RESUMO
Drug-tolerant persister cells (persisters) evade apoptosis upon targeted and conventional cancer therapies and represent a major non-genetic barrier to effective cancer treatment. Here, we show that cells that survive treatment with pro-apoptotic BH3 mimetics display a persister phenotype that includes colonization and metastasis in vivo and increased sensitivity toward ferroptosis by GPX4 inhibition. We found that sublethal mitochondrial outer membrane permeabilization (MOMP) and holocytochrome c release are key requirements for the generation of the persister phenotype. The generation of persisters is independent of apoptosome formation and caspase activation, but instead, cytosolic cytochrome c induces the activation of heme-regulated inhibitor (HRI) kinase and engagement of the integrated stress response (ISR) with the consequent synthesis of ATF4, all of which are required for the persister phenotype. Our results reveal that sublethal cytochrome c release couples sublethal MOMP to caspase-independent initiation of an ATF4-dependent, drug-tolerant persister phenotype.
Assuntos
Citocromos c , Neoplasias/tratamento farmacológico , Animais , Apoptose , Proteínas de Transporte , Caspases/metabolismo , Citocromos c/metabolismo , Resistencia a Medicamentos Antineoplásicos , Humanos , Camundongos , Mitocôndrias/metabolismoRESUMO
Through their many and varied metabolic functions, mitochondria power life. Paradoxically, mitochondria also have a central role in apoptotic cell death. Upon induction of mitochondrial apoptosis, mitochondrial outer membrane permeabilization (MOMP) usually commits a cell to die. Apoptotic signalling downstream of MOMP involves cytochrome c release from mitochondria and subsequent caspase activation. As such, targeting MOMP in order to manipulate cell death holds tremendous therapeutic potential across different diseases, including neurodegenerative diseases, autoimmune disorders and cancer. In this Review, we discuss new insights into how mitochondria regulate apoptotic cell death. Surprisingly, recent data demonstrate that besides eliciting caspase activation, MOMP engages various pro-inflammatory signalling functions. As we highlight, together with new findings demonstrating cell survival following MOMP, this pro-inflammatory role suggests that mitochondria-derived signalling downstream of pro-apoptotic cues may also have non-lethal functions. Finally, we discuss the importance and roles of mitochondria in other forms of regulated cell death, including necroptosis, ferroptosis and pyroptosis. Collectively, these new findings offer exciting, unexplored opportunities to target mitochondrial regulation of cell death for clinical benefit.
Assuntos
Apoptose/fisiologia , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Animais , Caspases/metabolismo , Citocromos c/metabolismo , Humanos , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/fisiologia , Transdução de SinaisRESUMO
Receptor-interacting protein kinase-3 (RIPK3) is an activator of necroptotic cell death, but recent work has implicated additional roles for RIPK3 in inflammatory signaling independent of cell death. However, while necroptosis has been shown to contribute to antiviral immunity, death-independent roles for RIPK3 in host defense have not been demonstrated. Using a mouse model of West Nile virus (WNV) encephalitis, we show that RIPK3 restricts WNV pathogenesis independently of cell death. Ripk3-/- mice exhibited enhanced mortality compared to wild-type (WT) controls, while mice lacking the necroptotic effector MLKL, or both MLKL and caspase-8, were unaffected. The enhanced susceptibility of Ripk3-/- mice arose from suppressed neuronal chemokine expression and decreased central nervous system (CNS) recruitment of T lymphocytes and inflammatory myeloid cells, while peripheral immunity remained intact. These data identify pleiotropic functions for RIPK3 in the restriction of viral pathogenesis and implicate RIPK3 as a key coordinator of immune responses within the CNS.
Assuntos
Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/fisiologia , Animais , Sistema Nervoso Central/metabolismo , Quimiocinas/imunologia , Leucócitos/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Neurônios/metabolismoRESUMO
Senescent cells drive age-related tissue dysfunction partially through the induction of a chronic senescence-associated secretory phenotype (SASP)1. Mitochondria are major regulators of the SASP; however, the underlying mechanisms have not been elucidated2. Mitochondria are often essential for apoptosis, a cell fate distinct from cellular senescence. During apoptosis, widespread mitochondrial outer membrane permeabilization (MOMP) commits a cell to die3. Here we find that MOMP occurring in a subset of mitochondria is a feature of cellular senescence. This process, called minority MOMP (miMOMP), requires BAX and BAK macropores enabling the release of mitochondrial DNA (mtDNA) into the cytosol. Cytosolic mtDNA in turn activates the cGAS-STING pathway, a major regulator of the SASP. We find that inhibition of MOMP in vivo decreases inflammatory markers and improves healthspan in aged mice. Our results reveal that apoptosis and senescence are regulated by similar mitochondria-dependent mechanisms and that sublethal mitochondrial apoptotic stress is a major driver of the SASP. We provide proof-of-concept that inhibition of miMOMP-induced inflammation may be a therapeutic route to improve healthspan.
Assuntos
Apoptose , Senescência Celular , Citosol , DNA Mitocondrial , Mitocôndrias , Animais , Camundongos , Citosol/metabolismo , DNA Mitocondrial/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Necrose Dirigida por Permeabilidade Transmembrânica da Mitocôndria , Estudo de Prova de Conceito , Inflamação/metabolismo , Fenótipo , Longevidade , Envelhecimento SaudávelRESUMO
Sensing nutrient availability is essential for appropriate cellular growth, and mTORC1 is a major regulator of this process. Mechanisms causing mTORC1 activation are, however, complex and diverse. We report here an additional important step in the activation of mTORC1, which regulates the efflux of amino acids from lysosomes into the cytoplasm. This process requires DRAM-1, which binds the membrane carrier protein SCAMP3 and the amino acid transporters SLC1A5 and LAT1, directing them to lysosomes and permitting efficient mTORC1 activation. Consequently, we show that loss of DRAM-1 also impacts pathways regulated by mTORC1, including insulin signaling, glycemic balance, and adipocyte differentiation. Interestingly, although DRAM-1 can promote autophagy, this effect on mTORC1 is autophagy independent, and autophagy only becomes important for mTORC1 activation when DRAM-1 is deleted. These findings provide important insights into mTORC1 activation and highlight the importance of DRAM-1 in growth control, metabolic homeostasis, and differentiation.
Assuntos
Aminoácidos/metabolismo , Proteína 7 Relacionada à Autofagia/metabolismo , Metabolismo Energético , Lisossomos/enzimologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Membrana/metabolismo , Células 3T3-L1 , Adipócitos/enzimologia , Adipogenia , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Sistema y+L de Transporte de Aminoácidos/genética , Sistema y+L de Transporte de Aminoácidos/metabolismo , Animais , Proteína 7 Relacionada à Autofagia/genética , Glicemia/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ativação Enzimática , Células HEK293 , Células HeLa , Humanos , Insulina/sangue , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Transporte ProteicoRESUMO
The anti-apoptotic BCL-2 protein family regulates cancer cell survival, thus it represents an important therapeutic target. Indeed, a drug class, called BH3-mimetics, have been developed to directly target BCL2 proteins and promote cancer cell death. Conventional wisdom suggests that the primary anti-cancer effect of BCL-2 inhibition is through induction of cancer cell death. However, a recent study by Zhao and colleagues describes that BCL-2 inhibition also enhances the function of classical dendritic cells, unleashing their role in immunosurveillance, promoting T cell immunity and tumour regression. Thus, inhibiting anti-apoptotic BCL-2 function may have a multi-pronged anti-tumour action.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas Reguladoras de Apoptose/farmacologia , Proteínas Reguladoras de Apoptose/uso terapêutico , Linhagem Celular TumoralRESUMO
Cytosolic DNA is recognized by the innate immune system as a potential threat. During apoptotic cell death, mitochondrial DNA (mtDNA) release activates the DNA sensor cyclic GMP-AMP synthase (cGAS) to promote a pro-inflammatory type I interferon response. Inflammation following mtDNA release during apoptotic cell death can be exploited to engage anti-tumor immunity and represents a potential avenue for cancer therapy. Additionally, various studies have described leakage of mtDNA, independent of cell death, with different underlying cues such as pathogenic infections, changes in mtDNA packaging, mtDNA stress or reduced mitochondrial clearance. The interferon response in these scenarios can be beneficial but also potentially disadvantageous, as suggested by a variety of disease phenotypes. In this review, we discuss mechanisms underlying mtDNA release governed by cell death pathways and summarize release mechanisms independent of cell death. We further highlight the similarities and differences in mtDNA release pathways, outlining gaps in our knowledge and questions for further research. Together, a deeper understanding of how and when mtDNA is released may enable the development of drugs to specifically target or inhibit mtDNA release in different disease settings.
Assuntos
DNA Mitocondrial , Mitocôndrias , Humanos , DNA Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Inflamação/metabolismo , Apoptose , Interferons/metabolismoRESUMO
During apoptosis, the mitochondrial outer membrane is permeabilized, leading to the release of cytochrome c that activates downstream caspases. Mitochondrial outer membrane permeabilization (MOMP) has historically been thought to occur synchronously and completely throughout a cell, leading to rapid caspase activation and apoptosis. Using a new imaging approach, we demonstrate that MOMP is not an all-or-nothing event. Rather, we find that a minority of mitochondria can undergo MOMP in a stress-regulated manner, a phenomenon we term "minority MOMP." Crucially, minority MOMP leads to limited caspase activation, which is insufficient to trigger cell death. Instead, this caspase activity leads to DNA damage that, in turn, promotes genomic instability, cellular transformation, and tumorigenesis. Our data demonstrate that, in contrast to its well-established tumor suppressor function, apoptosis also has oncogenic potential that is regulated by the extent of MOMP. These findings have important implications for oncogenesis following either physiological or therapeutic engagement of apoptosis.
Assuntos
Apoptose/fisiologia , Dano ao DNA , Instabilidade Genômica , Membranas Mitocondriais/fisiologia , Animais , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Western Blotting , Caspases/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p19/deficiência , Inibidor de Quinase Dependente de Ciclina p19/genética , Relação Dose-Resposta a Droga , Embrião de Mamíferos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HCT116 , Células HeLa , Histonas/metabolismo , Humanos , Células MCF-7 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Nitrofenóis/farmacologia , Permeabilidade , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Estaurosporina/farmacologia , Sulfonamidas/farmacologia , Fatores de TempoRESUMO
The use of bacteria as an alternative cancer therapy has been reinvestigated in recent years. SL7207: an auxotrophic Salmonella enterica serovar Typhimurium aroA mutant with immune-stimulatory potential has proven a promising strain for this purpose. Here, we show that systemic administration of SL7207 induces melanoma tumor growth arrest in vivo, with greater survival of the SL7207-treated group compared to control PBS-treated mice. Administration of SL7207 is accompanied by a change in the immune phenotype of the tumor-infiltrating cells toward pro-inflammatory, with expression of the TH 1 cytokines IFN-γ, TNF-α, and IL-12 significantly increased. Interestingly, Ly6C+ MHCII+ monocytes were recruited to the tumors following SL7207 treatment and were pro-inflammatory. Accordingly, the abrogation of these infiltrating monocytes using clodronate liposomes prevented SL7207-induced tumor growth inhibition. These data demonstrate a previously unappreciated role for infiltrating inflammatory monocytes underlying bacterial-mediated tumor growth inhibition. This information highlights a possible novel role for monocytes in controlling tumor growth, contributing to our understanding of the immune responses required for successful immunotherapy of cancer.
Assuntos
Imunoterapia , Melanoma Experimental , Monócitos/imunologia , Salmonella typhimurium/imunologia , Células Th1/imunologia , Animais , Citocinas/imunologia , Feminino , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Salmonella typhimurium/genéticaRESUMO
Mitochondrial outer membrane permeabilization (MOMP) is often required for activation of the caspase proteases that cause apoptotic cell death. Various intermembrane space (IMS) proteins, such as cytochrome c, promote caspase activation following their mitochondrial release. As a consequence, mitochondrial outer membrane integrity is highly controlled, primarily through interactions between pro- and anti-apoptotic members of the B cell lymphoma 2 (BCL-2) protein family. Following MOMP by pro-apoptotic BCL-2-associated X protein (BAX) or BCL-2 antagonist or killer (BAK), additional regulatory mechanisms govern the mitochondrial release of IMS proteins and caspase activity. MOMP typically leads to cell death irrespective of caspase activity by causing a progressive decline in mitochondrial function, although cells can survive this under certain circumstances, which may have pathophysiological consequences.
Assuntos
Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Animais , Caspases/metabolismo , Morte Celular , Humanos , Proteína X Associada a bcl-2/farmacocinéticaRESUMO
Mitochondria are organelles central to myriad cellular processes. To maintain mitochondrial health, various processes co-operate at both the molecular and organelle level. At the molecular level, mitochondria can sense imbalances in their homeostasis and adapt to these by signaling to the nucleus. This mito-nuclear communication leads to the expression of nuclear stress response genes. Upon external stimuli, mitochondria can also alter their morphology accordingly, by inducing fission or fusion. In an extreme situation, mitochondria are degraded by mitophagy. Adequate function and regulation of these mitochondrial quality control pathways are crucial for cellular homeostasis. As we discuss, alterations in these processes have been linked to several pathologies including neurodegenerative diseases and cancer.
Assuntos
Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Mitofagia , Animais , Humanos , Mitocôndrias/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Proteínas Quinases/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Cell senescence is an important tumour suppressor mechanism and driver of ageing. Both functions are dependent on the development of the senescent phenotype, which involves an overproduction of pro-inflammatory and pro-oxidant signals. However, the exact mechanisms regulating these phenotypes remain poorly understood. Here, we show the critical role of mitochondria in cellular senescence. In multiple models of senescence, absence of mitochondria reduced a spectrum of senescence effectors and phenotypes while preserving ATP production via enhanced glycolysis. Global transcriptomic analysis by RNA sequencing revealed that a vast number of senescent-associated changes are dependent on mitochondria, particularly the pro-inflammatory phenotype. Mechanistically, we show that the ATM, Akt and mTORC1 phosphorylation cascade integrates signals from the DNA damage response (DDR) towards PGC-1ß-dependent mitochondrial biogenesis, contributing to aROS-mediated activation of the DDR and cell cycle arrest. Finally, we demonstrate that the reduction in mitochondrial content in vivo, by either mTORC1 inhibition or PGC-1ß deletion, prevents senescence in the ageing mouse liver. Our results suggest that mitochondria are a candidate target for interventions to reduce the deleterious impact of senescence in ageing tissues.
Assuntos
Envelhecimento/fisiologia , Mitocôndrias/fisiologia , Animais , Linhagem Celular , Humanos , Camundongos , Modelos Biológicos , FenótipoRESUMO
Autophagy is a lysosomal degradation pathway that converts macromolecules into substrates for energy production during nutrient-scarce conditions such as those encountered in tumor microenvironments. Constitutive mitochondrial uptake of endoplasmic reticulum (ER) Ca²âº mediated by inositol triphosphate receptors (IP3Rs) maintains cellular bioenergetics, thus suppressing autophagy. We show that the ER membrane protein Bax inhibitor-1 (BI-1) promotes autophagy in an IP3R-dependent manner. By reducing steady-state levels of ER Ca²âº via IP3Rs, BI-1 influences mitochondrial bioenergetics, reducing oxygen consumption, impacting cellular ATP levels, and stimulating autophagy. Furthermore, BI-1-deficient mice show reduced basal autophagy, and experimentally reducing BI-1 expression impairs tumor xenograft growth in vivo. BI-1's ability to promote autophagy could be dissociated from its known function as a modulator of IRE1 signaling in the context of ER stress. The results reveal BI-1 as a novel autophagy regulator that bridges Ca²âº signaling between ER and mitochondria, reducing cellular oxygen consumption and contributing to cellular resilience in the face of metabolic stress.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/imunologia , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Metabolismo Energético , Proteínas de Membrana/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Autofagia/genética , Linhagem Celular Tumoral , Endorribonucleases/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/microbiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Consumo de Oxigênio , Proteínas Serina-Treonina Quinases/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Infecções Estreptocócicas/imunologia , Streptococcus/imunologia , Estresse Fisiológico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The Bcl-2 proteins Bax and Bak can permeabilize the outer mitochondrial membrane and commit cells to apoptosis. Pro-survival Bcl-2 proteins control Bax by constant retrotranslocation into the cytosol of healthy cells. The stabilization of cytosolic Bax raises the question whether the functionally redundant but largely mitochondrial Bak shares this level of regulation. Here we report that Bak is retrotranslocated from the mitochondria by pro-survival Bcl-2 proteins. Bak is present in the cytosol of human cells and tissues, but low shuttling rates cause predominant mitochondrial Bak localization. Interchanging the membrane anchors of Bax and Bak reverses their subcellular localization compared to the wild-type proteins. Strikingly, the reduction of Bax shuttling to the level of Bak retrotranslocation results in full Bax toxicity even in absence of apoptosis induction. Thus, fast Bax retrotranslocation is required to protect cells from commitment to programmed death.
Assuntos
Apoptose/fisiologia , Citosol/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Linhagem Celular , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Transporte Proteico/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Necroptosis is a form of programmed cell death defined by activation of the kinase receptor interacting protein kinase 3 and its downstream effector, the pseudokinase mixed lineage kinase domain-like (MLKL). Activated MLKL translocates to the cell membrane and disrupts it, leading to loss of cellular ion homeostasis. In this study, we use a system in which this event can be specifically triggered by a small-molecule ligand to show that MLKL activation is sufficient to induce the processing and release of bioactive IL-1ß. MLKL activation triggers potassium efflux and assembly of the NLRP3 inflammasome, which is required for the processing and activity of IL-1ß released during necroptosis. Notably, MLKL activation also causes cell membrane disruption, which allows efficient release of IL-1ß independently of the recently described pyroptotic effector gasdermin-D. Taken together, our findings indicate that MLKL is an endogenous activator of the NLRP3 inflammasome, and that MLKL activation provides a mechanism for concurrent processing and release of IL-1ß independently of gasdermin-D.
Assuntos
Apoptose , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Monócitos/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Necrose , Proteínas Quinases/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Homeostase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Fosfato , Potássio/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Atg7 is a noncanonical, homodimeric E1 enzyme that interacts with the noncanonical E2 enzyme, Atg3, to mediate conjugation of the ubiquitin-like protein (UBL) Atg8 during autophagy. Here we report that the unique N-terminal domain of Atg7 (Atg7(NTD)) recruits a unique "flexible region" from Atg3 (Atg3(FR)). The structure of an Atg7(NTD)-Atg3(FR) complex reveals hydrophobic residues from Atg3 engaging a conserved groove in Atg7, important for Atg8 conjugation. We also report the structure of the homodimeric Atg7 C-terminal domain, which is homologous to canonical E1s and bacterial antecedents. The structures, SAXS, and crosslinking data allow modeling of a full-length, dimeric (Atg7~Atg8-Atg3)(2) complex. The model and biochemical data provide a rationale for Atg7 dimerization: Atg8 is transferred in trans from the catalytic cysteine of one Atg7 protomer to Atg3 bound to the N-terminal domain of the opposite Atg7 protomer within the homodimer. The studies reveal a distinctive E1~UBL-E2 architecture for enzymes mediating autophagy.
Assuntos
Autofagia , Proteínas de Transporte/química , Fibroblastos/enzimologia , Proteínas Associadas aos Microtúbulos/química , Enzimas de Conjugação de Ubiquitina/química , Sequência de Aminoácidos , Animais , Proteína 7 Relacionada à Autofagia , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Cristalografia por Raios X , Fibroblastos/patologia , Interações Hidrofóbicas e Hidrofílicas , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos , Mutação , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Multimerização Proteica , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Transfecção , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
During apoptosis, the BCL-2 protein family controls mitochondrial outer membrane permeabilization (MOMP), but the dynamics of this regulation remain controversial. We employed chimeric proteins composed of exogenous BH3 domains inserted into a tBID backbone that can activate the proapoptotic effectors BAX and BAK to permeabilize membranes without being universally sequestered by all antiapoptotic BCL-2 proteins. We thus identified two "modes" whereby prosurvival BCL-2 proteins can block MOMP, by sequestering direct-activator BH3-only proteins ("MODE 1") or by binding active BAX and BAK ("MODE 2"). Notably, we found that MODE 1 sequestration is less efficient and more easily derepressed to promote MOMP than MODE 2. Further, MODE 2 sequestration prevents mitochondrial fusion. We provide a unified model of BCL-2 family function that helps to explain otherwise paradoxical observations relating to MOMP, apoptosis, and mitochondrial dynamics.
Assuntos
Apoptose , Regulação da Expressão Gênica , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Citocromos c/análise , Células HeLa , Humanos , Mamíferos , Camundongos , Camundongos Knockout , Anotação de Sequência Molecular , Permeabilidade , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Transfecção , 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 bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Caspase-2 is an initiator caspase activated in response to heat shock and other stressors that induce apoptosis. Activation of caspase-2 requires induced proximity resulting after recruitment to caspase-2 activation complexes such as the PIDDosome. We have adapted bimolecular fluorescence complementation (BiFC) to measure caspase-2 induced proximity in real time in single cells. Nonfluorescent fragments of the fluorescent protein Venus that can associate to reform the fluorescent complex were fused to caspase-2, allowing visualization and kinetic measurements of caspase-2 induced proximity after heat shock and other stresses. This revealed that the caspase-2 activation platform occurred in the cytosol and not in the nucleus in response to heat shock, DNA damage, cytoskeletal disruption, and other treatments. Activation, as measured by this approach, in response to heat shock was RAIDD dependent and upstream of mitochondrial outer-membrane permeabilization. Furthermore, we identify Hsp90alpha as a key negative regulator of heat shock-induced caspase-2 activation.
Assuntos
Apoptose , Caspase 2/metabolismo , Citoplasma/enzimologia , Estresse Fisiológico , Animais , Apoptose/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Proteínas de Bactérias/genética , Técnicas Biossensoriais , Proteína Adaptadora de Sinalização CRADD/metabolismo , Caspase 2/genética , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Proteína de Domínio de Morte Associada a Fas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Fatores de Transcrição de Choque Térmico , Temperatura Alta , Humanos , Cinética , Proteínas Luminescentes/genética , Camundongos , Camundongos Knockout , Microscopia Confocal , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mutagênese Sítio-Dirigida , Multimerização Proteica , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transfecção , Moduladores de Tubulina/farmacologiaRESUMO
Regulated, programmed cell death is crucial for all multicellular organisms. Cell death is essential in many processes, including tissue sculpting during embryogenesis, development of the immune system and destruction of damaged cells. The best-studied form of programmed cell death is apoptosis, a process that requires activation of caspase proteases. Recently it has been appreciated that various non-apoptotic forms of cell death also exist, such as necroptosis and pyroptosis. These non-apoptotic cell death modalities can be either triggered independently of apoptosis or are engaged should apoptosis fail to execute. In this Commentary, we discuss several regulated non-apoptotic forms of cell death including necroptosis, autophagic cell death, pyroptosis and caspase-independent cell death. We outline what we know about their mechanism, potential roles in vivo and define outstanding questions. Finally, we review data arguing that the means by which a cell dies actually matters, focusing our discussion on inflammatory aspects of cell death.