RESUMO
Mitochondrial stress, resulting from dysfunction and proteostasis disturbances, triggers the mitochondrial unfolded protein response (UPRMT), which activates gene encoding chaperones and proteases to restore mitochondrial function. Although ATFS-1 mediates mitochondrial stress UPRMT induction in C. elegans, the mechanisms relaying mitochondrial stress signals to the nucleus in mammals remain poorly defined. Here, we explored the role of protein kinase R (PKR), an eIF2α kinase activated by double-stranded RNAs (dsRNAs), in mitochondrial stress signaling. We found that UPRMT does not occur in cells lacking PKR, indicating its crucial role in this process. Mechanistically, we observed that dsRNAs accumulate within mitochondria under stress conditions, along with unprocessed mitochondrial transcripts. Furthermore, we demonstrated that accumulated mitochondrial dsRNAs in mouse embryonic fibroblasts (MEFs) deficient in the Bax/Bak channels are not released into the cytosol and do not induce the UPRMT upon mitochondrial stress, suggesting a potential role of the Bax/Bak channels in mediating the mitochondrial stress response. These discoveries enhance our understanding of how cells maintain mitochondrial integrity, respond to mitochondrial dysfunction, and communicate stress signals to the nucleus through retrograde signaling. This knowledge provides valuable insights into prospective therapeutic targets for diseases associated with mitochondrial stress.
Assuntos
Mitocôndrias , RNA de Cadeia Dupla , Resposta a Proteínas não Dobradas , eIF-2 Quinase , Animais , eIF-2 Quinase/metabolismo , eIF-2 Quinase/genética , Mitocôndrias/metabolismo , RNA de Cadeia Dupla/metabolismo , Camundongos , Estresse Fisiológico , Transdução de Sinais , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Fibroblastos/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , HumanosRESUMO
BAX and BAK are proapoptotic members of the BCL2 family that directly mediate mitochondrial outer membrane permeabilition (MOMP), a central step in apoptosis execution. However, the molecular architecture of the mitochondrial apoptotic pore remains a key open question and especially little is known about the contribution of lipids to MOMP. By performing a comparative lipidomics analysis of the proximal membrane environment of BAK isolated in lipid nanodiscs, we find a significant enrichment of unsaturated species nearby BAK and BAX in apoptotic conditions. We then demonstrate that unsaturated lipids promote BAX pore activity in model membranes, isolated mitochondria and cellular systems, which is further supported by molecular dynamics simulations. Accordingly, the fatty acid desaturase FADS2 not only enhances apoptosis sensitivity, but also the activation of the cGAS/STING pathway downstream mtDNA release. The correlation of FADS2 levels with the sensitization to apoptosis of different lung and kidney cancer cell lines by co-treatment with unsaturated fatty acids supports the relevance of our findings. Altogether, our work provides an insight on how local lipid environment affects BAX and BAK function during apoptosis.
Assuntos
Apoptose , Membranas Mitocondriais , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2 , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Humanos , Membranas Mitocondriais/metabolismo , Simulação de Dinâmica Molecular , Mitocôndrias/metabolismo , Linhagem Celular Tumoral , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos Insaturados/farmacologia , AnimaisRESUMO
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.
Assuntos
Apoptose , Canal de Ânion 2 Dependente de Voltagem , Proteína Killer-Antagonista Homóloga a bcl-2 , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Canal de Ânion 2 Dependente de Voltagem/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Humanos , Ligação Proteica , Mitocôndrias/metabolismo , Animais , Células HEK293RESUMO
The voltage-dependent anion channel 2 (VDAC2) is the abundant protein in the outer mitochondrial membrane. Opening VDAC2 pores leads to the induction of mitochondrial energy and material transport, facilitating interaction with various mitochondrial proteins implicated in essential processes such as cell apoptosis and proliferation. To investigate the VDAC2 in lower vertebrates, we identified Lr-VDAC2, a homologue of VDAC2 found in lamprey (Lethenteron reissneri), sharing a sequence identity of greater than 50 % with its counterparts. Phylogenetic analysis revealed that the position of Lr-VDAC2 aligns with the lamprey phylogeny, indicating its evolutionary relationship within the species. The Lr-VDAC2 protein was primarily located in the mitochondria of lamprey cells. The expression of the Lr-VDAC2 protein was elevated in high energy-demanding tissues, such as the gills, muscles, and myocardial tissue in normal lampreys. Lr-VDAC2 suppressed H2O2 (hydrogen peroxide)-induced 293 T cell apoptosis by reducing the expression levels of Caspase 3, Caspase 9, and Cyt C (cytochrome c). Further research into the mechanism indicated that the Lr-VDAC2 protein inhibited the pro-apoptotic activity of BAK (Bcl-2 antagonist/killer) protein by downregulating its expression at the protein translational level, thus exerting an anti-apoptotic function similar to the role of VDAC2 in humans.
Assuntos
Apoptose , Proteínas de Peixes , Lampreias , Canal de Ânion 2 Dependente de Voltagem , Animais , Humanos , Sequência de Aminoácidos , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Regulação para Baixo/efeitos dos fármacos , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica , Células HEK293 , Peróxido de Hidrogênio , Lampreias/genética , Lampreias/imunologia , Filogenia , Alinhamento de Sequência/veterinária , Canal de Ânion 2 Dependente de Voltagem/metabolismoRESUMO
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
Tamoxifen (TAM) resistance presents a major clinical obstacle in the management of estrogen-sensitive breast cancer, highlighting the need to understand the underlying mechanisms and potential therapeutic approaches. We showed that dysregulated mitochondrial dynamics were involved in TAM resistance by protecting against mitochondrial apoptosis. The dysregulated mitochondrial dynamics were associated with increased mitochondrial fusion and decreased fission, thus preventing the release of mitochondrial cytochrome c to the cytoplasm following TAM treatment. Dynamin-related GTPase protein mitofusin 1 (MFN1), which promotes fusion, was upregulated in TAM-resistant cells, and high MFN1 expression indicated a poor prognosis in TAM-treated patients. Mitochondrial translocation of MFN1 and interaction between MFN1 and mitofusin 2 (MFN2) were enhanced to promote mitochondrial outer membrane fusion. The interaction of MFN1 and cristae-shaping protein optic atrophy 1 (OPA1) and OPA1 oligomerization were reduced due to augmented OPA1 proteolytic cleavage, and their apoptosis-promoting function was reduced due to cristae remodeling. Furthermore, the interaction of MFN1 and BAK were increased, which restrained BAK activation following TAM treatment. Knockdown or pharmacological inhibition of MFN1 blocked mitochondrial fusion, restored BAK oligomerization and cytochrome c release, and amplified activation of caspase-3/9, thus sensitizing resistant cells to apoptosis and facilitating the therapeutic effects of TAM both in vivo and in vitro. Conversely, overexpression of MFN1 alleviated TAM-induced mitochondrial apoptosis and promoted TAM resistance in sensitive cells. These results revealed that dysregulated mitochondrial dynamics contributes to the development of TAM resistance, suggesting that targeting MFN1-mediated mitochondrial fusion is a promising strategy to circumvent TAM resistance.
Assuntos
Apoptose , Neoplasias da Mama , Resistencia a Medicamentos Antineoplásicos , GTP Fosfo-Hidrolases , Dinâmica Mitocondrial , Tamoxifeno , Humanos , Tamoxifeno/farmacologia , Dinâmica Mitocondrial/efeitos dos fármacos , Apoptose/efeitos dos fármacos , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Animais , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Linhagem Celular Tumoral , Antineoplásicos Hormonais/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Células MCF-7 , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BH3-mimetics represent promising anti-cancer agents in tumors that rely on the anti-apoptotic function of B-Cell Lymphoma 2 (BCL2) proteins, particularly in leukemia and lymphoma cells primed for apoptosis. Mechanistically, BH3-mimetics may displace pro-apoptotic binding partners thus inducing BAX/BAK-mediated mitochondrial permeabilization followed by cytochrome c release, activation of the caspase cascade and apoptosis. Here, we describe a novel mode of caspase-independent cell death (CICD) induced by BH3-mimetics in a subset of diffuse large B-cell lymphoma (DLBCL) cells. Of note, rather than occurring via necroptosis, CICD induced immediately after mitochondrial permeabilization was associated with transcriptional reprogramming mediated by activation of c-Jun N-terminal Kinase (JNK) signaling and Activator Protein 1 (AP1). Thereby, CICD resulted in the JNK/AP1-mediated upregulation of inflammatory chemokines and increased migration of cytotoxic Natural Killer (NK) cells. Taken together, our study describes a novel mode of CICD triggered by BH3-mimetics that may alter the immune response towards dying cells.
Assuntos
Antineoplásicos , Linfoma Difuso de Grandes Células B , Humanos , Proteína X Associada a bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Apoptose , Antineoplásicos/farmacologia , Caspases , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linhagem Celular , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
One hallmark of apoptosis is the oligomerization of BAX and BAK to form a pore in the mitochondrial outer membrane, which mediates the release of pro-apoptotic intermembrane space proteins into the cytosol. Cells overexpressing BAX or BAK fusion proteins are a powerful model system to study the dynamics and localization of these proteins in cells. However, it is unclear whether overexpressed BAX and BAK form the same ultrastructural assemblies following the same spatiotemporal hierarchy as endogenously expressed proteins. Combining live- and fixed-cell STED super-resolution microscopy, we show that overexpression of BAK results in novel BAK structures, which are virtually absent in non-overexpressing apoptotic cells. We further demonstrate that in wild type cells, BAK is recruited to apoptotic pores before BAX. Both proteins together form unordered, mosaic rings on apoptotic mitochondria in immortalized cell culture models as well as in human primary cells. In BAX- or BAK- single-knockout cells, the remaining protein is able to form rings independently. The heterogeneous nature of these rings in both wild type as well as single-knockout cells corroborates the toroidal apoptotic pore model.
Assuntos
Apoptose , Mitocôndrias , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2 , Animais , Humanos , Camundongos , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismoRESUMO
Bcl-2-associated X protein (BAX) and Blc-2 homologous antagonist killer 1 (BAK) are two pro-apoptotic members of BCL2 family. Here, two BAX/BAK double knock-out human induced pluripotent stem cell lines (iPSC) we generated using CRISPR-Cas9 to generate apoptosis incompetent cell lines. The resulting cell lines were karyotypically normal, had typical morphology and expressed typical markers for the undifferentiated state.
Assuntos
Células-Tronco Pluripotentes Induzidas , Proteínas Proto-Oncogênicas c-bcl-2 , Humanos , 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 , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Sistemas CRISPR-Cas/genética , Apoptose/genéticaRESUMO
Non-obstructive azoospermia affects more than 10% of infertile men with over 70% patients are idiopathic with uncharacterized molecular mechanisms, which is referred as idiopathic non-obstructive azoospermia. In this study, we checked the morphology of Sertoli cell mitochondria in testis biopsies from patients with idiopathic non-obstructive azoospermia and patients with obstructive azoospermia who have normal spermiogenesis. The expression of 104 genes controlling mitochondria fission and fusion were analyzed in three gene expression datasets including a total of 60 patients with non-obstructive azoospermia. The levels of 7 candidate genes were detected in testis biopsies from 38 patients with idiopathic non-obstructive azoospermia and 24 patients with obstructive azoospermia who have normal spermatogenesis by RT-qPCR. Cell viability, apoptosis, mitochondria membrane potential, adenosine triphosphate production, oxygen consumption, and mitochondria morphology were examined in primary human Sertoli cells. Mouse spermatogonial stem cells were used to detect the cell supporting capacity of Sertoli cells. We observed that patients with idiopathic non-obstructive azoospermia had elongated mitochondria. MTFR2 and ATP5IF1 were downregulated, whereas BAK1 was upregulated in idiopathic non-obstructive azoospermia testis and Sertoli cells. Sertoli cells from patients with idiopathic non-obstructive azoospermia had reduced viability, mitochondria membrane potential, adenosine triphosphate production, oxygen consumption rate, glycolysis and increased apoptosis. Knockdown MTFR2 in Sertoli cells increased the mitochondria size. Knockdown ATP5IF1 did not change mitochondrial morphology but increased adenosine triphosphate hydrolysis. Overexpression of BAK1 reduced membrane potential and upregulated cell apoptosis. The dysregulation of all these three genes contributed to the dysfunction of Sertoli cells, which provides a clue for idiopathic non-obstructive azoospermia treatment.
Assuntos
Azoospermia , Doenças Mitocondriais , Masculino , Humanos , Camundongos , Animais , Células de Sertoli/metabolismo , Azoospermia/genética , Dinâmica Mitocondrial , Testículo/metabolismo , Espermatogênese/genética , Trifosfato de Adenosina/metabolismo , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
The B-cell lymphoma-2 (BCL-2) family of proteins plays a crucial role in the regulation of apoptosis, offering a dual mechanism for its control. Numerous studies have established a strong association between gene disorders of these proteins and the proliferation of diverse cancer cell types. Consequently, the identification and development of drugs targeting BCL-2 family proteins have emerged as a prominent area in antitumor therapy. Over the last two decades, several small-molecules have been designed to modulate the protein-protein interactions between anti- and proapoptotic BCL-2 proteins, effectively suppressing tumor growth and metastasis in vivo. The primary focus of research has been on developing BCL-2 homology 3 (BH3) mimetics to target antiapoptotic BCL-2 proteins, thereby competitively releasing proapoptotic BCL-2 proteins and restoring the blocked intrinsic apoptotic program. Additionally, for proapoptotic BCL-2 proteins, exogenous small molecules have been explored to activate cell apoptosis by directly interacting with executioner proteins such as BCL-2-associated X protein (BAX) or BCL-2 homologous antagonist/killer protein (BAK). In this comprehensive review, we summarize the inhibitors and activators (sensitizers) of BCL-2 family proteins developed over the past decades, highlighting their discovery, optimization, preclinical and clinical status, and providing an overall landscape of drug development targeting these proteins for therapeutic purposes.
Assuntos
Neoplasias , Proteínas Proto-Oncogênicas , Humanos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/farmacologia , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Apoptose , Neoplasias/tratamento farmacológicoRESUMO
Proteins from the Bcl-2 family play an essential role in the regulation of apoptosis. However, they also possess cell death-unrelated activities that are less well understood. This prompted us to study apoptosis-unrelated activities of the Bax and Bak, pro-apoptotic members of the Bcl-2 family. We prepared Bax/Bak-deficient human cancer cells of different origin and found that while respiration in the glioblastoma U87 Bax/Bak-deficient cells was greatly enhanced, respiration of Bax/Bak-deficient B lymphoma HBL-2 cells was slightly suppressed. Bax/Bak-deficient U87 cells also proliferated faster in culture, formed tumours more rapidly in mice, and showed modulation of metabolism with a considerably increased NAD+/NADH ratio. Follow-up analyses documented increased/decreased expression of mitochondria-encoded subunits of respiratory complexes and stabilization/destabilization of the mitochondrial transcription elongation factor TEFM in Bax/Bak-deficient U87 and HBL-2 cells, respectively. TEFM downregulation using shRNAs attenuated mitochondrial respiration in Bax/Bak-deficient U87 as well as in parental HBL-2 cells. We propose that (post)translational regulation of TEFM levels in Bax/Bak-deficient cells modulates levels of subunits of mitochondrial respiratory complexes that, in turn, contribute to respiration and the accompanying changes in metabolism and proliferation in these cells.
Assuntos
Apoptose , Proteína Killer-Antagonista Homóloga a bcl-2 , Humanos , Animais , Camundongos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Apoptose/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , RespiraçãoRESUMO
In the present work, the mechanism of anticancer activity of some pyrrolopyrimidine derivatives was evaluated. Compounds 5 and 8 exhibiting significant antiproliferative activity against HT-29 cells with IC50 values of 4.17 µM and 2.96, arrested the cells at the G2/M phase and significantly induced apoptosis. The apoptotic potential of the compounds has been verified via ELISA assay, which resulted in increased BAX, PUMA, BIM, and cleaved caspase 3 expression and decreased BCL-XL and MCL-1 protein levels in HT-29 cells. Moreover, the immunofluorescence technique showing that compounds 5 and 8-treatment reduced Ki67 immunolocalization and increased the caspase 3 and p53 immunolocalization confirmed the apoptotic activity. While treatment of HT-29 cells to compounds 5 and 8 inhibited Akt and ERK1/2, there are no alterations in JNK and p38 signaling pathways. According to molecular docking results, compounds 5 and 8 occupied the active site of Akt kinase and showed important hydrogen bonding interactions with key amino acids. Also, siRNA-mediated depletion of BIM, PUMA, and BAX/BAK expression decreased apoptotic response in HT-29 cells upon exposure to compound 5 and compound 8. Compounds 5 and 8 trigger the activation of mitochondrial apoptosis in HT-29 cells. Additionally, we found that proapoptotic BH3-only proteins BIM and PUMA are required for the full engagement of mitochondrial apoptosis signaling. However, p53 was dispensable for compound 5- or compound 8-induced apoptosis in HT-29 cells.
Assuntos
Proteínas Reguladoras de Apoptose , Neoplasias do Colo , Pirimidinas , Pirróis , Humanos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/metabolismo , Proteína X Associada a bcl-2/metabolismo , Células HT29 , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Supressora de Tumor p53 , Simulação de Acoplamento Molecular , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/farmacologia , Linhagem Celular Tumoral , Apoptose , Neoplasias do Colo/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
The activation or inactivation of B-cell lymphoma-2 (Bcl-2) antagonist/killer (Bak) is critical for controlling mitochondrial outer membrane permeabilization-dependent apoptosis. Its pro-apoptotic activity is controlled by intermolecular interactions with the Bcl-2 homology 3 (BH3) domain, which is accommodated in the hydrophobic pocket of Bak. Bcl-2-interacting protein 5 (Bnip5) is a noncanonical BH3 domain-containing protein that interacts with Bak. Bnip5 is characterized by its controversial effects on the regulation of the pro-apoptotic activity of Bak. In the present study, we determined the crystal structure of Bak bound to Bnip5 BH3. The intermolecular association appeared to be typical at first glance, but we found that it is maintained by tight hydrophobic interactions together with hydrogen/ionic bonds, which accounts for their high binding affinity with a dissociation constant of 775 nM. Structural analysis of the complex showed that Bnip5 interacts with Bak in a manner similar to that of the Bak-activating pro-apoptotic factor peroxisomal testis-enriched protein 1, particularly in the destabilization of the intramolecular electrostatic network of Bak. Our structure is considered to reflect the initial point of drastic and consecutive conformational and stoichiometric changes in Bak induced by Bnip5 BH3, which helps in explaining the effects of Bnip5 in regulating Bak-mediated apoptosis.
Assuntos
Proteínas Proto-Oncogênicas c-bcl-2 , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Domínios Proteicos , Proteína bcl-X/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Apoptose/fisiologiaRESUMO
At least seven cell death programs are activated during myocardial infarction (MI), but which are most important in causing heart damage is not understood. Two of these programs are mitochondrial-dependent necrosis and apoptosis. The canonical function of the pro-cell death BCL-2 family proteins BAX and BAK is to mediate permeabilization of the outer mitochondrial membrane during apoptosis allowing apoptogen release. BAX has also been shown to sensitize cells to mitochondrial-dependent necrosis, although the underlying mechanisms remain ill-defined. Genetic deletion of Bax or both Bax and Bak in mice reduces infarct size following reperfused myocardial infarction (MI/R), but the contribution of BAK itself to cardiomyocyte apoptosis and necrosis and infarction has not been investigated. In this study, we use Bak-deficient mice and isolated adult cardiomyocytes to delineate the role of BAK in the pathogenesis of infarct generation and post-infarct remodeling during MI/R and non-reperfused MI. Generalized homozygous deletion of Bak reduced infarct size â¼50% in MI/R in vivo, which was attributable primarily to decreases in necrosis. Protection from necrosis was also observed in BAK-deficient isolated cardiomyocytes suggesting that the cardioprotection from BAK loss in vivo is at least partially cardiomyocyte-autonomous. Interestingly, heterozygous Bak deletion, in which the heart still retains â¼28% of wild type BAK levels, reduced infarct size to a similar extent as complete BAK absence. In contrast to MI/R, homozygous Bak deletion did not attenuate acute infarct size or long-term scar size, post-infarct remodeling, cardiac dysfunction, or mortality in non-reperfused MI. We conclude that BAK contributes significantly to cardiomyocyte necrosis and infarct generation during MI/R, while its absence does not appear to impact the pathogenesis of non-reperfused MI. These observations suggest BAK may be a therapeutic target for MI/R and that even partial pharmacological antagonism may provide benefit.
Assuntos
Infarto do Miocárdio , Proteína Killer-Antagonista Homóloga a bcl-2 , Animais , Camundongos , Apoptose/fisiologia , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Homozigoto , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Necrose/genética , Deleção de Sequência , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
Permeabilization of mitochondrial membrane by proteins of the BCL-2 family is a key decisive event in the induction of apoptosis in mammalian cells. Although yeast does not have homologs of the BCL-2 family, when these are expressed in yeast, they modulate the survival of cells in a way that corresponds to their activity in mammalian cells. The yeast gene, alternatively referred to as BXI1 or YBH3, encodes for membrane protein in the endoplasmic reticulum that was, contradictorily, shown to either inhibit Bax or to be required for Bax activity. We have tested the effect of the deletion of this gene on the pro-apoptotic activity of Bax and Bak and the anti-apoptotic activity of Bcl-XL and Bcl-2, as well on survival after treatment with inducers of regulated cell death in yeast, hydrogen peroxide and acetic acid. While deletion resulted in increased sensitivity to acetic acid, it did not affect the sensitivity to hydrogen peroxide nor to BCL-2 family members. Thus, our results do not support any model in which the activity of BCL-2 family members is directly affected by BXI1 but rather indicate that it may participate in modulating survival in response to some specific forms of stress.
Assuntos
Peróxido de Hidrogênio , Saccharomyces cerevisiae , Animais , Apoptose/genética , 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 , Sobrevivência Celular , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/metabolismo , Mamíferos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Genomic DNA sequences provide unique target sites, with high druggability value, for treatment of genetically-linked diseases like cancer. B-cell lymphoma protein-2 (BCL-2) prevents Bcl-2-associated X protein (BAX) and Bcl-2 antagonist killer 1 (BAK) oligomerization, which would otherwise lead to the release of several apoptogenic molecules from the mitochondrion. It is also known that BCL-2 binds to and inactivates BAX and other pro-apoptotic proteins, thereby inhibiting apoptosis. BCL-2 protein family, through its role in regulation of apoptotic pathways, is possibly related to chemo-resistance in almost half of all cancer types including breast cancer. Here for the first time, we have developed a nanohybrid using a peptide-based carrier and a Deoxyribonucleic acid inhibitor (DNAi) against BCL-2 oncogene to induce apoptosis in breast cancer cells. The genetically designed nanocarrier was functionalized with an internalizing RGD (iRGD) targeting motif and successfully produced by recombinant DNA technology. Gel retardation assay demonstrated that the peptide-based carrier binds single-stranded DNAi upon simple mixing. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) analyses further revealed the formation of nanohybrid particles with a size of 30 nm and a slightly positive charge. This hemocompatible nanohybrid efficiently delivered its contents into cancer cells using iRGD targeting moiety. Gene expression analysis demonstrated that the nanohybrids, which contained DNAi against BCL-2 proficiently suppressed the expression of this oncogene in a sequence specific manner. In addition, the nanohybrid, triggered release of cytochrome c (cyt c) and caspase3/7 activation with high efficiency. Although the DNAi and free nanocarrier were separately unable to affect the cell viability, the nanohybrid of 20 nM of DNAi showed outstanding antineoplastic potential, which was adjusted by the ratio of the MiRGD nanocarrier to DNAi. It should be noted that, the designed nanohybrid showed a suitable specificity profile and did not affect the viability of normal cells. The results suggest that this nanohybrid may be useful for robust breast cancer treatment through targeting the BCL-2 oncogene without any side effects.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Apoptose , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Nanopartículas , Oncogenes , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Genes bcl-2RESUMO
BH3 mimetics, targeting the Bcl-2 family anti-apoptotic proteins, represent a promising therapeutic opportunity in cancers. ABT-199, the first specific Bcl-2 inhibitor, was approved by FDA for the treatment of several hematological malignancies. We have recently discovered IS21, a novel pan BH3 mimetic with preclinical antitumor activity in several tumor types. Here, we evaluated the efficacy of IS21 and other BH3 mimetics, both as single agents and combined with the currently used antineoplastic agents in T-cell acute lymphoblastic leukemia, ovarian cancer, and melanoma. IS21 was found to be active in T-cell acute lymphoblastic leukemia, melanoma, lung, pancreatic, and ovarian cancer cell lines. Bcl-xL and Mcl-1 protein levels predicted IS21 sensitivity in melanoma and ovarian cancer, respectively. Exploring IS21 mechanism of action, we found that IS21 activity depends on the presence of BAX and BAK proteins: complexes between Bcl-2 and Bcl-xL proteins and their main binding partners were reduced after IS21 treatment. In combination experiments, BH3 mimetics sensitized leukemia cells to chemotherapy, ovarian cancer cells and melanoma models to PARP and MAPK inhibitors, respectively. We showed that this enhancing effect was related to the potentiation of the apoptotic pathway, both in hematologic and solid tumors. In conclusion, our data suggest the use of inhibitors of anti-apoptotic proteins as a therapeutic strategy to enhance the efficacy of anticancer treatment.
Assuntos
Antineoplásicos , Melanoma , Neoplasias Ovarianas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Feminino , Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteína bcl-X/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Melanoma/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Linhagem Celular TumoralRESUMO
BACKGROUND: Preeclampsia is a common pregnancy complication characterized by high blood pressure and damage to organs. Abnormal placenta and vascular function can lead to preeclampsia. Accumulating evidence has suggested a potential link between circular RNAs (circRNAs) and preeclampsia. As a placenta and endothelial-expressed circRNA, hsa_circ_0002348, may be promising to be the novel molecular target for preeclampsia. However, the function and mechanism of hsa_circ_0002348 in preeclampsia has not been elucidated. MATERIALS AND METHODS: An overlap analysis of two circRNA profiles from placenta and endothelial cells was used to identify a functionally unknown circRNA, hsa_circ_0002348. Quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH) were used to detect its expression in the trophoblast cells and placental tissues. The mouse model of lipopolysaccharide (LPS)-induced preeclampsia was established to determine the in vivo role of hsa_circ_0002348. RNA immunoprecipitation (RIP), Luciferase reporter assay, qRT-PCR, western blot, gain- and loss-of-function and rescue experiments were conducted to uncover the role of hsa_circ_0002348 and its interaction with miR-126-3p and BAK1 in regulating trophoblast proliferation and apoptosis. Fluorescence in situ hybridization (FISH) and Immunohistochemistry (IHC) were performed to examine the expression of miR-126-3p and BAK1 in mice and human placentas, respectively. RESULTS: Hsa_circ_0002348 was significantly increased in the preeclampsia placentas, and positively correlated with the severity of preeclampsia patients' clinical manifestations. Its overexpression exacerbated preeclampsia-like features in the mouse model of LPS-induced preeclampsia. Functionally, hsa_circ_0002348 was found to inhibit trophoblast proliferation and promote trophoblast apoptosis. Mechanistically, hsa_circ_0002348, as an endogenous miR-126-3p sponge, upregulated the expression of BAK1. Additionally, both hsa_circ_0002348 knockdown and miR-126-3p overexpression enhanced the mammalian target of rapamycin (mTOR) and ERK1/2 signaling pathway. CONCLUSIONS: Hsa_circ_0002348 might be a novel regulator of trophoblast proliferation and apoptosis through miR-126-3p/BAK1 axis in preeclampsia, which may serve as a potential target for detecting and treating preeclampsia.
Assuntos
MicroRNAs , Pré-Eclâmpsia , RNA Circular , Animais , Feminino , Humanos , Camundongos , Gravidez , Apoptose/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proliferação de Células/genética , Modelos Animais de Doenças , Células Endoteliais , Hibridização in Situ Fluorescente , Lipopolissacarídeos , Mamíferos , MicroRNAs/genética , Placenta , Pré-Eclâmpsia/genética , RNA Circular/genética , TrofoblastosRESUMO
Bak is a pro-apoptotic protein and a member of the Bcl-2 family that plays a key role in apoptosis, a programmed cell death mechanism of multicellular organisms. Its activation under death stimuli triggers the permeabilization of the mitochondrial outer membrane that represents a point of no return in the apoptotic pathway. This process is deregulated in many tumors where Bak is inactivated, whereas in other cases like in neurodegeneration, it exhibits an excessive response leading to disorders such as the Alzheimer disease. Members of the Bcl-2 family share a common 3D structure, exhibiting an extremely similar orthosteric binding site, a place where both pro and antiapoptotic proteins bind. This similarity raises a selectivity issue that hampers the identification of new drugs, capable of altering Bak activation in a selective manner. An alternative activation site triggered by antibodies has been recently identified, opening the opportunity to undertake new drug discovery studies. Despite this recent identification, an exhaustive study to identify cryptic pockets as prospective allosteric sites has not been yet performed. Thus, the present study aims to characterize novel hotspots in the Bak structure. For this purpose, we have carried out extensive molecular dynamics simulations using three different Bak systems including Bak in its apo form, Bak in complex with its endogen activator Bim and an intermediate form, set up by removing Bim from the previous complex. The results reported in the present work shed some light on future docking studies on Bak through the identification of new prospective allosteric sites, not previously described in this protein.