RESUMO
Mitochondrial permeability transition (mPT) is a phenomenon that abruptly causes the flux of low molecular weight solutes (molecular weight up to 1,500) across the generally impermeable inner mitochondrial membrane. The mPT is mediated by the so-called mitochondrial permeability transition pore (mPTP), a supramolecular entity assembled at the interface of the inner and outer mitochondrial membranes. In contrast to mitochondrial outer membrane permeabilization, which mostly activates apoptosis, mPT can trigger different cellular responses, from the physiological regulation of mitophagy to the activation of apoptosis or necrosis. Although there are several molecular candidates for the mPTP, its molecular nature remains contentious. This lack of molecular data was a significant setback that prevented mechanistic insight into the mPTP, pharmacological targeting and the generation of informative animal models. In recent years, experimental evidence has highlighted mitochondrial F1Fo ATP synthase as a participant in mPTP formation, although a molecular model for its transition to the mPTP is still lacking. Recently, the resolution of the F1Fo ATP synthase structure by cryogenic electron microscopy led to a model for mPTP gating. The elusive molecular nature of the mPTP is now being clarified, marking a turning point for understanding mitochondrial biology and its pathophysiological ramifications. This Review provides an up-to-date reference for the understanding of the mammalian mPTP and its cellular functions. We review current insights into the molecular mechanisms of mPT and validated observations - from studies in vivo or in artificial membranes - on mPTP activity and functions. We end with a discussion of the contribution of the mPTP to human disease. Throughout the Review, we highlight the multiple unanswered questions and, when applicable, we also provide alternative interpretations of the recent discoveries.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial , Necrose Dirigida por Permeabilidade Transmembrânica da Mitocôndria , Animais , Humanos , Trifosfato de Adenosina , Mamíferos , Proteínas de Transporte da Membrana Mitocondrial/química , Poro de Transição de Permeabilidade MitocondrialRESUMO
PTEN dysfunction plays a crucial role in the pathogenesis of hereditary and sporadic cancers. Here, we show that PTEN homodimerizes and, in this active conformation, exerts lipid phosphatase activity on PtdIns(3,4,5)P3. We demonstrate that catalytically inactive cancer-associated PTEN mutants heterodimerize with wild-type PTEN and constrain its phosphatase activity in a dominant-negative manner. To study the consequences of homo- and heterodimerization of wild-type and mutant PTEN in vivo, we generated Pten knockin mice harboring two cancer-associated PTEN mutations (PtenC124S and PtenG129E). Heterozygous Pten(C124S/+) and Pten(G129E/+) cells and tissues exhibit increased sensitivity to PI3-K/Akt activation compared to wild-type and Pten(+/-) counterparts, whereas this difference is no longer apparent between Pten(C124S/-) and Pten(-/-) cells. Notably, Pten KI mice are more tumor prone and display features reminiscent of complete Pten loss. Our findings reveal that PTEN loss and PTEN mutations are not synonymous and define a working model for the function and regulation of PTEN.
Assuntos
PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais , Animais , Embrião de Mamíferos/citologia , Feminino , Humanos , Perda de Heterozigosidade , Masculino , Camundongos , Mutação , Multimerização Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Endoplasmic reticulum (ER) calcium (Ca2+ ) stores are critical to proteostasis, intracellular signaling, and cellular bioenergetics. Through forward genetic screening in mice, we identified two members of a new complex, Pacs1 and Wdr37, which are required for normal ER Ca2+ handling in lymphocytes. Deletion of Pacs1 or Wdr37 caused peripheral lymphopenia that was linked to blunted Ca2+ release from the ER after antigen receptor stimulation. Pacs1-deficient cells showed diminished inositol triphosphate receptor expression together with increased ER and oxidative stress. Mature Pacs1-/- B cells proliferated and died in vivo under lymphocyte replete conditions, indicating spontaneous loss of cellular quiescence. Disruption of Pacs1-Wdr37 did not diminish adaptive immune responses, but potently suppressed lymphoproliferative disease models by forcing loss of quiescence. Thus, Pacs1-Wdr37 plays a critical role in stabilizing lymphocyte populations through ER Ca2+ handling and presents a new target for lymphoproliferative disease therapy.
Assuntos
Retículo Endoplasmático/metabolismo , Deleção de Genes , Linfopenia/genética , Transtornos Linfoproliferativos/genética , Proteínas Nucleares/genética , Proteínas de Transporte Vesicular/genética , Animais , Linfócitos B/metabolismo , Sinalização do Cálcio , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Linfopenia/metabolismo , Transtornos Linfoproliferativos/metabolismo , Masculino , Camundongos , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
The early secretory pathway and autophagy are two essential and evolutionarily conserved endomembrane processes that are finely interlinked. Although growing evidence suggests that intracellular trafficking is important for autophagosome biogenesis, the molecular regulatory network involved is still not fully defined. In this study, we demonstrate a crucial effect of the COPII vesicle-related protein TFG (Trk-fused gene) on ULK1 puncta number and localization during autophagy induction. This, in turn, affects formation of the isolation membrane, as well as the correct dynamics of association between LC3B and early ATG proteins, leading to the proper formation of both omegasomes and autophagosomes. Consistently, fibroblasts derived from a hereditary spastic paraparesis (HSP) patient carrying mutated TFG (R106C) show defects in both autophagy and ULK1 puncta accumulation. In addition, we demonstrate that TFG activity in autophagy depends on its interaction with the ATG8 protein LC3C through a canonical LIR motif, thereby favouring LC3C-ULK1 binding. Altogether, our results uncover a link between TFG and autophagy and identify TFG as a molecular scaffold linking the early secretion pathway to autophagy.
Assuntos
Autofagossomos/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Western Blotting , Imunofluorescência , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/genética , Proteínas/genética , Interferência de RNARESUMO
OBJECTIVES: To evaluate the in vitro effect of tofacitinib on autophagy activity of psoriatic arthritis (PsA) fibroblast-like synoviocytes (FLS), and to confirm its activity on inflammatory and invasive properties of FLS and synovial cells, deepening the impact on mitochondrial function. METHODS: FLS, peripheral blood mononuclear cells (PBMCs), and synovial cells from active PsA patients were cultured with tofacitinib 1 µM or vehicle control for 24 h. Autophagy was measured by Western blot and by fluorescence microscopy. Chemokines/cytokines released into culture supernatants were quantified by ELISA, while invasive properties of FLS by migration assays. Specific mitochondrial probes were adopted to measure intracellular reactive oxygen species (ROS), mitochondrial potential, morphology, turnover and mitophagy. Oxygen consumption rate (OCR), reflecting oxidative phosphorylation, was quantified using the Seahorse technology. Differences were determined by adopting the non-parametric Wilcoxon signed rank test. RESULTS: 18 patients with moderately-to-severely active PsA were enrolled. Tofacitinib significantly increased the levels of the autophagy markers LC3-II and ATG7 in PsA FLS compared to vehicle control, suggesting an increase in spontaneous autophagy activity; no effect was highlighted in PBMCs and synovial cells cultures. Tofacitinib reduced migration properties of PsA FLS, and reduced MCP-1 and IL-6 release into FLS and synovial cells cultures supernatants. Furthermore, tofacitinib decreased intracellular ROS production, increased basal OCR, ATP production and maximal respiratory capacity, and enhanced mitophagy and mitochondrial turnover. CONCLUSIONS: The JAK inhibitor tofacitinib reduces the pro-invasive and pro-inflammatory properties of PsA FLS. Autophagy induction and mitochondrial quality control modulation by tofacitinib might contribute to FLS function restoration.
Assuntos
Artrite Psoriásica , Piperidinas , Pirimidinas , Sinoviócitos , Humanos , Artrite Psoriásica/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Leucócitos Mononucleares , Transdução de Sinais , Autofagia , Fibroblastos/metabolismo , Mitocôndrias , Células Cultivadas , Membrana Sinovial/metabolismoRESUMO
BACKGROUND: Plant-derived nanovesicles (PDNVs) are a novelty in medical and agrifood environments, with several studies exploring their functions and potential applications. Among fruits, apples (sp. Malus domestica) have great potential as PDNVs source, given their widespread consumption, substantial waste production, and recognized health benefits. Notably, apple-derived nanovesicles (ADNVs) can interact with human cell lines, triggering anti-inflammatory and antioxidant responses. This work is dedicated to the comprehensive biochemical characterization of apple-derived nanovesicles (ADNVs) through proteomic and lipidomic analysis, and small RNAs sequencing. This research also aims to shed light on the underlying mechanism of action (MOA) when ADNVs interface with human cells, through observation of intracellular calcium signalling in human fibroblasts, and to tackles differences in ADNVs content when isolated from fruits derived from integrated and organic production methods cultivars. RESULTS: The ADNVs fraction is mainly composed of exocyst-positive organelles (EXPOs) and MVB-derived exosomes, identified through size and molecular markers (Exo70 and TET-3-like proteins). ADNVs' protein cargo is heterogeneous and exhibits a diverse array of functions, especially in plant's protection (favouring ABA stress-induced signalling, pathogen resistance and Reactive Oxygen Species (ROS) metabolism). Noteworthy plant miRNAs also contribute to phytoprotection. In relation with human cells lines, ADNVs elicit spikes of intracellular Ca2+ levels, utilizing the cation as second messenger, and produce an antioxidant effect. Lastly, organic samples yield a substantial increase in ADNV production and are particularly enriched in bioactive lysophospholipids. CONCLUSIONS: We have conclusively demonstrated that ADNVs confer an antioxidant effect upon human cells, through the initiation of a molecular pathway triggered by Ca2+ signalling. Within ADNVs, a plethora of bioactive proteins, small RNAs, and lipids have been identified, each possessing well-established functions within the realm of plant biology. While ADNVs predominantly function in plants, to safeguard against pathogenic agents and abiotic stressors, it is noteworthy that proteins with antioxidant power might act as antioxidants within human cells.
Assuntos
Antioxidantes , Malus , Humanos , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Cálcio/metabolismo , Verduras , Proteômica , Malus/metabolismo , Transdução de SinaisRESUMO
Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease characterized by myelin damage followed by axonal and ultimately neuronal loss. The etiology and physiopathology of MS are still elusive, and no fully effective therapy is yet available. We investigated the role in MS of autophagy (physiologically, a controlled intracellular pathway regulating the degradation of cellular components) and of mitophagy (a specific form of autophagy that removes dysfunctional mitochondria). We found that the levels of autophagy and mitophagy markers are significantly increased in the biofluids of MS patients during the active phase of the disease, indicating activation of these processes. In keeping with this idea, in vitro and in vivo MS models (induced by proinflammatory cytokines, lysolecithin, and cuprizone) are associated with strongly impaired mitochondrial activity, inducing a lactic acid metabolism and prompting an increase in the autophagic flux and in mitophagy. Multiple structurally and mechanistically unrelated inhibitors of autophagy improved myelin production and normalized axonal myelination, and two such inhibitors, the widely used antipsychotic drugs haloperidol and clozapine, also significantly improved cuprizone-induced motor impairment. These data suggest that autophagy has a causal role in MS; its inhibition strongly attenuates behavioral signs in an experimental model of the disease. Therefore, haloperidol and clozapine may represent additional therapeutic tools against MS.
Assuntos
Antipsicóticos/uso terapêutico , Autofagia , Mitofagia , Esclerose Múltipla/tratamento farmacológico , Animais , Antipsicóticos/farmacologia , Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/sangue , Proteínas Relacionadas à Autofagia/líquido cefalorraquidiano , Axônios/efeitos dos fármacos , Axônios/metabolismo , Biomarcadores/metabolismo , Clozapina/farmacologia , Citocinas/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Glucose/metabolismo , Haloperidol/farmacologia , Inflamação/patologia , Interleucina-1beta/metabolismo , Mitocôndrias/metabolismo , Mitofagia/efeitos dos fármacos , Modelos Biológicos , Atividade Motora/efeitos dos fármacos , Esclerose Múltipla/sangue , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/fisiopatologia , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Pulsed electromagnetic fields (PEMFs) are recognized for their potential in regenerative medicine, offering a non-invasive avenue for tissue rejuvenation. While prior research has mainly focused on their effects on bone and dermo-epidermal tissues, the impact of PEMFs on nervous tissue, particularly in the context of neuropathy associated with the diabetic foot, remains relatively unexplored. Addressing this gap, our preliminary in vitro study investigates the effects of complex magnetic fields (CMFs) on glial-like cells derived from mesenchymal cell differentiation, serving as a model for neuropathy of the diabetic foot. Through assessments of cellular proliferation, hemocompatibility, mutagenicity, and mitochondrial membrane potential, we have established the safety profile of the system. Furthermore, the analysis of microRNAs (miRNAs) suggests that CMFs may exert beneficial effects on cell cycle regulation, as evidenced by the upregulation of the miRNAs within the 121, 127, and 142 families, which are known to be associated with mitochondrial function and cell cycle control. This exploration holds promise for potential applications in mitigating neuropathic complications in diabetic foot conditions.
Assuntos
Neuropatias Diabéticas , Campos Eletromagnéticos , MicroRNAs , Mitocôndrias , Estresse Oxidativo , Mitocôndrias/metabolismo , Neuropatias Diabéticas/terapia , Neuropatias Diabéticas/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/terapia , Doenças Neuroinflamatórias/etiologia , Potencial da Membrana Mitocondrial , Proliferação de Células , Magnetoterapia/métodosRESUMO
Mitochondrial F1/FO ATP synthase participation in the mitochondrial permeability transition pore complex (PTPC) remains controversial. Neginskaya et al. (Cell Rep. 2019;26:11-17) reported an unexpected current with PTPC-like properties in F1/FO ATP synthase C subunit knockout cells that could explain part of the conflictual literature.
Assuntos
Ciclosporina , Necrose Dirigida por Permeabilidade Transmembrânica da Mitocôndria , Trifosfato de Adenosina , Mitocôndrias , PermeabilidadeRESUMO
Intracellular calcium signaling is a universal language source shared by the most part of biological entities inside cells that, all together, give rise to physiological and functional anatomical units, the organ. Although preferentially recognized as signaling between cell life and death processes, in the heart it assumes additional relevance considered the importance of calcium cycling coupled to ATP consumption in excitation-contraction coupling. The concerted action of a plethora of exchangers, channels and pumps inward and outward calcium fluxes where needed, to convert energy and electric impulses in muscle contraction. All this without realizing it, thousands of times, every day. An improper function of those proteins (i.e., variation in expression, mutations onset, dysregulated channeling, differential protein-protein interactions) being part of this signaling network triggers a short circuit with severe acute and chronic pathological consequences reported as arrhythmias, cardiac remodeling, heart failure, reperfusion injury and cardiomyopathies. By acting with chemical, peptide-based and pharmacological modulators of these players, a correction of calcium homeostasis can be achieved accompanied by an amelioration of clinical symptoms. This review will focus on all those defects in calcium homeostasis which occur in the most common cardiac diseases, including myocardial infarction, arrhythmia, hypertrophy, heart failure and cardiomyopathies. This part will be introduced by the state of the art on the proteins involved in calcium homeostasis in cardiomyocytes and followed by the therapeutic treatments that to date, are able to target them and to revert the pathological phenotype.
Assuntos
Cardiomiopatias , Insuficiência Cardíaca , Arritmias Cardíacas/metabolismo , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cardiomiopatias/metabolismo , Homeostase , Humanos , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Retículo SarcoplasmáticoRESUMO
BACKGROUND: An alteration of autophagy and mitophagy, two highly conserved lysosome-dependent degradation pathways involved in the maintenance of cellular homeostasis, has been associated with multiple sclerosis (MS). OBJECTIVE: To search the level of autophagy-related 5 (ATG5) and Parkin proteins, as markers of autophagy and mitophagy respectively, and lactate in a cohort of MS patients. METHODS: Cerebrospinal fluid (CSF) and serum samples from 60 MS patients were analyzed: 30 with magnetic resonance imaging (MRI) evidence of disease activity, gadolinium (Gd)-based contrast agent positive (Gd+), and 30 without MRI evidence of disease activity (Gd-). ATG5, Parkin, and lactate were measured using commercially available products. RESULTS AND CONCLUSIONS: Serum levels of ATG5, Parkin, and lactate were more elevated in Gd+ than in Gd- MS patients (p < 0.0001), and CSF concentrations of ATG5 and Parkin were greater in Gd+ than in Gd- MS (p < 0.0001). Our results demonstrated that molecular markers of autophagy and mitophagy are increased in CSF of MS patients during the active phases of the disease and that these catabolic markers, together with lactate, are also remarkably augmented in blood suggesting a role of these processes in MS pathogenesis and the possible use of these molecules as biomarkers of disease activity.
Assuntos
Autofagia/fisiologia , Imageamento por Ressonância Magnética , Mitofagia/fisiologia , Esclerose Múltipla Recidivante-Remitente/diagnóstico por imagem , Esclerose Múltipla Recidivante-Remitente/metabolismo , Adulto , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Feminino , Humanos , Imageamento por Ressonância Magnética/tendências , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
The impact of the mitochondrial permeability transition (MPT) on cellular physiology is well characterized. In contrast, the composition and mode of action of the permeability transition pore complex (PTPC), the supramolecular entity that initiates MPT, remain to be elucidated. Specifically, the precise contribution of the mitochondrial F1FO ATP synthase (or subunits thereof) to MPT is a matter of debate. We demonstrate that F1FO ATP synthase dimers dissociate as the PTPC opens upon MPT induction. Stabilizing F1FO ATP synthase dimers by genetic approaches inhibits PTPC opening and MPT Specific mutations in the F1FO ATP synthase c subunit that alter C-ring conformation sensitize cells to MPT induction, which can be reverted by stabilizing F1FO ATP synthase dimers. Destabilizing F1FO ATP synthase dimers fails to trigger PTPC opening in the presence of mutants of the c subunit that inhibit MPT The current study does not provide direct evidence that the C-ring is the long-sought pore-forming subunit of the PTPC, but reveals that PTPC opening requires the dissociation of F1FO ATP synthase dimers and involves the C-ring.
Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico , Ciclosporina/farmacologia , Células HEK293 , Humanos , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/química , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/genética , Necrose , Permeabilidade , Conformação Proteica , Multimerização Proteica , RatosRESUMO
The tumor suppressor p53 is a key protein in preventing cell transformation and tumor progression. Activated by a variety of stimuli, p53 regulates cell-cycle arrest and apoptosis. Along with its well-documented transcriptional control over cell-death programs within the nucleus, p53 exerts crucial although still poorly understood functions in the cytoplasm, directly modulating the apoptotic response at the mitochondrial level. Calcium (Ca(2+)) transfer between the endoplasmic reticulum (ER) and mitochondria represents a critical signal in the induction of apoptosis. However, the mechanism controlling this flux in response to stress stimuli remains largely unknown. Here we show that, in the cytoplasm, WT p53 localizes at the ER and at specialized contact domains between the ER and mitochondria (mitochondria-associated membranes). We demonstrate that, upon stress stimuli, WT p53 accumulates at these sites and modulates Ca(2+) homeostasis. Mechanistically, upon activation, WT p53 directly binds to the sarco/ER Ca(2+)-ATPase (SERCA) pump at the ER, changing its oxidative state and thus leading to an increased Ca(2+) load, followed by an enhanced transfer to mitochondria. The consequent mitochondrial Ca(2+) overload causes in turn alterations in the morphology of this organelle and induction of apoptosis. Pharmacological inactivation of WT p53 or naturally occurring p53 missense mutants inhibits SERCA pump activity at the ER, leading to a reduction of the Ca(2+) signaling from the ER to mitochondria. These findings define a critical nonnuclear function of p53 in regulating Ca(2+) signal-dependent apoptosis.
Assuntos
Apoptose/fisiologia , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Equorina/metabolismo , Animais , Western Blotting , Linhagem Celular , Citosol/metabolismo , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Fura-2 , Técnicas de Silenciamento de Genes , Humanos , Imunoprecipitação , Camundongos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
Until 1972, the term 'apoptosis' was used to differentiate the programmed cell death that naturally occurs in organismal development from the acute tissue death referred to as necrosis. Many studies on cell death and programmed cell death have been published and most are, at least to some degree, related to cancer. Some key proteins and molecular pathways implicated in cell death have been analyzed, whereas others are still being actively researched; therefore, an increasing number of cellular compartments and organelles are being implicated in cell death and cancer. Here, we discuss the mitochondria and subdomains of the endoplasmic reticulum (ER) that interact with mitochondria, the mitochondria-associated membranes (MAMs), which have been identified as critical hubs in the regulation of cell death and tumor growth. MAMs-dependent calcium (Ca2+) release from the ER allows selective Ca2+ uptake by the mitochondria. The perturbation of Ca2+ homeostasis in cancer cells is correlated with sustained cell proliferation and the inhibition of cell death through the modulation of Ca2+ signaling. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.
Assuntos
Cálcio/fisiologia , Mitocôndrias/fisiologia , Membranas Mitocondriais/fisiologia , Animais , Canais de Cálcio/fisiologia , Sinalização do Cálcio/fisiologia , Morte Celular , Divisão Celular , Transformação Celular Neoplásica , Progressão da Doença , Retículo Endoplasmático/metabolismo , Homeostase , Humanos , Proteínas de Membrana/fisiologia , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas Oncogênicas/fisiologia , Transdução de SinaisRESUMO
Acute myocardial infarction (MI) is a major cause of death and disability worldwide. The treatment of choice for reducing ischemic injury and limiting infarct size (IS) in patients with ST-segment elevation MI (STEMI) is timely and effective myocardial reperfusion via primary percutaneous coronary intervention (PCI). However, myocardial reperfusion itself may induce further cardiomyocyte death, a phenomenon known as reperfusion injury (RI). The opening of a large pore in the mitochondrial membrane, namely, the mitochondrial permeability transition pore (mPTP), is widely recognized as the final step of RI and is responsible for mitochondrial and cardiomyocyte death. Although myocardial reperfusion interventions continue to improve, there remain no effective therapies for preventing RI due to incomplete knowledge regarding RI components and mechanisms and to premature translations of findings from animals to humans. In the last year, increasing amounts of data describing mPTP components, structure, regulation and function have surfaced. These data may be crucial for gaining a better understanding of RI genesis and for planning future trials evaluating new cardioprotective strategies. In this chapter, we review the role of the mPTP in RI pathophysiology and report on recent studies investigating its structure and components. Finally, we provide a brief overview of principal cardioprotective strategies and their pitfalls.
Assuntos
Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Intervenção Coronária Percutânea/efeitos adversos , Infarto do Miocárdio com Supradesnível do Segmento ST/terapia , Transdução de Sinais , Animais , Circulação Coronária , Humanos , Mitocôndrias Cardíacas/patologia , Proteínas de Transporte da Membrana Mitocondrial/química , Poro de Transição de Permeabilidade Mitocondrial , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/patologia , Conformação Proteica , Fatores de Risco , Infarto do Miocárdio com Supradesnível do Segmento ST/metabolismo , Infarto do Miocárdio com Supradesnível do Segmento ST/patologia , Infarto do Miocárdio com Supradesnível do Segmento ST/fisiopatologia , Volume Sistólico , Relação Estrutura-Atividade , Função Ventricular EsquerdaRESUMO
The P2X7 receptor (P2X7R) is a known and powerful activator of the NOD-like receptor (NLR)P3 inflammasome; however, the underlying pathways are poorly understood. Thus, we investigated the molecular mechanisms involved. The effect of P2X7R expression and activation on NLRP3 expression and recruitment was investigated by Western blot, RT-PCR, coimmunoprecipitation, and confocal microscopy in microglial mouse cell lines selected for reduced P2X7R expression and in primary cells from P2X7R(-/-) C57BL/6 mice. We show here that P2X7R activation by ATP (EC50 = 1 mM) or benzoyl-ATP (EC50 = 300 µM) and P2X7R down-modulation caused a 2- to 8-fold up-regulation of NLRP3 mRNA in mouse N13 microglial cells. Moreover, NLRP3 mRNA was also up-regulated in primary microglial and macrophage cells from P2X7R(-/-) mice. Confocal microscopy and immunoprecipitation assays showed that P2X7R and NLRP3 closely interacted at discrete subplasmalemmal sites. Finally, P2X7R stimulation caused a transient (3-4 min) cytoplasmic Ca(2+) increase localized to small (2-3 µm wide) discrete subplasmalemmal regions. The Ca(2+) increase drove P2X7R recruitment and a 4-fold increase in P2X7R/NLRP3 association within 1-2 min. These data show a close P2X7R and NLRP3 interaction and highlight the role of P2X7R in the localized cytoplasmic ion changes responsible for both NLRP3 recruitment and activation.
Assuntos
Proteínas de Transporte/genética , Regulação da Expressão Gênica , Inflamassomos/metabolismo , Receptores Purinérgicos P2X7/genética , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Western Blotting , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Células Cultivadas , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Células HEK293 , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microscopia Confocal , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ligação Proteica , Receptores Purinérgicos P2X7/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/efeitos dos fármacosRESUMO
The mitochondrial permeability transition is a key event in cell death. Intense research efforts have been focused on elucidating the molecular components of the mitochondrial permeability transition pore (mPTP) to improve the understanding and treatment of various pathologies, including neurodegenerative disorders, cancer and cardiac diseases. Several molecular factors have been proposed as core components of the mPTP; however, further investigation has indicated that these factors are among a wide range of regulators. Thus, the scientific community lacks a clear model of the mPTP. Here, we review the molecular factors involved in the regulation and formation of the mPTP. Furthermore, we propose that the mitochondrial ATP synthase, specifically its c subunit, is the central core component of the mPTP complex. Moreover, we discuss the involvement of the mPTP in ischemia and reperfusion as well as the results of clinical studies targeting the mPTP to ameliorate ischemia-reperfusion injury. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".
Assuntos
Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Animais , Apoptose , Cálcio/metabolismo , Cardiotônicos/farmacologia , Cardiotônicos/uso terapêutico , Humanos , Mitocôndrias Cardíacas/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/química , Poro de Transição de Permeabilidade Mitocondrial , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Terapia de Alvo Molecular , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Necrose/metabolismo , Subunidades Proteicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Intracellular calcium (Ca(2+)) is largely known as a second messenger that is able to drive effects ranging from vesicle formation to muscle contraction, energy production and much more. In spite of its physiological regulation, Ca(2+) is a strategic tool for regulating apoptosis, especially during transmission between the endoplasmic reticulum and the mitochondria. Contact sites between these organelles are well-defined as signaling platforms where oncogenes and oncosuppressors can exert anti/pro-apoptotic activities. Recent advances from in vivo investigations into these regions highlight the role of the master oncosuppressor p53 in regulating Ca(2+) transmission and apoptosis, and we propose that Ca(2+) signals are relevant targets when developing new therapeutic approaches.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Animais , Apoptose/fisiologia , Descoberta de Drogas , Humanos , Modelos Biológicos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Fotoquimioterapia , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Mitochondrial metabolism plays a central role in the regulation of hematopoietic stem cell (HSC) biology. Mitochondrial fatty acid oxidation (FAO) is pivotal in controlling HSC self-renewal and differentiation. Herein, we discuss recent evidence suggesting that NADPH generated in the mitochondria can influence the fate of HSCs. Although NADPH has multiple functions, HSCs show high levels of NADPH that are preferentially used for cholesterol biosynthesis. Endogenous cholesterol supports the biogenesis of extracellular vesicles (EVs), which are essential for maintaining HSC properties. We also highlight the significance of EVs in hematopoiesis through autocrine signaling. Elucidating the mitochondrial NADPH-cholesterol axis as part of the metabolic requirements of healthy HSCs will facilitate the development of new therapies for hematological disorders.
RESUMO
One open question in the biology of growth factor receptors is how a quantitative input (i.e., ligand concentration) is decoded by the cell to produce specific response(s). Here, we show that an EGFR endocytic mechanism, non-clathrin endocytosis (NCE), which is activated only at high ligand concentrations and targets receptor to degradation, requires a tripartite organelle platform involving the plasma membrane (PM), endoplasmic reticulum (ER) and mitochondria. At these contact sites, EGFR-dependent, ER-generated Ca2+ oscillations are sensed by mitochondria, leading to increased metabolism and ATP production. Locally released ATP is required for cortical actin remodeling and EGFR-NCE vesicle fission. The same biochemical circuitry is also needed for an effector function of EGFR, i.e., collective motility. The multiorganelle signaling platform herein described mediates direct communication between EGFR signaling and mitochondrial metabolism, and is predicted to have a broad impact on cell physiology as it is activated by another growth factor receptor, HGFR/MET.