BIM-Mediated Membrane Insertion of the BAK Pore Domain Is an Essential Requirement for Apoptosis

Weber, Kathrin; Harper, Nicholas W.; Schwabe, John W. R.; Cohen, Gerald M.

doi:10.1016/j.celrep.2013.09.010

Cited by 36 publications

(33 citation statements)

References 38 publications

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“…4c). These data support a recent report that tethering WT Bak blocks pore formation in response to heat 25 , and defines a1 dissociation as required for Bak pore formation. To assess which Bak conformation changes were hindered by these tethers, epitope exposure was quantified by flow cytometry.…”

Section: Most Bak Antibodies Have Linear Epitopessupporting

confidence: 91%

“…G317-2 failed to bind V34C, F35C and R36C, consistent with its epitope being at EEVFR. Ab-1 and Ab-2 failed to bind Y38C, Y41C and R42C, consistent with their epitope being within YVFYRHQQ, and with a recent report that Y38 is critical for Ab-1 binding 25 . Thus, the anti-Bak antibodies tested bound to three distinct sites (residues 28, 34-36 and 37-44) along a1.…”

Section: Most Bak Antibodies Have Linear Epitopessupporting

confidence: 88%

“…A similar argument holds for the start of a1, as the NT antibody recognizes only activated Bak 13 and its epitope includes both A28 (Fig. 2d) and T31 25 , which are also buried in non-activated Bak (Supplementary Fig. 3).…”

Section: Most Bak Antibodies Have Linear Epitopesmentioning

confidence: 54%

“…32). Where Bcl-x L has apparently prevented exposure of N-terminal epitopes in Bak or Bax (as measured by immunoprecipitation assays) 17,25,27 , it is likely that Bcl-x L prevented Bak/Bax activation via Mode 1 interactions with BH3-only proteins 27 or that it masked the Bak or Bax epitopes. Three factors hint that a1 may not retain the helical conformation evident in the structure of non-activated Bak 3 .…”

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: Most Bak Antibodies Have Linear Epitopessupporting

confidence: 91%

Section: Most Bak Antibodies Have Linear Epitopessupporting

confidence: 88%

Section: Most Bak Antibodies Have Linear Epitopesmentioning

confidence: 54%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

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

et al. 2015

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“…Very recently, another study reported IASD labeling results on Bak (38). That study used a buffer system similar to ours but used transiently transfected Bak cDNA rather than stable transfection followed by apoptotic signaling.…”

Section: Discussionmentioning

confidence: 99%

Apoptotic pore formation is associated with in-plane insertion of Bak or Bax central helices into the mitochondrial outer membrane

Westphal

Dewson

Ménard

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

106

151

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The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores.cell death | mitochondrial permeabilization | protein-membrane topology | membrane pores | cysteine-scanning mutagenesis C ommitment of cells to apoptosis is determined primarily by interactions within the B-cell lymphoma-2 (Bcl-2) protein family on the mitochondrial outer membrane (MOM) (1-4). The proapoptotic members Bcl-2 antagonist/killer (Bak) and Bcl-2-associated X protein (Bax) mediate the pivotal step of MOM permeabilization, which releases proteins, such as cytochrome c, that promote the proteolytic demolition by caspases. Two other Bcl-2 subfamilies tightly control Bak and Bax activation. Their activation is promoted by the Bcl-2 homology domain 3 (BH3)-only proteins, such as BH3-interacting domain death agonist (Bid), the truncated form of which (tBid) can directly bind both. Conversely, prosurvival family members can bind and inhibit activated Bak and Bax, as well as the BH3-only proteins.Like their prosurvival relatives, Bak and Bax in healthy cells are globular monomers, comprising similar helical bundles with a hydrophobic α-helix (α5) surrounded by amphipathic helices (5, 6). Their C-terminal helix (α9) is a hydrophobic transmembrane (TM) domain that anchors them in the MOM. In healthy cells Bak is already anchored there, presumably solely by α9, whereas Bax is primarily cytosolic (5), accumulating at the MOM after an apoptotic signal and inserting its α9. Other major conformational changes in both Bak and Bax, reviewed in ref 4, include exposure of their BH3 (α2) and its reburial within the surface groove of another activated Bak or Bax molecule (7-10). These novel "symmetric" homo-dimers can multimerize via association of α6 helices (8,11,12).Although oligomeric Bak and Bax are highly implicated in MOM permeabilization, how they interact with the membrane to form pores remains a mystery. The first structure of a Bcl-2 family member, the prosurvival protein Bcl-x L (13), and later those of Bax (5) and Bak (6), provided a tantalizing clue: similarities with the pore-forming domains of bacterial toxins, such as diphtheria toxin or colicin A. To form pores, these toxins are thought to insert their two...

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UMI-77 primes glioma cells for TRAIL-induced apoptosis by unsequestering Bim and Bak from Mcl-1

Liu

Zhu

et al. 2017

Mol Cell Biochem

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Malignant glioma is the most common and aggressive form of brain tumor with poor prognosis of survival. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent but is insufficient of inducing apoptosis in some types of gliomas. In this study, we showed that the small-molecule Mcl-1 inhibitor UMI-77 sensitized glioma cells to TRAIL treatment, as evidenced by cell viability assay, Annexin V staining and JC-1 staining. Combination of UMI-77 and TRAIL in glioma cells led to the activation of caspase-8 and Bid, cleavage of caspase-3 and poly-ADP ribose polymerase (PARP), accumulation of tBid in the mitochondria and release of cytochrome c into the cytosol. UMI-77 alone or in combination with TRAIL untethered pro-apoptotic Bcl-2 proteins Bim and Bak from the sequestration of Mcl-1 and promoted the conformational activation of Bak. Small hairpin RNA (shRNA) of Bid attenuated the cleavage of caspase-8, Bid, caspase-3 and PARP, and reduced the cytotoxicity of UMI-77 plus TRAIL as compared with control shRNA cells, indicating this synergy entails the crosstalk between extrinsic and intrinsic apoptotic signaling. Taken together, UMI-77 enhances TRAIL-induced apoptosis by unsequestering Bim and Bak, which provides a novel therapeutic strategy for the treatment of gliomas.

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BIM-Mediated Membrane Insertion of the BAK Pore Domain Is an Essential Requirement for Apoptosis

Cited by 36 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

Apoptotic pore formation is associated with in-plane insertion of Bak or Bax central helices into the mitochondrial outer membrane

UMI-77 primes glioma cells for TRAIL-induced apoptosis by unsequestering Bim and Bak from Mcl-1

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