Elucidation of some Bax conformational changes through crystallization of an antibody–peptide complex

Peyerl, Fred W.; Dai, Shaodong; Murphy, Guinevere A.; Crawford, Frances; White, Janicé; Marrack, Philippa; Kappler, John W.

doi:10.1038/sj.cdd.4402025

Cited by 50 publications

(40 citation statements)

References 38 publications

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“…Moreover, the epitopes are in the region of Bax a1 (residues 13-19, 1-21 and 11-30, respectively) that abuts the a6-a8 latch domain [34][35][36] , and the epitopes of polyclonal Bax N20 may even include residues of the Bax BH4 domain ( 26 LLQGFI 31 ). As Bax N-terminal exposure is akin to that for Bak, a1 dissociation is probably also required for Bax activation and pore formation.…”

Section: Discussionmentioning

confidence: 99%

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Dissociation of Bak α1 helix from the core and latch domains is required for apoptosis

et al. 2015

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During apoptosis, Bak permeabilizes mitochondria after undergoing major conformational changes, including poorly defined N-terminal changes. Here, we characterize those changes using 11 antibodies that were epitope mapped using peptide arrays and mutagenesis. After Bak activation by Bid, epitopes throughout the a1 helix are exposed indicating complete dissociation of a1 from a2 in the core and from a6-a8 in the latch. Moreover, disulfide tethering of a1 to a2 or a6 blocks cytochrome c release, suggesting that a1 dissociation is required for further conformational changes during apoptosis. Assaying epitope exposure when a1 is tethered shows that Bid triggers a2 movement, followed by a1 dissociation. However, a2 reaches its final position only after a1 dissociates from the latch. Thus, a1 dissociation is a key step in unfolding Bak into three major components, the N terminus, the core (a2-a5) and the latch (a6-a8).

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Section: Discussionmentioning

confidence: 99%

“…1a). For three of these antibodies , the immunogen contained a stretch of residues (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36) conserved in mouse and human Bak (Table 1). Another two antibodies (4B5 and G23) had previously been mapped to a conserved region 9 .…”

Section: Most Bak Antibodies Have Linear Epitopesmentioning

confidence: 99%

Dissociation of Bak α1 helix from the core and latch domains is required for apoptosis

et al. 2015

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“…17 This was evidenced by confocal immunofluorescence analysis using the conformation-specific 6A7 Bax-antibody (Figure 2a) raised against the NH 2 -terminal epitope of the Bax protein, which is exposed on 'full' Bax activation. 7,18 In all individual cells analysed, YFP-Bax activation (as evidenced by the 6A7 antibody and detected in the red channel, Alexa 594) was associated with a significant clustering of YFP-Bax fluorescence detected in the YFP channel. Similar results were obtained in DU145-Bax cells expressing untagged Bax (Figure 2a; Supplementary Figure 1a).…”

Section: Resultsmentioning

confidence: 99%

Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation

et al. 2009

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Mitochondrial outer membrane permeabilisation (MOMP) during apoptosis is triggered by the activation and oligomerisation of Bax and Bak, but a quantification of these processes in individual cells has not yet been performed. Single-cell imaging of Bax translocation and oligomerisation in Bax-deficient DU-145 cells expressing CFP-Bax and YFP-Bax revealed that both processes started only minutes before or concomitantly with MOMP, with the majority of Bax translocation and oligomerisation occurring downstream of MOMP. Quantification of YFP-Bax concentrations at mitochondria revealed an increase of only 1.8 ± 1.5% at MOMP onset. This was increased to 11.2±3.6% in bak-silenced cells. These data suggested that Bax activation exceeded by far the quantities required for MOMP induction, and that minimal Bax or Bak activation may be sufficient to trigger rapid pore formation. In a cellular automaton modelling approach that incorporated the quantities and movement probabilities of Bax and its inhibitors, activators and enablers in the mitochondrial membrane, we could re-model rapid pore formation kinetics at submaximal Bax activation. Apoptosis is an important physiological cell death process during development, and a key cell death pathway for the elimination of damaged or superfluous cells in the adult. In the 'mitochondrial' or 'intrinsic' apoptosis pathway, the permeabilisation of the mitochondrial outer membrane permeabilisation (MOMP) represents the key decisive process. 1 MOMP enables the release of mitochondrial intermembrane proteins into the cytosol which then activate a family of cytosolic cysteine proteases, the caspases. Bax and Bak are multidomain proapoptotic Bcl-2 family proteins required for the process of MOMP. 2 Although Bak is localised to mitochondria in nonapoptotic cells, large quantities of Bax are found in the cytosol and only translocate to mitochondria during apoptosis. 3 To activate MOMP, Bax and Bak undergo specific conformational changes that enable both proteins to anchor in the mitochondrial outer membrane and to oligomerise. [4][5][6] Although the mechanisms of Bax and Bak activation during apoptosis is still an area of intensive research, evidence is growing that specific proapoptotic proteins of the Bcl-2 protein super-family, in particular the Bcl-2 homology-domain 3 (BH3)-only proteins tBid and Bim, may be able to directly activate Bax and Bak. [5][6][7] Bax and Bak homo-tetramers and higher oligomers are believed to physically form the release channels in the mitochondrial outer membrane large enough to allow for release of intermembrane proteins. 7,8

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“…51 On a different note, one should consider that Bax has an essential, non-apoptotic, homeostatic activity, since it can associate with mitochondria under normal conditions, regulating mitochondrial energy production. 25,26 Indeed, cells deficient in Bax display decreased mitochondrial oxidative capacity and reduced levels of intracellular ATP. 26 These observations would be consistent with the weak or absent mitochondrial-dependent production of ROS in HCT116 Bax -/-.…”

Section: Discussionmentioning

confidence: 99%

“…24 Recently it has been also shown that Bax plays a role in mitochondrial energy production, where its deficiency leads to decreased mitochondrial oxidative capacity and reduced levels of intracellular ATP. 25,26 Also, cysteine residues in Bax can chemically react with ROS, leading to a change in its conformation and subsequent activation. 25,27,28 In this study, using HCT116 Bax +/-and HCT116 Bax -/-cells, 29 we show that PGC1α overexpression induces Bax translocation to mitochondria with a consequent release of cyt c and …”

Section: Pgc1α Induces Bax Activationmentioning

confidence: 99%

Bax is necessary for PGC1α pro-apoptotic effect in colorectal cancer cells

D'Errico¹,

Sasso²,

Salvatore³

et al. 2011

Cell Cycle

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Elucidation of some Bax conformational changes through crystallization of an antibody–peptide complex

Cited by 50 publications

References 38 publications

Dissociation of Bak α1 helix from the core and latch domains is required for apoptosis

Dissociation of Bak α1 helix from the core and latch domains is required for apoptosis

Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation

Bax is necessary for PGC1α pro-apoptotic effect in colorectal cancer cells

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