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Nature Communications Jul 2020Many cellular stresses are transduced into apoptotic signals through modification or up-regulation of the BH3-only subfamily of BCL2 proteins. Through direct or indirect...
Many cellular stresses are transduced into apoptotic signals through modification or up-regulation of the BH3-only subfamily of BCL2 proteins. Through direct or indirect mechanisms, these proteins activate BAK and BAX to permeabilize the mitochondrial outer membrane. While the BH3-only proteins BIM, PUMA, and tBID have been confirmed to directly activate BAK through its canonical BH3 binding groove, whether the BH3-only proteins BMF, HRK or BIK can directly activate BAK is less clear. Here we show that BMF and HRK bind and directly activate BAK. Through NMR studies, site-directed mutagenesis, and advanced molecular dynamics simulations, we also find that BAK activation by BMF and possibly HRK involves a previously unrecognized binding groove formed by BAK α4, α6, and α7 helices. Alterations in this groove decrease the ability of BMF and HRK to bind BAK, permeabilize membranes and induce apoptosis, suggesting a potential role for this BH3-binding site in BAK activation.
Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Animals; Apoptosis Regulatory Proteins; Binding Sites; Cells, Cultured; Humans; Jurkat Cells; Magnetic Resonance Spectroscopy; Mice, Knockout; Mitochondrial Membranes; Molecular Dynamics Simulation; Mutation; Protein Binding; Protein Domains; Proto-Oncogene Proteins c-bcl-2; Sequence Homology, Amino Acid; bcl-2 Homologous Antagonist-Killer Protein
PubMed: 32620849
DOI: 10.1038/s41467-020-17074-y -
Oncotarget Oct 2016
Topics: Cell Death; Proto-Oncogene Proteins c-bcl-2; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 27626173
DOI: 10.18632/oncotarget.11948 -
The EMBO Journal Aug 2022The intrinsic apoptosis pathway, regulated by the BCL-2 protein family, is essential for embryonic development. Using mice lacking all known apoptosis effectors, BAX,...
The intrinsic apoptosis pathway, regulated by the BCL-2 protein family, is essential for embryonic development. Using mice lacking all known apoptosis effectors, BAX, BAK and BOK, we have previously defined the processes during development that require apoptosis. Rare Bok Bax Bak triple knockout (TKO) mice developed to adulthood and several tissues that were thought to require apoptosis during development appeared normal. This raises the question if all apoptosis had been abolished in the TKO mice or if other BCL-2 family members could act as effectors of apoptosis. Here, we investigated the role of BID, generally considered to link the extrinsic and intrinsic apoptosis pathways, acting as a BH3-only protein initiating apoptosis upstream of BAX and BAK. We found that Bok Bax Bak Bid quadruple knockout (QKO) mice have additional developmental anomalies compared to TKO mice, consistent with a role of BID, not only upstream but also in parallel to BAX, BAK and BOK. Mitochondrial experiments identified a small cytochrome c-releasing activity of full-length BID. Collectively, these findings suggest a new effector role for BID in the intrinsic apoptosis pathway.
Topics: Animals; Mice; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Embryonic Development; Mice, Knockout; Proto-Oncogene Proteins c-bcl-2
PubMed: 35758142
DOI: 10.15252/embj.2021110300 -
Cell Death and Differentiation Sep 2022Pro-apoptotic BAK and BAX are activated by BH3-only proteins to permeabilise the outer mitochondrial membrane. The antibody 7D10 also activates BAK on mitochondria and...
Pro-apoptotic BAK and BAX are activated by BH3-only proteins to permeabilise the outer mitochondrial membrane. The antibody 7D10 also activates BAK on mitochondria and its epitope has previously been mapped to BAK residues in the loop connecting helices α1 and α2 of BAK. A crystal structure of the complex between the Fv fragment of 7D10 and the BAK mutant L100A suggests a possible mechanism of activation involving the α1-α2 loop residue M60. M60 mutants of BAK have reduced stability and elevated sensitivity to activation by BID, illustrating that M60, through its contacts with residues in helices α1, α5 and α6, is a linchpin stabilising the inert, monomeric structure of BAK. Our data demonstrate that BAK's α1-α2 loop is not a passive covalent connector between secondary structure elements, but a direct restraint on BAK's activation.
Topics: Antibodies; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Mitochondrial Membranes; Protein Structure, Secondary; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 35279694
DOI: 10.1038/s41418-022-00961-w -
Cell Death and Differentiation Nov 2018Bax is a Bcl-2 protein critical for apoptosis induction. In healthy cells, Bax is mostly a monomeric, cytosolic protein, while upon apoptosis initiation it inserts into...
Bax is a Bcl-2 protein critical for apoptosis induction. In healthy cells, Bax is mostly a monomeric, cytosolic protein, while upon apoptosis initiation it inserts into the outer mitochondrial membrane, oligomerizes, and forms pores that release proapoptotic factors like Cytochrome c into the cytosol. The structures of active Bax and its homolog Bak are only partially understood and the topology of the proteins with respect to the membrane bilayer is controversially described in the literature. Here, we systematically review and examine the protein-membrane, protein-water, and protein-protein contacts of the nine helices of active Bax and Bak, and add a new set of topology data obtained by fluorescence and EPR methods. We conclude based on the consistent part of the datasets that the core/dimerization domain of Bax (Bak) is water exposed with only helices 4 and 5 in membrane contact, whereas the piercing/latch domain is in peripheral membrane contact, with helix 9 being transmembrane. Among the available structural models, those considering the dimerization/core domain at the rim of a toroidal pore are the most plausible to describe the active state of the proteins, although the structural flexibility of the piercing/latch domain does not allow unambiguous discrimination between the existing models.
Topics: Dimerization; Humans; Mitochondrial Membranes; Protein Conformation, alpha-Helical; Protein Structure, Tertiary; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 30185826
DOI: 10.1038/s41418-018-0184-6 -
Cell Death & Disease Feb 2020Despite the introduction of novel targeted therapies, chemotherapy still remains the primary treatment for metastatic melanoma in poorly funded healthcare environments...
Despite the introduction of novel targeted therapies, chemotherapy still remains the primary treatment for metastatic melanoma in poorly funded healthcare environments or in case of disease relapse, with no reliable molecular markers for progression-free survival (PFS) available. As chemotherapy primarily eliminates cancer cells by apoptosis, we here evaluated if the expression of key apoptosis regulators (Bax, Bak, Bcl-2, Bcl-xL, Smac, Procaspase-9, Apaf-1, Procaspase-3 and XIAP) allows prognosticating PFS in stage III/IV melanoma patients. Following antibody validation, marker expression was determined by automated and manual scoring of immunohistochemically stained tissue microarrays (TMAs) constructed from treatment-naive metastatic melanoma biopsies. Interestingly and counter-intuitively, low expression of the pro-apoptotic proteins Bax, Bak and Smac indicated better prognosis (log-rank p < 0.0001, p = 0.0301 and p = 0.0227 for automated and p = 0.0422, p = 0.0410 and p = 0.0073 for manual scoring). These findings were independently validated in the cancer genome atlas (TCGA) metastatic melanoma cohort (TCGA-SKCM) at transcript level (log-rank p = 0.0004, p = 0.0104 and p = 0.0377). Taking expression heterogeneity between the markers in individual tumour samples into account allowed defining combinatorial Bax, Bak, Smac signatures that were associated with significantly increased PFS (p = 0.0002 and p = 0.0028 at protein and transcript level, respectively). Furthermore, combined low expression of Bax, Bak and Smac allowed predicting prolonged PFS (> 12 months) on a case-by-case basis (area under the receiver operating characteristic curve (ROC AUC) = 0.79). Taken together, our results therefore suggest that Bax, Bak and Smac jointly define a signature with potential clinical utility in chemotherapy-treated metastatic melanoma.
Topics: Aged; Antineoplastic Agents; Apoptosis Regulatory Proteins; Biomarkers, Tumor; Down-Regulation; Female; Gene Expression Profiling; Humans; Image Interpretation, Computer-Assisted; Immunohistochemistry; Male; Melanoma; Middle Aged; Mitochondrial Proteins; Pattern Recognition, Automated; Predictive Value of Tests; Progression-Free Survival; Skin Neoplasms; Time Factors; Tissue Array Analysis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 32054850
DOI: 10.1038/s41419-020-2309-3 -
Neurotoxicity Research Aug 2021Paraquat, a superoxide generator, can damage the cochlea causing an ototoxic hearing loss. The purpose of the study was to determine if deletion of Bak, a pro-apoptotic...
Paraquat, a superoxide generator, can damage the cochlea causing an ototoxic hearing loss. The purpose of the study was to determine if deletion of Bak, a pro-apoptotic gene, would reduce paraquat ototoxicity or if deletion of Sirt3, which delays age-related hearing loss under caloric restriction, would increase paraquat ototoxicity. We tested these two hypotheses by treating postnatal day 3 cochlear cultures from Bak, Bak, Sirt3, Sirt3, and WT mice with paraquat and compared the results to a standard rat model of paraquat ototoxicity. Paraquat damaged nerve fibers and dose-dependently destroyed rat outer hair cells (OHCs) and inner hair cells (IHCs). Rat hair cell loss began in the base of the cochlea with a 10 μM dose and as the dose increased from 50 to 500 μM, the hair cell loss increased near the base of the cochlea and spread toward the apex of the cochlea. Rat OHC losses were consistently greater than IHC losses. Unexpectedly, in all mouse genotypes, paraquat-induced hair cell lesions were maximal near the apex of the cochlea and minimal near the base. This unusual damage gradient is opposite to that seen in paraquat-treated rats and in mice and rats treated with other ototoxic drugs. However, paraquat always induced greater OHC loss than IHC loss in all mouse strains. Contrary to our hypothesis, Bak deficient mice were more vulnerable to paraquat ototoxicity than WT mice (Bak > Bak > WT), suggesting that Bak plays a protective role against hair cell stress. Also, contrary to expectation, Sirt3-deficient mice did not differ significantly from WT mice, possibly due to the fact that Sirt3 was not experimentally upregulated in Sirt3-expressing mice prior to paraquat treatment. Our results show for the first time a gradient of ototoxic damage in mice that is greater in the apex than the base of the cochlea.
Topics: Animals; Animals, Newborn; Cells, Cultured; Cochlea; Dose-Response Relationship, Drug; Female; Hair Cells, Auditory, Inner; Hair Cells, Auditory, Outer; Herbicides; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Organ Culture Techniques; Paraquat; Rats; Rats, Sprague-Dawley; Sirtuin 3; bcl-2 Homologous Antagonist-Killer Protein
PubMed: 33900547
DOI: 10.1007/s12640-021-00366-6 -
Cell Death and Differentiation Mar 2023Intrinsic apoptosis is principally governed by the BCL-2 family of proteins, but some non-BCL-2 proteins are also critical to control this process. To identify novel...
Intrinsic apoptosis is principally governed by the BCL-2 family of proteins, but some non-BCL-2 proteins are also critical to control this process. To identify novel apoptosis regulators, we performed a genome-wide CRISPR-Cas9 library screen, and it identified the mitochondrial E3 ubiquitin ligase MARCHF5/MITOL/RNF153 as an important regulator of BAK apoptotic function. Deleting MARCHF5 in diverse cell lines dependent on BAK conferred profound resistance to BH3-mimetic drugs. The loss of MARCHF5 or its E3 ubiquitin ligase activity surprisingly drove BAK to adopt an activated conformation, with resistance to BH3-mimetics afforded by the formation of inhibitory complexes with pro-survival proteins MCL-1 and BCL-XL. Importantly, these changes to BAK conformation and pro-survival association occurred independently of BH3-only proteins and influence on pro-survival proteins. This study identifies a new mechanism by which MARCHF5 regulates apoptotic cell death by restraining BAK activating conformation change and provides new insight into how cancer cells respond to BH3-mimetic drugs. These data also highlight the emerging role of ubiquitin signalling in apoptosis that may be exploited therapeutically.
Topics: bcl-X Protein; bcl-2 Homologous Antagonist-Killer Protein; Ubiquitin-Protein Ligases; Myeloid Cell Leukemia Sequence 1 Protein; Apoptosis; Proto-Oncogene Proteins c-bcl-2
PubMed: 36171332
DOI: 10.1038/s41418-022-01067-z -
Cell Death and Differentiation Nov 2012The Bcl-2 family of proteins is formed by pro- and antiapoptotic members. Together they regulate the permeabilization of the mitochondrial outer membrane, a key step in... (Review)
Review
The Bcl-2 family of proteins is formed by pro- and antiapoptotic members. Together they regulate the permeabilization of the mitochondrial outer membrane, a key step in apoptosis. Their complex network of interactions both in the cytosol and on mitochondria determines the fate of the cell. In the past 2 decades, the members of the family have been identified and classified according to their function. Several competing models have been proposed to explain how the Blc-2 proteins orchestrate apoptosis signaling. However, basic aspects of the action of these proteins remain elusive. This review is focused on the biophysical mechanisms that are relevant for their action in apoptosis and on the challenging gaps in our knowledge that necessitate further exploration to finally understand how the Bcl-2 family regulates apoptosis.
Topics: Apoptosis; Cell Membrane Permeability; Mitochondrial Membranes; Proto-Oncogene Proteins c-bcl-2; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 22935609
DOI: 10.1038/cdd.2012.105 -
Cell Death & Disease Apr 2023Chimeric antigen receptor (CAR) T cell therapy is an FDA-approved treatment for several hematologic malignancies, yet not all patients respond to this treatment. While...
Chimeric antigen receptor (CAR) T cell therapy is an FDA-approved treatment for several hematologic malignancies, yet not all patients respond to this treatment. While some resistance mechanisms have been identified, cell death pathways in target cancer cells remain underexplored. Impairing mitochondrial apoptosis via knockout of Bak and Bax, forced Bcl-2 and Bcl-XL expression, or caspase inhibition protected several tumor models from CAR T killing. However, impairing mitochondrial apoptosis in two liquid tumor cell lines did not protect target cells from CAR T killing. We found that whether a cell was Type I or Type II in response to death ligands explained the divergence of these results, so that mitochondrial apoptosis was dispensable for CART killing of cells that were Type I but not Type II. This suggests that the apoptotic signaling induced by CAR T cells bears important similarities to that induced by drugs. Combinations of drug and CAR T therapies will therefore require tailoring to the specific cell death pathways activated by CAR T cells in different types of cancer cells.
Topics: Humans; Receptors, Chimeric Antigen; Apoptosis; Caspases; Cell Line, Tumor; bcl-2 Homologous Antagonist-Killer Protein; Neoplasms
PubMed: 37055388
DOI: 10.1038/s41419-023-05727-x