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. 2019 Jun 28;38(15):e101964. doi: 10.15252/embj.2019101964

Figure 3. GSTZ1‐1 deficiency activates the KEAP1/NRF2 pathway via succinylacetone accumulation.

Figure 3

  • A
    Luciferase (LUC) activity of ARE promoter in GSTZ1‐1‐overexpressing (GSTZ1‐OE) Huh7 cells (top) and GSTZ1 knockout (GSTZ1‐KO) HepG2 cells (bottom). The antioxidant N‐acetylcysteine (NAC, 20 mM for 12 h) and the NRF2 activator tertiary butylhydroquinone (tBHQ, 40 μM for 3 h) were used as negative and positive controls, respectively.
  • B, C
    Relative mRNA (B) and protein (C) expression of NRF2 downstream target genes (such as NQO1, TXNRD1, and HO1), and cytoplasmic (Cyto) and nuclear (Nuc) expression of NRF2 (C) in GSTZ1‐OE Huh7 cells (top) and GSTZ1‐KO HepG2 cells (bottom).
  • D
    Phenylalanine and tyrosine (Phe/Tyr) catabolic pathway (left) and relative levels of metabolites in tyrosine catabolism in murine liver as measured by mass spectrometry (right).
  • E
    LUC activity of ARE promoter in HepG2 cells treated with Phe (2.0 mM) and succinylacetone (SA, 200 μΜ) for 36 h. NAC and tBHQ were described as above.
  • F
    Western blotting shows NQO1 expression and cytoplasmic and nuclear expression of NRF2 in HepG2 cells treated with Phe (2.0 mM, left) and SA (200 μM, right) for 48 h.
  • G
    SA alkylation of human KEAP1 cysteine residues on Cys406 was identified in GSTZ1‐KO HepG2 cells infected with adenoviruses expressing KEAP1 (AdKEAP1) by MS/MS analysis.
  • H
    Western blotting shows NRF2 and KEAP1 expression in HepG2 cells cotransfected with NRF2‐Myc and wild‐type (WT) or mutant KEAP1 (C23S, C151, C319S, C406S, or C513S).
Data information: Values are shown as means ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001, Student's t‐test (two groups) or one‐way ANOVA followed by the Tukey test (more than two groups). Abbreviations: NQO1, NAD(P):H quinone oxidoreductase 1; MRP2, multidrug resistance protein 2; GCLM, glutamate‐cysteine ligase modifier subunit; TXNRD1, thioredoxin reductase 1; G6PD, glucose‐6‐phosphate dehydrogenase; HO1, heme oxygenase 1; ns, not significant; 4HPP, 4‐hydroxyphenylpyruvate; HGA, homogentisic acid; MAA, maleylacetoacetate; FAA, fumarylacetoacetate; FUMA, fumarate; ACA, acetoacetate; SAA, succinylacetoacetate; Vec, vector.Source data are available online for this figure.