RESUMEN

Optic atrophy 1 (OPA1) is a dynamin protein that mediates mitochondrial fusion at the inner membrane. OPA1 is also necessary for maintaining the cristae and thus essential for supporting cellular energetics. OPA1 exists as membrane-anchored long form (L-OPA1) and short form (S-OPA1) that lacks the transmembrane region and is generated by cleavage of L-OPA1. Mitochondrial dysfunction and cellular stresses activate the inner membrane-associated zinc metallopeptidase OMA1 that cleaves L-OPA1, causing S-OPA1 accumulation. The prevailing notion has been that L-OPA1 is the functional form, whereas S-OPA1 is an inactive cleavage product in mammals, and that stress-induced OPA1 cleavage causes mitochondrial fragmentation and sensitizes cells to death. However, S-OPA1 contains all functional domains of dynamin proteins, suggesting that it has a physiological role. Indeed, we recently demonstrated that S-OPA1 can maintain cristae and energetics through its GTPase activity, despite lacking fusion activity. Here, applying oxidant insult that induces OPA1 cleavage, we show that cells unable to generate S-OPA1 are more sensitive to this stress under obligatory respiratory conditions, leading to necrotic death. These findings indicate that L-OPA1 and S-OPA1 differ in maintaining mitochondrial function. Mechanistically, we found that cells that exclusively express L-OPA1 generate more superoxide and are more sensitive to Ca2+-induced mitochondrial permeability transition, suggesting that S-OPA1, and not L-OPA1, protects against cellular stress. Importantly, silencing of OMA1 expression increased oxidant-induced cell death, indicating that stress-induced OPA1 cleavage supports cell survival. Our findings suggest that S-OPA1 generation by OPA1 cleavage is a survival mechanism in stressed cells.

Asunto(s)

GTP Fosfohidrolasas/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/enzimología , Estrés Oxidativo , Animales , Calcio/metabolismo , Línea Celular , Supervivencia Celular , GTP Fosfohidrolasas/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/genética , Permeabilidad , Superóxidos/metabolismo

RESUMEN

Phosphatidylserine (PS), an anionic phospholipid enriched in the inner leaflet of the plasma membrane, is exposed to the outer leaflet during apoptosis. PS exposure was recently shown to be induced during tumor necrosis factor-induced necroptosis. We herein demonstrated that interferon (IFN)-γ induced necroptosis in Caspase-8-knockout mouse-derived embryonic fibroblasts (C8KO MEFs), as well as in WT MEFs co-treated with the pan-caspase inhibitor, z-VAD-fmk. PS exposure and necroptosis were significant after 6- and 24-h treatments with IFN-γ, respectively. To elucidate the molecular mechanisms underlying IFN-γ-induced PS exposure, we generated C8KO MEF-derived cell lines without the expression of RIPK3 (receptor-interacting protein kinase 3), an essential molecule in tumor necrosis factor-induced necroptosis, and IFN-γ-induced PS exposure and necrotic cell death were shown to be specifically inhibited by the loss of RIPK3 expression. Furthermore, the down-regulated expression of MLKL (mixed lineage kinase domain-like protein), a key molecule for inducing membrane rupture downstream of RIPK3 in necroptosis, abolished IFN-γ-induced PS exposure in C8KO MEFs. In human colorectal adenocarcinoma-derived HT29 cells, PS exposure and necroptosis were similarly induced by treatment with IFN-γ in the presence of Smac mimetics and z-VAD-fmk. The removal of IFN-γ from PS-exposing MEFs after a 6-h treatment completely inhibited necroptotic cell death but not the subsequent increase in the number of PS-exposing cells. Therefore, PS exposure mediated by RIPK3-activated MLKL oligomers was induced by a treatment with IFN-γ for a significant interval of time before the induction of necroptosis by membrane rupture.

Asunto(s)

Caspasa 8/genética , Interferón gamma/farmacología , Necroptosis/efectos de los fármacos , Fosfatidilserinas/metabolismo , Proteínas Quinasas/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Animales , Caspasa 8/metabolismo , Línea Celular , Células HT29 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas/química , Proteínas Quinasas/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo

RESUMEN

Formation of membrane pores/channels regulates various cellular processes, such as necroptosis or stem cell niche signaling. However, the roles of membrane lipids in the formation of pores and their biological functions are largely unknown. Here, using the cellular stress model evoked by the sphingolipid analog drug FTY720, we show that formation of ceramide-enriched membrane pores, referred to here as ceramidosomes, is initiated by a receptor-interacting Ser/Thr kinase 1 (RIPK1)-ceramide complex transported to the plasma membrane by nonmuscle myosin IIA-dependent trafficking in human lung cancer cells. Molecular modeling/simulation coupled with site-directed mutagenesis revealed that Asp147 or Asn169 of RIPK1 are key for ceramide binding and that Arg258 or Leu293 residues are involved in the myosin IIA interaction, leading to ceramidosome formation and necroptosis. Moreover, generation of ceramidosomes independently of any external drug/stress stimuli was also detected in the plasma membrane of germ line stem cells in ovaries during the early stages of oogenesis in Drosophila melanogaster Inhibition of ceramidosome formation via myosin IIA silencing limited germ line stem cell signaling and abrogated oogenesis. In conclusion, our findings indicate that the RIPK1-ceramide complex forms large membrane pores we named ceramidosomes. They further suggest that, in addition to their roles in stress-mediated necroptosis, these ceramide-enriched pores also regulate membrane integrity and signaling and might also play a role in D. melanogaster ovary development.

Asunto(s)

Membrana Celular/metabolismo , Ceramidas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Motoras Moleculares/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Necrosis/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Células A549 , Animales , Línea Celular , Membrana Celular/patología , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Humanos , Neoplasias Pulmonares/patología , Simulación del Acoplamiento Molecular , Necrosis/patología , Oogénesis , Ovario/crecimiento & desarrollo

RESUMEN

Immune therapy of cancer is among the most promising recent advances in medicine. Whether the immune system can keep cancer in check depends on, among other factors, the efficiency of immune cells to recognize and eliminate cancer cells. We describe a time-resolved single-cell assay that reports the quality, quantity, and kinetics of target cell death induced by single primary human natural killer (NK) cells. The assay reveals that single NK cells induce cancer cell death by apoptosis and necrosis but also by mixed forms. Inhibition of either one of the two major cytotoxic pathways, perforin/granzyme release or FasL/FasR interaction, unmasked the parallel activity of the other one. Ca2+ influx through Orai channels is important for tuning killer cell function. We found that the apoptosis/necrosis ratio of cancer cell death by NK cells is controlled by the magnitude of Ca2+ entry and furthermore by the relative concentrations of perforin and granzyme B. The possibility to change the apoptosis/necrosis ratio employed by NK cells offers an intriguing possibility to modulate the immunogenicity of the tumor microenvironment.

Asunto(s)

Células Asesinas Naturales/inmunología , Neoplasias/inmunología , Calcio/metabolismo , Calcio/farmacología , Muerte Celular , Granzimas/análisis , Humanos , Neoplasias/patología , Perforina/análisis , Análisis de la Célula Individual

RESUMEN

Monocytes differentiate into macrophages, which deactivate invading pathogens. Macrophages can be resistant to cell death mechanisms in some situations, and the mechanisms involved are not clear. Here, using mouse immune cells, we investigated whether the differentiation of macrophages affects their susceptibility to cell death by the ripoptosome/necrosome pathways. We show that treatment of macrophages with a mimetic of second mitochondrial activator of caspases (SMAC) resulted in ripoptosome-driven cell death that specifically depended on tumor necrosis factor α (TNFα) expression and the receptor-interacting serine/threonine protein kinase 1 (RipK1)-RipK3-caspase-8 interaction in activated and cycling macrophages. Differentiation of macrophages increased the expression of pro-inflammatory cytokines but reduced RipK1-dependent cell death and the RipK3-caspase-8 interaction. The expression of the anti-apoptotic mediators, X-linked inhibitor of apoptosis protein (XIAP) and caspase-like apoptosis regulatory protein (cFLIPL), also increased in differentiated macrophages, which inhibited caspase activation. The resistance to cell death was abrogated in XIAP-deficient macrophages. However, even in the presence of increased XIAP expression, inhibition of the mitogen-activated protein kinase (MAPK) p38 and MAPK-activated protein kinase 2 (MK2) made differentiated macrophages susceptible to cell death. These results suggest that the p38/MK2 pathway overrides apoptosis inhibition by XIAP and that acquisition of resistance to cell death by increased expression of XIAP and cFLIPL may allow inflammatory macrophages to participate in pathogen control for a longer duration.

Asunto(s)

Inflamación/inmunología , Proteínas Inhibidoras de la Apoptosis/inmunología , Macrófagos/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Animales , Apoptosis , Diferenciación Celular , Células Cultivadas , Macrófagos/citología , Ratones Endogámicos C57BL

RESUMEN

Mitochondrial dysfunction lies at the core of acute pancreatitis (AP). Diverse AP stimuli induce Ca2+-dependent formation of the mitochondrial permeability transition pore (MPTP), a solute channel modulated by cyclophilin D (CypD), the formation of which causes ATP depletion and necrosis. Oxidative stress reportedly triggers MPTP formation and is elevated in clinical AP, but how reactive oxygen species influence cell death is unclear. Here, we assessed potential MPTP involvement in oxidant-induced effects on pancreatic acinar cell bioenergetics and fate. H2O2 application promoted acinar cell apoptosis at low concentrations (1-10 µm), whereas higher levels (0.5-1 mm) elicited rapid necrosis. H2O2 also decreased the mitochondrial NADH/FAD+ redox ratio and ΔΨm in a concentration-dependent manner (10 µm to 1 mm H2O2), with maximal effects at 500 µm H2O2 H2O2 decreased the basal O2 consumption rate of acinar cells, with no alteration of ATP turnover at <50 µm H2O2 However, higher H2O2 levels (≥50 µm) diminished spare respiratory capacity and ATP turnover, and bioenergetic collapse, ATP depletion, and cell death ensued. Menadione exerted detrimental bioenergetic effects similar to those of H2O2, which were inhibited by the antioxidant N-acetylcysteine. Oxidant-induced bioenergetic changes, loss of ΔΨm, and cell death were not ameliorated by genetic deletion of CypD or by its acute inhibition with cyclosporine A. These results indicate that oxidative stress alters mitochondrial bioenergetics and modifies pancreatic acinar cell death. A shift from apoptosis to necrosis appears to be associated with decreased mitochondrial spare respiratory capacity and ATP production, effects that are independent of CypD-sensitive MPTP formation.

Asunto(s)

Apoptosis , Ciclofilinas/fisiología , Mitocondrias/fisiología , Proteínas de Transporte de Membrana Mitocondrial/fisiología , Necrosis , Estrés Oxidativo , Páncreas/patología , Células Acinares/metabolismo , Células Acinares/patología , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Peptidil-Prolil Isomerasa F , Metabolismo Energético , Potencial de la Membrana Mitocondrial , Ratones Endogámicos C57BL , Ratones Noqueados , Poro de Transición de la Permeabilidad Mitocondrial , Páncreas/metabolismo , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Necroptosis is an immunogenic cell death program that is associated with a host of human diseases, including inflammation, infections, and cancer. Receptor-interacting protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like protein (MLKL) are required for necroptosis activation. Specifically, RIPK3-dependent MLKL phosphorylation promotes the assembly of disulfide bond-dependent MLKL polymers that drive the execution of necroptosis. However, how MLKL disulfide bond formation is regulated is not clear. In this study we discovered that the MLKL-modifying compound necrosulfonamide cross-links cysteine 86 of human MLKL to cysteine 32 of the thiol oxidoreductase thioredoxin-1 (Trx1). Recombinant Trx1 preferentially binds to monomeric MLKL and blocks MLKL disulfide bond formation and polymerization in vitro Inhibition of MLKL polymer formation requires the reducing activity of Trx1. Importantly, shRNA-mediated knockdown of Trx1 promotes MLKL polymerization and sensitizes cells to necroptosis. Furthermore, pharmacological inhibition of Trx1 with compound PX-12 induces necroptosis in multiple cancer cell lines. Altogether, these findings demonstrate that Trx1 is a critical regulator of necroptosis that suppresses cell death by maintaining MLKL in a reduced inactive state. Our results further suggest new directions for targeted cancer therapy in which thioredoxin inhibitors like PX-12 could potentially be used to specifically target cancers expressing high levels of MLKL or MLKL short isoforms.

Asunto(s)

Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas Quinasas/metabolismo , Multimerización de Proteína , Tiorredoxinas/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Disulfuros/farmacología , Células HeLa , Humanos , Imidazoles/farmacología , Proteínas de Neoplasias/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Proteínas Quinasas/genética , Tiorredoxinas/genética

RESUMEN

We investigated the in vitro and in vivo anticancer effect of combining lysosomal membrane permeabilization (LMP)-inducing agent N-dodecylimidazole (NDI) with glycolytic inhibitor 2-deoxy-d-glucose (2DG). NDI-triggered LMP and 2DG-mediated glycolysis block synergized in inducing rapid ATP depletion, mitochondrial damage, and reactive oxygen species production, eventually leading to necrotic death of U251 glioma cells but not primary astrocytes. NDI/2DG-induced death of glioma cells was partly prevented by lysosomal cathepsin inhibitor E64 and antioxidant α-tocopherol, suggesting the involvement of LMP and oxidative stress in the observed cytotoxicity. LMP-inducing agent chloroquine also displayed a synergistic anticancer effect with 2DG, whereas glucose deprivation or glycolytic inhibitors iodoacetate and sodium fluoride synergistically cooperated with NDI, thus further indicating that the anticancer effect of NDI/2DG combination was indeed due to LMP and glycolysis block. The two agents synergistically induced ATP depletion, mitochondrial depolarization, oxidative stress, and necrotic death also in B16 mouse melanoma cells. Moreover, the combined oral administration of NDI and 2DG reduced in vivo melanoma growth in C57BL/6 mice by inducing necrotic death of tumor cells, without causing liver, spleen, or kidney toxicity. Based on these results, we propose that NDI-triggered LMP causes initial mitochondrial damage that is further increased by 2DG due to the lack of glycolytic ATP required to maintain mitochondrial health. This leads to a positive feedback cycle of mitochondrial dysfunction, ATP loss, and reactive oxygen species production, culminating in necrotic cell death. Therefore, the combination of LMP-inducing agents and glycolysis inhibitors seems worthy of further exploration as an anticancer strategy.

Asunto(s)

Desoxiglucosa/farmacología , Glioma/metabolismo , Glucólisis/efectos de los fármacos , Imidazoles/farmacología , Lisosomas/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Sinergismo Farmacológico , Glioma/tratamiento farmacológico , Glioma/fisiopatología , Humanos , Lisosomas/genética , Lisosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos

RESUMEN

Recent studies have shown that myocardial ischemia/reperfusion (I/R)-induced necrosis can be controlled by multiple genes. In this study, we observed that both strands (5p and 3p) of miR-223 were remarkably dysregulated in mouse hearts upon I/R. Precursor miR-223 (pre-miR-223) transgenic mouse hearts exhibited better recovery of contractile performance over reperfusion period and lesser degree of myocardial necrosis than wild type hearts upon ex vivo and in vivo myocardial ischemia. Conversely, pre-miR-223 knock-out (KO) mouse hearts displayed opposite effects. Furthermore, we found that the RIP1/RIP3/MLKL necroptotic pathway and inflammatory response were suppressed in transgenic hearts, whereas they were activated in pre-miR-223 KO hearts upon I/R compared with wild type controls. Accordingly, treatment of pre-miR-223 KO mice with necrostatin-1s, a potent necroptosis inhibitor, significantly decreased I/R-triggered cardiac necroptosis, infarction size, and dysfunction. Mechanistically, we identified two critical cell death receptors, TNFR1 and DR6, as direct targets of miR-223-5p, whereas miR-223-3p directly suppressed the expression of NLRP3 and IκB kinase α, two important mediators known to be involved in I/R-induced inflammation and cell necroptosis. Our findings indicate that miR-223-5p/-3p duplex works together and cooperatively inhibits I/R-induced cardiac necroptosis at multiple layers. Thus, pre-miR-223 may constitute a new therapeutic agent for the treatment of ischemic heart disease.

Asunto(s)

MicroARNs/biosíntesis , Daño por Reperfusión Miocárdica/metabolismo , Animales , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Imidazoles/farmacología , Indoles/farmacología , Ratones , Ratones Noqueados , MicroARNs/genética , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/patología , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Necrosis , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/biosíntesis , Receptores Tipo I de Factores de Necrosis Tumoral/genética

RESUMEN

Injury to the barrier tissue initiates a rapid distribution of myeloid immune cells from bone marrow, which guide sound wound healing. Bisphosphonates, a widely used anti-bone resorptive drug with minimal systemic side effects, have been linked to an abnormal wound healing in the oral barrier tissue leading to, in some cases, osteonecrosis of the jaw (ONJ). Here we report that the development of ONJ may involve abnormal phenotypic plasticity of Ly6G+/Gr1+ myeloid cells in the oral barrier tissue undergoing tooth extraction wound healing. A bolus intravenous zoledronate (ZOL) injection to female C57Bl/6 mice followed by maxillary first molar extraction resulted in the development of ONJ-like lesion during the second week of wound healing. The multiplex assay of dissociated oral barrier cells exhibited the secretion of cytokines and chemokines, which was significantly modulated in ZOL mice. Tooth extraction-induced distribution of Ly6G+/Gr1+ cells in the oral barrier tissue increased in ZOL mice at week 2. ONJ-like lesion in ZOL mice contained Ly6G+/Gr1+ cells with abnormal size and morphology as well as different flow cytometric staining intensity. When anti-Ly6G (Gr1) antibody was intraperitoneally injected for 5 days during the second week of tooth extraction, CD11b+GR1(hi) cells in bone marrow and Ly6G+ cells in the oral barrier tissue were depleted, and the development of ONJ-like lesion was significantly attenuated. This study suggests that local modulation of myeloid cell plasticity in the oral barrier tissue may provide the basis for pathogenesis and thus therapeutic as well as preventive strategy of ONJ.

Asunto(s)

Osteonecrosis de los Maxilares Asociada a Difosfonatos/inmunología , Células Mieloides/inmunología , Cicatrización de Heridas/inmunología , Animales , Antígenos Ly/inmunología , Osteonecrosis de los Maxilares Asociada a Difosfonatos/patología , Médula Ósea/inmunología , Médula Ósea/patología , Femenino , Ratones , Boca/patología , Células Mieloides/patología , Extracción Dental

RESUMEN

Necroptosis is a RIP1-dependent programmed cell death (PCD) pathway that is distinct from apoptosis. Downstream effector pathways of necroptosis include formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS), both of which depend on glycolysis. This suggests that increased cellular glucose may prime necroptosis. Here we show that exposure to hyperglycemic levels of glucose enhances necroptosis in primary red blood cells (RBCs), Jurkat T cells, and U937 monocytes. Pharmacologic or siRNA inhibition of RIP1 prevented the enhanced death, confirming it as RIP1-dependent necroptosis. Hyperglycemic enhancement of necroptosis depends upon glycolysis with AGEs and ROS playing a role. Total levels of RIP1, RIP3, and mixed lineage kinase domain-like (MLKL) proteins were increased following treatment with high levels of glucose in Jurkat and U937 cells and was not due to transcriptional regulation. The observed increase in RIP1, RIP3, and MLKL protein levels suggests a potential positive feedback mechanism in nucleated cell types. Enhanced PCD due to hyperglycemia was specific to necroptosis as extrinsic apoptosis was inhibited by exposure to high levels of glucose. Hyperglycemia resulted in increased infarct size in a mouse model of brain hypoxia-ischemia injury. The increased infarct size was prevented by treatment with nec-1s, strongly suggesting that increased necroptosis accounts for exacerbation of this injury in conditions of hyperglycemia. This work reveals that hyperglycemia represents a condition in which cells are extraordinarily susceptible to necroptosis, that local glucose levels alter the balance of PCD pathways, and that clinically relevant outcomes may depend on glucose-mediated effects on PCD.

Asunto(s)

Eritrocitos/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Hiperglucemia/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Muerte Celular , Modelos Animales de Enfermedad , Eritrocitos/patología , Proteínas Activadoras de GTPasa/genética , Productos Finales de Glicación Avanzada/genética , Productos Finales de Glicación Avanzada/metabolismo , Humanos , Hiperglucemia/genética , Hiperglucemia/patología , Células Jurkat , Ratones , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Unión al ARN/genética , Células U937

RESUMEN

Receptor-interacting protein kinase 3 (RIPK3) is a serine/threonine kinase with essential function in necroptosis. The activity of RIPK3 is controlled by phosphorylation. Once activated, RIPK3 phosphorylates and activates the downstream effector mixed lineage kinase domain-like (MLKL) to induce necroptosis. In certain situations, RIPK3 has also been shown to promote apoptosis or cytokine expression in a necroptosis and kinase-independent manner. The ubiquitin-proteasome system is the major pathway for selective degradation of cellular proteins and thus has a critical role in many cellular processes such as cell survival and cell death. Clinically, proteasome inhibition has shown promise as an anti-cancer agent. Here we show that the proteasome inhibitors MG132 and bortezomib activate the RIPK3-MLKL necroptotic pathway in mouse fibroblasts as well as human leukemia cells. Unlike necroptosis induced by classical TNF-like cytokines, necroptosis induced by proteasome inhibitors does not require caspase inhibition. However, an intact RIP homotypic interaction motif (RHIM) is essential. Surprisingly, when recruitment of MLKL to RIPK3 is restricted, proteasome inhibitors induced RIPK3-dependent apoptosis. Proteasome inhibition led to accumulation of K48-linked ubiquitinated RIPK3, which was partially reduced when Lys-264 was mutated. Taken together, these results reveal the ubiquitin-proteasome system as a novel regulatory mechanism for RIPK3-dependent necroptosis.

Asunto(s)

Muerte Celular/efectos de los fármacos , Leupeptinas/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Antineoplásicos/farmacología , Bortezomib/farmacología , Línea Celular , Línea Celular Tumoral , Humanos , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Quinasas/metabolismo , Ubiquitinación/efectos de los fármacos

RESUMEN

Inflammation is a key instigator of the immune responses that drive atherosclerosis and allograft rejection. IL-1α, a powerful cytokine that activates both innate and adaptive immunity, induces vessel inflammation after release from necrotic vascular smooth muscle cells (VSMCs). Similarly, IL-1α released from endothelial cells (ECs) damaged during transplant drives allograft rejection. However, IL-1α requires cleavage for full cytokine activity, and what controls cleavage in necrotic ECs is currently unknown. We find that ECs have very low levels of IL-1α activity upon necrosis. However, TNFα or IL-1 induces significant levels of active IL-1α in EC necrotic lysates without alteration in protein levels. Increased activity requires cleavage of IL-1α by calpain to the more active mature form. Immunofluorescence and proximity ligation assays show that IL-1α associates with interleukin-1 receptor-2, and this association is decreased by TNFα or IL-1 and requires caspase activity. Thus, TNFα or IL-1 treatment of ECs leads to caspase proteolytic activity that cleaves interleukin-1 receptor-2, allowing IL-1α dissociation and subsequent processing by calpain. Importantly, ECs could be primed by IL-1α from adjacent damaged VSMCs, and necrotic ECs could activate neighboring normal ECs and VSMCs, causing them to release inflammatory cytokines and up-regulate adhesion molecules, thus amplifying inflammation. These data unravel the molecular mechanisms and interplay between damaged ECs and VSMCs that lead to activation of IL-1α and, thus, initiation of adaptive responses that cause graft rejection.

Asunto(s)

Aloinjertos/inmunología , Caspasa 1/metabolismo , Rechazo de Injerto/metabolismo , Rechazo de Injerto/patología , Células Endoteliales de la Vena Umbilical Humana/patología , Interleucina-1alfa/metabolismo , Receptores Tipo II de Interleucina-1/metabolismo , Calpaína/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Interleucina-1/farmacología , Necrosis/inmunología , Proteolisis , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage, and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidence suggests that both processes can be morphological expressions of a common biochemical network known as "apoptosis-necrosis continuum." To gain insight into the events driving the apoptosis-necrosis continuum, apoptotically proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After using a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase activation and necrotic-like cell death. Lower amounts of thiolic antioxidants partially prevented chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death.

Asunto(s)

Antineoplásicos/farmacología , Caspasas/metabolismo , Cromatina/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Necrosis/enzimología , Clorometilcetonas de Aminoácidos/farmacología , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Benzofenantridinas/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Caspasas/genética , Línea Celular Tumoral , Cromatina/metabolismo , Cromatina/ultraestructura , Colchicina/farmacología , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Necrosis/inducido químicamente , Necrosis/genética , Neuronas , Nocodazol/farmacología , Peptidomiméticos/farmacología , Quinolinas/farmacología , Rotenona/farmacología , Transducción de Señal , Estaurosporina/farmacología , Tapsigargina/farmacología

RESUMEN

It is widely accepted that overactivation of NMDA receptors, resulting in calcium overload and consequent mitochondrial dysfunction in retinal ganglion neurons, plays a significant role in promoting neurodegenerative disorders such as glaucoma. Calcium has been shown to initiate a transient hyperpolarization of the mitochondrial membrane potential triggering a burst of reactive oxygen species leading to apoptosis. Strategies that enhance cell survival signaling pathways aimed at preventing this adverse hyperpolarization of the mitochondrial membrane potential may provide a novel therapeutic intervention in retinal disease. In the retina, brain-derived neurotrophic factor has been shown to be neuroprotective, and our group previously reported a PSD-95/PDZ-binding cyclic peptide (CN2097) that augments brain-derived neurotrophic factor-induced pro-survival signaling. Here, we examined the neuroprotective properties of CN2097 using an established retinal in vivo NMDA toxicity model. CN2097 completely attenuated NMDA-induced caspase 3-dependent and -independent cell death and PARP-1 activation pathways, blocked necrosis, and fully prevented the loss of long term ganglion cell viability. Although neuroprotection was partially dependent upon CN2097 binding to the PDZ domain of PSD-95, our results show that the polyarginine-rich transport moiety C-R(7), linked to the PDZ-PSD-95-binding cyclic peptide, was sufficient to mediate short and long term protection via a mitochondrial targeting mechanism. C-R(7) localized to mitochondria and was found to reduce mitochondrial respiration, mitochondrial membrane hyperpolarization, and the generation of reactive oxygen species, promoting survival of retinal neurons.

Asunto(s)

Potencial de la Membrana Mitocondrial/efectos de los fármacos , N-Metilaspartato/farmacología , Péptidos/farmacología , Neuronas Retinianas/efectos de los fármacos , Animales , Western Blotting , Muerte Celular/efectos de los fármacos , Homólogo 4 de la Proteína Discs Large , Agonistas de Aminoácidos Excitadores/farmacología , Guanilato-Quinasas/metabolismo , Células HEK293 , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/fisiología , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/fisiología , Fármacos Neuroprotectores/metabolismo , Fármacos Neuroprotectores/farmacología , Péptidos/metabolismo , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Unión Proteica , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Retina/citología , Retina/efectos de los fármacos , Retina/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Neuronas Retinianas/metabolismo , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/fisiología

RESUMEN

Necroptosis is an alternate programmed cell death pathway that is unleashed by caspase-8 compromise and mediated by receptor-interacting protein kinase 3 (RIP3). Murine cytomegalovirus (CMV) and herpes simplex virus (HSV) encode caspase-8 inhibitors that prevent apoptosis together with competitors of RIP homotypic interaction motif (RHIM)-dependent signal transduction to interrupt the necroptosis. Here, we show that pro-necrotic murine CMV M45 mutant virus drives virus-induced necroptosis during nonproductive infection of RIP3-expressing human fibroblasts, whereas WT virus does not. Thus, M45-encoded RHIM competitor, viral inhibitor of RIP activation, sustains viability of human cells like it is known to function in infected mouse cells. Importantly, human CMV is shown to block necroptosis induced by either TNF or M45 mutant murine CMV in RIP3-expressing human cells. Human CMV blocks TNF-induced necroptosis after RIP3 activation and phosphorylation of the mixed lineage kinase domain-like (MLKL) pseudokinase. An early, IE1-regulated viral gene product acts on a necroptosis step that follows MLKL phosphorylation prior to membrane leakage. This suppression strategy is distinct from RHIM signaling competition by murine CMV or HSV and interrupts an execution process that has not yet been fully elaborated.

Asunto(s)

Citomegalovirus/fisiología , Necrosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Citomegalovirus/metabolismo , Evolución Molecular , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/virología , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Ratones , Muromegalovirus/fisiología , Fosforilación , Transporte de Proteínas , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal , Transducción Genética

RESUMEN

In reply to internal or external danger stimuli, the body orchestrates an inflammatory response. The endogenous triggers of this process are the damage-associated molecular patterns (DAMPs). DAMPs represent a heterogeneous group of molecules that draw their origin either from inside the various compartments of the cell or from the extracellular space. Following interaction with pattern recognition receptors in cross-talk with various non-immune receptors, DAMPs determine the downstream signaling outcome of septic and aseptic inflammatory responses. In this review, the diverse nature, structural characteristics, and signaling pathways elicited by DAMPs will be critically evaluated.

Asunto(s)

Citosol/inmunología , Matriz Extracelular/inmunología , Inflamación/inmunología , Receptores de Reconocimiento de Patrones/inmunología , Animales , Citosol/metabolismo , Matriz Extracelular/metabolismo , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Modelos Inmunológicos , Proteoglicanos/inmunología , Proteoglicanos/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Transducción de Señal/inmunología

RESUMEN

Glycogen synthase kinase-3ß (GSK-3ß) is a major positive regulator of the mitochondrial permeability transition pore (mPTP), a principle trigger of cell death, under the condition of oxidative stress. However, the mechanism by which cytosolic GSK-3ß translocates to mitochondria, promoting mPTP opening, remains unclear. Here we addressed this issue by analyses of the effect of site-directed mutations in GSK-3ß on mitochondrial translocation and protein/protein interactions upon oxidative stress. H9c2 cardiomyoblasts were transfected with GFP-tagged GSK-3ß (WT), a mutant GSK-3ß insensitive to inhibitory phosphorylation (S9A), or kinase-deficient GSK-3ß (K85R). Time lapse observation revealed that WT and S9A translocated from the cytosol to the mitochondria more promptly than did K85R after exposure to oxidative stress. H2O2 increased the density of nine spots on two-dimensional gel electrophoresis of anti-GSK-3ß-immunoprecipitates by more than 3-fold. MALDI-TOF/MS analysis revealed that one of the spots contained voltage-dependent anion channel 2 (VDAC2). Knockdown of VDAC2, but not VDAC1 or VDAC3, by siRNA attenuated both the mitochondrial translocation of GSK-3ß and mPTP opening under stress conditions. The mitochondrial translocation of GSK-3ß was attenuated also when Lys-15, but not Arg-4 or Arg-6, in the N-terminal domain of GSK-3ß was replaced with alanine. The oxidative stress-induced mitochondrial translocation of GSK-3ß was associated with an increase in cell death, which was suppressed by lithium chloride (LiCl), a GSK-3ß inhibitor. These results demonstrate that GSK-3ß translocates from the cytosol to mitochondria in a kinase activity- and VDAC2-dependent manner in which an N-terminal domain of GSK-3ß may function as a mitochondrial targeting sequence.

Asunto(s)

Glucógeno Sintasa Quinasa 3/metabolismo , Canal Aniónico 2 Dependiente del Voltaje/metabolismo , Transporte Biológico , Muerte Celular , Citosol/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Peróxido de Hidrógeno/química , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Necrosis , Estrés Oxidativo , Permeabilidad , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

Cell death by necrosis is emerging not merely as a passive phenomenon but as a cell-regulated process. Here, by using different necrotic triggers, we prove the existence of two distinct necrotic pathways. The mitochondrial reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone elicits necrosis characterized by the involvement of RIP1 and Drp1. However, G5, a non-selective isopeptidase inhibitor, triggers a distinct necrotic pathway that depends on the protein phosphatase PP2A and the actin cytoskeleton. PP2A catalytic subunit is stabilized by G5 treatment, and its activity is increased. Furthermore, PP2Ac accumulates into the cytoplasm during necrosis similarly to HMGB1. We have also defined in the actin-binding protein cofilin-1 a link between PP2A, actin cytoskeleton, and necrotic death. Cofilin-1-severing/depolymerization activity is negatively regulated by phosphorylation of serine 3. PP2A contributes to the dephosphorylation of serine 3 elicited by G5. Finally, a cofilin mutant that mimics phosphorylated Ser-3 can partially rescue necrosis in response to G5.

Asunto(s)

Citoesqueleto de Actina/metabolismo , Cofilina 1/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas de Unión al ARN/metabolismo , Citoesqueleto de Actina/ultraestructura , Factores Despolimerizantes de la Actina/química , Estructuras de la Membrana Celular/química , Estructuras de la Membrana Celular/efectos de los fármacos , Cofilina 1/química , Células HT29 , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Necrosis/genética , Necrosis/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteína Fosfatasa 2/genética , Proteolisis , Piranos/farmacología , Proteínas de Unión al ARN/química , Especies Reactivas de Oxígeno/metabolismo , Compuestos de Sulfhidrilo/farmacología

RESUMEN

ALKBH7 is the mitochondrial AlkB family member that is required for alkylation- and oxidation-induced programmed necrosis. In contrast to the protective role of other AlkB family members after suffering alkylation-induced DNA damage, ALKBH7 triggers the collapse of mitochondrial membrane potential and promotes cell death. Moreover, genetic ablation of mouse Alkbh7 dramatically increases body weight and fat mass. Here, we present crystal structures of human ALKBH7 in complex with Mn(II) and α-ketoglutarate at 1.35 Å or N-oxalylglycine at 2.0 Å resolution. ALKBH7 possesses the conserved double-stranded ß-helix fold that coordinates a catalytically active iron by a conserved HX(D/E) … Xn … H motif. Self-hydroxylation of Leu-110 was observed, indicating that ALKBH7 has the potential to catalyze hydroxylation of its substrate. Unlike other AlkB family members whose substrates are DNA or RNA, ALKBH7 is devoid of the "nucleotide recognition lid" which is essential for binding nucleobases, and thus exhibits a solvent-exposed active site; two loops between ß-strands ß6 and ß7 and between ß9 and ß10 create a special outer wall of the minor ß-sheet of the double-stranded ß-helix and form a negatively charged groove. These distinct features suggest that ALKBH7 may act on protein substrate rather than nucleic acids. Taken together, our findings provide a structural basis for understanding the distinct function of ALKBH7 in the AlkB family and offer a foundation for drug design in treating cell death-related diseases and metabolic diseases.

Asunto(s)

Proteínas Mitocondriales/química , Enzimas AlkB , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Grasas/metabolismo , Humanos , Ácidos Cetoglutáricos , Manganeso/química , Manganeso/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Necrosis , Multimerización de Proteína , Estructura Secundaria de Proteína , Alineación de Secuencia , Difracción de Rayos X