Detergent-activated BAX Protein Is a Monomer

Ivashyna, Olena; García‐Sáez, Ana J.; Ries, Jonas; Christenson, Eric T.; Schwille, Petra; Schlesinger, Paul H.

doi:10.1074/jbc.m109.023853

Cited by 27 publications

(22 citation statements)

References 37 publications

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“…This indicates that BAK maintains its global fold with the detergent-induced changes restricted to the residues surrounding its putative BH3-binding hydrophobic groove. This result is consistent with reports that CHAPS has little effect on BAX oligomerization (45).…”

Section: Discussionsupporting

confidence: 83%

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Heterodimerization of BAK and MCL-1 Activated by Detergent Micelles

Liu

Gehring

2010

Journal of Biological Chemistry

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BAK is a key protein mediating mitochondrial outer membrane permeabilization; however, its behavior in the membrane is poorly understood. Here, we characterize the conformational changes in BAK and MCL-1 using detergents to mimic the membrane environment and study their interaction by in vitro pulldown experiments, size exclusion chromatography, titration calorimetry, and NMR spectroscopy. The nonionic detergent IGEPAL has little impact on the structure of MCL-1 but induces a conformational change in BAK, whereby its BH3 region is able to engage the hydrophobic groove of MCL-1. Although the zwitterionic detergent CHAPS induces only minor conformational changes in both proteins, it is still able to initiate heterodimerization. The complex of MCL-1 and BAK can be disrupted by a BID-BH3 peptide, which acts through binding to MCL-1, but a mutant peptide, BAK-BH3-L78A, with low affinity for MCL-1 failed to dissociate the complex. The mutation L78A in BAK prevented binding to MCL-1, thus demonstrating the essential role of the BH3 region of BAK in its regulation by MCL-1. Our results validate the current models for the activation of BAK and highlight the potential value of small molecule inhibitors that target MCL-1 directly.

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Section: Discussionsupporting

confidence: 83%

“…To gain insight into this process, detergents have been used to mimic the native membrane environment of the protein. These studies have shown that BAX can be specifically activated by detergents to form higher order aggregates (24,39,45). Here, we describe the behavior of BAK in the presence of detergents.…”

Section: Discussionmentioning

confidence: 99%

Heterodimerization of BAK and MCL-1 Activated by Detergent Micelles

Liu

Gehring

2010

Journal of Biological Chemistry

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“…However, comparing Bax and Bid under similar conditions and performing concomitantly blue native PAGE and AFM experiments, we showed that detergent dilution provoked disassembly of Bax oligomers down to dimers and that oligomerization is an intrinsic property of Bax. These results clearly prove that the protein to detergent ratio is crucial for oligomer formation and maintenance, consistent with a recent study (46) performed on a truncated version of Bax (Bax⌬C, lacking ␣9). However, these authors concluded that Bax⌬C is mainly monomeric in the presence of detergents or could be at least dimeric in dodecyl maltoside and Triton X-100 (46).…”

Section: Discussionsupporting

confidence: 79%

“…SEC, used extensively to follow oligomerization in Bax and Bak (8,13,23,32,46), showed that the effect of the micelle on oligomer size estimation is difficult to quantify. However, comparing Bax and Bid under similar conditions and performing concomitantly blue native PAGE and AFM experiments, we showed that detergent dilution provoked disassembly of Bax oligomers down to dimers and that oligomerization is an intrinsic property of Bax.…”

Section: Discussionmentioning

confidence: 99%

Molecular Details of Bax Activation, Oligomerization, and Membrane Insertion

Bleicken

Classen

Padmavathi

et al. 2010

Journal of Biological Chemistry

164

179

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Bax and Bid are pro-apoptotic members of the Bcl-2 protein family. Upon cleavage by caspase-8, Bid activates Bax. Activated Bax inserts into the mitochondrial outer membrane forming oligomers which lead to membrane poration, release of cytochrome c, and apoptosis. The detailed mechanism of Bax activation and the topology and composition of the oligomers are still under debate. Here molecular details of Bax activation and oligomerization were obtained by application of several biophysical techniques, including atomic force microscopy, cryoelectron microscopy, and particularly electron paramagnetic resonance (EPR) spectroscopy performed on spin-labeled Bax. Incubation with detergents, reconstitution, and Bid-triggered insertion into liposomes were found to be effective in inducing Bax oligomerization. Bid was shown to activate Bax independently of the stoichiometric ratio, suggesting that Bid has a catalytic function and that the interaction with Bax is transient. The formation of a stable dimerization interface involving two Bcl-2 homology 3 (BH3) domains was found to be the nucleation event for Bax homo-oligomerization. Based on intermolecular distance determined by EPR, a model of six adjacent Bax molecules in the oligomer is presented where the hydrophobic hairpins (helices ␣5 and ␣6) are equally spaced in the membrane and the two BH3 domains are in close vicinity in the dimer interface, separated by >5 nm from the next BH3 pairs.Members of the Bcl-2 protein family are essential players in the complex regulation of apoptosis (1, 2). They are divided into three subgroups: the anti-apoptotic Bcl-2-like proteins, the pro-apoptotic multidomain proteins (Bax and Bak), and the pro-apoptotic BH3 3 -only proteins. To keep programmed cell death under control, Bax activation needs to be strictly regulated, as abnormal cell death is disadvantageous for multicellular organisms.Following cleavage by caspase-8, the BH3-only protein, Bid, is known to activate Bax (3-6). Recently the events involved in BaxBid interaction were investigated by fluorescent techniques (7). Bax is activated through a cascade of conformational changes from being inactive and cytosolic to an oligomeric, membrane-inserted state. In the mitochondrial outer membrane (8, 9) activated Bax is responsible for cytochrome c release and apoptosis initiation (10). Bax oligomerization has been shown to occur also in vitro by incubation with detergents (10 -14).The structures of monomeric Bax and Bid were solved by NMR (14 -16). Bax has a globular fold composed of nine ␣-helices (␣1 to ␣9), with ␣2 representing the BH3 domain and ␣5/␣6 the hydrophobic hairpin (see Fig. 1A). Similarities in structure and function with the monomeric inactive form of the channel-forming domain of bacterial colicins or diphtheria toxins are evident (14, 17). The BH3-only protein, Bid, shows a similar globular fold but lacks ␣8 and ␣9 and has an additional short helix (␣1/2) between ␣1 and ␣2. The structure of active Bax is still unknown, but helices ␣5, ␣6, and ␣9 are reported to in...

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“…During the process of apoptosis induction, Bax undergoes a conformational change (7), permitting Bax to translocate to the mitochondrion, oligomerize, and form pores in the outer mitochondrial membrane, thereby allowing cytochrome c and other pro-apoptotic molecules to escape into the cytoplasm (1,20,23,24,26,47). In the cytosol, these factors activate caspases, which then execute the apoptotic cascade (24,49).…”

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confidence: 99%

p38 MAPK regulates Bax activity and apoptosis in enterocytes at baseline and after intestinal resection

Wakeman

Guo

Santos

et al. 2012

American Journal of Physiology-Gastrointestinal and Liver Physi

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Increased apoptosis in crypt enterocytes is a key feature of intestinal adaptation following massive small bowel resection (SBR). Expression of the proapoptotic factor Bax has been shown to be required for resection-induced apoptosis. It has also been demonstrated that p38-α MAPK (p38) is necessary for Bax activation and apoptosis in vitro. The present studies were designed to test the hypothesis that p38 is a key regulator of Bax activation during adaptation after SBR in vivo. Enterocyte expression of p38 was deleted by tamoxifen administration to activate villin-Cre in adult mice with a floxed Mapk14 (p38-α) gene. Proximal 50% SBR or sham operations were performed on wild-type (WT) and p38 intestinal knockout (p38-IKO) mice under isoflurane anesthesia. Mice were killed 3 or 7 days after operation, and adaptation was analyzed by measuring intestinal morphology, proliferation, and apoptosis. Bax activity was quantified by immunoprecipitation, followed by Western blotting. After SBR, p38-IKO mice had deeper crypts, longer villi, and accelerated proliferation compared with WT controls. Rates of crypt apoptosis were significantly lower in p38-IKO mice, both at baseline and after SBR. Levels of activated Bax were twofold higher in WT mice after SBR relative to sham. In contrast, activated Bax levels were reduced by 67% in mice after p38 MAPK deletion. Deleted p38 expression within the intestinal epithelium leads to enhanced adaptation and reduced levels of enterocyte apoptosis after massive intestinal resection. p38-regulated Bax activation appears to be an important mechanism underlying resection-induced apoptosis.

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Detergent-activated BAX Protein Is a Monomer

Cited by 27 publications

References 37 publications

Heterodimerization of BAK and MCL-1 Activated by Detergent Micelles

Heterodimerization of BAK and MCL-1 Activated by Detergent Micelles

Molecular Details of Bax Activation, Oligomerization, and Membrane Insertion

p38 MAPK regulates Bax activity and apoptosis in enterocytes at baseline and after intestinal resection

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