Identification of hyperoxidized PRDX3 as a ferroptosis marker reveals ferroptotic damage in chronic liver diseases

Abstract

Ferroptosis, a regulated cell death pathway driven by accumulation of phospholipid peroxides, has been challenging to identify in physiological conditions owing to the lack of a specific marker. Here, we identify hyperoxidized peroxiredoxin 3 (PRDX3) as a marker for ferroptosis both in vitro and in vivo. During ferroptosis, mitochondrial lipid peroxides trigger PRDX3 hyperoxidation, a posttranslational modification that converts a Cys thiol to sulfinic or sulfonic acid. Once hyperoxidized, PRDX3 translocates from mitochondria to plasma membranes, where it inhibits cystine uptake, thereby causing ferroptosis. Applying hyperoxidized PRDX3 as a marker, we determined that ferroptosis is responsible for death of hepatocytes in mouse models of both alcoholic and nonalcoholic fatty liver diseases, the most prevalent chronic liver disorders. Our study highlights the importance of ferroptosis in pathophysiological conditions and opens the possibility to treat these liver diseases with drugs that inhibit ferroptosis.

Keywords: AFLD; NAFLD; PRDX3; cell death marker; ferroptosis; hyperoxidation.

Figure 1.. PRDX3 is hyperoxidized during ferroptosis.

(A) Catalytic cycle of PRDXs. (B-D) Amounts of hyperoxidized and total PRDX1-4 in SV589 cells treated with indicated concentrations of erastin (B), RSL3 (C) or FIN56 (D) for 12 h were determined by immunoblot analysis. (E) Lysate of cells treated with or without 200 nM erastin2 for 12 h was immunoblotted by anti-PRDX3 and anti-SO_2/3-PRDX1-4 in the same blot followed by detection with red and green fluorescence-conjugated secondary antibodies, respectively. (F) Lysate of cells treated with or without 2 μM erastin for 12 h was immunoprecipitated with anti-PRDX3 or control IgG. The input and pellet fractions were analyzed by immunoblot analysis with the indicated antibodies. Asterisk denotes the light chain of the antibodies. (G) Immunoblot analysis of the indicated cells treated with indicated concentration of erastin for 12 h with anti-SO_2/3-PRDX1-4 or anti-PRDX3. (H-K) The amount of hyperoxidized PRDX3 in HT1080 (H), A549 (I), Huh7 (J), and HT29 cells (K) treated with erastin or RSL3 was determined as described in (B) and (C). See also Figure S1.

Figure 2.. Hyperoxidized PRDX3 stimulates ferroptosis by inhibiting cystine uptake.

(A) The amount of hyperoxidized PRDX3 in indicated cells treated with 2 μM erastin for the indicated time was determined by immunoblot analysis (upper panel). Viability of indicated cells treated with 2 μM erastin for the indicated time was determined as described in Star Methods, with the value of the untreated WT cells set at 100% (lower panel). (B-D) Viability of cells treated with indicated concentrations of erastin (B), RSL3 (C) or FIN56 (D) for 24 h was measured as shown in (A). (E) The total amount of GSH in the indicated cells treated with or without 2 μM erastin for 9 h. (F) BODIPY 581/591 C11 flow cytometry of the green fluorescence of indicated cells treated with or without 2 μM erastin for 12 h. (G) Indicated cells treated with or without 200 nM erastin2 for 12 h were subject to immunofluorescent microscopy with indicated antibodies and MitoTracker. Scale bar=10 μm. Lower magnification of the images was present in Figure S2J. (H and I) Cystine uptake (H) and glutamate export (I) of the indicated cells was measured as described in Star Methods in the absence or presence of 2 μM erastin pretreatment that prolonged the erastin treatment time to 9 h. (J) Viability of the indicated cells starved for cystine in the absence or presence of deferoxamine (DFO, 50 μM) or ferrostatin-1 (Fer, 1 μM) for the indicated time was analyzed as described in (A). (A-E and H-J) Results are reported as mean ± S.E.M. from three independent experiments. Statistical significance was calculated by unpaired, two-tailed t-test. See also Figure S2.

Figure 3.. Hyperoxidized PRDX3 is specifically produced in ferroptotic cells.

(A and B) The amount of hyperoxidized PRDX3 in cells treated with 2 μM erastin (A) or 30 nM RSL3 (B) together with deferoxamine (DFO, 10 μM), ferrostatin-1 (Fer, 200 nM), vitamin E (VE, 10 μM), or oleate (OA, 200 μM) for 12 h was determined by immunoblot analysis. (C) The amounts of hyperoxidized PRDX3 and cleaved caspase 3 (C-Caspase3) in SV589 cells treated with indicated concentration of erastin or camptothecin for 12 h were determined by immunoblot analysis. (D) The amount of hyperoxidized PRDX3 and p-MLKL in HT29 cells treated with 20 ng/ml TNF-α, 100 nM SM164 and 20 μM z-VAD (T/S/Z) for the indicated time, or 2 μM erastin for 12 h was determined by immunoblot analysis. (E) The amount of hyperoxidized PRDX3 in SV589 cells treated with 2 μM erastin or indicated concentration of elesclomol plus 2 μM CuCl₂ for 12 h were determined by immunoblot analysis. (F) The amount of hyperoxidized PRDX3 in SV589 cells treated with 2 μM erastin or 4 μM CCCP for 12 h were determined by immunoblot analysis. (G) Immunofluorescence of bone tissues containing xenograft multiple myeloma from mice subject to indicated therapy with the antibody that detects hyperoxidized PRDX3. Scale bar=20 μm. Lower magnification of the images was present in Figure S3D. (H) The amount of hyperoxidized PRDX3 in livers from AA-fed control or L-Faf1^−/− mice injected with Lip-1 or the control vehicle PBS. (I) Immunofluorescence of livers from mice shown in (H) with the antibody that detects hyperoxidized PRDX3. Scale bar=20 μm. Lower magnification of the images was present in Figure S3E. See also Figure S3.

Figure 4.. Hepatic damage in mouse models of AFLD and NAFLD is caused by ferroptosis.

(A and B) AST (A) and ALT (B) activities in serum of indicated female (n = 5) or male mice (n = 5-7) fed with alcohol or control liquid diets. Statistical significance was calculated by unpaired, two-tailed t-test. (C) The amount of hyperoxidized PRDX3, p-MLKL, and C-caspase3 in livers from mice shown in (A) was determined by immunoblot analysis. (D and E) AST (D) and ALT (E) activities in serum of mice fed with a chow (n = 8) or HFD diet (Research Diet D12451) (n = 23) for 12 weeks. Statistical significance was calculated by unpaired, two-tailed t-test. (F) The amount of hyperoxidized PRDX3, p-MLKL, and C-caspase3 in livers from mice shown in (D) was determined by immunoblot analysis. For the HFD-fed group, each lane contained samples taken from different mice, and the results from all 23 mice were shown in three panels. For the chow-fed group, samples from all 8 mice were loaded on the first panel, from which those loaded in the first 6 lanes were reloaded in the second and third panels for comparison purposes. (G and H) The correlation between AST (G) or ALT (H) values and that of the Li-COR-quantified hyperoxidized PRDX3 immunoblot signal (normalized against calnexin) in all the mice analyzed in A-F was determined by Pearson’s analysis.