p97 Negatively Regulates NRF2 by Extracting Ubiquitylated NRF2 from the KEAP1-CUL3 E3 Complex

Abstract

Activation of the stress-responsive transcription factor NRF2 is the major line of defense to combat oxidative or electrophilic insults. Under basal conditions, NRF2 is continuously ubiquitylated by the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex and is targeted to the proteasome for degradation (the canonical mechanism). However, the path from the CUL3 complex to ultimate proteasomal degradation was previously unknown. p97 is a ubiquitin-targeted ATP-dependent segregase that extracts ubiquitylated client proteins from membranes, protein complexes, or chromatin and has an essential role in autophagy and the ubiquitin proteasome system (UPS). In this study, we show that p97 negatively regulates NRF2 through the canonical pathway by extracting ubiquitylated NRF2 from the KEAP1-CUL3 E3 complex, with the aid of the heterodimeric cofactor UFD1/NPL4 and the UBA-UBX-containing protein UBXN7, for efficient proteasomal degradation. Given the role of NRF2 in chemoresistance and the surging interest in p97 inhibitors to treat cancers, our results indicate that dual p97/NRF2 inhibitors may offer a more potent and long-term avenue of p97-targeted treatment.

Keywords: KEAP1; NRF2; autophagy; cancer chemoresistance; oxidative stress; p97; proteasome; protein quality control; ubiquitylation.

FIG 1

Reduction of the p97 level or activity leads to increased levels of NRF2 and its target genes. (a) Protein levels of NRF2 and p97 were examined by immunoblot analysis for two lung adenocarcinoma cell lines (A549 and H1299), a normal bronchial epithelial cell line (HBE), and an osteosarcoma cell line (U2OS). (b) H1299 cells were transfected with a scrambled siRNA (control [Ctrl]) or one of four different p97 siRNAs (#6, #7, #9, and #10; see Materials and Methods for details) for 48 h. Total cell lysates were used for immunoblot analysis with antibodies against the indicated proteins. In this panel, NRF2 appears as a single band because the protein lysates were resolved in 4 to 12% gradient gels. All other NRF2 bands appear as two bands because they were resolved in 7.5% gels. (c) H1299 cells were treated for 8 h (left) or 16 h (right) with the indicated doses of the p97 inhibitor CB-5083. Total cell lysates were subjected to immunoblot analyses. (d) HBE, A549, and U2OS cells were treated with the p97 inhibitor CB-5083 for 8 h. NRF2 expression was detected by immunoblot analysis. (e) H1299 KEAP1 knockout (KEAP1^−/−) cells were generated using CRISPR-Cas9 gene editing. Wild-type (WT) and KEAP1^−/− cells were transfected with Ctrl or p97 siRNA for 48 h. Total cell lysates were subjected to immunoblot analyses.

FIG 2

p97 interacts with NRF2 in a ubiquitin-dependent manner. (a) H1299 cells were first transfected with p97 siRNA, NRF2 siRNA (to knock down endogenous NRF2), or both siRNAs for 24 h and then transfected with a plasmid containing either HA-tagged NRF2-WT or HA-tagged NRF2-K7 for another 24 h. Total cell lysates were subjected to immunoblot analysis. (b) H1299 cells were transfected with a plasmid containing FLAG-tagged p97 and a plasmid for either HA-tagged NRF2-WT or HA-tagged NRF2-K7 for 24 h. Different amounts of NRF2-WT and NRF2-K7 were used in the presence or absence of p97 to ensure equal expression of total NRF2. Cell lysates were used for reciprocal immunoprecipitation (IP) and immunoblot (IB) analyses. (Left) p97 was immunoprecipitated with an anti-FLAG antibody, and NRF2 was detected using an anti-HA antibody. (Right) NRF2-WT or NRF2-K7 was immunoprecipitated using an anti-HA antibody, and p97 was detected using an anti-FLAG antibody. (Bottom) An aliquot of total cell lysate was used for immunoblot analysis with an anti-FLAG (p97) or anti-HA (NRF2) antibody. (c) H1299 WT and KEAP1^−/− cells were treated with 10 μM MG132 for 4 h to block degradation of ubiquitylated NRF2 before harvest. Cell lysates were subjected to immunoprecipitation analyses with anti-p97 or anti-NRF2 antibodies. Isotype IgG was used as a negative control, and anti-KEAP1 antibody was used as a positive control.

FIG 3

p97 facilitates ubiquitin-mediated degradation of NRF2. (a) Ubiquitylation of ectopically expressed NRF2. H1299 cells were first transfected with p97 siRNA for 24 h and then transfected with expression vectors for FLAG-NRF2 and HA-ubiquitin (HA-Ub) for an additional 24 h. Before harvest, cells were treated with 10 μM MG132 for 4 h to block degradation of ubiquitylated NRF2. NRF2 was immunoprecipitated using an anti-FLAG antibody, and immunoprecipitated NRF2 was subjected to immunoblot analysis with an anti-HA antibody. (Bottom) An aliquot of total cell lysate was used for immunoblot analysis. (b) Ubiquitylation of endogenous NRF2. H1299 cells were transfected with p97 siRNA for 48 h. NRF2 was immunoprecipitated with an anti-NRF2 antibody, and immunoprecipitated NRF2 was subjected to immunoblot analysis with an antiubiquitin (anti-Ub) antibody for detection of endogenous ubiquitylated NRF2. (Bottom) An aliquot of total cell lysate was used for immunoblot analysis. (c) H1299 cells were transfected with a scrambled siRNA (Ctrl siRNA) or p97 siRNA for 48 h. Cells were treated with cycloheximide for the indicated time before harvest. (Top) Total cell lysates were subjected to immunoblot analysis. (Bottom) NRF2 and GAPDH levels were determined by densitometry, and the level of NRF2 relative to that of GAPDH was plotted as a function of time to determine the half-life of NRF2. NRF2:S, short exposure; NRF2:L, long exposure.

FIG 4

p97 is involved in the canonical NRF2 pathway. (a and b) H1299 cells were transfected with the indicated siRNAs for 48 h. Total cell lysates were subjected to immunoblot analysis (a) and qRT-PCR analysis of GCLM and HMOX-1 (the gene encoding HO-1) (b). (c and d) H1299 cells were transfected with p97 siRNA for 48 h, followed by a 4-h or 16-h treatment with the autophagy blocker bafilomycin (Baf) (100 nM for 4 h or 50 nM for 16 h) or the canonical NRF2 activator sulforaphane (SF) (5 μM for 4 h or 2.5 μM for 8 h). Total cell lysates were subjected to immunoblot analysis (c) and qRT-PCR analysis of GCLM and HMOX-1 (for the 16-h treatment only) (d). Results were obtained from three independent experiments. *, P < 0.05 compared to control.

FIG 5

Decreased levels of p97, UFD1/NPL4, and UBXN7 increase NRF2 signaling. (a to c) H1299 cells were transfected with the indicated siRNA for 48 h. (a) Total cell lysates were subjected to immunoblot analysis. (b) Cells were grown on glass coverslips for 48 h and then were subjected to indirect immunofluorescence analysis using an anti-NRF2 antibody. Hoechst 33258 was included to label nuclei. (c) Total RNA was extracted and reverse transcribed, and equal amounts of cDNA were used for qRT-PCR. *, P < 0.05 compared to Ctrl siRNA. The experiments were repeated three times, each with duplicate samples. (d) Model of the p97-mediated extraction of NRF2 from the KEAP1-CUL3-RBX1 complex for proteasomal degradation in the canonical NRF2 pathway. NRF2 is recruited to the KEAP1-CUL3-RBX1 E3 ligase complex by KEAP1, leading to ubiquitylation of NRF2, which is segregated from the complex by p97. UBXN7 acts as a scaffold protein that brings together all the components, which we propose to take place by the following model. The UIM domain binds to NEDDylated (NEDD8) CUL3 (in green), the UBA domain binds to ubiquitylated NRF2, and the UBX domain recruits p97 through interaction with the p97 N-terminal domain. The UFD1/NPL4 heterodimer likely binds to both the p97 N-terminal domain and NRF2, bringing these two proteins into close proximity. This allows p97 to act as an ATP-driven motor to extract ubiquitylated NRF2 from the KEAP1-CUL3 complex to deliver it to the 26S proteasome for degradation.