RESUMO
The mitochondrial permeability transition (PT) is a permeability increase of the inner mitochondrial membrane mediated by a channel, the permeability transition pore (PTP). After a brief historical introduction, we cover the key regulatory features of the PTP and provide a critical assessment of putative protein components that have been tested by genetic analysis. The discovery that under conditions of oxidative stress the F-ATP synthases of mammals, yeast, and Drosophila can be turned into Ca(2+)-dependent channels, whose electrophysiological properties match those of the corresponding PTPs, opens new perspectives to the field. We discuss structural and functional features of F-ATP synthases that may provide clues to its transition from an energy-conserving into an energy-dissipating device as well as recent advances on signal transduction to the PTP and on its role in cellular pathophysiology.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos , Poro de Transição de Permeabilidade Mitocondrial , Estresse Oxidativo/fisiologiaRESUMO
BACKGROUND: Cigarette smoking is a risk factor for interstitial lung abnormalities (ILAs) and interstitial lung diseases (ILDs). Investigation defining the relationships between ILAs/ILDs and clinical, radiographic, and pathologic findings in smokers have been incomplete. Employing a cohort undergoing surgical resection for lung nodules/masses, we (1) define the prevalence of ILAs/ILDs, (2) delineate their clinical, radiographic and pathologic predictors, and (3) determine their associations with mortality. METHODS: Patients undergoing resection of lung nodules/masses between 2017 and 2020 at a rural Appalachian, tertiary medical center were retrospectively investigated. Predictors for ILAs/ILDs and mortality were assessed using multivariate logistic regression analysis. RESULTS: In the total study cohort of 352 patients, radiographic ILAs and ILDs were observed in 35.2% and 17.6%, respectively. Among ILA patterns, subpleural reticular changes (14.8%), non-emphysematous cysts, centrilobular (CL) ground glass opacities (GGOs) (8% each), and mixed CL-GGO and subpleural reticular changes (7.4%) were common. ILD patterns included combined pulmonary fibrosis emphysema (CPFE) (3.1%), respiratory bronchiolitis (RB)-ILD (3.1%), organizing pneumonitis (2.8%) and unclassifiable (4.8%). The group with radiographic ILAs/ILDs had a significantly higher proportion of ever smokers (49% vs. 39.9%), pack years of smoking (44.57 ± 36.21 vs. 34.96 ± 26.22), clinical comorbidities of COPD (35% vs. 26.5%) and mildly reduced diffusion capacity (% predicated 66.29 ± 20.55 vs. 71.84 ± 23). Radiographic centrilobular and paraseptal emphysema (40% vs. 22.2% and 17.6% vs. 9.6%, respectively) and isolated traction bronchiectasis (10.2% vs. 4.2%) were associated with ILAs/ILDs. Pathological variables of emphysema (34.9% vs. 18.5%), any fibrosis (15.9% vs. 4.6%), peribronchiolar metaplasia (PBM, 8% vs. 1.1%), RB (10.3% vs. 2.5%), and anthracosis (21.6% vs. 14.5%) were associated with ILAs/ILDs. Histologic emphysema showed positive correlations with any fibrosis, RB, anthracosis and ≥ 30 pack year of smoking. The group with ILAs/ILDs had significantly higher mortality (9.1% vs. 2.2%, OR 4.13, [95% CI of 1.84-9.25]). CONCLUSIONS: In a rural cohort undergoing surgical resection, radiographic subclinical ILAs/ILDs patterns were highly prevalent and associated with ever smoking and intensity of smoking. The presence of radiographic ILA/ILD patterns and isolated honeycomb changes were associated with increased mortality. Subclinical ILAs/ILDs and histologic fibrosis correlated with clinical COPD as well as radiographic and pathologic emphysema emphasizing the co-existence of these pulmonary injuries in a heavily smoking population.
Assuntos
Antracose , Bronquiolite , Fumar Cigarros , Enfisema , Doenças Pulmonares Intersticiais , Enfisema Pulmonar , Fibrose Pulmonar , Anormalidades do Sistema Respiratório , Antracose/complicações , Antracose/patologia , Bronquiolite/complicações , Humanos , Pulmão/diagnóstico por imagem , Pulmão/patologia , Doenças Pulmonares Intersticiais/complicações , Enfisema Pulmonar/complicações , Fibrose Pulmonar/patologia , Anormalidades do Sistema Respiratório/complicações , Estudos RetrospectivosRESUMO
High-throughput screening identified isoxazoles as potent but metabolically unstable inhibitors of the mitochondrial permeability transition pore (PTP). Here we have studied the effects of a metabolically stable triazole analog, TR001, which maintains the PTP inhibitory properties with an in vitro potency in the nanomolar range. We show that TR001 leads to recovery of muscle structure and function of sapje zebrafish, a severe model of Duchenne muscular dystrophy (DMD). PTP inhibition fully restores the otherwise defective respiration in vivo, allowing normal development of sapje individuals in spite of lack of dystrophin. About 80 % sapje zebrafish treated with TR001 are alive and normal at 18 days post fertilization (dpf), a point in time when not a single untreated sapje individual survives. Time to 50 % death of treated zebrafish increases from 5 to 28 dpf, a sizeable number of individuals becoming young adults in spite of the persistent lack of dystrophin expression. TR001 improves respiration of myoblasts and myotubes from DMD patients, suggesting that PTP-dependent dysfunction also occurs in the human disease and that mitochondrial therapy of DMD with PTP-inhibiting triazoles is a viable treatment option.
Assuntos
Proteínas de Membrana/deficiência , Poro de Transição de Permeabilidade Mitocondrial/antagonistas & inibidores , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Proteínas Musculares/deficiência , Triazóis/farmacologia , Proteínas de Peixe-Zebra/deficiência , Animais , Animais Geneticamente Modificados , Linhagem Celular Transformada , Relação Dose-Resposta a Droga , Humanos , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Proteínas de Membrana/genética , Proteínas Musculares/genética , Rodaminas/farmacologia , Triazóis/química , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Ischemia/reperfusion (I/R) injury is mediated in large part by opening of the mitochondrial permeability transition pore (PTP). Consequently, inhibitors of the PTP hold great promise for the treatment of a variety of cardiovascular disorders. At present, PTP inhibition is obtained only through the use of drugs (e.g. cyclosporine A, CsA) targeting cyclophilin D (CyPD) which is a key modulator, but not a structural component of the PTP. This limitation might explain controversial findings in clinical studies. Therefore, we investigated the protective effects against I/R injury of small-molecule inhibitors of the PTP (63 and TR002) that do not target CyPD. Both compounds exhibited a dose-dependent inhibition of PTP opening in isolated mitochondria and were more potent than CsA. Notably, PTP inhibition was observed also in mitochondria devoid of CyPD. Compounds 63 and TR002 prevented PTP opening and mitochondrial depolarization induced by Ca2+ overload and by reactive oxygen species in neonatal rat ventricular myocytes (NRVMs). Remarkably, both compounds prevented cell death, contractile dysfunction and sarcomeric derangement induced by anoxia/reoxygenation injury in NRVMs at sub-micromolar concentrations, and were more potent than CsA. Cardioprotection was observed also in adult mouse ventricular myocytes and human iPSc-derived cardiomyocytes, as well as ex vivo in perfused hearts. Thus, this study demonstrates that 63 and TR002 represent novel cardioprotective agents that inhibit PTP opening independent of CyPD targeting.
Assuntos
Cardiotônicos/uso terapêutico , Poro de Transição de Permeabilidade Mitocondrial/antagonistas & inibidores , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Cardiotônicos/farmacologia , Linhagem Celular , Células Cultivadas , Humanos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/efeitos dos fármacos , Ratos Sprague-Dawley , Ratos Wistar , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
The permeability transition pore (PTP) is a Ca2+-dependent mitochondrial channel whose opening causes a permeability increase in the inner membrane to ions and solutes. The most potent inhibitors are matrix protons, with channel block at pH 6.5. Inhibition is reversible, mediated by histidyl residue(s), and prevented by their carbethoxylation by diethylpyrocarbonate (DPC), but their assignment is unsolved. We show that PTP inhibition by H+ is mediated by the highly conserved histidyl residue (H112 in the human mature protein) of oligomycin sensitivity conferral protein (OSCP) subunit of mitochondrial F1FO (F)-ATP synthase, which we also show to undergo carbethoxylation after reaction of mitochondria with DPC. Mitochondrial PTP-dependent swelling cannot be inhibited by acidic pH in H112Q and H112Y OSCP mutants, and the corresponding megachannels (the electrophysiological counterpart of the PTP) are insensitive to inhibition by acidic pH in patch-clamp recordings of mitoplasts. Cells harboring the H112Q and H112Y mutations are sensitized to anoxic cell death at acidic pH. These results demonstrate that PTP channel formation and its inhibition by H+ are mediated by the F-ATP synthase.
Assuntos
Histidina/metabolismo , Concentração de Íons de Hidrogênio , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Bovinos , Linhagem Celular , Permeabilidade da Membrana Celular , Histidina/química , Humanos , Hidrólise , Hipóxia/metabolismo , Camundongos , Mitocôndrias Hepáticas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , ATPases Mitocondriais Próton-Translocadoras/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Consumo de Oxigênio , Conformação Proteica , Subunidades ProteicasRESUMO
F-ATP synthases convert the electrochemical energy of the H+ gradient into the chemical energy of ATP with remarkable efficiency. Mitochondrial F-ATP synthases can also undergo a Ca2+-dependent transformation to form channels with properties matching those of the permeability transition pore (PTP), a key player in cell death. The Ca2+ binding site and the mechanism(s) through which Ca2+ can transform the energy-conserving enzyme into a dissipative structure promoting cell death remain unknown. Through in vitro, in vivo and in silico studies we (i) pinpoint the "Ca2+-trigger site" of the PTP to the catalytic site of the F-ATP synthase ß subunit and (ii) define a conformational change that propagates from the catalytic site through OSCP and the lateral stalk to the inner membrane. T163S mutants of the ß subunit, which show a selective decrease in Ca2+-ATP hydrolysis, confer resistance to Ca2+-induced, PTP-dependent death in cells and developing zebrafish embryos. These findings are a major advance in the molecular definition of the transition of F-ATP synthase to a channel and of its role in cell death.
Assuntos
Cálcio/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Transporte Biológico , Domínio Catalítico , Morte Celular , Diferenciação Celular , Embrião não Mamífero/citologia , Células HeLa , Humanos , Hidrólise , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Poro de Transição de Permeabilidade Mitocondrial , ATPases Mitocondriais Próton-Translocadoras/química , Permeabilidade , Ligação Proteica , Conformação Proteica , Peixe-Zebra/embriologiaRESUMO
The mitochondrial permeability transition pore (PTP) is now recognized as playing a key role in a wide variety of human diseases whose common pathology may be based in mitochondrial dysfunction. Recently, PTP assays have been adapted to high-throughput screening approaches to identify small molecules specifically inhibiting the PTP. Following extensive secondary chemistry, the most potent inhibitors of the PTP described to date have been developed. This review will provide an overview of each of these screening efforts, use of resulting compounds in animal models of PTP-based diseases, and problems that will require further study. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
Assuntos
Mitocôndrias/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Membranas Mitocondriais/efeitos dos fármacos , Anilidas/síntese química , Anilidas/farmacologia , Benzamidinas/síntese química , Benzamidinas/farmacologia , Ciclosporina/química , Ciclosporina/farmacologia , Desenho de Fármacos , Ensaios de Triagem em Larga Escala , Isoxazóis/síntese química , Isoxazóis/farmacologia , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Oximas/química , Oximas/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Compostos de Quinolínio/química , Compostos de Quinolínio/farmacologia , Secoesteroides/química , Secoesteroides/farmacologia , Relação Estrutura-AtividadeRESUMO
Mitochondria-associated ER membranes, or MAMs, define the sites of endoplasmic reticulum/mitochondria juxtaposition that control Ca(2+) flux between these organelles. We found that in a mouse model of the human lysosomal storage disease GM1-gangliosidosis, GM1-ganglioside accumulates in the glycosphingolipid-enriched microdomain (GEM) fractions of MAMs, where it interacts with the phosphorylated form of IP3 receptor-1, influencing the activity of this channel. Ca(2+) depleted from the ER is then taken up by the mitochondria, leading to Ca(2+) overload in this organelle. The latter induces mitochondrial membrane permeabilization (MMP), opening of the permeability transition pore, and activation of the mitochondrial apoptotic pathway. This study identifies the GEMs as the sites of Ca(2+) diffusion between the ER and the mitochondria. We propose a new mechanism of Ca(2+)-mediated apoptotic signaling whereby GM1 accumulation at the GEMs alters Ca(2+) dynamics and acts as a molecular effector of both ER stress-induced and mitochondria-mediated apoptosis of neuronal cells.
Assuntos
Apoptose , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Gangliosídeo G(M1)/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Encéfalo/metabolismo , Cálcio/farmacologia , Células Cultivadas , Citocromos c/metabolismo , Modelos Animais de Doenças , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Gangliosídeo G(M1)/farmacologia , Gangliosidose GM1/genética , Gangliosidose GM1/metabolismo , Gangliosidose GM1/patologia , Glicoesfingolipídeos/metabolismo , Humanos , Immunoblotting , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Microdomínios da Membrana/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Mitochondria of Drosophila melanogaster undergo Ca(2+)-induced Ca(2+) release through a putative channel (mCrC) that has several regulatory features of the permeability transition pore (PTP). The PTP is an inner membrane channel that forms from F-ATPase, possessing a conductance of 500 picosiemens (pS) in mammals and of 300 pS in yeast. In contrast to the PTP, the mCrC of Drosophila is not permeable to sucrose and appears to be selective for Ca(2+) and H(+). We show (i) that like the PTP, the mCrC is affected by the sense of rotation of F-ATPase, by Bz-423, and by Mg(2+)/ADP; (ii) that expression of human cyclophilin D in mitochondria of Drosophila S2R(+) cells sensitizes the mCrC to Ca(2+) but does not increase its apparent size; and (iii) that purified dimers of D. melanogaster F-ATPase reconstituted into lipid bilayers form 53-pS channels activated by Ca(2+) and thiol oxidants and inhibited by Mg(2+)/γ-imino ATP. These findings indicate that the mCrC is the PTP of D. melanogaster and that the signature conductance of F-ATPase channels depends on unique structural features that may underscore specific roles in different species.
Assuntos
Adenosina Trifosfatases/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Adenosina Trifosfatases/genética , Animais , Canais de Cálcio/genética , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Poro de Transição de Permeabilidade MitocondrialAssuntos
Cálcio/química , Cardiopatias/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Sistema Cardiovascular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Peptidil-Prolil Isomerase F/metabolismo , Fundações , Cardiopatias/tratamento farmacológico , Humanos , Serviços de Informação , Cooperação Internacional , Mitocôndrias Cardíacas/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Poro de Transição de Permeabilidade Mitocondrial , Modelos Cardiovasculares , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca(2+) like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca(2+). Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca(2+), addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (γ-imino ATP, a nonhydrolyzable ATP analog) and Mg(2+)/ADP. These results indicate that the PTP forms from dimers of the ATP synthase.
Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Apoptose , Cálcio/metabolismo , Bovinos , Linhagem Celular Tumoral , Dimerização , Humanos , Hidrólise , Potenciais da Membrana , Camundongos , Mitocôndrias Hepáticas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , RNA Interferente Pequeno/metabolismo , TransfecçãoRESUMO
Purified F-ATP synthase dimers of yeast mitochondria display Ca(2+)-dependent channel activity with properties resembling those of the permeability transition pore (PTP) of mammals. After treatment with the Ca(2+) ionophore ETH129, which allows electrophoretic Ca(2+) uptake, isolated yeast mitochondria undergo inner membrane permeabilization due to PTP opening. Yeast mutant strains ΔTIM11 and ΔATP20 (lacking the e and g F-ATP synthase subunits, respectively, which are necessary for dimer formation) display a striking resistance to PTP opening. These results show that the yeast PTP originates from F-ATP synthase and indicate that dimerization is required for pore formation in situ.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial/fisiologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Saccharomyces cerevisiae/enzimologia , Western Blotting , Dimerização , Eletroforese em Gel de Poliacrilamida , Proteínas de Transporte da Membrana Mitocondrial/química , Poro de Transição de Permeabilidade MitocondrialRESUMO
Translocator protein of 18 kDa (TSPO) is a highly conserved, ubiquitous protein localized in the outer mitochondrial membrane, where it is thought to play a key role in the mitochondrial transport of cholesterol, a key step in the generation of steroid hormones. However, it was first characterized as the peripheral benzodiazepine receptor because it appears to be responsible for high affinity binding of a number of benzodiazepines to non-neuronal tissues. Ensuing studies have employed natural and synthetic ligands to assess the role of TSPO function in a number of natural and pathological circumstances. Largely through the use of these compounds and biochemical associations, TSPO has been proposed to play a role in the mitochondrial permeability transition pore (PTP), which has been associated with cell death in many human pathological conditions. Here, we critically assess the role of TSPO in the function of the PTP through the generation of mice in which the Tspo gene has been conditionally eliminated. Our results show that 1) TSPO plays no role in the regulation or structure of the PTP, 2) endogenous and synthetic ligands of TSPO do not regulate PTP activity through TSPO, 3) outer mitochondrial membrane regulation of PTP activity occurs though a mechanism that does not require TSPO, and 4) hearts lacking TSPO are as sensitive to ischemia-reperfusion injury as hearts from control mice. These results call into question a wide variety of studies implicating TSPO in a number of pathological processes through its actions on the PTP.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Receptores de GABA/metabolismo , Animais , Feminino , Deleção de Genes , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Poro de Transição de Permeabilidade Mitocondrial , Miocárdio/citologia , Miocárdio/metabolismo , Permeabilidade , Receptores de GABA/deficiência , Receptores de GABA/genéticaRESUMO
Atopic dermatitis (AD) is a pervasive and multifaceted dermatological disorder causing daily distress to afflicted individuals worldwide. This comprehensive review synthesizes the historical and contemporary advancements in therapeutic strategies, offering a critical analysis of their efficacy, safety profiles, and adaptability. The enduring role of topical corticosteroids in managing AD is examined, acknowledging their potent anti-inflammatory properties alongside their potential adverse side effects, particularly in extended usage. The article explores the utilization of topical calcineurin inhibitors like tacrolimus and pimecrolimus, highlighting their novel anti-inflammatory pathways while also scrutinizing concerns over potential malignancies that relegate them to second-line therapy. The present investigation features the emergence of crisaborole, a phosphodiesterase four inhibitor. Its innovative mode of action, benign safety profile, and applicability to mild and moderate AD are thoroughly evaluated. The review also includes challenges, particularly cost considerations, which constrain accessibility and necessitate nuanced implementation in therapeutic regimens. This study underscores the need for persistent investigation, teamwork, and innovations in managing AD. In this regard, AD requires a united approach between clinicians, researchers, affected individuals, and policymakers to refine patient-focused treatment and develop precise, economical strategies to address this chronic and frequently life-altering health condition.
RESUMO
Axonal spheroids occur as part of the pathology of a variety of neurologic diseases. Reactive oxygen species (ROS) trigger formation of spheroids, axonal severing, and Ca(2+) overload. The mechanisms by which ROS lead to the spheroid formation at specific axonal sites remain elusive. Here, using adult mouse primary neurons, we investigate the role of Ca(2+), its regulating systems, and cytoskeletal changes in formation of axonal spheroids triggered by ROS. The results reveal that dramatically higher axoplasmic Ca(2+) levels occur at the sites of axonal spheroids than in the rest of the axon. High focal axoplasmic Ca(2+) levels correlate with focal aggregation of the reverse Na(+)/Ca(2+) exchanger 1, voltage-gated N-type Ca(2+) channel α1B subunit, and actin at the sites of spheroids in individual axons. This study provides new insights into the mechanism of a spheroid formation at specific sites along axons undergoing oxidative stress and a basis for new neuroprotective strategies.
Assuntos
Actinas/metabolismo , Axônios/metabolismo , Canais de Cálcio Tipo N/metabolismo , Cálcio/metabolismo , Estresse Oxidativo/fisiologia , Trocador de Sódio e Cálcio/metabolismo , Esferoides Celulares/metabolismo , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regulação para Cima/fisiologiaRESUMO
BH3-only proteins of the Bcl-2 family regulate programmed cell death in mammals through activation of multidomain proapoptotic proteins Bax and Bak in response to various proapoptotic stimuli by mechanism that remains under dispute. Here, we report that the cell death-promoting activity of BH3-only proteins Bik, Bmf, Noxa, and tBid can only be reconstituted in yeast when both multidomain proapoptotic and antiapoptotic Bcl-2 family proteins are present. Inability of these proteins to induce cell death in the absence of antiapoptotic proteins suggests that all tested BH3-only proteins likely activate Bax and Bak indirectly by inhibiting antiapoptotic proteins.
Assuntos
Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Expressão Gênica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
We have studied the pathways for Ca(2+) transport in mitochondria of the fruit fly Drosophila melanogaster. We demonstrate the presence of ruthenium red (RR)-sensitive Ca(2+) uptake, of RR-insensitive Ca(2+) release, and of Na(+)-stimulated Ca(2+) release in energized mitochondria, which match well characterized Ca(2+) transport pathways of mammalian mitochondria. Following larger matrix Ca(2+) loading Drosophila mitochondria underwent spontaneous RR-insensitive Ca(2+) release, an event that in mammals is due to opening of the permeability transition pore (PTP). Like the PTP of mammals, Drosophila Ca(2+)-induced Ca(2+) release could be triggered by uncoupler, diamide, and N-ethylmaleimide, indicating the existence of regulatory voltage- and redox-sensitive sites and was inhibited by tetracaine. Unlike PTP-mediated Ca(2+) release in mammals, however, it was (i) insensitive to cyclosporin A, ubiquinone 0, and ADP; (ii) inhibited by P(i), as is the PTP of yeast mitochondria; and (iii) not accompanied by matrix swelling and cytochrome c release even in KCl-based medium. We conclude that Drosophila mitochondria possess a selective Ca(2+) release channel with features intermediate between the PTP of yeast and mammals.
Assuntos
Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Animais , Antifúngicos/farmacologia , Benzoquinonas/farmacologia , Ciclosporina/farmacologia , Drosophila melanogaster , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/fisiologia , Indicadores e Reagentes/farmacologia , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Mamíferos/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Rutênio Vermelho/farmacologia , Especificidade da Espécie , Leveduras/metabolismoRESUMO
Although multiple sclerosis (MS) has traditionally been considered to be an inflammatory disease, recent evidence has brought neurodegeneration into the spotlight, suggesting that accumulated damage and loss of axons is critical to disease progression and the associated irreversible disability. Proposed mechanisms of axonal degeneration in MS posit cytosolic and subsequent mitochondrial Ca(2+) overload, accumulation of pathologic reactive oxygen species (ROS), and mitochondrial dysfunction leading to cell death. In this context, the role of the p66 isoform of ShcA protein (p66) may be significant. The ShcA isoform is uniquely targeted to the mitochondrial intermembrane space in response to elevated oxidative stress, and serves as a redox enzyme amplifying ROS generation in a positive feedforward loop that eventually mediates cell death by activation of the mitochondrial permeability transition pore. Consequently, we tested the hypothesis that genetic inactivation of p66 would reduce axonal injury in a murine model of MS, experimental autoimmune encephalomyelitis (EAE). As predicted, the p66-knockout (p66-KO) mice developed typical signs of EAE, but had less severe clinical impairment and paralysis than wild-type (WT) mice. Histologic examination of spinal cords and optic nerves showed significant axonal protection in the p66-KO tissue, despite similar levels of inflammation. Furthermore, cultured p66-KO neurons treated with agents implicated in MS neurodegenerative pathways showed greater viability than WT neurons. These results confirm the critical role of ROS-mediated mitochondrial dysfunction in the axonal loss that accompanies EAE, and identify p66 as a new pharmacologic target for MS neuroprotective therapeutics.
Assuntos
Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/prevenção & controle , Proteínas Adaptadoras da Sinalização Shc/deficiência , Animais , Axônios/patologia , Axônios/ultraestrutura , Proliferação de Células , Células Cultivadas , Córtex Cerebral/citologia , Peptidil-Prolil Isomerase F , Ciclofilinas/deficiência , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/patologia , Adjuvante de Freund/efeitos adversos , Glicoproteínas/efeitos adversos , Peróxido de Hidrogênio/farmacologia , Infiltração Leucêmica/tratamento farmacológico , Infiltração Leucêmica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Glicoproteína Mielina-Oligodendrócito , Fibras Nervosas Mielinizadas/patologia , Neurônios/metabolismo , Neurônios/ultraestrutura , Nervo Óptico/imunologia , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Nervo Óptico/ultraestrutura , Fragmentos de Peptídeos/efeitos adversos , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Medula Espinal/ultraestrutura , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Objectives Thrombotic and bleeding complications are common in COVID-19 disease. In a prospective study, we performed a comprehensive panel of tests to predict the risk of bleeding and thrombosis in patients admitted with hypoxic respiratory failure due to severe COVID-19 infection. Methods We performed a single center (step down and intensive care unit [ICU] at a quaternary care academic hospital) prospective study. Sequentially enrolled adult (≥18 years) patients were admitted with acute hypoxic respiratory failure due to COVID-19 between June 2020 and November 2020. Several laboratory markers of coagulopathy were tested after informed and written consent. Results Thirty-three patients were enrolled. In addition to platelet counts, prothrombin time, and activated partial thromboplastin time, a series of protocol laboratories were collected within 24 hours of admission. These included Protein C, Protein S, Antithrombin III, ADAMTS13, fibrinogen, ferritin, haptoglobin, and peripheral Giemsa smear. Patients were then monitored for the development of hematological (thrombotic and bleeding) events and followed for 30 days after discharge. Twenty-four patients (73%) required ICU admissions. At least one laboratory abnormality was detected in 100% of study patients. Nine patients (27%) suffered from significant hematological events, and four patients had a clinically significant bleeding event requiring transfusion. No significant association was observed between abnormalities of coagulation parameters and the incidence of hematologic events. However, a higher SOFA score (10.89 ± 3.48 vs. 6.92 ± 4.10, p = 0.016) and CKD (5/9 [22.2%] vs. 2/24 [12.5%] p = 0.009) at baseline were associated with the development of hematologic events. 33.3% of patients died at 30 days. Mortality was similar in those with and without hematological events. Reduced ADAMTS13 level was significantly associated with mortality. Conclusion Routine extensive testing of coagulation parameters did not predict the risk of bleeding and thrombosis in COVID-19 patients. Thrombotic and bleeding events in COVID-19 patients are not associated with a higher risk of mortality. Interestingly, renal dysfunction and a high SOFA score were found to be associated with increased risk of hematological events.
RESUMO
Cyclophilins are a family of peptidyl-prolyl cis-trans isomerases whose enzymatic activity can be inhibited by cyclosporin A. Sixteen cyclophilins have been identified in humans, and cyclophilin D is a unique isoform that is imported into the mitochondrial matrix. Here we shall (i) review the best characterized functions of cyclophilin D in mitochondria, i.e. regulation of the permeability transition pore, an inner membrane channel that plays an important role in the execution of cell death; (ii) highlight new regulatory interactions that are emerging in the literature, including the modulation of the mitochondrial F1FO ATP synthase through an interaction with the lateral stalk of the enzyme complex; and (iii) discuss diseases where cyclophilin D plays a pathogenetic role that makes it a suitable target for pharmacologic intervention.