RESUMEN
Changes in mitochondrial membrane permeability are closely associated with mitochondria-mediated apoptosis. Antimicrobial peptides (AMPs), which have been found to enter cells to exert physiological effects, cause damage to the mitochondria. This paper reviews the molecular mechanisms of AMP-mediated apoptosis by changing the permeability of the mitochondrial membrane through three pathways: the outer mitochondrial membrane (OMM), inner mitochondrial membrane (IMM), and mitochondrial permeability transition pore (MPTP). The roles of AMPs in inducing changes in membrane permeability and apoptosis are also discussed. Combined with recent research results, the possible application prospects of AMPs are proposed to provide a theoretical reference for the development of AMPs as therapeutic agents for human diseases.
Asunto(s)
Péptidos Antimicrobianos , Membranas Mitocondriales , Humanos , Membranas Mitocondriales/metabolismo , Mitocondrias/fisiología , Apoptosis/fisiología , Permeabilidad , Proteínas de Transporte de Membrana Mitocondrial/metabolismoRESUMEN
Several drug targets have been amenable to drug discovery pursuit not until the characterization of the mitochondrial permeability transition pore (MPTP), a pore with an undefined molecular identity that forms on the inner mitochondrial membrane upon mitochondrial permeability transition (MPT) under the influence of calcium overload and oxidative stress. The opening of the pore which is presumed to cause cell death in certain human diseases also has implications under physiological parlance. Different models for this pore have been postulated following its first identification in the last six decades. The mitochondrial community has witnessed many protein candidates such as; voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), Mitochondrial phosphate carrier (PiC), Spastic Paralegin (SPG7), disordered proteins, and F1Fo ATPase. However, genetic studies have cast out most of these candidates with only F1Fo ATPase currently under intense argument. Cyclophilin D (CyPD) remains the widely accepted positive regulator of the MPTP known to date, but no drug candidate has emerged as its inhibitor, raising concern issues for therapeutics. Thus, in this review, we discuss various models of MPTP reported with the hope of stimulating further research in this field. We went beyond the classical description of the MPTP to ascribe a 'two-edged sword property' to the pore for therapeutic function in human disease because its inhibition and activation have pharmacological relevance. We suggested putative proteins upstream to CyPD that can regulate its activity and prevent cell deaths in neurodegenerative disease and ischemia-reperfusion injury.
Asunto(s)
Poro de Transición de la Permeabilidad Mitocondrial , Enfermedades Neurodegenerativas , Humanos , Adenosina Trifosfatasas , Calcio/metabolismo , Peptidil-Prolil Isomerasa F , Descubrimiento de Drogas , Proteínas de Transporte de Membrana Mitocondrial/metabolismoRESUMEN
A new series of conjugates of aminoadamantane and γ-carboline, which are basic scaffolds of the known neuroactive agents, memantine and dimebon (Latrepirdine) was synthesized and characterized. Conjugates act simultaneously on several biological structures and processes involved in the pathogenesis of Alzheimer's disease and some other neurodegenerative disorders. In particular, these compounds inhibit enzymes of the cholinesterase family, exhibiting higher inhibitory activity against butyrylcholinesterase (BChE), but having almost no effect on the activity of carboxylesterase (anti-target). The compounds serve as NMDA-subtype glutamate receptor ligands, show mitoprotective properties by preventing opening of the mitochondrial permeability transition (MPT) pore, and act as microtubule stabilizers, stimulating the polymerization of tubulin and microtubule-associated proteins. Structure-activity relationships were studied, with particular attention to the effect of the spacer on biological activity. The synthesized conjugates showed new properties compared to their prototypes (memantine and dimebon), including the ability to bind to the ifenprodil-binding site of the NMDA receptor and to occupy the peripheral anionic site of acetylcholinesterase (AChE), which indicates that these compounds can act as blockers of AChE-induced ß-amyloid aggregation. These new attributes of the conjugates represent improvements to the pharmacological profiles of the separate components by conferring the potential to act as neuroprotectants and cognition enhancers with a multifunctional mode of action.
Asunto(s)
Amantadina/química , Amantadina/farmacología , Carbolinas/química , Carbolinas/farmacología , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Acetilcolinesterasa/química , Amantadina/análogos & derivados , Animales , Butirilcolinesterasa/química , Carboxilesterasa/química , Dominio Catalítico , Línea Celular , Inhibidores de la Colinesterasa/síntesis química , Caballos , Humanos , Cinética , Ligandos , Memantina/química , Memantina/farmacología , Necrosis por Permeabilidad de la Transmembrana Mitocondrial/efectos de los fármacos , Simulación del Acoplamiento Molecular , Propidio/química , Ratas , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Relación Estructura-Actividad , Porcinos , Tubulina (Proteína)/efectos de los fármacos , Moduladores de Tubulina/síntesis química , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologíaRESUMEN
Mitochondrial dysfunction is associated with a variety of human diseases including neurodegeneration, diabetes, nonalcohol fatty liver disease (NAFLD), and cancer, but its underlying causes are incompletely understood. Using the human hepatic cell line HepG2 as a model, we show here that endoplasmic reticulum-associated degradation (ERAD), an ER protein quality control process, is critically required for mitochondrial function in mammalian cells. Pharmacological inhibition or genetic ablation of key proteins involved in ERAD increased cell death under both basal conditions and in response to proinflammatory cytokines, a situation frequently found in NAFLD. Decreased viability of ERAD-deficient HepG2 cells was traced to impaired mitochondrial functions including reduced ATP production, enhanced reactive oxygen species (ROS) accumulation, and increased mitochondrial outer membrane permeability. Transcriptome profiling revealed widespread down-regulation of genes underpinning mitochondrial functions, and up-regulation of genes associated with tumor growth and aggression. These results highlight a critical role for ERAD in maintaining mitochondrial functional and structural integrity and raise the possibility of improving cellular and organismal mitochondrial function via enhancing cellular ERAD capacity.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Transcriptoma , Adenosina Trifosfato/metabolismo , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Edición Génica , Células Hep G2 , Humanos , Interleucina-12/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/genética , Proteínas/genética , Proteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia ArribaRESUMEN
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/metabolismoRESUMEN
Calcium homeostasis is essential for cell survival and is precisely controlled by several cellular actors such as the sarco/endoplasmic reticulum and mitochondria. Upon stress induction, Ca2+ released from sarco/endoplasmic reticulum stores and from extracellular Ca2+ pools accumulates in the cytosol and in the mitochondria. This induces Ca2+ overload and ultimately the opening of the mitochondrial permeability transition pore (mPTP), promoting cell death. Currently, it is unclear whether intracellular Ca2+ stores are sufficient to promote the mPTP opening. Ca2+ retention capacity (CRC) corresponds to the maximal Ca2+ uptake by the mitochondria before mPTP opening. In this study, using permeabilized cardiomyocytes isolated from adult mice, we modified the standard CRC assay by specifically inducing reticular Ca2+ release to investigate the respective contributions of reticular Ca2+ and extracellular Ca2+ to mPTP opening in normoxic conditions or after anoxia-reoxygenation. Our experiments revealed that Ca2+ released from the sarco/endoplasmic reticulum is not sufficient to trigger mPTP opening and corresponds to â¼50% of the total Ca2+ levels required to open the mPTP. We also studied mPTP opening after anoxia-reoxygenation in the presence or absence of extracellular Ca2+ In both conditions, Ca2+ leakage from internal stores could not trigger mPTP opening by itself but significantly decreased the CRC. Our findings highlight how a modified CRC assay enables the investigation of the role of reticular and extracellular Ca2+ pools in the regulation of the mPTP. We propose that this method may be useful for screening molecules of interest implicated in mPTP regulation.
Asunto(s)
Calcio/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Muerte Celular , Células Cultivadas , Retículo Endoplásmico/metabolismo , Humanos , Hipoxia/metabolismo , Hipoxia/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Poro de Transición de la Permeabilidad Mitocondrial , Miocitos Cardíacos/citologíaRESUMEN
The mitochondrial F-ATP synthase is a complex molecular motor arranged in V-shaped dimers that is responsible for most cellular ATP synthesis in aerobic conditions. In the yeast F-ATP synthase, subunits e and g of the FO sector constitute a lateral domain, which is required for dimer stability and cristae formation. Here, by using site-directed mutagenesis, we identified Arg-8 of subunit e as a critical residue in mediating interactions between subunits e and g, most likely through an interaction with Glu-83 of subunit g. Consistent with this hypothesis, (i) the substitution of Arg-8 in subunit e (eArg-8) with Ala or Glu or of Glu-83 in subunit g (gGlu-83) with Ala or Lys destabilized the digitonin-extracted F-ATP synthase, resulting in decreased dimer formation as revealed by blue-native electrophoresis; and (ii) simultaneous substitution of eArg-8 with Glu and of gGlu-83 with Lys rescued digitonin-stable F-ATP synthase dimers. When tested in lipid bilayers for generation of Ca2+-dependent channels, WT dimers displayed the high-conductance channel activity expected for the mitochondrial megachannel/permeability transition pore, whereas dimers obtained at low digitonin concentrations from the Arg-8 variants displayed currents of strikingly small conductance. Remarkably, double replacement of eArg-8 with Glu and of gGlu-83 with Lys restored high-conductance channels indistinguishable from those seen in WT enzymes. These findings suggest that the interaction of subunit e with subunit g is important for generation of the full-conductance megachannel from F-ATP synthase.
Asunto(s)
Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Dimerización , Potencial de la Membrana Mitocondrial , Poro de Transición de la Permeabilidad Mitocondrial , ATPasas de Translocación de Protón Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/genética , Mutagénesis Sitio-Dirigida , Estabilidad Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The mitochondrial matrix ATPase associated with diverse cellular activities (m-AAA) protease spastic paraplegia 7 (SPG7) has been recently implicated as either a negative or positive regulatory component of the mitochondrial permeability transition pore (mPTP) by two research groups. To address this controversy, we investigated possible mechanisms that explain the discrepancies between these two studies. We found that loss of the SPG7 gene increased resistance to Ca2+-induced mPTP opening. However, this occurs independently of cyclophilin D (cyclosporine A insensitive) rather it is through decreased mitochondrial Ca2+ concentrations and subsequent adaptations mediated by impaired formation of functional mitochondrial Ca2+ uniporter complexes. We found that SPG7 directs the m-AAA complex to favor association with the mitochondrial Ca2+ uniporter (MCU) and MCU processing regulates higher order MCU-complex formation. The results suggest that SPG7 does not constitute a core component of the mPTP but can modulate mPTP through regulation of the basal mitochondrial Ca2+ concentration.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Canales de Calcio/metabolismo , Metaloendopeptidasas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/fisiología , Calcio/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Células HEK293 , Humanos , Metaloendopeptidasas/fisiología , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/fisiología , Membranas Mitocondriales/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Necrosis por Permeabilidad de la Transmembrana Mitocondrial/fisiología , Paraplejía/metabolismo , ATPasas de Translocación de Protón/metabolismo , Paraplejía Espástica Hereditaria/metabolismoRESUMEN
Oxidative stress (OS) has been implicated in a variety of pathological conditions, including diabetes mellitus, characterized by hyperglycemia. In the present study, OS induced by hyperglycemia and the effect of trolox, a vitamin E analog, were studied in cardiomyocytes and H9c2 cells exposed to 15 to 33 mM glucose (HG) for 24 to 72 hours in Dulbecco modified Eagle medium. Cells treated wirh 24 or 33 mM glucose for 24 hours or above showed decreased viability and adenosine triphosphate (ATP) content with a concomitant increase in radicals of oxygen species, calcium (Ca2+ ), mitochondrial permeability transition, and oxidative markers, confirming that the cells were under stress. However, upon exposure to 15 mM glucose for 24 hours, H9c2 cells maintained homeostasis and ATP generation. Pretreatment of cells with trolox reduced HG-induced OS to control levels. Here, we report that the toxic effect of HG is highly regulated and that OS induction can be prevented with Trolox, a potential inhibitor of membrane damage.
Asunto(s)
Antioxidantes/farmacología , Cromanos/farmacología , Glucosa/administración & dosificación , Mitocondrias/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Línea Celular , Glucosa/toxicidad , Miocitos Cardíacos/metabolismo , RatasRESUMEN
Ca2+ influx into mitochondria is mediated by the mitochondrial calcium uniporter (MCU), whose identity was recently revealed as a 40-kDa protein that along with other proteins forms the mitochondrial Ca2+ uptake machinery. The MCU is a Ca2+-conducting channel spanning the inner mitochondrial membrane. Here, deletion of the MCU completely inhibited Ca2+ uptake in liver, heart, and skeletal muscle mitochondria. However, in brain nonsynaptic and synaptic mitochondria from neuronal somata/glial cells and nerve terminals, respectively, the MCU deletion slowed, but did not completely block, Ca2+ uptake. Under resting conditions, brain MCU-KO mitochondria remained polarized, and in brain MCU-KO mitochondria, the electrophoretic Ca2+ ionophore ETH129 significantly accelerated Ca2+ uptake. The residual Ca2+ uptake in brain MCU-KO mitochondria was insensitive to inhibitors of mitochondrial Na+/Ca2+ exchanger and ryanodine receptor (CGP37157 and dantrolene, respectively), but was blocked by the MCU inhibitor Ru360. Respiration of WT and MCU-KO brain mitochondria was similar except that for mitochondria that oxidized pyruvate and malate, Ca2+ more strongly inhibited respiration in WT than in MCU-KO mitochondria. Of note, the MCU deletion significantly attenuated but did not completely prevent induction of the permeability transition pore (PTP) in brain mitochondria. Expression level of cyclophilin D and ATP content in mitochondria, two factors that modulate PTP induction, were unaffected by MCU-KO, whereas ADP was lower in MCU-KO than in WT brain mitochondria. Our results suggest the presence of an MCU-independent Ca2+ uptake pathway in brain mitochondria that mediates residual Ca2+ influx and induction of PTP in a fraction of the mitochondrial population.
Asunto(s)
Encéfalo/metabolismo , Canales de Calcio/genética , Calcio/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Neuronas/metabolismo , Animales , Encéfalo/efectos de los fármacos , Canales de Calcio/deficiencia , Ciclohexanos/farmacología , Dantroleno/farmacología , Femenino , Eliminación de Gen , Transporte Iónico/efectos de los fármacos , Ionóforos/farmacología , Malatos/metabolismo , Malatos/farmacología , Masculino , Ratones , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Neuronas/efectos de los fármacos , Ácido Pirúvico/metabolismo , Ácido Pirúvico/farmacología , Compuestos de Rutenio/farmacología , Tiazepinas/farmacologíaRESUMEN
Modification with arginine-specific glyoxals modulates the permeability transition (PT) of rat liver mitochondria, with inhibitory or inducing effects that depend on the net charge of the adduct(s). Here, we show that phenylglyoxal (PGO) affects the PT in a species-specific manner (inhibition in mouse and yeast, induction in human and Drosophila mitochondria). Following the hypotheses (i) that the effects are mediated by conserved arginine(s) and (ii) that the PT is mediated by the F-ATP synthase, we have narrowed the search to 60 arginines. Most of these residues are located in subunits α, ß, γ, ϵ, a, and c and were excluded because PGO modification did not significantly affect enzyme catalysis. On the other hand, yeast mitochondria lacking subunit g or bearing a subunit g R107A mutation were totally resistant to PT inhibition by PGO. Thus, the effect of PGO on the PT is specifically mediated by Arg-107, the only subunit g arginine that has been conserved across species. These findings are evidence that the PT is mediated by F-ATP synthase.
Asunto(s)
Arginina/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Fenilglioxal/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Calcio/metabolismo , Catálisis , Dimerización , Drosophila , Células HEK293 , Humanos , Ratones , Mitocondrias/enzimología , Poro de Transición de la Permeabilidad Mitocondrial , ATPasas de Translocación de Protón Mitocondriales/química , Especificidad de la EspecieRESUMEN
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/metabolismoRESUMEN
A decade of research has established the phospholipase iPLA2γ as being involved in cardiomyocyte dysfunction and necrosis leading to heart failure, but the mechanisms by which iPLA2γ acts and its interaction with the mitochondrial permeability transition pore (mPTP) that is critical for cardiac homeostasis are unclear. New investigations by Moon et al. demonstrate that mitochondria in failing hearts undergo dynamic shifts in PLA2 isoform expression, leading to a redistribution of eicosanoid composition that contributes to pathologic mPTP opening.
Asunto(s)
Insuficiencia Cardíaca , Ácidos Hidroxieicosatetraenoicos , Calcio , Humanos , Mitocondrias , Proteínas de Transporte de Membrana Mitocondrial , Poro de Transición de la Permeabilidad Mitocondrial , FosfolipasasRESUMEN
As a novel delocalized lipophilic cation, F16 selectively accumulates in mitochondria of carcinoma cells and shows a broad spectrum of antiproliferative action towards cancer cell lines. In order to reveal the mode of action and molecular mechanism of F16 inducing cytotoxicity, we investigated the effects of F16 on cancer cells and isolated mitochondria relative to its precursor compound (E)-3-(2-(pyridine-4yl)vinyl)-1 H-indole (PVI), which has a similar structure without positive charge. It was found that PVI did not accumulate in mitochondria, and exhibited lower cytotoxicity compared to F16. However, when they were directly incubated with mitochondria, both F16 and PVI were observed to induce damage to mitochondrial structure and function. Moreover, it was found that F16 as well as PVI acted as uncouplers on mitochondria, and further rescue experiments revealed that the addition of adenosine 5'-triphosphate was the most effective way to recover the cell viability decreased by F16. Thus it was concluded that the decreased intracellular adenosine 5'-triphosphate availability induced by the uncoupling effect of F16 was a major factor in F16-mediated cytotoxicity. Futhermore, the results indicated that the uncoupling effect of F16 is attributed to its chemical stucture in common with PVI but independent of its positive charge. The study may shed light on understanding the underlying mechanism of action for F16, and providing suggestions for the design of new mitochondria-targeted antitumor molecules.
Asunto(s)
Antineoplásicos/farmacología , Indoles/farmacología , Mitocondrias/efectos de los fármacos , Piridinas/farmacología , Compuestos de Piridinio/farmacología , Desacopladores/farmacología , Adenosina Trifosfato/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Células HEK293 , Humanos , Células MCF-7 , Fluidez de la Membrana/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Dilatación Mitocondrial/efectos de los fármacos , Ratas Wistar , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Cyclophilin D (CyPD), a mitochondrial matrix protein, has been widely studied for its role in mitochondrial-mediated cell death. Unexpectedly, we previously discovered that overexpression of CyPD in a stable cell line, increased mitochondrial membrane potentials and enhanced cell survival under conditions of oxidative stress. Here, we investigated the underlying mechanisms responsible for these findings. Spectrophotometric measurements in isolated mitochondria revealed that overexpression of CyPD in HEK293 cells increased respiratory chain activity, but only for Complex III (CIII). Acute treatment of mitochondria with the immumosupressant cyclosporine A did not affect CIII activity. Expression levels of the CIII subunits cytochrome b and Rieske-FeS were elevated in HEK293 cells overexpressing CyPD. However, CIII activity was still significantly higher compared to control mitochondria, even when normalized by protein expression. Blue native gel electrophoresis and Western blot assays revealed a molecular interaction of CyPD with CIII and increased levels of supercomplexes in mitochondrial protein extracts. Radiolabeled protein synthesis in mitochondria showed that CIII assembly and formation of supercomplexes containing CIII were significantly faster when CyPD was overexpressed. Taken together, these data indicate that CyPD regulates mitochondrial metabolism, and likely cell survival, by promoting more efficient electrons flow through the respiratory chain via increased supercomplex formation.
Asunto(s)
Ciclofilinas/metabolismo , Mitocondrias/metabolismo , Ciclosporina/química , Transporte de Electrón , Regulación de la Expresión Génica , Células HEK293 , Humanos , Potencial de la Membrana Mitocondrial , Membranas Mitocondriales/metabolismo , Estrés Oxidativo , Oxígeno/química , Unión Proteica , Conformación Proteica , EspectrofotometríaRESUMEN
Calcium-independent phospholipase A2γ (iPLA2γ) is a mitochondrial enzyme that produces lipid second messengers that facilitate opening of the mitochondrial permeability transition pore (mPTP) and contribute to the production of oxidized fatty acids in myocardium. To specifically identify the roles of iPLA2γ in cardiac myocytes, we generated cardiac myocyte-specific iPLA2γ knock-out (CMiPLA2γKO) mice by removing the exon encoding the active site serine (Ser-477). Hearts of CMiPLA2γKO mice exhibited normal hemodynamic function, glycerophospholipid molecular species composition, and normal rates of mitochondrial respiration and ATP production. In contrast, CMiPLA2γKO mice demonstrated attenuated Ca(2+)-induced mPTP opening that could be rapidly restored by the addition of palmitate and substantially reduced production of oxidized polyunsaturated fatty acids (PUFAs). Furthermore, myocardial ischemia/reperfusion (I/R) in CMiPLA2γKO mice (30 min of ischemia followed by 30 min of reperfusion in vivo) dramatically decreased oxidized fatty acid production in the ischemic border zones. Moreover, CMiPLA2γKO mice subjected to 30 min of ischemia followed by 24 h of reperfusion in vivo developed substantially less cardiac necrosis in the area-at-risk in comparison with their WT littermates. Furthermore, we found that membrane depolarization in murine heart mitochondria was sensitized to Ca(2+) by the presence of oxidized PUFAs. Because mitochondrial membrane depolarization and calcium are known to activate iPLA2γ, these results are consistent with salvage of myocardium after I/R by iPLA2γ loss of function through decreasing mPTP opening, diminishing production of proinflammatory oxidized fatty acids, and attenuating the deleterious effects of abrupt increases in calcium ion on membrane potential during reperfusion.
Asunto(s)
Ácidos Grasos Insaturados/metabolismo , Fosfolipasas A2 Grupo VI/metabolismo , Potencial de la Membrana Mitocondrial , Mitocondrias Cardíacas/enzimología , Daño por Reperfusión Miocárdica/enzimología , Miocardio/enzimología , Miocitos Cardíacos/enzimología , Animales , Calcio/metabolismo , Fosfolipasas A2 Grupo VI/genética , Ratones , Ratones Noqueados , Mitocondrias Cardíacas/genética , Daño por Reperfusión Miocárdica/genética , Especificidad de Órganos , Oxidación-ReducciónRESUMEN
Manganese-induced toxicity has been linked to mitochondrial dysfunction and an increased generation of reactive oxygen species (ROS). We could recently show in mechanistic studies that Mn2+ ions induce hydrogen peroxide (H2O2) production from the ubiquinone binding site of mitochondrial complex II (IIQ) and generally enhance H2O2 formation by accelerating the rate of superoxide dismutation. The present study with intact mitochondria reveals that manganese additionally enhances H2O2 emission by inducing mitochondrial permeability transition (mPT). In mitochondria fed by NADH-generating substrates, the combination of Mn2+ and different respiratory chain inhibitors led to a dynamically increasing H2O2emission which was sensitive to the mPT inhibitor cyclosporine A (CsA) as well as Ru-360, an inhibitor of the mitochondrial calcium uniporter (MCU). Under these conditions, flavin-containing enzymes of the mitochondrial matrix, e.g. the mitochondrial 2-oxoglutaratedehydrogenase (OGDH), were major sources of ROS. With succinate as substrate, Mn2+ stimulated ROS production mainly at complex II, whereby the applied succinate concentration had a marked effect on the tendency for mPT. Also Ca2+ increased the rate of H2O2 emission by mPT, while no direct effect on ROS-production of complex II was observed. The present study reveals a complex scenario through which manganese affects mitochondrial H2O2 emission: stimulating its production from distinct sites (e.g. site IIQ), accelerating superoxide dismutation and enhancing the emission via mPT which also leads to the loss of soluble components of the mitochondrial antioxidant systems and favors the ROS production from flavin-containing oxidoreductases of the Krebs cycle.
Asunto(s)
Cloruros/farmacología , Ciclo del Ácido Cítrico/efectos de los fármacos , Complejo II de Transporte de Electrones/metabolismo , Peróxido de Hidrógeno/metabolismo , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Compuestos de Manganeso/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Animales , Canales de Calcio/metabolismo , Cloruro de Calcio/farmacología , Ciclo del Ácido Cítrico/fisiología , Ciclosporina/farmacología , Transporte de Electrón/efectos de los fármacos , Transporte de Electrón/fisiología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Oxidorreductasas/metabolismo , Permeabilidad/efectos de los fármacos , Ratas , Compuestos de Rutenio/farmacología , Ácido Succínico/metabolismo , Ácido Succínico/farmacología , Superóxidos/metabolismo , Ubiquinona/metabolismoRESUMEN
Cardiac arrest induces the cessation of cerebral blood flow, which can result in brain damage. The primary intervention to salvage the brain under such a pathological condition is to restore the cerebral blood flow to the ischemic region. Ischemia is defined as a reduction in blood flow to a level that is sufficient to alter normal cellular function. Brain tissue is highly sensitive to ischemia, such that even brief ischemic periods in neurons can initiate a complex sequence of events that may ultimately culminate in cell death. However, paradoxically, restoration of blood flow can cause additional damage and exacerbate the neurocognitive deficits in patients who suffered a brain ischemic event, which is a phenomenon referred to as "reperfusion injury." Transient brain ischemia following cardiac arrest results from the complex interplay of multiple pathways including excitotoxicity, acidotoxicity, ionic imbalance, peri-infarct depolarization, oxidative and nitrative stress, inflammation, and apoptosis. The pathophysiology of post-cardiac arrest brain injury involves a complex cascade of molecular events, most of which remain unknown. Many lines of evidence have shown that mitochondria suffer severe damage in response to ischemic injury. Mitochondrial dysfunction based on the mitochondrial permeability transition after reperfusion, particularly involving the calcineurin/immunophilin signal transduction pathway, appears to play a pivotal role in the induction of neuronal cell death. The aim of this article is to discuss the underlying pathophysiology of brain damage, which is a devastating pathological condition, and highlight the central signal transduction pathway involved in brain damage, which reveals potential targets for therapeutic intervention.
RESUMEN
Mitochondrial dysfunction is one of the major contributors to neurodegenerative disorders including Parkinson disease. The mitochondrial permeability transition pore is a protein complex located on the mitochondrial membrane. Under cellular stress, the pore opens, increasing the release of pro-apoptotic proteins, and ultimately resulting in cell death. MicroRNA-7 (miR-7) is a small non-coding RNA that has been found to exhibit a protective role in the cellular models of Parkinson disease. In the present study, miR-7 was predicted to regulate the function of mitochondria, according to gene ontology analysis of proteins that are down-regulated by miR-7. Indeed, miR-7 overexpression inhibited mitochondrial fragmentation, mitochondrial depolarization, cytochrome c release, reactive oxygen species generation, and release of mitochondrial calcium in response to 1-methyl-4-phenylpyridinium (MPP(+)) in human neuroblastoma SH-SY5Y cells. In addition, several of these findings were confirmed in mouse primary neurons. Among the mitochondrial proteins identified by gene ontology analysis, the expression of voltage-dependent anion channel 1 (VDAC1), a constituent of the mitochondrial permeability transition pore, was down-regulated by miR-7 through targeting 3'-untranslated region of VDAC1 mRNA. Similar to miR-7 overexpression, knockdown of VDAC1 also led to a decrease in intracellular reactive oxygen species generation and subsequent cellular protection against MPP(+). Notably, overexpression of VDAC1 without the 3'-UTR significantly abolished the protective effects of miR-7 against MPP(+)-induced cytotoxicity and mitochondrial dysfunction, suggesting that the protective effect of miR-7 is partly exerted through promoting mitochondrial function by targeting VDAC1 expression. These findings point to a novel mechanism by which miR-7 accomplishes neuroprotection by improving mitochondrial health.
Asunto(s)
MicroARNs/genética , Interferencia de ARN , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Expresión Génica , Ontología de Genes , Humanos , Potencial de la Membrana Mitocondrial , Ratones Endogámicos C57BL , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Tamaño Mitocondrial , Especies Reactivas de Oxígeno/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/genéticaRESUMEN
The mitochondrial permeability transition pore is a recognized drug target for neurodegenerative conditions such as multiple sclerosis and for ischemia-reperfusion injury in the brain and heart. The peptidylprolyl isomerase, cyclophilin D (CypD, PPIF), is a positive regulator of the pore, and genetic down-regulation or knock-out improves outcomes in disease models. Current inhibitors of peptidylprolyl isomerases show no selectivity between the tightly conserved cyclophilin paralogs and exhibit significant off-target effects, immunosuppression, and toxicity. We therefore designed and synthesized a new mitochondrially targeted CypD inhibitor, JW47, using a quinolinium cation tethered to cyclosporine. X-ray analysis was used to validate the design concept, and biological evaluation revealed selective cellular inhibition of CypD and the permeability transition pore with reduced cellular toxicity compared with cyclosporine. In an experimental autoimmune encephalomyelitis disease model of neurodegeneration in multiple sclerosis, JW47 demonstrated significant protection of axons and improved motor assessments with minimal immunosuppression. These findings suggest that selective CypD inhibition may represent a viable therapeutic strategy for MS and identify quinolinium as a mitochondrial targeting group for in vivo use.