Mechanistic basis for impaired ferroptosis in cells expressing the African-centric S47 variant of p53

Abstract

A population-restricted single-nucleotide coding region polymorphism (SNP) at codon 47 exists in the human TP53 gene (P47S, hereafter P47 and S47). In studies aimed at identifying functional differences between these variants, we found that the African-specific S47 variant associates with an impaired response to agents that induce the oxidative stress-dependent, nonapoptotic cell death process of ferroptosis. This phenotype is manifested as a greater resistance to glutamate-induced cytotoxicity in cultured cells as well as increased carbon tetrachloride-mediated liver damage in a mouse model. The differential ferroptotic responses associate with intracellular antioxidant differences between P47 and S47 cells, including elevated abundance of the low molecular weight thiols coenzyme A (CoA) and glutathione in S47 cells. Importantly, the disparate ferroptosis phenotypes related to the P47S polymorphism are reversible. Exogenous administration of CoA provides protection against ferroptosis in cultured mouse and human cells, as well as in a mouse model. The combined data support a positive role for p53 in ferroptosis and identify CoA as a regulator of this cell death process. Together, these findings provide mechanistic insight linking redox regulation of p53 to small molecule antioxidants and stress signaling pathways. They also identify potential therapeutic approaches to redox-related pathologies.

Keywords: S-thiolation; coenzyme A; ferroptosis; p53; polymorphism.

Fig. 1.

Effect of codon 47 variants on sensitivity to glutamate toxicity and redox environment. (A) Cells, as indicated, were treated for 24 h with increasing concentrations of glutamate, and cell viability was assayed. Data represent the mean ± SD of multiple experiments (*P < 0.05, **P < 0.01). (B) P47 iMEFs were treated with PBS or 9.8 mM glutamate, with or without the indicated compounds, for 24 h, and analyzed for viability. Means and SD are shown (n = 4, *P < 0.05). (C) P47 iMEFs were treated with DMSO or 0.5 μM erastin, with or without the indicated compounds, for 24 h, and analyzed for viability. Means and SD are shown (n = 4, *P < 0.05). (D) qRT-PCR analyses of CARS in primary MEFs, iMEFs, and livers, as indicated. Relative expression data are plotted as the mean ± SD of multiple experiments (**P < 0.01). (E–G) iMEFs were treated with PBS or 6.4 mM glutamate for 24 h, as indicated. Cells were collected, washed with PBS, and analyzed for reduced GSH, GSH/GSSG ratio, and CoA abundance. Means and SD are shown (n = 4, *P < 0.05, **P < 0.01). (H–J) P47 iMEFs were treated with PBS, 9.8 mM glutamate, or 9.8 mM glutamate plus 0.5 mM CoA for 5 h or 24 h, as indicated. Intracellular levels of reduced GSH, GSH/GSSG ratio, and CoA abundance were determined. Means and SD are shown (n = 4, *P < 0.05, **P < 0.01). (K) P47 iMEFs were treated with PBS or 6.4 mM glutamate, with or without indicated 0.5 mM CoA, for 24 h, and analyzed for viability. Means and SD are shown (n = 4, *P < 0.05). (L) P47 iMEFs were treated with DMSO or 0.5 μM erastin, with or without indicated 0.5 mM CoA, for 24 h, and analyzed for viability. Means and SD are shown (n = 4, *P < 0.05).

Fig. 2.

Effect of GSH or CoA change on gene expression in response to glutamate. (A–J) qRT-PCR analyses of the indicated genes in P47, S47, and p53-null (p53^−/−) iMEFs. P47 iMEFs were pretreated with 0.5 mM CoA or 1 mM GSH for 2 h followed by 6.4 mM glutamate for 5 h. S47 and p53-null (p53^−/−) iMEFs were treated with 6.4 mM glutamate, 50 μM DEM, or 6.4 mM glutamate + 50 μM DEM, as indicated, for 5 h. P47, S47, and p53^−/− iMEFs treated with PBS served as controls. Relative expression data are plotted as the mean ± SD (n = 3, *P < 0.05, **P < 0.01).

Fig. 3.

Effect of GSH and CoA modulation on p53 oligomerization potential. (A) P47 and S47 iMEFs were treated with PBS, 6.4 mM glutamate, 0.5 mM CoA, 6.4 mM glutamate + 0.5 mM CoA, 50 μM DEM, or 6.4 mM glutamate + 50 μM DEM, as indicated, for 5 h. Protein lysates were analyzed by Western blot analysis for the proteins indicated (Top). p53 proteins were cross-linked with BMH, resolved by SDS/PAGE, and detected by Western blotting with a p53-specific antibody (Lower). (NS, nonspecific band). (B) P47 and S47 iMEFs were treated with DMSO or 10 μM Nutlin-3 for 24 h. Proteins were prepared and analyzed as described in A. (C) Proposed model for the influence of LMW thiols, such as GSH and CoA, on p53 tetramerization and function.

Fig. 4.

Effect of P47S polymorphism and redox environment on liver response to CCl₄. (A) P47 and S47 Hupki mice were treated, as indicated. Liver sections from the proposed cohorts (n = 7 per genotype per treatment) were stained with 4HNE. The brown staining indicates positive 4HNE staining. (Scale bars, 50 μM.) (B and C) Representative Prussian Blue (B)- and Sirius Red (C)-stained liver sections from P47 and S47 mice that were treated as indicated with one of the following: Three injections of CCl₄ (once a day for 3 d), with or without cotreatment with DEM or CoA, as indicated, and examined at 72 h; challenged with a chronic CCl₄ treatment for 5 wk (5W); or chronic CCl₄ treatment, with or without cotreatment with DEM or CoA, as indicated, for 5 wk followed by a 2-wk recovery (5W:O2W). (Scale bars, 50 μM.)