RESUMO
Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543-559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.
Assuntos
Fator de Indução de Apoptose/metabolismo , Caspases/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Necrose/metabolismo , Animais , Fator de Indução de Apoptose/química , Calpaína/metabolismo , Linhagem Celular , Ciclofilina A/genética , Ciclofilina A/metabolismo , Dano ao DNA , Regulação para Baixo , Fibroblastos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Histonas/química , Histonas/genética , Metilnitronitrosoguanidina/farmacologia , Camundongos , Modelos Moleculares , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Cell death has been initially divided into apoptosis, in which the cell plays an active role, and necrosis, which is considered a passive cell death program. Intense research performed in the last decades has concluded that "programmed" cell death (PCD) is a more complex physiological process than initially thought. Indeed, although in most cases the PCD process is achieved via a family of Cys proteases known as caspases, an important number of regulated PCD pathways do not involve this family of proteases. As a consequence, active forms of PCD are initially referred to as caspase-dependent and caspase-independent. More recent data has revealed that there are also active caspase-independent necrotic pathways defined as necroptosis (programmed necrosis). The existence of necroptotic forms of death was corroborated by the discovery of key executioners such as the kinase RIP1 or the mitochondrial protein apoptosis-inducing factor (AIF). AIF is a Janus protein with a redox activity in the mitochondria and a pro-apoptotic function in the nucleus. We have recently described a particular form of AIF-mediated caspase-independent necroptosis that also implicates other molecules such as PARP-1, calpains, Bax, Bcl-2, histone H2AX, and cyclophilin A. From a therapeutic point of view, the unraveling of this new form of PCD poses a question: is it possible to modulate this necroptotic pathway independently of the classical apoptotic paths? Because the answer is yes, a wider understanding of AIF-mediated necroptosis could theoretically pave the way for the development of new drugs that modulate PCD. To this end, we present here an overview of the current knowledge of AIF and AIF-mediated necroptosis. We also summarize the state of the art in some of the most interesting therapeutic strategies that could target AIF or the AIF-mediated necroptotic pathway.
Assuntos
Fator de Indução de Apoptose/metabolismo , Caspases/metabolismo , Morte Celular/fisiologia , Terapia de Alvo Molecular , Sequência de Aminoácidos , Animais , Fator de Indução de Apoptose/genética , Morte Celular/efeitos dos fármacos , Sequência Conservada , Humanos , Mitocôndrias/metabolismoRESUMO
Introduction: Focal and segmental glomerulosclerosis (FSGS) is a frequent form of glomerulonephritis that may be caused by a soluble permeability factor and regulated by the immune system. We previously described a soluble form of calcium/calmodulin-dependent serine/threonine kinase (CASK) acting as a permeability factor in patients with recurrent FSGS (rFSGS). Here, we aimed to identify the immune cells associated with CASK secretion in patients with rFSGS. Methods: FACS, western blotting and immunoprecipitation were performed to detect CASK in peripheral blood mononuclear cells, including CD3+, CD20+, and CD14+subsets, from patients with rFSGS, healthy donors, transplant patients and patients with nephrotic syndrome due to diabetes mellitus, and in KHM2 cells. Results: CASK was produced mostly by monocytes in patients with rFSGS but not by T or B lymphocytes. It was not detectein cells from control patients. CASK was also produced and secreted by M2 polarized macrophages and KMH2 cells, but not by M1 polarized macrophages. CASK secretion was not not inhibited by brefeldin A, suggesting an absence of classical secretion pathway involvement. Within cells, CASK was partly colocalized with ALIX, a molecule involved in exosome development, and these two molecules were coprecipitated from M2 macrophages. Moreover, exosomes derived from M2 macrophages induced podocyte cytoskeleton alterations and increased podocyte motility. Conclusion: These results suggest that the soluble permeability factor CASK is secreted by monocytes and M2 macrophages, via exosomes, to alter the glomerular filtration barrier in rFSGS.
Assuntos
Glomerulosclerose Segmentar e Focal/imunologia , Guanilato Quinases/imunologia , Macrófagos/metabolismo , Adulto , Idoso , Animais , Transporte Biológico , Exossomos , Feminino , Glomerulosclerose Segmentar e Focal/sangue , Guanilato Quinases/metabolismo , Humanos , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , RecidivaRESUMO
INTRODUCTION: Focal and Segmental GlomeruloSclerosis (FSGS) can cause nephrotic syndrome with a risk of progression to end-stage renal disease. The idiopathic form has a high rate of recurrence after transplantation, suggesting the presence of a systemic circulating factor that causes glomerular permeability and can be removed by plasmapheresis or protein-A immunoadsorption. RESULTS: To identify this circulating factor, the eluate proteins bound on therapeutic immunoadsorption with protein-A columns were analyzed by comparative electrophoresis and mass spectrometry. A soluble form of calcium/calmodulin-dependent serine protein kinase (CASK) was identified. CASK was immunoprecipitated only in the sera of patients with recurrent FSGS after transplantation and not in control patients. Recombinant-CASK (rCASK) induced the reorganization of the actin cytoskeleton in immortalized podocytes, a redistribution of synaptopodin, ZO-1,vinculin and ENA. rCASK also induced alterations in the permeability of a monolayer of podocytes and increased the motility of pdodocytes in vitro. The extracellular domain of CD98, a transmembrane receptor expressed on renal epithelial cells, has been found to co-immunoprecipitated with rCASK. The invalidation of CD98 with siRNA avoided the structural changes of rCask treated cells suggesting its involvement in physiopathology of the disease. In mice, recombinant CASK induced proteinuria and foot process effacement in podocytes. CONCLUSION: Our results suggest that CASK can induce the recurrence of FSGS after renal transplantation.
Assuntos
Glomerulosclerose Segmentar e Focal/sangue , Guanilato Quinases/sangue , Transplante de Rim , Adulto , Animais , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Feminino , Proteína-1 Reguladora de Fusão/metabolismo , Glomerulosclerose Segmentar e Focal/complicações , Humanos , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Masculino , Membranas/metabolismo , Membranas/ultraestrutura , Camundongos , Pessoa de Meia-Idade , Podócitos/metabolismo , Podócitos/patologia , Podócitos/ultraestrutura , Ligação Proteica , Proteinúria/complicações , RecidivaRESUMO
Resistance to anticancer drugs is often related to deficient cell death execution pathways in cancer cells. Apoptosis, which denotes a form of cell death executed by caspases, was traditionally considered as the only physiological and programmed form of cell death. However, recent evidence indicates that programmed cell death (PCD) can occur in complete absence of caspase activation. Indeed, a large number of caspase-independent models are now defined and a key protein implicated in this type of PCD, apoptosis-inducing factor (AIF), has been identified. AIF is a mitochondrial protein with two faces looking in opposite life/death directions. Recently, the identification of five different isoforms allowed a better characterization of AIFs life/mitochondrial versus death/nuclear functions, as well as definition of its pro-apoptotic region and some of its nuclear partners. Importantly, much work on caspase-independent PCD has revealed that AIF participates in more PCD systems than initially thought. A wider molecular knowledge of AIF, and of the caspase-independent PCDs in which it is involved, are key to provide new insights into the role of PCD. There is no doubt that these insights will lead to the development of more selective and efficient drugs against cancer, degenerative diseases, and other pathological disorders implicating AIF.
Assuntos
Antineoplásicos/uso terapêutico , Fator de Indução de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Fármacos Cardiovasculares/uso terapêutico , Caspases/metabolismo , Núcleo Celular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacologia , Fator de Indução de Apoptose/química , Fármacos Cardiovasculares/farmacologia , Núcleo Celular/enzimologia , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Humanos , Isquemia/tratamento farmacológico , Isquemia/metabolismo , Isquemia/patologia , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Modelos Moleculares , Dados de Sequência Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Conformação Proteica , Isoformas de Proteínas/metabolismoRESUMO
Activation of caspases is recognized as a key element in the apoptotic process. However, new evidence is drawing attention to the emergent role of cell death pathways where caspases are not involved. Recent advances in the molecular understanding of these new ways to die, called caspase-independent, have revealed that mitochondria play an important role via the release of proapoptotic proteins. The purpose of this review is to integrate, from a biological and structural point of view, the most recent advances in the knowledge of the main mitochondrial proapoptotic proteins involved in this cell death cascade. The origin of programmed cell death is discussed through these strongly conserved effectors.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Morte Celular/fisiologia , Mitocôndrias/fisiologia , Sequência de Aminoácidos , Animais , Domínio Catalítico , Sequência Conservada , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
We have established a new method to purify Müllerian inhibiting substance (MIS) with higher purity and recovery over existing procedures. Recombinant human MIS was expressed in Chinese hamster ovary cells and secreted into chemically defined serum-free media. The secreted products were concentrated by either precipitation with ammonium sulfate or lectin-affinity chromatography, each of which was followed by anion-exchange chromatography. Further separation of the active carboxy-terminal domain of MIS was achieved after cleavage by plasmin followed by lectin-affinity chromatography. This method may be applicable to other members of the transforming growth factor beta family with which MIS shares sequence homology.
Assuntos
Glicoproteínas , Inibidores do Crescimento/isolamento & purificação , Hormônios Testiculares/isolamento & purificação , Animais , Hormônio Antimülleriano , Células CHO , Cromatografia de Afinidade/métodos , Cromatografia por Troca Iônica/métodos , Cricetinae , Inibidores do Crescimento/farmacologia , Humanos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Hormônios Testiculares/farmacologiaRESUMO
Caspase-independent programmed necrosis is a highly regulated cellular demise that displays morphological and biochemical necrotic hallmarks, such as an earlier permeability of the plasma membrane and lactate dehydrogenase (LDH) leakiness. This form of programmed cell death (PCD) is regulated by AIF, a FAD-dependent oxidoreductase, which is released from the mitochondria to the nucleus where it induces chromatin tcondensation and DNA fragmentation. Some years ago, it was established that the sequential activation of poly(ADP-ribose) polymerase- 1 (PARP-1), calpains and Bax regulates the mitochondrial AIF release associated to programmed necrosis. But, what happens when AIF is in the nucleus? How does this protein induce chromatinolysis and programmed necrosis? Recently, we have unraveled some of the mechanisms underlying the nuclear action of AIF in this type of caspase-independent cell death. Indeed, AIF plays a key role in programmed necrosis by its ability to organize a DNA-degrading complex with H2AX and Cyclophiline A (CypA). The AIF/H2AX link is indeed a critical event and explains the nuclear AIF apoptogenic action. In the present article, we outline the current knowledge on cell death by programmed necrosis and discuss the relevance of the AIF/H2AX/CypA DNA-degrading complex in the regulation of this original form of cell death.
Assuntos
Fator de Indução de Apoptose/metabolismo , Caspases/metabolismo , Histonas/metabolismo , Necrose/metabolismo , Apoptose , Fator de Indução de Apoptose/química , Calpaína/metabolismo , Ciclofilinas/química , Histonas/química , Humanos , Proteína X Associada a bcl-2/metabolismoRESUMO
The roles of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinases-1 and -2 (ERK-1/2) in fetal lung development have not been extensively characterized. To determine if ERK-1/2 signaling plays a role in fetal lung branching morphogenesis, U-0126, an inhibitor of the upstream kinase MAP ERK kinase (MEK), was added to fetal lung explants in vitro. Morphometry as measured by branching, area, perimeter, and complexity were significantly reduced in U-0126-treated lungs. At the same time, U-0126 treatment reduced ERK-1/2, slightly increased p38 kinase, but did not change c-Jun NH(2)-terminal kinase activities, indicating that U-0126 specifically inhibited the ERK-1/2 enzymes. These changes were associated with increased apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunofluorescent labeling of anti-active caspase-3 in the mesenchyme of explants after U-0126 treatment compared with the control. Mitosis characterized by immunolocalization of proliferating cell nuclear antigen was found predominantly in the epithelium and was reduced in U-0126-treated explants. Thus U-0126 causes specific inhibition of ERK-1/2 signaling, diminished branching morphogenesis, characterized by increased mesenchymal apoptosis, and decreased epithelial proliferation in fetal lung explants.
Assuntos
Butadienos/farmacologia , Inibidores Enzimáticos/farmacologia , Pulmão/embriologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Nitrilas/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Técnicas de Cultura , Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Feto/citologia , Feto/fisiologia , MAP Quinase Quinase 1 , MAP Quinase Quinase 2 , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Mesoderma/fisiologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , RatosRESUMO
Activated human T lymphocytes exposed to apoptotic stimuli targeting mitochondria (i.e. staurosporine), enter an early, caspase-independent phase of commitment to apoptosis characterized by cell shrinkage and peripheral chromatin condensation. We show that during this phase, AIF is selectively released from the intermembrane space of mitochondria, and that Bax undergo conformational change, relocation to mitochondria, and insertion into the outer mitochondrial membrane, in a Bid-independent manner. We analyzed the subcellular distribution of cathepsins (Cat) B, D, and L, in a search for caspase-independent factors responsible for Bax activation and AIF release. All were translocated from lysosomes to the cytosol, in correlation with limited destabilization of the lysosomes and release of lysosomal molecules in a size selective manner. However, only inhibition of Cat D activity by pepstatin A inhibited the early apoptotic events and delayed cell death, even in the presence of bafilomycin A1, an inhibitor of vacuolar type H+-ATPase, which inhibits acidification in lysosomes. Small interfering RNA-mediated gene silencing was used to inactivate Cat D, Bax, and AIF gene expression. This allowed us to define a novel sequence of events in which Cat D triggers Bax activation, Bax induces the selective release of mitochondrial AIF, and the latter is responsible for the early apoptotic phenotype.