RESUMO
BAX and BAK are pro-apoptotic members of the BCL2 family that are required to permeabilize the mitochondrial outer membrane. The proteins can adopt a non-activated monomeric conformation, or an activated conformation in which the exposed BH3 domain facilitates binding either to a prosurvival protein or to another activated BAK or BAX protein to promote pore formation. Certain cancer cells are proposed to have high levels of activated BAK sequestered by MCL1 or BCLXL, thus priming these cells to undergo apoptosis in response to BH3 mimetic compounds that target MCL1 or BCLXL. Here we report the first antibody, 14G6, that is specific for the non-activated BAK conformer. A crystal structure of 14G6 Fab bound to BAK revealed a binding site encompassing both the α1 helix and α5-α6 hinge regions of BAK, two sites involved in the unfolding of BAK during its activation. In mitochondrial experiments, 14G6 inhibited BAK unfolding triggered by three diverse BAK activators, supporting crucial roles for both α1 dissociation and separation of the core (α2-α5) and latch (α6-α9) regions in BAK activation. 14G6 bound the majority of BAK in several leukaemia cell lines, and binding decreased following treatment with BH3 mimetics, indicating only minor levels of constitutively activated BAK in those cells. In summary, 14G6 provides a new means of assessing BAK status in response to anti-cancer treatments.
Assuntos
Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Humanos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias/patologia , Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Animais , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidoresRESUMO
BH3-mimetic drugs are an anti-cancer therapy that can induce apoptosis in malignant cells by directly binding and inhibiting pro-survival proteins of the BCL-2 family. The BH3-mimetic drug venetoclax, which targets BCL-2, has been approved for the treatment of chronic lymphocytic leukaemia and acute myeloid leukaemia by regulatory authorities worldwide. However, while most patients initially respond well, resistance and relapse while on this drug is an emerging and critical issue in the clinic. Though some studies have begun uncovering the factors involved in resistance to BCL-2-targeting BH3-mimetic drugs, little focus has been applied to pre-emptively tackle resistance for the next generation of BH3-mimetic drugs targeting MCL-1, which are now in clinical trials for diverse blood cancers. Therefore, using pre-clinical mouse and human models of aggressive lymphoma, we sought to predict factors likely to contribute to the development of resistance in patients receiving MCL-1-targeting BH3-mimetic drugs. First, we performed multiple whole genome CRISPR/Cas9 KO screens and identified that loss of the pro-apoptotic effector protein BAX, but not its close relative BAK, could confer resistance to MCL-1-targeting BH3-mimetic drugs in both short-term and long-term treatment regimens, even in lymphoma cells lacking the tumour suppressor TRP53. Furthermore, we found that mouse Eµ-Myc lymphoma cells selected for loss of BAX, as well as upregulation of the untargeted pro-survival BCL-2 family proteins BCL-XL and A1, when made naturally resistant to MCL-1 inhibitors by culturing them in increasing doses of drug over time, a situation mimicking the clinical application of these drugs. Finally, we identified therapeutic approaches which could overcome these two methods of resistance: the use of chemotherapeutic drugs or combined BH3-mimetic treatment, respectively. Collectively, these results uncover some key factors likely to cause resistance to MCL-1 inhibition in the clinic and suggest rational therapeutic strategies to overcome resistance that should be investigated further.
Assuntos
Antineoplásicos , Proteínas Proto-Oncogênicas c-bcl-2 , Humanos , Animais , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proteína bcl-X/metabolismoRESUMO
Despite their common use in research, monoclonal antibodies are currently not systematically sequenced. This can lead to issues with reproducibility and the occasional loss of antibodies with loss of cell lines. Hybridoma cell lines have been the primary means of generating monoclonal antibodies from immunized animals, including mice, rats, rabbits, and alpacas. Excluding therapeutic antibodies, few hybridoma-derived antibody sequences are known. Sanger sequencing has been "the gold standard" for antibody gene sequencing, but this method relies on the availability of species-specific degenerate primer sets for amplification of light and heavy antibody genes and it requires lengthy and expensive cDNA preparation. Here, we leveraged recent improvements in long-read Oxford Nanopore Technologies (ONT) sequencing to develop Nanopore Antibody sequencing (NAb-seq): a three-day, species-independent, and cost-effective workflow to characterize paired full-length immunoglobulin light- and heavy-chain genes from hybridoma cell lines. When compared to Sanger sequencing of two hybridoma cell lines, long-read ONT sequencing was highly accurate, reliable, and amenable to high throughput. We further show that the method is applicable to single cells, allowing efficient antibody discovery in rare populations such as memory B cells. In summary, NAb-seq promises to accelerate identification and validation of hybridoma antibodies as well as antibodies from single B cells used in research, diagnostics, and therapeutics.
Assuntos
Anticorpos Monoclonais , Sequenciamento de Nucleotídeos em Larga Escala , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Linhagem Celular , Análise Custo-Benefício , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hibridomas/metabolismo , Camundongos , Coelhos , Ratos , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteínas Proto-Oncogênicas c-bcl-2 , Proteína Killer-Antagonista Homóloga a bcl-2 , Animais , Camundongos , Apoptose , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Desenvolvimento Embrionário/genética , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Glioblastoma multiforme (GBM) is the most common and aggressive form of brain cancer, with treatment options often constrained due to inherent resistance of malignant cells to conventional therapy. We investigated the impact of triggering programmed cell death (PCD) by using BH3 mimetic drugs in human GBM cell lines. We demonstrate that co-targeting the pro-survival proteins BCL-XL and MCL-1 was more potent at killing six GBM cell lines compared to conventional therapy with Temozolomide or the bromodomain inhibitor JQ1 in vitro. Enhanced cell killing was observed in U251 and SNB-19 cells in response to dual treatment with TMZ or JQ1 combined with a BCL-XL inhibitor, compared to single agent treatment. This was reflected in abundant cleavage/activation of caspase-3 and cleavage of PARP1, markers of apoptosis. U251 and SNB-19 cells were more readily killed by a combination of BH3 mimetics targeting BCL-XL and MCL-1 as opposed to dual treatment with the BCL-2 inhibitor Venetoclax and a BCL-XL inhibitor. The combined loss of BAX and BAK, the essential executioners of intrinsic apoptosis, rendered U251 and SNB-19 cells refractory to any of the drug combinations tested, demonstrating that apoptosis is responsible for their killing. In an orthotopic mouse model of GBM, we demonstrate that the BCL-XL inhibitor A1331852 can penetrate the brain, with A1331852 detected in both tumour and healthy brain regions. We also investigated the impact of combining small molecule inducers of ferroptosis, erastin and RSL3, with BH3 mimetic drugs. We found that a BCL-XL or an MCL-1 inhibitor potently cooperates with inducers of ferroptosis in killing U251 cells. Overall, these findings demonstrate the potential of dual targeting of distinct PCD signalling pathways in GBM and may guide the utility of BCL-XL inhibitors and inducers of ferroptosis with standard of care treatment for improved therapies for GBM.
Assuntos
Antineoplásicos , Ferroptose , Glioblastoma , Animais , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Humanos , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Temozolomida/farmacologia , Proteína bcl-X/metabolismoRESUMO
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.
Assuntos
Apoptose , Proteína Killer-Antagonista Homóloga a bcl-2 , Anticorpos , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Membranas Mitocondriais/metabolismo , Estrutura Secundária de Proteína , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genéticaRESUMO
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.
Assuntos
Detergentes/química , Multimerização Proteica , Proteína Killer-Antagonista Homóloga a bcl-2/química , Sequência de Aminoácidos , Animais , Lipossomos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Estrutura Secundária de Proteína , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
Selective targeting of BCL-2 with the BH3-mimetic venetoclax has been a transformative treatment for patients with various leukemias. TP-53 controls apoptosis upstream of where BCL-2 and its prosurvival relatives, such as MCL-1, act. Therefore, targeting these prosurvival proteins could trigger apoptosis across diverse blood cancers, irrespective of TP53 mutation status. Indeed, targeting BCL-2 has produced clinically relevant responses in blood cancers with aberrant TP-53. However, in our study, TP53-mutated or -deficient myeloid and lymphoid leukemias outcompeted isogenic controls with intact TP-53, unless sufficient concentrations of BH3-mimetics targeting BCL-2 or MCL-1 were applied. Strikingly, tumor cells with TP-53 dysfunction escaped and thrived over time if inhibition of BCL-2 or MCL-1 was sublethal, in part because of an increased threshold for BAX/BAK activation in these cells. Our study revealed the key role of TP-53 in shaping long-term responses to BH3-mimetic drugs and reconciled the disparate pattern of initial clinical response to venetoclax, followed by subsequent treatment failure among patients with TP53-mutant chronic lymphocytic leukemia or acute myeloid leukemia. In contrast to BH3-mimetics targeting just BCL-2 or MCL-1 at doses that are individually sublethal, a combined BH3-mimetic approach targeting both prosurvival proteins enhanced lethality and durably suppressed the leukemia burden, regardless of TP53 mutation status. Our findings highlight the importance of using sufficiently lethal treatment strategies to maximize outcomes of patients with TP53-mutant disease. In addition, our findings caution against use of sublethal BH3-mimetic drug regimens that may enhance the risk of disease progression driven by emergent TP53-mutant clones.
Assuntos
Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Indolizinas/farmacologia , Isoquinolinas/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Mieloide Aguda/tratamento farmacológico , Morfolinas/farmacologia , Proteínas de Neoplasias/fisiologia , Fragmentos de Peptídeos/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Sulfonamidas/farmacologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Dano ao DNA , Genes p53 , Humanos , Indolizinas/uso terapêutico , Subunidade alfa de Receptor de Interleucina-2/deficiência , Isoquinolinas/uso terapêutico , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Morfolinas/uso terapêutico , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Fosforilação Oxidativa/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/administração & dosagem , Sulfonamidas/uso terapêutico , Proteína Supressora de Tumor p53/deficiência , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BAK and BAX, which drive commitment to apoptosis, are activated principally by certain BH3-only proteins that bind them and trigger major rearrangements. One crucial conformation change is exposure of their BH3 domain which allows BAK or BAX to form homodimers, and potentially to autoactivate other BAK and BAX molecules to ensure robust pore formation and cell death. Here, we test whether full-length BAK or mitochondrial BAX that are specifically activated by antibodies can then activate other BAK or BAX molecules. We found that antibody-activated BAK efficiently activated BAK as well as mitochondrial or cytosolic BAX, but antibody-activated BAX unexpectedly proved a poor activator. Notably, autoactivation by BAK involved transient interactions, as BAK and BAX molecules it activated could dissociate and homodimerize. The results suggest that BAK-driven autoactivation may play a substantial role in apoptosis, including recruitment of BAX to the mitochondria. Hence, directly targeting BAK rather than BAX may prove particularly effective in inhibiting unwanted apoptosis, or alternatively, inducing apoptosis in cancer cells.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose , Humanos , Camundongos , Dobramento de ProteínaRESUMO
Mitochondrial permeabilization is a key event in the intrinsic pathway of apoptosis, and is mediated by either of the BCL-2 family members BAK or BAX. These two proteins generate pores in the mitochondrial outer membrane that release factors such as cytochrome c into the cytosol to trigger caspase activation and apoptotic cell death. To generate pores, BAK and BAX undergo major changes including BAX translocation to the outer membrane, and partial unfolding, dimerization, and oligomerization. Here we describe biochemical protocols that can be used on most cell types to gain a population overview of BAK and BAX status.
Assuntos
Citocromos c/metabolismo , Oxidantes/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Células HeLa , Humanos , Camundongos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , ProteóliseRESUMO
Intrinsic apoptosis is critical to prevent tumor formation and is engaged by many anti-cancer agents to eliminate tumor cells. BAX and BAK, the two essential mediators of apoptosis, are thought to be regulated through similar mechanisms and act redundantly to drive apoptotic cell death. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as important for BAX, but not BAK, to function. Genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes containing VDAC1, VDAC2, and VDAC3, but only inhibited BAX apoptotic function. Deleting VDAC2 phenocopied the loss of BAX in impairing both the killing of tumor cells by anti-cancer agents and the ability to suppress tumor formation. Together, our studies show that efficient BAX-mediated apoptosis depends on VDAC2, and reveal a striking difference in how BAX and BAK are functionally impacted by their interactions with VDAC2.
Assuntos
Apoptose , Carcinogênese/metabolismo , Carcinogênese/patologia , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Desenvolvimento Embrionário , Células HCT116 , Células HeLa , Humanos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Regiões Promotoras Genéticas/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
BAX and BAK are essential mediators of intrinsic apoptosis that permeabilize the mitochondrial outer membrane. BAX activation requires its translocation from cytosol to mitochondria where conformational changes cause its oligomerization. To better understand the critical step of translocation, we examined its blockade by mutation near the C terminus (P168G) or by antibody binding near the N terminus. Similarities in the crystal structures of wild-type and BAX P168G but significant other differences suggest that cytosolic BAX exists as an ensemble of conformers, and that the distribution of conformers within the ensemble determines the different functions of wild-type and mutant proteins. We also describe the structure of BAX in complex with an antibody, 3C10, that inhibits cytosolic BAX by limiting exposure of the membrane-associating helix α9, as does the P168G mutation. Our data for both means of BAX inhibition argue for an allosteric model of BAX regulation that derives from properties of the ensemble of conformers.
Assuntos
Mutação , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Regulação Alostérica , Animais , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Citosol/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Humanos , Ictaluridae/metabolismo , Camundongos , Modelos Moleculares , Conformação Proteica , Proteína X Associada a bcl-2/genéticaRESUMO
The prosurvival Bcl-2 family proteins Mcl-1 and Bcl-xL 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-xL 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-xL preferentially sequestered Bid while Mcl-1 preferentially sequestered Bak. Notably, Bcl-xL could sequester Bak in response to the BH3 mimetic ABT-737, despite this molecule targeting Bcl-xL. These findings highlight the importance of Bak sequestration in resistance to anti-cancer treatments, including BH3 mimetics.
Assuntos
Compostos de Bifenilo/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Nitrofenóis/farmacologia , Sulfonamidas/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína bcl-X/metabolismo , Animais , Camundongos , Camundongos Knockout , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Piperazinas/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína bcl-X/genéticaRESUMO
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.
Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Animais , Apoptose , Sítios de Ligação , Linhagem Celular , Citocromos c/metabolismo , Dissulfetos/química , Epitopos/química , Fibroblastos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Multimerização Proteica , Proteína X Associada a bcl-2/metabolismoRESUMO
Apoptotic cell death via the mitochondrial pathway occurs in all vertebrate cells and requires the formation of pores in the mitochondrial outer membrane. Two Bcl-2 protein family members, Bak and Bax, form these pores during apoptosis, and how they do so has been investigated for the last two decades. Many of the conformation changes that occur during their transition to pore-forming proteins have now been delineated. Notably, biochemical, biophysical and structural studies indicate that symmetric homodimers are the basic unit of pore formation. Each dimer contains an extended hydrophobic surface that lies on the outer membrane, and is anchored at either end by a transmembrane domain. Membrane-remodelling events such as positive membrane curvature have been reported to accompany apoptotic pore formation, suggesting Bak and Bax form lipidic pores rather than proteinaceous pores. However, it remains unclear how symmetric dimers assemble to porate the membrane. Here, we review how clusters of dimers and their lipid-mediated interactions provide a molecular explanation for the heterogeneous assemblies of Bak and Bax observed during apoptosis.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'.
Assuntos
Apoptose , Membranas Mitocondriais/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Multimerização ProteicaRESUMO
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.
Assuntos
Apoptose , Mitocôndrias/fisiologia , Membranas Mitocondriais/metabolismo , Multimerização Proteica , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Animais , Humanos , CamundongosRESUMO
PCAF and ADA3 associate within the same macromolecular complexes to control the transcription of many genes, including some that regulate apoptosis. Here we show that PCAF and ADA3 regulate the expression of PACS1, whose protein product is a key component of the machinery that sorts proteins among the trans-Golgi network and the endosomal compartment. We describe a novel role for PACS1 as a regulator of the intrinsic pathway of apoptosis and mitochondrial outer membrane permeabilization. Cells with decreased PACS1 expression were refractory to cell death mediated by a variety of stimuli that operate through the mitochondrial pathway, including human granzyme B, staurosporine, ultraviolet radiation and etoposide, but remained sensitive to TRAIL receptor ligation. The mitochondria of protected cells failed to release cytochrome c as a result of perturbed oligomerization of BAX and BAK. We conclude that PCAF and ADA3 transcriptionally regulate PACS1 and that PACS1 is a key regulator of BAX/BAK oligomerization and the intrinsic (mitochondrial) pathway to apoptosis.
Assuntos
Apoptose , Epigênese Genética , Mitocôndrias/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Linhagem Celular , Granzimas , Humanos , Mitocôndrias/genética , Mitocôndrias/fisiologia , Multimerização Proteica , Estaurosporina , Raios Ultravioleta , Proteínas de Transporte Vesicular/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
During apoptosis, Bak and Bax are activated by BH3-only proteins binding to the α2-α5 hydrophobic groove; Bax is also activated via a rear pocket. Here we report that antibodies can directly activate Bak and mitochondrial Bax by binding to the α1-α2 loop. A monoclonal antibody (clone 7D10) binds close to α1 in non-activated Bak to induce conformational change, oligomerization, and cytochrome c release. Anti-FLAG antibodies also activate Bak containing a FLAG epitope close to α1. An antibody (clone 3C10) to the Bax α1-α2 loop activates mitochondrial Bax, but blocks translocation of cytosolic Bax. Tethers within Bak show that 7D10 binding directly extricates α1; a structural model of the 7D10 Fab bound to Bak reveals the formation of a cavity under α1. Our identification of the α1-α2 loop as an activation site in Bak paves the way to develop intrabodies or small molecules that directly and selectively regulate these proteins.
Assuntos
Anticorpos Monoclonais/metabolismo , Apoptose/fisiologia , Epitopos/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Células Cultivadas , Citocromos c/metabolismo , Citosol/metabolismo , Mapeamento de Epitopos/métodos , Feminino , Fibroblastos , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oócitos , Ligação Proteica/fisiologia , Conformação Proteica em alfa-Hélice , Multimerização Proteica/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genéticaRESUMO
Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-xL Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-xL via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-xL in apoptotic regulation. In vitro, Bak(Q75L) cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-xL binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak.