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

Alsop, Amber E.; Fennell, Stephanie; Bartolo, Ray C.; Tan, Iris; Dewson, Grant; Kluck, Ruth M.

doi:10.1038/ncomms7841

Cited by 54 publications

(82 citation statements)

References 41 publications

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“…1F). We have previously shown that tethering V142C (α5) to F150C (α6) to constrain "core/latch" dissociation still shows evidence of cBID-induced N-terminal epitope exposure, indicating that cBID still interacts with the tethered form to induce early steps in BAK conformation change (19). However, in contrast with the α1:α2 and α1/2 loop:α6 tethers, tethering α5:6 failed to stabilize the cBID:BAK complex (Fig.…”

Section: Bak Conformation Change Destabilizes the Cbid:bak Interactiomentioning

confidence: 81%

“…To define the step in BAK activation at which cBID dissociates we used additional constraints between the α1-α2, α1-α6, and α5-α6 that we have shown impair defined steps in BAK activation to block cytochrome c release (7,19) (Fig. 1E).…”

Section: Bak Conformation Change Destabilizes the Cbid:bak Interactiomentioning

confidence: 99%

“…Characterizing the interaction of BH3-only proteins with fulllength BAK at mitochondria has proven difficult because the bound BH3-only protein dissociates during consequent BAK conformation changes, including exposure of the N-terminus (amino terminus) and BH3-domain (16)(17)(18)(19) and dissociation of their α2-5 helices ("core") from their α6-8 helices ("latch") (6,7) to facilitate selfassociation. Additionally, structural studies have been largely confined to truncated protein or peptides in the absence of a membrane where interactions between BH3-only proteins and BAK occur.…”

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confidence: 99%

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BAK α6 permits activation by BH3-only proteins and homooligomerization via the canonical hydrophobic groove

Tan

Stephen

et al. 2017

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

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BAK and BAX are the essential effectors of apoptosis because without them a cell is resistant to most apoptotic stimuli. BAK and BAX undergo conformation changes to homooligomerize then permeabilize the mitochondrial outer membrane during apoptosis. How BCL-2 homology 3 (BH3)-only proteins bind to activate BAK and BAX is unclear. We report that BH3-only proteins bind inactive full-length BAK at mitochondria and then dissociate following exposure of the BAK BH3 and BH4 domains before BAK homodimerization. Using a functional obstructive labeling approach, we show that activation of BAK involves important interactions of BH3-only proteins with both the canonical hydrophobic binding groove (α2-5) and α6 at the rear of BAK, with interaction at α6 promoting an open groove to receive a BH3-only protein. Once activated, how BAK homodimers multimerize to form the putative apoptotic pore is unknown. Obstructive labeling of BAK beyond the BH3 domain and hydrophobic groove did not inhibit multimerization and mitochondrial damage, indicating that critical protein-protein interfaces in BAK self-association are limited to the α2-5 homodimerization domain.AK and BAX are the pivotal effectors of intrinsic apoptosis, with one or the other being required for mitochondrial damage and cell death (1, 2). They are activated by interaction with BCL-2 homology 3 (BH3)-only proteins including BID and BIM (3). Structures show that peptides based on the BH3 domains of activator BH3-only proteins can bind directly to BAK and BAX via a hydrophobic groove (comprising α-helices 2-5) that is also shared with their prosurvival homologs (4-8). BAX has been proposed to have an additional activation site, distinct from the hydrophobic groove, at the rear of the molecule comprising α-helices 1 and 6 (9). Binding of stapled BH3 peptides at this site induced the dissociation of the BAX C-terminal transmembrane domain from the hydrophobic groove to facilitate mitochondrial localization (10, 11). Interaction of BH3-only proteins at this noncanonical site is not thought to be necessary for BAK activity because BAK is constitutively anchored in the mitochondrial outer membrane (MOM) via its transmembrane domain (12). However, recent reports that both BAX (13, 14) and BAK (15) are constantly "retrotranslocated" from the mitochondria to the cytosol suggest conserved mechanisms of activation for BAK and BAX.Characterizing the interaction of BH3-only proteins with fulllength BAK at mitochondria has proven difficult because the bound BH3-only protein dissociates during consequent BAK conformation changes, including exposure of the N-terminus (amino terminus) and and dissociation of their α2-5 helices ("core") from their α6-8 helices ("latch") (6, 7) to facilitate selfassociation. Additionally, structural studies have been largely confined to truncated protein or peptides in the absence of a membrane where interactions between BH3-only proteins and BAK occur.Once activated BAK and BAX self-associate to form pores that damage the MOM to release cytochrome c and...

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Section: Bak Conformation Change Destabilizes the Cbid:bak Interactiomentioning

confidence: 81%

Section: Bak Conformation Change Destabilizes the Cbid:bak Interactiomentioning

confidence: 99%

mentioning

confidence: 99%

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BAK α6 permits activation by BH3-only proteins and homooligomerization via the canonical hydrophobic groove

Tan

Stephen

et al. 2017

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

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“…These changes suggest that the α1-α2 loop separates from α1, consistent with increased labelling of A54C in the α1-α2 loop that opposes α1. Thus, increased labelling of cysteine residues placed throughout the N-segment, α1 and the α1-α2 loop, together with exposure of several N-terminal antibody epitopes (Alsop et al, 2015), indicate that the Bak N-terminus becomes completely solvent-exposed after activation ( Figure 1D).…”

Section: The Bakmentioning

confidence: 99%

“…In contrast, nonactivated Bax is largely cytosolic due to binding of α9 to its own hydrophobic groove (Wolter et al, 1997, Gahl et al, 2014. Following binding of BH3-only proteins, both Bak and Bax undergo similar conformation changes including α1 dissociation (Weber et al, 2013, Alsop et al, 2015 and separation of the "latch" domain (α6-α8) from the "core" domain (α2-α5) (Czabotar et al, 2013, Brouwer et al, 2014. The core and α6 then collapse onto the membrane surface and become shallowly inserted into the membrane to lie in-plane (Aluvila, 2014, Bleicken et al, 2014.…”

Section: Introductionmentioning

confidence: 99%

Disordered clusters of Bak dimers rupture mitochondria during apoptosis

Uren

O’Hely

Iyer

et al. 2016

Preprint

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During apoptosis, Bak and Bax undergo major conformational change and form symmetric dimers that coalesce to perforate the mitochondrial outer membrane via an unknown mechanism. We have employed cysteine labelling and linkage analysis to the full length of Bak in mitochondria. This comprehensive survey showed that in each Bak dimer the N-termini are fully solvent-exposed and mobile, the core is highly structured, and the C-termini are flexible but restrained by their contact with the membrane. Dimer-dimer interactions were more labile than the BH3:groove interaction within dimers, suggesting there is no extensive protein interface between dimers. In addition, linkage in the mobile Bak N-terminus (V61C) specifically quantified association between dimers, allowing mathematical simulations of dimer arrangement. Together, our data show that Bak dimers form disordered clusters to generate lipidic pores. These findings provide a molecular explanation for the observed structural heterogeneity of the apoptotic pore.

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Targeted Apoptosis of Ductular Reactive Cells Reduces Hepatic Fibrosis in a Mouse Model of Cholestasis

et al. 2020

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Background and Aims In cholestatic liver diseases, ductular reactive (DR) cells extend into the hepatic parenchyma and promote inflammation and fibrosis. We have previously observed that multidrug‐resistant 2 (Mdr2−/−) double knockout (DKO) mice lacking tumor necrosis factor–related apoptosis‐inducing ligand receptor (Tr−/−) display a more extensive ductular reaction and hepatic fibrosis compared to Mdr2−/− mice. This observation suggests that the magnitude of the DR‐cell population may be regulated by apoptosis. Approach and Results To examine this concept, we cultured epithelial cell adhesion molecule–positive reactive cholangioids (ERCs) obtained from wild‐type (WT), Tr−/−, Mdr2−/− and DKO mice. Single‐cell transcriptomics and immunostaining of both WT and DKO ERCs confirmed their DR‐cell phenotype. Moreover, DKO ERCs displayed a unique translational cluster with expression of chemokines, indicating a reactive state. Incubation with the myeloid cell leukemia 1 (MCL1) inhibitor S63845, a proapoptotic BH3‐mimetic therapy, significantly decreased DKO and Mdr2−/− ERC viability compared to WT. Intravenous administration of S63845 significantly reduced the DR‐cell population and markers of inflammation and liver fibrosis in Mdr2−/− and DKO mice. Furthermore, DKO mice treated with S63845 displayed a significant decrease in hepatic B lymphocytes compared to untreated mice as assessed by high‐definition mass cytometry by time‐of‐flight. Coculture of bone marrow–derived macrophages with ERCs from DKO mouse livers up‐regulated expression of the B cell–directed chemokine (C‐C motif) ligand 5. Finally, DR cells were noted to be primed for apoptosis with Bcl‐2 homologous antagonist/killer activation in vitro and in vivo in primary sclerosing cholangitis liver specimens. Conclusions DR cells appear to play a key role in recruiting immune cells to the liver to actively create an inflammatory and profibrogenic microenvironment. Pharmacologic targeting of MCL1 in a mouse model of chronic cholestasis reduces DR‐cell and B‐cell populations and hepatic fibrosis.

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

Cited by 54 publications

References 41 publications

BAK α6 permits activation by BH3-only proteins and homooligomerization via the canonical hydrophobic groove

BAK α6 permits activation by BH3-only proteins and homooligomerization via the canonical hydrophobic groove

Disordered clusters of Bak dimers rupture mitochondria during apoptosis

Targeted Apoptosis of Ductular Reactive Cells Reduces Hepatic Fibrosis in a Mouse Model of Cholestasis

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