Funding InformationThis work was supported by NIH grant SC1CA143985 and by CTSA award No. ULITR000445 from the National Center for Advancing Translational Sciences. +Supported by NIH grant # 2 S21MD000104This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The cellular abundance of the redox‐sensitive transcription factor Nrf2 is controlled by Keap1, an inhibitory protein that functions as a substrate adaptor for ubiquitylation of Nrf2 by the Cullin3/RING box1 (Cul3‐Rbx1) E3 ubiquitin ligase complex, thereby targeting Nrf2 for proteasome‐dependent degradation in the cytoplasm. Nrf2 can also be degraded in the nucleus but mechanistic details are lacking. Here, we show that promyelocytic leukemia‐nuclear body (PML‐NB)‐enriched preparations of HepG2 cells treated with arsenic trioxide (ATO) contained modified (sumoylated) Nrf2 and the poly‐SUMO‐specific ubiquitin E3 ligase RNF4, but not Keap1. Overexpressing RNF4 decreased the steady‐state levels of Nrf2 whereas the catalytically inactive RNF4 did not. The proteasomal inhibitor MG‐132 interfered with this RNF4‐induced decrease. In MG‐132‐treated cells, the content of ubiquitylated Nrf2 was higher in cells transfected with wild‐type RNF4 than in cells transfected with catalytically inactive RNF4. RNF4 also decreased the half‐life of Nrf2, measured in PML‐NB‐enriched fractions prepared from ATO‐treated cells. Given that treatment with ATO enhances SUMO‐2/3 attachment to Nrf2, these data suggest that RNF4 induces poly‐ubiquitylation of poly‐sumoylated Nrf2, leading to its degradation by proteasomes in PML‐NBs.Supported by NIH grants #5T32HL007737, 2 SD1MD000104 and SC1CA143985
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