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1.
Clin Sci (Lond) ; 134(2): 239-259, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-31943002

RESUMO

Mitochondrial stress has been widely observed in diabetic kidney disease (DKD). Cyclophilin D (CypD) is a functional component of the mitochondrial permeability transition pore (mPTP) which allows the exchange of ions and solutes between the mitochondrial matrix to induce mitochondrial swelling and activation of cell death pathways. CypD has been successfully targeted in other disease contexts to improve mitochondrial function and reduced pathology. Two approaches were used to elucidate the role of CypD and the mPTP in DKD. Firstly, mice with a deletion of the gene encoding CypD (Ppif-/-) were rendered diabetic with streptozotocin (STZ) and followed for 24 weeks. Secondly, Alisporivir, a CypD inhibitor was administered to the db/db mouse model (5 mg/kg/day oral gavage for 16 weeks). Ppif-/- mice were not protected against diabetes-induced albuminuria and had greater glomerulosclerosis than their WT diabetic littermates. Renal hyperfiltration was lower in diabetic Ppif-/- as compared with WT mice. Similarly, Alisporivir did not improve renal function nor pathology in db/db mice as assessed by no change in albuminuria, KIM-1 excretion and glomerulosclerosis. Db/db mice exhibited changes in mitochondrial function, including elevated respiratory control ratio (RCR), reduced mitochondrial H2O2 generation and increased proximal tubular mitochondrial volume, but these were unaffected by Alisporivir treatment. Taken together, these studies indicate that CypD has a complex role in DKD and direct targeting of this component of the mPTP will likely not improve renal outcomes.


Assuntos
/metabolismo , Diabetes Mellitus Experimental/metabolismo , Nefropatias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Albuminúria/genética , Albuminúria/metabolismo , Animais , /genética , Ciclosporina/farmacologia , Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Peróxido de Hidrogênio/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Nefropatias/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , ATPases Translocadoras de Prótons/metabolismo
2.
Cell Physiol Biochem ; 53(S1): 11-43, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31834993

RESUMO

Ion channels residing in the inner (IMM) and outer (OMM) mitochondrial membranes are emerging as noteworthy pharmacological targets in oncology. While these aspects have not been investigated for all of them, a role in cancer growth and/or metastasis and/or drug resistance has been shown at least for the IMM-residing Ca2+ uniporter complex and K+- selective mtKV1.3, mtIKCa, mtSKCa and mtTASK-3, and for the OMM Voltage-Dependent Anion Channel (mitochondrial porin). A special case is that of the Mitochondrial Permeability Transition Pore, a large pore which forms in the IMM of severely stressed cells, and which may be exploited to precipitate the death of cancerous cells. Here we briefly discuss the oncological relevance of mitochondria and their channels, and summarize the methods that can be adopted to selectively target these intracellular organelles. We then present an updated list of known mitochondrial channels, and review the pharmacology of those with proven relevance for cancer.


Assuntos
Antineoplásicos/química , Canais Iônicos/metabolismo , Mitocôndrias/metabolismo , Bibliotecas de Moléculas Pequenas/química , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Humanos , Canais Iônicos/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Canais de Potássio/química , Canais de Potássio/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/uso terapêutico , Canais de Ânion Dependentes de Voltagem/química , Canais de Ânion Dependentes de Voltagem/metabolismo
3.
Life Sci ; 237: 116941, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31606382

RESUMO

AIMS: Podocytes play an important role in the development of diabetic kidney disease (DKD). Mitochondria are the source of energy for cell survival, and mitochondrial abnormalities have been shown to contribute to podocyte injury in DKD. In high glucose (HG)-treated podocytes, mitochondrial function and dynamics are abnormal, and intracellular metabolism is often disrupted. However, the molecular mechanism is still unclear. Mitochondrial pyruvate carrier 2 (MPC2) mediates pyruvate transport from the cytoplasm to the mitochondrial matrix, which determines the cellular energy supply and cell survival. Here, we hypothesize that MPC2 damages mitochondria and induces apoptosis in HG-treated podocytes. MAIN METHODS: We used Western blotting, immunofluorescence and immunoprecipitation to detect the expression of MPC2 in HG-treated podocytes. Pyruvate levels were measured to evaluate metabolic station. Mitochondrial membrane potential (MMP) was measured by inverted fluorescence microscopy and flow cytometry. Mitochondrial morphology was assayed by MitoTracker Red staining, and cellular apoptosis was examined by flow cytometry. Furthermore, we treated podocytes with UK5099 and MPC2 siRNA to assess the outcomes of UK5099 treatment and MPC2 knockdown. KEY FINDINGS: Intracellular pyruvate accumulated, the mitochondria were damaged and cellular apoptosis increased in podocytes cultured with HG compared to that in control podocytes. MPC2 acetylation was significantly increased in HG-treated podocytes. Furthermore, the mitochondrial morphology changed, the MMP decreased, and cellular apoptosis increased. Inhibition of MPC2 function by UK5099 or MPC2 knockdown by siRNA produced the same abnormal effects observed following treatment with HG. SIGNIFICANCE: MPC2 may mediate mitochondrial dysfunction in HG-treated podocytes, ultimately leading to cell apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Glucose/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Podócitos/patologia , Ácido Pirúvico/metabolismo , Células Cultivadas , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Podócitos/efeitos dos fármacos , Podócitos/metabolismo
4.
Nat Commun ; 10(1): 4341, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31554800

RESUMO

The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. By combining highly purified, fully active bovine F-ATP synthase with preformed liposomes we show that Ca2+ dissipates the H+ gradient generated by ATP hydrolysis. After incorporation of the same preparation into planar lipid bilayers Ca2+ elicits currents matching those of the MMC/PTP. Currents were fully reversible, were stabilized by benzodiazepine 423, a ligand of the OSCP subunit of F-ATP synthase that activates the MMC/PTP, and were inhibited by Mg2+ and adenine nucleotides, which also inhibit the PTP. Channel activity was insensitive to inhibitors of the adenine nucleotide translocase (ANT) and of the voltage-dependent anion channel (VDAC). Native gel-purified oligomers and dimers, but not monomers, gave rise to channel activity. These findings resolve the long-standing mystery of the MMC/PTP and demonstrate that Ca2+ can transform the energy-conserving F-ATP synthase into an energy-dissipating device.


Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Bovinos , Microscopia Crioeletrônica , Hidrólise , Magnésio/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/ultraestrutura , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
5.
Nat Commun ; 10(1): 3732, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31427612

RESUMO

Recently identified core proteins (MICU1, MCU, EMRE) forming the mitochondrial Ca2+ uniporter complex propelled investigations into its physiological workings. Here, we apply structured illumination microscopy to visualize and localize these proteins in living cells. Our data show that MICU1 localizes at the inner boundary membrane (IBM) due to electrostatic interaction of its polybasic domain. Moreover, this exclusive localization of MICU1 is important for the stability of cristae junctions (CJ), cytochrome c release and mitochondrial membrane potential. In contrast to MICU1, MCU and EMRE are homogeneously distributed at the inner mitochondrial membrane under resting conditions. However, upon Ca2+ elevation MCU and EMRE dynamically accumulate at the IBM in a MICU1-dependent manner. Eventually, our findings unveil an essential function of MICU1 in CJ stabilization and provide mechanistic insights of how sophistically MICU1 controls the MCU-Complex while maintaining the structural mitochondrial membrane framework.


Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Sinalização do Cálcio/fisiologia , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Membranas Mitocondriais/metabolismo
6.
Cell Physiol Biochem ; 53(3): 465-479, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31464387

RESUMO

BACKGROUND/AIMS: Cyclophilin D (CypD) mediates the mitochondrial permeability transition pore (mPTP) opening that contributes to mitochondrial dysfunction. CypD is regulated by its acetylation/deacetylation state that depends on Sirtuin-3 (SIRT3) mitochondrial deacetylase. Since obesity and metabolic syndrome decrease SIRT3 activity and expression, we tested the hypothesis that CypD hyperacetylation promotes mitochondrial dysfunction under this pathophysiological state, which is associated with ventricular dysfunction and heart failure. METHODS: Myocardial tissue samples from patients with left ventricular heart failure, with either obesity or normal weight, were processed for the expression of SIRT3 and acetylation profile by Western Blot (WB). In addition, a rat model of obesity and metabolic syndrome induced by 30% (w/v) of sucrose was conducted. The WB analysis was used to determine the levels of mitochondrial expression of SIRT3, Adenine Nucleotide Translocator (ANT), CypD and the acetylation profile, as well as immunoprecipitation to establish the acetylation levels of CypD. Mitochondrial function was assessed by oxygen consumption analysis and maximum Ca2+ retention capacity. Oxidative stress was assessed by aconitase activity, protein carbonyl and thiol groups content. RESULTS: SIRT3 expression in the biopsies of the failing human hearts showed a 46% decrease in the expression levels of obese patients in comparison to the non-obese patients (p=0.0219). Remarkably, body mass index was associated with protein acetylation (0.627; p = 0.035), suggesting that the acetylation profiles of the failing hearts of obese patients are partly mediated by a reduction in SIRT3, which is also associated with higher BNP levels, indicating a more severe ventricular dysfunction (-0.636; p = 0.043). Accordingly, obese rats demonstrated a SIRT3 mitochondrial expression decrease of 22% concomitantly with a hyperacetylated mitochondrial profile, including CypD. Cardiac mitochondria from obese animals were 2.5-fold more prone to mPTP opening than the controls. CONCLUSION: Our results indicate that obesity reduces SIRT3 expression and that CypD hyperacetylation increases mPTP opening, suggesting that the activation of SIRT3 might be a potential target to decrease ventricular dysfunction and slow the progression of heart failure.


Assuntos
Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Obesidade/metabolismo , Sirtuína 3/metabolismo , Acetilação , Adulto , Idoso , Animais , Índice de Massa Corporal , Cálcio/metabolismo , Ciclofilinas/metabolismo , Feminino , Insuficiência Cardíaca/metabolismo , Humanos , Imunoprecipitação , Técnicas In Vitro , Masculino , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Translocases Mitocondriais de ADP e ATP/metabolismo , Consumo de Oxigênio/fisiologia , Ratos , Ratos Wistar
7.
Gastroenterology ; 157(5): 1368-1382, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31336123

RESUMO

BACKGROUND & AIMS: Hepatic ischemia/reperfusion injury is a complication of liver surgery that involves mitochondrial dysfunction resulting from mitochondrial permeability transition pore (mPTP) opening. Cyclophilin D (PPIF or CypD) is a peptidyl-prolyl cis-trans isomerase that regulates mPTP opening in the inner mitochondrial membrane. We investigated whether and how recently created small-molecule inhibitors of CypD prevent opening of the mPTP in hepatocytes and the resulting effects in cell models and livers of mice undergoing ischemia/reperfusion injury. METHODS: We measured the activity of 9 small-molecule inhibitors of cyclophilins in an assay of CypD activity. The effects of the small-molecule CypD inhibitors or vehicle on mPTP opening were assessed by measuring mitochondrial swelling and calcium retention in isolated liver mitochondria from C57BL/6J (wild-type) and Ppif-/- (CypD knockout) mice and in primary mouse and human hepatocytes by fluorescence microscopy. We induced ischemia/reperfusion injury in livers of mice given a small-molecule CypD inhibitor or vehicle before and during reperfusion and collected samples of blood and liver for histologic analysis. RESULTS: The compounds inhibited peptidyl-prolyl isomerase activity (half maximal inhibitory concentration values, 0.2-16.2 µmol/L) and, as a result, calcium-induced mitochondrial swelling, by preventing mPTP opening (half maximal inhibitory concentration values, 1.4-132 µmol/L) in a concentration-dependent manner. The most potent inhibitor (C31) bound CypD with high affinity and inhibited swelling in mitochondria from livers of wild-type and Ppif-/- mice (indicating an additional, CypD-independent effect on mPTP opening) and in primary human and mouse hepatocytes. Administration of C31 in mice with ischemia/reperfusion injury before and during reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage compared with vehicle. CONCLUSIONS: Recently created small-molecule inhibitors of CypD reduced calcium-induced swelling in mitochondria from mouse and human liver tissues. Administration of these compounds to mice during ischemia/reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage. These compounds might be developed to protect patients from ischemia/reperfusion injury after liver surgery or for other hepatic or nonhepatic disorders related to abnormal mPTP opening.


Assuntos
/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Hepatopatias/prevenção & controle , Fígado/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Traumatismo por Reperfusão/prevenção & controle , Animais , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , /metabolismo , Citoproteção , Modelos Animais de Doenças , Humanos , Fígado/enzimologia , Fígado/patologia , Hepatopatias/enzimologia , Hepatopatias/genética , Hepatopatias/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais
8.
Oxid Med Cell Longev ; 2019: 3403075, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31191798

RESUMO

Mitochondrial permeability transition pore (PTP), a (patho)physiological phenomenon discovered over 40 years ago, is still not completely understood. PTP activation results in a formation of a nonspecific channel within the inner mitochondrial membrane with an exclusion size of 1.5 kDa. PTP openings can be transient and are thought to serve a physiological role to allow quick Ca2+ release and/or metabolite exchange between mitochondrial matrix and cytosol or long-lasting openings that are associated with pathological conditions. While matrix Ca2+ and oxidative stress are crucial in its activation, the consequence of prolonged PTP opening is dissipation of the inner mitochondrial membrane potential, cessation of ATP synthesis, bioenergetic crisis, and cell death-a primary characteristic of mitochondrial disorders. PTP involvement in mitochondrial and cellular demise in a variety of disease paradigms has been long appreciated, yet the exact molecular entity of the PTP and the development of potent and specific PTP inhibitors remain areas of active investigation. In this review, we will (i) summarize recent advances made in elucidating the molecular nature of the PTP focusing on evidence pointing to mitochondrial FoF1-ATP synthase, (ii) summarize studies aimed at discovering novel PTP inhibitors, and (iii) review data supporting compromised PTP activity in specific mitochondrial diseases.


Assuntos
Doenças Mitocondriais/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Animais , Humanos , Membranas Mitocondriais/metabolismo
9.
Environ Toxicol ; 34(10): 1074-1084, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31157505

RESUMO

Microcystin-LR (MC-LR), a potent endotoxin, can induce reproductive toxicity. In order to investigate the role and mechanisms of apoptosis (p53-dependent and mitochondrial pathways) of germ cells induced by MC-LR, the co-cultured primary Sertoli-germ cells from Sprague-Dawley rats were used for the experiments. Expression levels of proteins, genes, and mitochondrial membrane potential (MMP) were obtained after exposing co-cultured Sertoli-germ cells to MC-LR with or without the addition of the p53 inhibitor, pifithrin-α (PFT-α), and MMP inhibitor, cyclosporin A (CsA). Results indicated that MC-LR could activate p53-dependent pathway-associated proteins in Sertoli-germ cells, leading to a decrease in MMP (indicating the opening of mitochondrial permeability transition pore [mPTP] and the release of Cytochrome-c [Cyt-c]) from the mitochondria into the cytoplasm and eventually the induction of apoptosis. PFT-α inhibited the expression ofp53, ameliorated the MMP of the co-cultured Sertoli-germ cells, and prevented the release of Cyt-c from the mitochondria into the cytoplasm, which reduces the occurrence of apoptosis. Similarly, the decreased release of Cyt-c from the mitochondria into the cytoplasm and the declined level of apoptosis in Sertoli-germ cells induced by MC-LR were observed after the addition of CsA. These results indicated that the apoptosis of the co-cultured Sertoli-germ cells induced by MC-LR was mediated by the p53-dependent pathway, with the involvement of the opening of mPTP.


Assuntos
Células Germinativas/efeitos dos fármacos , Microcistinas/toxicidade , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Células de Sertoli/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Técnicas de Cocultura , Células Germinativas/citologia , Células Germinativas/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos , Ratos Sprague-Dawley , Células de Sertoli/citologia , Células de Sertoli/metabolismo
10.
Food Chem ; 293: 396-407, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151627

RESUMO

To explore the involvement of protein lysine acetylation in the conversion of muscle to meat, a quantitative analysis of the acetylome in postmortem porcine muscle with or without antemortem stress was conducted. In total, 771 acetylpeptides containing 681 lysine acetylation sites mapping to 176 acetylproteins were identified. Acetylproteins were enriched in muscle contraction, carbohydrate metabolism, cell apoptosis and calcium signaling. Bioinformatic analysis suggests that preslaughter handling may be associated with glycolysis in postmortem muscle and the overall meat quality, via acetylation of multiple enzymes of glycogenolysis/glycolysis, regulate rigor mortis via acetylation of contractile, ATP production and calcium signaling-related proteins, and regulate stress response, cell apoptosis and meat tenderization via regulating the functions of heat shock proteins and permeability transition pore complex. This study provides the first overview of the acetylome in postmortem muscle as affected by preslaughter handling and broadens knowledge of the biochemistry regulating meat quality development.


Assuntos
Qualidade dos Alimentos , Lisina/metabolismo , Músculo Esquelético/metabolismo , Proteômica/métodos , Carne Vermelha/análise , Acetilação , Animais , Biologia Computacional/métodos , Glicólise , Proteínas de Choque Térmico/metabolismo , Proteínas de Carne/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mudanças Depois da Morte , Estresse Psicológico , Suínos
11.
Chem Biol Interact ; 309: 108723, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31228469

RESUMO

Ischemic preconditioning and pharmacological preconditioning are common strategies to prevent lethal myocardial injury, especially nutritional preconditioning (NPC). In this study, we investigated the effects of astragaloside IV (Ast), as an NPC agent, on myocardium suffered anoxia/reoxygenation (A/R) injury. Rats received 5 mg/kg Ast daily for 3 weeks by intragastric administration. Then, hearts were harvested and underwent A/R treatment using a Langendorff apparatus. Ast- pretreatment significantly promoted functional recovery of the myocardium, reduced infarct size, and oxidative stress, and decreased the apoptotic index. Similar findings were demonstrated in H9c2 cardiomyocytes that were pretreated with Ast for 24 h. Moreover, Ast-pretreatment significantly upregulated Bcl-2 expression, especially in mitochondria. The effects of Ast treatment against A/R injury were also reflected by increased antioxidant potential, inhibited reactive oxygen species (ROS) burst, increased oxygen consumption rate, maintained mitochondrial membrane potential (MMP), inhibited mitochondrial permeability transition pore (mPTP) opening, and prevented apoptosis. Selective inhibition of Bcl-2 by ABT-737 decreased myocardial injury protection of Ast. Ast-pretreatment resulted in NPC- related effects against A/R, and mitochondria may be the target of a cascade of events elicited by upregulating Bcl-2 expression, promoting translocation of Bcl-2 into mitochondria, maintaining MMP, inhibiting ROS bursts, thereby leading to recovery of mitochondrial respiration, preventing mPTP opening, decreasing cytochrome C release, preventing apoptosis, and ultimately alleviating myocardial injury.


Assuntos
Mitocôndrias/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologia , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Compostos de Bifenilo/uso terapêutico , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Nitrofenóis/farmacologia , Nitrofenóis/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Superóxido Dismutase/metabolismo
12.
Oxid Med Cell Longev ; 2019: 4508762, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31236191

RESUMO

Much evidence demonstrates that mitochondrial dysfunction plays a crucial role in the pathogenesis of vascular complications of diabetes. However, the signaling pathways through which hyperglycemia leads to mitochondrial dysfunction of endothelial cells are not fully understood. Here, we treated human umbilical vein endothelial cells (HUVECs) with high glucose and examined the role of translocase of mitochondrial outer membrane (Tom) 22 on mitochondrial dynamics and cellular function. Impaired Tom22 expression and protein expression of oxidative phosphorylation (OXPHOS) as well as decreased mitochondrial fusion were observed in HUVECs treated with high glucose. The deletion of Tom22 resulted in reduced mitochondrial fusion and ATP production and increased apoptosis in HUVECs. The overexpression of Tom22 restored the balance of mitochondrial dynamics and OXPHOS disrupted by high glucose. Importantly, we found that Tom22 modulates mitochondrial dynamics and OXPHOS by interacting with mitofusin (Mfn) 1. Taken together, our findings demonstrate for the first time that Tom22 is a novel regulator of both mitochondrial dynamics and bioenergetic function and contributes to cell survival following high-glucose exposure.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Células Endoteliais da Veia Umbilical Humana/patologia , Mitocôndrias/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Apoptose , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Transdução de Sinais
13.
mBio ; 10(3)2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064825

RESUMO

The mitochondrial Ca2+ uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used Trypanosoma cruzi to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca2+ uptake. T. cruzi MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated TcMICU1 and TcMICU2 knockout (-KO) cell lines. Ablation of either TcMICU1 or TcMICU2 showed a significantly reduced mitochondrial Ca2+ uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca2+ concentrations ([Ca2+]cyt), as occurs with their mammalian orthologs. TcMICU1-KO and TcMICU2-KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles.IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca2+ sensors and gatekeepers of the MCU, preventing Ca2+ uptake under resting conditions and favoring it at high cytosolic Ca2+ concentrations ([Ca2+]cyt). Using the CRISPR/Cas9 technique, we generated TcMICU1 and TcMICU2 knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca2+]cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/genética , Adaptação Fisiológica , Transporte Biológico , Sistemas CRISPR-Cas , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte de Cátions , Citosol/química , Citosol/metabolismo , Técnicas de Inativação de Genes , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/patogenicidade
14.
Oxid Med Cell Longev ; 2019: 4353791, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31093314

RESUMO

Targeting mitochondria as a hepatic-protective strategy has gained attention, because of their important roles in energy production, adjustment of apoptosis, and generation of reactive oxygen species. To promote the discovery of natural mitochondria-targeted hepatic-protectants, we established a hepatocellular mitochondria-based capturing method by coupling affinity ultrafiltration with liquid chromatography/mass spectrometry (LC/MS), which is suitable for identifying mitochondrial ligands from medicinal herbs (MHs). After evaluating the feasibility of the method, it was applied for capturing mitochondria-targeting constituents from Peucedani Radix extract. A total of 10 active compounds were identified by LC/MS, all of which were newly identified mitochondrial ligands. The mitochondria-remedying activity of 4 of the 10 hits was confirmed by pharmacological tests in vitro. Additionally, the hepatic-protective abilities of 4 hits were verified in both carbon tetrachloride-damaged liver L02 cells and mice. These results indicated that the method could be used for identifying hepatic mitochondria-targeting constituents in MHs, which might be beneficial for hepatic-protective development.


Assuntos
Fígado/metabolismo , Mitocôndrias Hepáticas/metabolismo , Plantas Medicinais/química , Substâncias Protetoras/farmacologia , Animais , Fígado/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Extratos Vegetais/farmacologia , Ratos Sprague-Dawley , Padrões de Referência
15.
EMBO J ; 38(10)2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30979775

RESUMO

The mitochondrial pyruvate carrier (MPC) is critical for cellular homeostasis, as it is required in central metabolism for transporting pyruvate from the cytosol into the mitochondrial matrix. MPC has been implicated in many diseases and is being investigated as a drug target. A few years ago, small membrane proteins, called MPC1 and MPC2 in mammals and Mpc1, Mpc2 and Mpc3 in yeast, were proposed to form large protein complexes responsible for this function. However, the MPC complexes have never been isolated and their composition, oligomeric state and functional properties have not been defined. Here, we identify the functional unit of MPC from Saccharomyces cerevisiae In contrast to earlier hypotheses, we demonstrate that MPC is a hetero-dimer, not a multimeric complex. When not engaged in hetero-dimers, the yeast Mpc proteins can also form homo-dimers that are, however, inactive. We show that the earlier described substrate transport properties and inhibitor profiles are embodied by the hetero-dimer. This work provides a foundation for elucidating the structure of the functional complex and the mechanism of substrate transport and inhibition.


Assuntos
Proteínas de Transporte de Ânions , Proteínas de Transporte da Membrana Mitocondrial , Transportadores de Ácidos Monocarboxílicos , Complexos Multiproteicos/fisiologia , Multimerização Proteica/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Transporte de Ânions/química , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Transportadores de Ácidos Monocarboxílicos/química , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Organismos Geneticamente Modificados , Estrutura Quaternária de Proteína/fisiologia , Ácido Pirúvico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Temperatura Ambiente
16.
Neurol Res ; 41(7): 594-608, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30973085

RESUMO

Recent evidence has suggested that cadmium (Cd) ions-induced neurotoxicity is associated with increased oxidative stress and mitochondrial-dependent and endoplasmic reticulum (ER) stress-induced apoptosis. This study aimed to investigate if rutin hydrate (RH), a well-reported neuroprotective and an antioxidant flavonoid, can ameliorate cadmium chloride (CdCl2)-induced neurotoxicity by inhibiting the resultant ER stress. Rats were divided into 4 groups (n = 16/group) of control, control + RH (100 mg/kg), CdCl2 (5 mg/kg), and CdCl2 + RH. All treatments were administered orally for 30 days, on daily basis. Brain homogenates from CdCl2-treated rats showed increased oxidative stress and induced activation of ER stress characterized by increasing mRNA and protein levels of GRP78, ATF-6, CHOP and Xbp-1 and protein levels of p-elF2α, p-JNK1/2 and cleaved caspase-12. Also, CdCl2 significantly reduced Bcl-2, enhanced Bax translocation to the mitochondrial membrane, increased cytoplasmic levels of cytochrome-C and caspase-3, and reduced mitochondrial membrane potential (Δψm) (increased Vmax and reduced time to Vmax). In contrast, RH significantly enhanced levels GSH and activities of SOD, GSH-Px, decreased levels of MDA and inhibited mitochondrial permeability transition pore (mtPTP) in the brains of both control and CdCl2-treated rats. Interestingly, in brain homogenates of CdCl2-treated rats only, RH reduced all markers of ER stress, increased Bcl-2, reduced mitochondrial Bax translocation and improved mitochondrial coupling. It also reduced cytosolic levels of cytochrome-C, cleaved caspase-3, and cleaved caspase-12. Overall, these findings support the efficiency of RH to inhibit ER stress in brains CdCl2-treated rats which is added to its existing mechanisms of neuroprotection. Abbreviations: ATF-6: activating transcription factor-6; Bax: Bcl-associated x; BBB: blood-brain barrier; Bcl-2: B-cell lymphoma 2; BiP: immunoglobulin heavy-chain-binding protein; [Ca2+]i: intracellular free Ca2+ concentration; Cd: cadmium; CdCl2: cadmium chloride; CHOP: CCAAT/enhancer-binding protein-homologous protein; CMC: carboxymethyl cellulose; Δψm: mitochondrial membrane potential; elF2α: phospho-eukaryotic translation initiation factor 2-alpha; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERK1/2: extracellular signal-regulated kinases 1 and 2; GADD 153: growth arrest and DNA damage-inducible protein 153; GRP78, 78 kDa glucose-regulated protein; GSH: reduced glutathione; GSH: reduced glutathione; GSH-Px: glutathione peroxidase; GSSG: glutathione disulfide (oxidized glutathione); IRE-1: inositol-requiring enzyme-1; JNK: c-Jun N-terminal kinase; MAPK: mitogen-activated protein kinase; MDA: malondialdehyde; mTOR: Akt/mammalian target of rapamycin; mtPTP: mitochondrial permeability transition pore; ONOO-: peroxynitrite; PCR: polymerase chain reaction; PERK: protein kinase RNA-like ER kinase; p-JNK: phospho-JNK; qPCR: quantitative PCR; RCR: respiratory control ratio; RH: rutin hydrate; RHoGDI: Rho-GDP-dissociation inhibitor; ROS: reactive oxygen species; SOD: superoxide dismutase; UPR: unfolded protein response; VDAC: voltage-dependent anion channel; Vmax: maximal rate of pore opening; Xbp-1: X-box binding protein 1.


Assuntos
Apoptose/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Cloreto de Cádmio/toxicidade , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/efeitos dos fármacos , Rutina/farmacologia , Animais , Encéfalo/metabolismo , Cloreto de Cádmio/antagonistas & inibidores , Caspase 3/metabolismo , Citocromos c/metabolismo , Masculino , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Proteína X Associada a bcl-2/metabolismo
17.
Cell ; 177(2): 299-314.e16, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30929899

RESUMO

Autophagy is required in diverse paradigms of lifespan extension, leading to the prevailing notion that autophagy is beneficial for longevity. However, why autophagy is harmful in certain contexts remains unexplained. Here, we show that mitochondrial permeability defines the impact of autophagy on aging. Elevated autophagy unexpectedly shortens lifespan in C. elegans lacking serum/glucocorticoid regulated kinase-1 (sgk-1) because of increased mitochondrial permeability. In sgk-1 mutants, reducing levels of autophagy or mitochondrial permeability transition pore (mPTP) opening restores normal lifespan. Remarkably, low mitochondrial permeability is required across all paradigms examined of autophagy-dependent lifespan extension. Genetically induced mPTP opening blocks autophagy-dependent lifespan extension resulting from caloric restriction or loss of germline stem cells. Mitochondrial permeability similarly transforms autophagy into a destructive force in mammals, as liver-specific Sgk knockout mice demonstrate marked enhancement of hepatocyte autophagy, mPTP opening, and death with ischemia/reperfusion injury. Targeting mitochondrial permeability may maximize benefits of autophagy in aging.


Assuntos
Envelhecimento/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Membranas Mitocondriais/fisiologia , Animais , Autofagia/fisiologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Restrição Calórica , Células HEK293 , Humanos , Longevidade/fisiologia , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Permeabilidade , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais
18.
Nature ; 567(7748): 341-346, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30842654

RESUMO

Cancer-specific inhibitors that reflect the unique metabolic needs of cancer cells are rare. Here we describe Gboxin, a small molecule that specifically inhibits the growth of primary mouse and human glioblastoma cells but not that of mouse embryonic fibroblasts or neonatal astrocytes. Gboxin rapidly and irreversibly compromises oxygen consumption in glioblastoma cells. Gboxin relies on its positive charge to associate with mitochondrial oxidative phosphorylation complexes in a manner that is dependent on the proton gradient of the inner mitochondrial membrane, and it inhibits the activity of F0F1 ATP synthase. Gboxin-resistant cells require a functional mitochondrial permeability transition pore that regulates pH and thus impedes the accumulation of Gboxin in the mitochondrial matrix. Administration of a metabolically stable Gboxin analogue inhibits glioblastoma allografts and patient-derived xenografts. Gboxin toxicity extends to established human cancer cell lines of diverse organ origin, and shows that the increased proton gradient and pH in cancer cell mitochondria is a mode of action that can be targeted in the development of antitumour reagents.


Assuntos
Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Aloenxertos , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Linhagem Celular Tumoral , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , Transplante de Neoplasias , Especificidade de Órgãos , Força Próton-Motriz/efeitos dos fármacos , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Basic Res Cardiol ; 114(3): 17, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874894

RESUMO

We and others have reported that calpain-1 was increased in myocardial mitochondria from various animal models of heart disease. This study investigated whether constitutive up-regulation of calpain-1 restricted to mitochondria induced myocardial injury and heart failure and, if so, whether these phenotypes could be rescued by selective inhibition of mitochondrial superoxide production. Transgenic mice with human CAPN1 up-regulation restricted to mitochondria in cardiomyocytes (Tg-mtCapn1/tTA) were generated and characterized with low and high over-expression of transgenic human CAPN1 restricted to mitochondria, respectively. Transgenic up-regulation of mitochondria-targeted CAPN1 dose-dependently induced cardiac cell death, adverse myocardial remodeling, heart failure, and early death in mice, the changes of which were associated with mitochondrial dysfunction and mitochondrial superoxide generation. Importantly, a daily injection of mitochondria-targeted superoxide dismutase mimetics mito-TEMPO for 1 month starting from age 2 months attenuated cardiac cell death, adverse myocardial remodeling and heart failure, and reduced mortality in Tg-mtCapn1/tTA mice. In contrast, administration of TEMPO did not achieve similar cardiac protection in transgenic mice. Furthermore, transgenic up-regulation of mitochondria-targeted CAPN1 induced a reduction of ATP5A1 protein and ATP synthase activity in hearts. In cultured cardiomyocytes, increased calpain-1 in mitochondria promoted mitochondrial permeability transition pore (mPTP) opening and induced cell death, which were prevented by over-expression of ATP5A1, mito-TEMPO or cyclosporin A, an inhibitor of mPTP opening. In conclusion, this study has provided direct evidence demonstrating that increased mitochondrial calpain-1 is an important mechanism contributing to myocardial injury and heart failure by disrupting ATP synthase, and promoting mitochondrial superoxide generation and mPTP opening.


Assuntos
Calpaína/metabolismo , Cardiomiopatia Dilatada/etiologia , Insuficiência Cardíaca/etiologia , Mitocôndrias/metabolismo , Animais , Cardiomiopatia Dilatada/metabolismo , Morte Celular , Óxidos N-Cíclicos , Modelos Animais de Doenças , Insuficiência Cardíaca/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Miócitos Cardíacos/metabolismo , Superóxidos/metabolismo
20.
Protein J ; 38(3): 330-342, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30868341

RESUMO

Mitochondria are essential organelles of eukaryotic cells. They consist of hundreds of different proteins that exhibit crucial activities in respiration, catabolic metabolism and the synthesis of amino acids, lipids, heme and iron-sulfur clusters. With the exception of a handful of hydrophobic mitochondrially encoded membrane proteins, all these proteins are synthesized on cytosolic ribosomes, targeted to receptors on the mitochondrial surface, and transported across or inserted into the outer and inner mitochondrial membrane before they are folded and assembled into their final native structure. This review article provides a comprehensive overview of the mechanisms and components of the mitochondrial protein import systems with a particular focus on recent developments in the field.


Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Chaperonas Moleculares/metabolismo , Humanos , Transporte Proteico , Leveduras/metabolismo
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