SUMO-Modification of Human Nrf2 at K and K Regulates Its Nucleocytoplasmic Localization, Stability and Transcriptional Activity.

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 LKDE of human (h) and its homologous LKDE of mouse (m) Nrf2, exactly matches the SUMO-binding consensus motif. The other high probability SUMO-acceptor site identified was residue K, in the motifs PKSD and PKQD of human and mouse Nrf2, respectively. Mutational analysis of putative sumoylation sites (human (h)/mouse (m)
K, hK and mK) 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 K and K in human Nrf2 regulates in part its transcriptional activity by enhancing its stabilization and nuclear localization.