Suppression of the pro-apoptotic function of cytochrome c by singlet oxygen via a haem redox state-independent mechanism

Abstract

Stimuli for apoptotic signalling typically induce release of cyt c (cytochrome c) from mitochondria. Cyt c then initiates the formation of the apoptosome, comprising Apaf-1 (apoptotic protease-activating factor 1), caspase-9 and other cofactors. The issue of whether the redox state of the haem in cyt c affects the initiation of the apoptotic pathway is currently a subject of debate. In a cell-free reconstitution system, we found that only oxidized cyt c was capable of activating the caspase cascade. Oxidized cyt c was reduced by the physiological reductants cysteine and glutathione, after which it was unable to activate the caspase cascade. It is thus likely that cyt c with oxidized haem is in a conformation capable of interaction with Apaf-1 and forming apoptosomes. When either oxidized or reduced cyt c was treated with submillimolar concentrations of endoperoxide, which affected less than 3% of the redox state of haem, the ability of the oxidized cyt c to activate the caspase cascade was abolished. Higher amounts of singlet oxygen were required to affect the optical spectral change of haem, suggesting that the suppressed pro-apoptotic function of oxidized cyt c is a mechanism that is separate from the redox state of haem. Oxidative protein modification of cyt c by singlet oxygen was evident, on the basis of elevated contents of carbonyl compounds. Our data suggest that singlet oxygen eliminates the pro-apoptotic ability of oxidized cyt c not via the reduction of haem, but via the modification of amino acid residues that are required for apoptosome formation.

Figure 1. Effects of oxidized and reduced cyt c on activation of caspases

After incubation of cytosolic fractions from HepG2 cells with oxidized or reduced cyt c, the activities of caspase-9 (A) and -3 (B) were measured. An immunoblot analysis was performed for the caspase assay mixture containing either oxidized (C) or reduced (D) cyt c using specific antibodies against caspase-9, -3 or cyt c (1:1000 dilution). The arrowheads in (C) indicate the processed, active form of the caspases. Abbreviation: U, units.

Figure 2. Effects of GSH and cysteine on redox state of cyt c

(A) After incubation of oxidized cyt c with 1 mM DTT, GSH or cysteine for 1 h at 37 °C, spectral changes between 370 and 600 nm were measured. The dotted line shows the spectrum of oxidized cyt c in the absence of a reductant; the solid line shows superimposed spectra of oxidized cyt c treated with GSH or cysteine. (B) Absorbance at 550 nm of oxidized cyt c was continuously recorded in the presence of 1 mM GSH or cysteine for 1 h. The spectra corresponded to oxidized cyt c (9 μM) that was incubated with GSH (top trace), cysteine (middle trace) or no reductant (bottom trace).

Figure 3. Dose-dependent inhibition by GSH and cysteine of the caspase-activating ability of oxidized cyt c

After pre-incubation of oxidized cyt c with various concentrations of GSH or cysteine for 1 h at 37 °C, a cell-free caspase (Casp)-activation assay was performed. The activities of caspase-9 (A) and -3 (B) are shown. An immunoblot analysis was performed for the caspase assay mixture containing cyt c treated with either GSH (C) or cysteine (D) using specific antibodies against caspase-9, -3 or cyt c (1:1000 dilution). The arrowheads indicate the processed, active form of the enzymes.

Figure 4. Effects of MNPE treatment on the caspase-activating ability of oxidized cyt c

After incubation of oxidized cyt c with various concentrations of MNPE or MNP for 1 h at 37 °C, a cell-free caspase (Casp)-activation assay was performed. The activities of caspase-9 (A) and -3 (B) are shown. An immunoblot analysis was performed for the caspase assay mixture containing cyt c treated with MNPE (C) or MNP (D) using specific antibodies against caspase-9, -3 or cyt c (1:1000 dilution). The arrowheads indicate the processed, active form of the enzymes.

Figure 5. Effects of singlet oxygen ‘quenchers’ on pro-apoptotic ability of reduced cyt c

Cyt c in PBS was first oxidized and treated with 1 mM MNPE or the control condition (CTR) in the presence of 20 mM NaN₃, 20 mM histidine, 5 mM DABCO or 10 μM β-carotene at 37 °C for 1 h. After gel filtration of cyt c on the PD10 column, caspase activity was measured in a cell-free reconstitution system. Data represent relative activities of caspase-9 and -3 to control experiments that were performed with fully oxidized cyt c (CTR).

Figure 6. Effects of singlet oxygen on redox state of cyt c

After incubation of either oxidized (A) or reduced (B) cyt c (8 μM) with 0, 1, 3 or 5 mM MNPE for 1 h at 37 °C, spectral changes between 370 and 800 nm were measured. Informative spectral ranges are shown. (A) Spectral traces shown from bottom to top (↑) correspond to oxidized cyt c that was treated with 0, 1, 3 or 5 mM MNPE. (B) Spectral traces shown from top to bottom (↓) correspond to reduced cyt c that was treated with 0, 1, 3 or 5 mM MNPE. (C) Absorbance changes at 550 nm of oxidized or reduced cyt c (8 μM) in the presence of 1 mM MNPE were continuously recorded for 60 min. (D) Spectral changes between 600 and 720 nm are shown. Spectral traces shown from top to bottom (↓) correspond to oxidized cyt c (50 μM) that was incubated with 0 (top), 1 (middle) or 5 (bottom) mM MNPE for 1 h at 37 °C.

Figure 7. EPR spectra of oxidized cyt c treated with or without MNPE at 77 K

(A) The spectrum of oxidized cyt c (4 mM) without MNPE, (B) the spectrum of oxidized cyt c with 1 mM MNPE, and (C) the spectrum of oxidized cyt c with 5 mM MNPE are shown. Cyt c (4 mM) was incubated in PBS. Instrumental conditions were: sweep time, 8 min; time constant, 0.3 s; modulation amplitude, 0.5 mT; modulation frequency, 100 KHz; and microwave frequency, ≈9.2 GHz.

Figure 8. Irreversible dysfunction of cyt c by treatment with singlet oxygen

Cyt c was first fully oxidized, and then treated as indicated. After the treatments, a cell-free caspase-activation assay was performed. Data represent relative activities of caspase-9 and -3 to control experiments that were performed with fully oxidized (Ox) cyt c. CTR, control.

Figure 9. Elevated carbonyl contents in singlet oxygen-treated cyt c

(A) Carbonyl contents of oxidized cyt c (100 ng) that were treated with or without 1 mM MNPE for 1 h at 37 °C were assessed by an Oxyblot kit. Protein staining by Coomassie Brilliant Blue (CBB) was performed to show equal protein loading. (B) Quantification of the band intensity was performed for five assays by densitometric scanning of X-ray films. Data represent the mean values±S.D. relative to the control sample (without MNPE treatment).