Abstract

Ubiquitylation of Nrf2 by the Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase complex targets Nrf2 for proteasomal degradation in the cytoplasm and is an extensively studied mechanism for regulating the cellular level of Nrf2. Although mechanistic details are lacking, reports abound that Nrf2 can also be degraded in the nucleus. Here, we demonstrate that Nrf2 is a target for sumoylation by both SUMO-1 and SUMO-2. HepG2 cells treated with As2O3, which enhances attachment of SUMO-2/3 to target proteins, increased SUMO-2/3-modification (polysumoylation) of Nrf2. We show that Nrf2 traffics, in part, to promyelocytic leukemia-nuclear bodies (PML-NBs). Cell fractions harboring key components of PML-NBs did not contain biologically active Keap1 but contained modified Nrf2 as well as RING finger protein 4 (RNF4), a poly-SUMO-specific E3 ubiquitin ligase. Overexpression of wild-type RNF4, but not the catalytically inactive mutant, decreased the steady-state levels of Nrf2, measured in the PML-NB-enriched cell fraction. The proteasome inhibitor MG-132 interfered with this decrease, resulting in elevated levels of polysumoylated Nrf2 that was also ubiquitylated. Wild-type RNF4 accelerated the half-life (t½) of Nrf2, measured in PML-NB-enriched cell fractions. These results suggest that RNF4 mediates polyubiquitylation of polysumoylated Nrf2, leading to its subsequent degradation in PML-NBs. Overall, this work identifies Nrf2 as a target for sumoylation and provides a novel mechanism for its degradation in the nucleus, independent of Keap1.

Highlights

  • Nrf2 enables cells to mount a cytoprotective response to oxidative stress

  • Wild-type RING finger protein 4 (RNF4) accelerated the half-life (t1⁄2) of Nrf2, measured in promyelocytic leukemia-nuclear bodies (PML-NBs)-enriched cell fractions. These results suggest that RNF4 mediates polyubiquitylation of polysumoylated Nrf2, leading to its subsequent degradation in PML-NBs

  • We show that RNF4, a poly-SUMO-specific E3 ubiquitin ligase ubiquitylates polysumoylated Nrf2, leading to degradation of the modified Nrf2 in PML-NB domains

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Summary

Background

Nrf (nuclear factor erythroid 2-related factor 2) enables cells to mount a cytoprotective response to oxidative stress. Wild-type RNF4 accelerated the half-life (t1⁄2) of Nrf, measured in PML-NB-enriched cell fractions These results suggest that RNF4 mediates polyubiquitylation of polysumoylated Nrf, leading to its subsequent degradation in PML-NBs. Overall, this work identifies Nrf as a target for sumoylation and provides a novel mech-. Its abundance is restricted by Keap, a redoxregulated inhibitory protein that functions as a substrate adaptor for ubiquitylation of Nrf by the Cullin3/RING box (Cul3-Rbx1) E3 ubiquitin ligase complex. This ubiquitylation targets Nrf for proteasome-dependent degradation in the cytoplasm (4 – 6). They posited that the Keap1-dependent modality occurs only under homeostatic

The abbreviations used are
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