Cysteine 62 of Bax Is Critical for Its Conformational Activation and Its Proapoptotic Activity in Response to H2O2-induced Apoptosis

Chunlai Nie,Changhai Tian,Lixia Zhao,Patrice Xavier Petit,Maryam Mehrpour,Quan Chen

doi:10.1074/jbc.m800847200

Abstract

Bax is activated and translocated onto mitochondria to mediate cytochrome c release and apoptosis. The molecular mechanisms of Bax activation during apoptosis remain a subject of debate. We addressed the question of whether reactive oxygen species could directly activate Bax for its subsequent translocation and apoptosis. Using the SW480 human colon adenocarcinoma cell line stably expressing Bax fused to GFP, we showed that H2O2 induces Bax conformational change, mitochondrial translocation, and subsequent oligomerization at mitochondria. We found that H2O2-induced Bax activation is dependent on the conserved cysteine residue 62 of Bax. Mutation of cysteine 62, but not cysteine 126, to serine or alanine abolished its activation by H2O2 but not other death stimuli, both in SW480 and Bax-deficient HCT116 cells, whereas wild type Bax sensitizes these cells to apoptosis. Cysteines of Bax could chemically react with H2O2. Mutation of Bax BH3 domain in the presence of cysteine 62 also abolished Bax proapoptotic activity. We conclude that reactive oxygen species could be a direct signal for Bax activation by reacting with cysteine residues. Our results identify a critical role of cysteine 62 in oxidative stress-induced Bax activation and subsequent apoptosis.

Highlights

Critical questions remain regarding how apoptotic stimuli and the intracellular environmental cues trigger Bax conformational change and subsequent activation during the onset of apoptosis
Mitochondria are a major source of superoxide anion that can be converted into H2O2; the latter can be released from mitochondria into cytosol
Our results may explain how overexpressing Bcl-xL, which mainly localized at the outer membrane of mitochondria, effectively prevented the Bax conformational change and oligomerization, which only occur in the mitochondrial outer membrane (MOM) in our system

Summary

EXPERIMENTAL PROCEDURES

Materials—H2O2, xanthine (X), xanthine oxidase (XO), N-acetyl-L-cysteine (NAC), campothecin, S-nitroso-Nacetylpenicillamine anti-actin (A-5411), and anti-Bax 6A7 monoclonal antibody were purchased from Sigma. The cell lysates were normalized for protein content, and 250 ␮g of total protein was incubated with 1 ␮g of anti-Bax 6A7 monoclonal antibody in 500 ␮l of Chaps lysis buffer at 4 °C for 3 h or overnight. The beads were washed three times in Chaps lysis buffer, boiled in loading buffer, and the conformationally changed Bax protein in the immunoprecipitates was subjected to SDS-PAGE and immunoblot analysis with anti-Bax polyclonal antibody as described above. Precipitated cells were collected by centrifugation, and the pellet was dissolved in 40 ␮l of reaction buffer (0.1% SDS, 0.67 M Tris-HCl, pH 7.5) supplemented with a mixture of protease inhibitors with 15 mM freshly prepared 4-acetamido-4Ј-maleimidylstilbene-2,2Ј-disulfonic acid (AMS; Molecular Probes). When run on nonreducing SDS-PAGE, proteins in their reduced (alkylated) and oxidized (nonalkylated) forms can be resolved by the increase in molecular weight due to the addition of AMS

RESULTS

B Medium

1: GFP 2: GFP 3: GFP 4

DISCUSSION