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PLoS Biology May 2024BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2...
BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial channel protein VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, while interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK and inhibited BAK apoptotic activity. Thus, this VDAC2-BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease or to promote BAK-mediated apoptosis for cancer therapy.
Topics: Voltage-Dependent Anion Channel 2; bcl-2 Homologous Antagonist-Killer Protein; Apoptosis; Humans; Protein Binding; Mitochondria; Animals; HEK293 Cells
PubMed: 38696533
DOI: 10.1371/journal.pbio.3002617 -
International Journal of Molecular... Mar 2023The B-cell lymphoma 2 (Bcl-2) family of proteins is the main regulator of apoptosis. However, multiple emerging evidence has revealed that Bcl-2 family proteins are also... (Review)
Review
The B-cell lymphoma 2 (Bcl-2) family of proteins is the main regulator of apoptosis. However, multiple emerging evidence has revealed that Bcl-2 family proteins are also involved in cellular senescence. On the one hand, the different expression of these proteins determines the entry into senescence. On the other hand, entry into senescence modulates the expression of these proteins, generally conferring resistance to apoptosis. With some exceptions, senescent cells are characterized by the upregulation of antiapoptotic proteins and downregulation of proapoptotic proteins. Under physiological conditions, freshly formed tetraploid cells die by apoptosis due to the tetraploidy checkpoint. However, suppression of Bcl-2 associated x protein (Bax), as well as overexpression of Bcl-2, favors the appearance and survival of tetraploid cells. Furthermore, it is noteworthy that our laboratory has shown that the joint absence of Bax and Bcl-2 antagonist/killer (Bak) favors the entry into senescence of tetraploid cells. Certain microtubule inhibitory chemotherapies, such as taxanes and vinca alkaloids, induce the generation of tetraploid cells. Moreover, the combined use of inhibitors of antiapoptotic proteins of the Bcl-2 family with microtubule inhibitors increases their efficacy. In this review, we aim to shed light on the involvement of the Bcl-2 family of proteins in the senescence program activated after tetraploidization and the possibility of using this knowledge to create a new therapeutic strategy targeting cancer cells.
Topics: Humans; Proto-Oncogene Proteins c-bcl-2; bcl-2-Associated X Protein; bcl-2 Homologous Antagonist-Killer Protein; Tetraploidy; Apoptosis Regulatory Proteins; Lymphoma, B-Cell; Apoptosis; bcl-X Protein
PubMed: 37047342
DOI: 10.3390/ijms24076374 -
Cell Death and Differentiation Feb 2014The central role of the Bcl-2 family in regulating apoptotic cell death was first identified in the 1980s. Since then, significant in-roads have been made in identifying... (Review)
Review
The central role of the Bcl-2 family in regulating apoptotic cell death was first identified in the 1980s. Since then, significant in-roads have been made in identifying the multiple members of this family, characterizing their form and function and understanding how their interactions determine whether a cell lives or dies. In this review we focus on the recent progress made in characterizing the proapoptotic Bcl-2 family members, Bax and Bak. This progress has resolved longstanding controversies, but has also challenged established theories in the apoptosis field. We will discuss different models of how these two proteins become activated and different 'modes' by which they are inhibited by other Bcl-2 family members. We will also discuss novel conformation changes leading to Bak and Bax oligomerization and speculate how these oligomers might permeabilize the mitochondrial outer membrane.
Topics: Apoptosis; Humans; Mitochondrial Membranes; Protein Binding; Protein Multimerization; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 24162660
DOI: 10.1038/cdd.2013.139 -
Structure (London, England : 1993) Mar 2023Apoptosis is important for development and tissue homeostasis, and its dysregulation can lead to diseases, including cancer. As an apoptotic effector, BAK undergoes...
Apoptosis is important for development and tissue homeostasis, and its dysregulation can lead to diseases, including cancer. As an apoptotic effector, BAK undergoes conformational changes that promote mitochondrial outer membrane disruption, leading to cell death. This is termed "activation" and can be induced by peptides from the human proteins BID, BIM, and PUMA. To identify additional peptides that can regulate BAK, we used computational protein design, yeast surface display screening, and structure-based energy scoring to identify 10 diverse new binders. We discovered peptides from the human proteins BNIP5 and PXT1 and three non-native peptides that activate BAK in liposome assays and induce cytochrome c release from mitochondria. Crystal structures and binding studies reveal a high degree of similarity among peptide activators and inhibitors, ruling out a simple function-determining property. Our results shed light on the vast peptide sequence space that can regulate BAK function and will guide the design of BAK-modulating tools and therapeutics.
Topics: Humans; Proto-Oncogene Proteins; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; bcl-X Protein; bcl-2 Homologous Antagonist-Killer Protein; Apoptosis; Peptides; bcl-2-Associated X Protein; Proto-Oncogene Proteins c-bcl-2
PubMed: 36706751
DOI: 10.1016/j.str.2023.01.001 -
Scientific Reports Feb 2018Pressure-induced injury (PI), such as a pressure ulcer, in patients with limited mobility is a healthcare issue worldwide. PI is an injury to skin and its underlying...
Pressure-induced injury (PI), such as a pressure ulcer, in patients with limited mobility is a healthcare issue worldwide. PI is an injury to skin and its underlying tissue such as skeletal muscle. Muscle compression, composed of mechanical deformation of muscle and external load, leads to localized ischemia and subsequent unloading reperfusion and, hence, a pressure ulcer in bed-bound patients. Although the gross factors involved in PI have been identified, little is known about the exact disease mechanism or its links to apoptosis, autophagy and inflammation. Here, we report that PI is mediated by intrinsic apoptosis and exacerbated by autophagy. Conditional ablation of Bax and Bak activates the Akt-mTOR pathway and Bnip3-mediated mitophagy and preserves mitochondrial contents in compressed muscle. Moreover, we find that the presence/absence of Bax and Bak alters the roles and functions of autophagy in PI. Our results suggest that manipulating apoptosis and autophagy are potential therapeutic targets for treatment and prevention of PI.
Topics: Animals; Blotting, Western; Cell Death; Immunoprecipitation; Male; Mice; Mice, Knockout; Muscle, Skeletal; Pressure; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 29487339
DOI: 10.1038/s41598-018-21853-5 -
Proceedings of the National Academy of... Mar 2013Proapoptotic B-cell lymphoma 2 (BCL-2) antagonist/killer (BAK) and BCL-2-associated X (BAX) form toxic mitochondrial pores in response to cellular stress. Whereas BAX...
Proapoptotic B-cell lymphoma 2 (BCL-2) antagonist/killer (BAK) and BCL-2-associated X (BAX) form toxic mitochondrial pores in response to cellular stress. Whereas BAX resides predominantly in the cytosol, BAK is constitutively localized to the outer mitochondrial membrane. Select BCL-2 homology domain 3 (BH3) helices activate BAX directly by engaging an α1/α6 trigger site. The inability to express full-length BAK has hampered full dissection of its activation mechanism. Here, we report the production of full-length, monomeric BAK by mutagenesis-based solubilization of its C-terminal α-helical surface. Recombinant BAK autotranslocates to mitochondria but only releases cytochrome c upon BH3 triggering. A direct activation mechanism was explicitly demonstrated using a liposomal system that recapitulates BAK-mediated release upon addition of BH3 ligands. Photoreactive BH3 helices mapped both triggering and autointeractions to the canonical BH3-binding pocket of BAK, whereas the same ligands crosslinked to the α1/α6 site of BAX. Thus, activation of both BAK and BAX is initiated by direct BH3-interaction but at distinct trigger sites. These structural and biochemical insights provide opportunities for developing proapoptotic agents that activate the death pathway through direct but differential engagement of BAK and BAX.
Topics: Animals; Apoptosis; Humans; Mice; Mice, Knockout; Mitochondria, Liver; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Transport; Recombinant Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 23404709
DOI: 10.1073/pnas.1214313110 -
Cell Death and Differentiation Mar 2018The prosurvival Bcl-2 family proteins Mcl-1 and Bcl-x inhibit apoptosis by sequestering BH3-only proteins such as Bid and Bim (MODE 1) or the effector proteins Bak and...
The prosurvival Bcl-2 family proteins Mcl-1 and Bcl-x inhibit apoptosis by sequestering BH3-only proteins such as Bid and Bim (MODE 1) or the effector proteins Bak and Bax (MODE 2). To better understand the contributions of MODE 1 and MODE 2 in blocking cell death, and thus how to bypass resistance to cell death, we examined prescribed mixtures of Bcl-2 family proteins. In a Bim and Bak mixture, Bcl-x and Mcl-1 each sequestered not only Bim but also Bak as it became activated by Bim. In contrast, in a Bid and Bak mixture, Bcl-x preferentially sequestered Bid while Mcl-1 preferentially sequestered Bak. Notably, Bcl-x could sequester Bak in response to the BH3 mimetic ABT-737, despite this molecule targeting Bcl-x. These findings highlight the importance of Bak sequestration in resistance to anti-cancer treatments, including BH3 mimetics.
Topics: Animals; Biphenyl Compounds; Mice; Mice, Knockout; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Sulfonamides; bcl-2 Homologous Antagonist-Killer Protein; bcl-X Protein
PubMed: 29459767
DOI: 10.1038/s41418-017-0010-6 -
Cell Death & Disease Sep 2020Intrinsic apoptosis relies on the ability of the BCL-2 family to induce the formation of pores on the outer mitochondrial membrane. Previous studies have shown that both...
Intrinsic apoptosis relies on the ability of the BCL-2 family to induce the formation of pores on the outer mitochondrial membrane. Previous studies have shown that both BAX and BAK are essential during murine embryogenesis, and reports in human cancer cell lines identified non-canonical roles for BAX and BAK in mitochondrial fission during apoptosis. BAX and BAK function in human brain development remains elusive due to the lack of appropriate model systems. Here, we generated BAX/BAK double knockout human-induced pluripotent stem cells (hiPSCs), hiPSC-derived neural progenitor cells (hNPCs), neural rosettes, and cerebral organoids to uncover the effects of BAX and BAK deletion in an in vitro model of early human brain development. We found that BAX and BAK-deficient cells have abnormal mitochondrial morphology and give rise to aberrant cortical structures. We suggest crucial functions for BAX and BAK during human development, including maintenance of homeostatic mitochondrial morphology, which is crucial for proper development of progenitors and neurons of the cortex. Human pluripotent stem cell-derived systems can be useful platforms to reveal novel functions of the apoptotic machinery in neural development.
Topics: Animals; Apoptosis; Brain; Cell Differentiation; Disease Models, Animal; Humans; Induced Pluripotent Stem Cells; Male; Mice; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 32978370
DOI: 10.1038/s41419-020-03002-x -
Molecular Cell May 2021A body of data supports the existence of core (α2-α5) dimers of BAK and BAX in the oligomeric, membrane-perturbing conformation of these essential apoptotic effector...
A body of data supports the existence of core (α2-α5) dimers of BAK and BAX in the oligomeric, membrane-perturbing conformation of these essential apoptotic effector molecules. Molecular structures for these dimers have only been captured for truncated constructs encompassing the core domain alone. Here, we report a crystal structure of BAK α2-α8 dimers (i.e., minus its flexible N-terminal helix and membrane-anchoring C-terminal segment) that has been obtained through the activation of monomeric BAK with the detergent C12E8. Core dimers are evident, linked through the crystal by contacts via latch (α6-α8) domains. This crystal structure shows activated BAK dimers with the extended latch domain present. Our data provide direct evidence for the conformational change converting BAK from inert monomer to the functional dimer that destroys mitochondrial integrity. This dimer is the smallest functional unit for recombinant BAK or BAX described so far.
Topics: Amino Acid Sequence; Animals; Detergents; Liposomes; Mice, Inbred C57BL; Mice, Knockout; Models, Molecular; Protein Multimerization; Protein Structure, Secondary; bcl-2 Homologous Antagonist-Killer Protein; Mice
PubMed: 33794146
DOI: 10.1016/j.molcel.2021.03.014 -
The EMBO Journal Feb 2007Mitochondrial outer membrane permeabilization (MOMP) and release of mitochondrial intermembrane proteins like cytochrome c are critical steps in the control of... (Comparative Study)
Comparative Study
Mitochondrial outer membrane permeabilization (MOMP) and release of mitochondrial intermembrane proteins like cytochrome c are critical steps in the control of apoptosis. Previous work has shown that MOMP depends on the functionally redundant multidomain proapoptotic proteins, Bak and Bax. Here we demonstrate that Bak and Bax are functionally non-redundant during Neisseria gonorrhoeae (Ngo)- and cisplatin-induced apoptosis. While the activation of Bak is caspase independent Bax activation needs Bak and active caspases. Silencing of either Bak or Bax resists both Ngo- and cisplatin- but not TNFalpha-induced apoptosis. Activation of Bak is required to release cytochrome c from the mitochondria; however, Bax is still required to activate effector caspases. Thus, both Bak and Bax are necessary to accomplish DNA damage and Ngo-induced apoptosis.
Topics: Apoptosis; Blotting, Western; Caspases; Cell Fractionation; DNA Damage; DNA Primers; Flow Cytometry; HeLa Cells; Humans; Immunoprecipitation; Intracellular Membranes; Microscopy, Fluorescence; Mitochondria; Neisseria gonorrhoeae; Permeability; RNA Interference; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 17235284
DOI: 10.1038/sj.emboj.7601533