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

Nie, Chao; Tian, Changhai; Zhao, Lingjuan; Petit, Patrice X.; Mehrpour, Maryam; Chen, Quan

doi:10.1074/jbc.m800847200

Cited by 86 publications

(83 citation statements)

References 35 publications

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“…In silico models predict that homodimerization between cysteine 62 and cysteine 126 allows exposure of the hydrophobic helix 9 (D' Alessio et al, 2005), possibly allowing membrane insertion; this would provide a functional role to oxidative dimerization. In colon adenocarcinoma cells, substitution of cysteine 62, but not 126, abolishes pro-apoptotic activity of Bax in response to H 2 O 2 -induced stress, but not to non-oxidative damage (Nie et al, 2008). Interestingly, in colorectal cancer cells both cysteines are required for Bax activation in selenite-induced apoptosis ).…”

Section: Bax As a Sensor Of Physico-chemical Alterationsmentioning

confidence: 97%

Multistep and multitask Bax activation

Ghibelli

Diederich

2010

Mitochondrion

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Bax is a pro-apoptotic protein allowing apoptosis to occur through the intrinsic, damage-induced pathway, and amplifying that one occurring via the extrinsic, receptor mediated pathway. Bax is present in viable cells and activated by pro-apoptotic stimuli. Activation implies structural changes, consisting of exposure of the N terminus and hydrophobic domains; changes in localization, consisting in migration from cytosol to mitochondria and endoplasmic reticulum membranes; changes in the aggregation status, from monomer to dimer and multimer. Bax has multiple critical domains, namely the N terminus exposed after activation; two hydrophobic stretches exposed for membrane anchorage; two reactive cysteines allowing multimerization; the BH3 domain for interactions with the Bcl-2 family members; alpha helix 1 for t-Bid interaction. Bax has also multiple functions: it releases different mitochondrial factors such as cytochrome c, SMAC/diablo; it regulates mitochondrial fission, the mitochondrial permeability transition pore; it promotes Ca 2+ leakage through ER membrane. Altogether, Bax activation is a complex multi-step phenomenon. Here, we analyze these events as logically separable or alternative steps, attempting to assess their role, timing and reciprocal relation.

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Section: Bax As a Sensor Of Physico-chemical Alterationsmentioning

confidence: 97%

Multistep and multitask Bax activation

Ghibelli

Diederich

2010

Mitochondrion

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“…Early apoptosis and late apoptosis/necrosis were measured using flow cytometry and Annexin V/7-AAD staining. As HCT116 and SW480 cells show low basal levels of apoptosis, measurements were made for cells growing under standard conditions and following incubation with hydrogen peroxide, a potent inducer of programmed cell death (27). As anticipated, treatment with hydrogen peroxide resulted in a significant increase in the proportion of early apoptotic and late apoptotic/necrotic cells when compared with no treatment, but PHLDA1 suppression had no detectable influence under either experimental condition (Fig.…”

Section: Phlda1 Suppression In Colon Cancer Cells Inhibits Anchorage-mentioning

confidence: 99%

PHLDA1 Expression Marks the Putative Epithelial Stem Cells and Contributes to Intestinal Tumorigenesis

et al. 2011

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Studies employing mouse models have identified crypt base and position þ4 cells as strong candidates for intestinal epithelial stem cells. Equivalent cell populations are thought to exist in the human intestine; however robust and specific protein markers are lacking. Here, we show that in the human small and large intestine, PHLDA1 is expressed in discrete crypt base and some position þ4 cells. In small adenomas, PHLDA1 was expressed in a subset of undifferentiated and predominantly Ki-67-negative neoplastic cells, suggesting that a basic hierarchy of differentiation is retained in early tumorigenesis. In large adenomas, carcinomas, and metastases PHLDA1 expression became widespread, with increased expression and nuclear localization at invasive margins. siRNA-mediated suppression of PHLDA1 in colon cancer cells inhibited migration and anchorage-independent growth in vitro and tumor growth in vivo. The integrins ITGA2 and ITGA6 were downregulated in response to PHLDA1 suppression, and accordingly cell adhesion to laminin and collagen was significantly reduced. We conclude that PHLDA1 is a putative epithelial stem cell marker in the human small and large intestine and contributes to migration and proliferation in colon cancer cells. Cancer Res; 71(10);

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“…[3][4][5] For example, in the intrinsic apoptotic pathway, the apoptosis regulator protein Bax has been shown to undergo oxidative activation through disulfide formation; and activation of the initiator caspase Casp9 has been shown to occur via intermolecular disulfide formation with apoptotic protease activating factor 1 (APAF1). 6,7 Interestingly, redox regulation seems to be particularly prevalent amongst transcription factors (TF), where cysteine modifications can control function via several mechanisms. For example, in the AP-1 family of TFs, Cys oxidation has been shown to induce conformational change, inhibiting DNA-binding.…”

mentioning

confidence: 99%

Determination of Protein Thiol Reduction Potential by Isotope Labeling and Intact Mass Measurement

Thurlow

Kilgour

Campopiano³

et al. 2016

Anal. Chem.

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Oxidation/reduction of thiol residues in proteins is an important type of post-translational modification that is implicated in regulating a range of biological processes. The nature of the modification makes it possible to define a quantifiable electrochemical potential, ⊕ , for oxidation/reduction that allows cysteine-containing proteins to be ranked based on their propensity to be oxidized. Measuring oxidation of cysteine residues in proteins is difficult using standard electrochemical methods but recently top-down mass-spectrometry has been shown to enable the quantification of ⊕ for thiol oxidations. In this paper we demonstrate that mass spectrometry of intact proteins can be used in combination with an isotopic labeling strategy and an automated data analysis algorithm to measure ⊕ for the thiols in both E Coli Thioredoxin 1 and Human Thioredoxin 1. Our methodology relies on accurate mass measurement of proteins using LC-MS analyses and does not necessarily require top-down fragmentation. As well as analyzing homogeneous protein samples, we also demonstrate that our methodology can be used to determine thiol ⊕ measurements in samples which contain mixtures of proteins. Thus the combination of experiential methodology and data analysis regime have the potential to make such measurements in a high-throughput manner and in a manner more accessible to a broad community of protein scientists.Over the past three decades oxidative thiol modification has emerged as a central mechanism for dynamic post-translational regulation of protein activity and protein signaling pathways. In 1992, Stamler et al. investigated the S-nitrosylation of protein cysteine thiols as a reversible covalent modification important in vasodilation. 1 In 2000, Finkel published a review article in which he suggested 'the covalent addition of glutathione to reactive cysteines may be one mechanism for achieving reversible redox-dependent signaling as well as a potential means for identifying relevant redox-dependent signaling molecules. 2 Since then, it has become apparent that many important biological pathways are subject to redox regulation through such oxidation-reduction reactions. [3][4][5] For example, in the intrinsic apoptotic pathway, the apoptosis regulator protein Bax has been shown to undergo oxidative activation through disulfide formation; and activation of the initiator caspase Casp9 has been shown to occur via intermolecular disulfide formation with apoptotic protease activating factor 1 (APAF1). 6,7 Interestingly, redox regulation seems to be particularly prevalent amongst transcription factors (TF), where cysteine modifications can control function via several mechanisms. For example, in the AP-1 family of TFs, Cys oxidation has been shown to induce conformational change, inhibiting DNA-binding. 8 In contrast, Nuclear factor-like 2 (NRF2) activity is controlled by oxidation of its partner protein KEAP1; disulfide formation in KEAP1 results in a reduced affinity for NRF2, allowing translocation of NRF2 into the nuc...

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

Cited by 86 publications

References 35 publications

Multistep and multitask Bax activation

Multistep and multitask Bax activation

PHLDA1 Expression Marks the Putative Epithelial Stem Cells and Contributes to Intestinal Tumorigenesis

Determination of Protein Thiol Reduction Potential by Isotope Labeling and Intact Mass Measurement

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