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1.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806352

RESUMO

Lipids play essential roles in numerous cellular processes, including membrane remodeling, signal transduction, the modulation of hormone activity, and steroidogenesis. We chose steroidogenic MA-10 mouse tumor Leydig cells to investigate subcellular lipid localization during steroidogenesis. Electron microscopy showed that cAMP stimulation increased associations between the plasma membrane (PM) and the endoplasmic reticulum (ER) and between the ER and mitochondria. cAMP stimulation also increased the movement of cholesterol from the PM compared to untreated cells, which was partially inhibited when ATPase family AAA-domain containing protein 3 A (ATAD3A), which functions in ER and mitochondria interactions, was knocked down. Mitochondria, ER, cytoplasm, PM, PM-associated membranes (PAMs), and mitochondria-associated membranes (MAMs) were isolated from control and hormone-stimulated cells. Lipidomic analyses revealed that each isolated compartment had a unique lipid composition, and the induction of steroidogenesis caused the significant remodeling of its lipidome. cAMP-induced changes in lipid composition included an increase in phosphatidylserine and cardiolipin levels in PAM and PM compartments, respectively; an increase in phosphatidylinositol in the ER, mitochondria, and MAMs; and a reorganization of phosphatidic acid, cholesterol ester, ceramide, and phosphatidylethanolamine. Abundant lipids, such as phosphatidylcholine, were not affected by hormone treatment. Our data suggested that PM-ER-mitochondria tethering may be involved in lipid trafficking between organelles and indicated that hormone-induced acute steroid production involves extensive organelle remodeling.


Assuntos
Tumor de Células de Leydig/metabolismo , Lipídeos de Membrana/metabolismo , Esteroides/biossíntese , Neoplasias Testiculares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Bucladesina/farmacologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Colesterol/metabolismo , AMP Cíclico/farmacologia , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Tumor de Células de Leydig/ultraestrutura , Lipidômica , Masculino , Camundongos , Microscopia Eletrônica de Transmissão , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Neoplasias Testiculares/ultraestrutura
2.
Arch Biochem Biophys ; 692: 108535, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32781052

RESUMO

NAD(P)+ transhydrogenase (NNT) is located in the inner mitochondrial membrane and catalyzes a reversible hydride transfer between NAD(H) and NADP(H) that is coupled to proton translocation between the intermembrane space and mitochondrial matrix. NNT activity has an essential role in maintaining the NADPH supply for antioxidant defense and biosynthetic pathways. In the present report, we evaluated the effects of chemical compounds used as inhibitors of NNT over the last five decades, namely, 4-chloro-7-nitrobenzofurazan (NBD-Cl), N,N'-dicyclohexylcarbodiimide (DCC), palmitoyl-CoA, palmitoyl-l-carnitine, and rhein, on NNT activity and mitochondrial respiratory function. Concentrations of these compounds that partially inhibited the forward and reverse NNT reactions in detergent-solubilized mouse liver mitochondria significantly impaired mitochondrial respiratory function, as estimated by ADP-stimulated and nonphosphorylating respiration. Among the tested compounds, NBD-Cl showed the best relationship between NNT inhibition and low impact on respiratory function. Despite this, NBD-Cl concentrations that partially inhibited NNT activity impaired mitochondrial respiratory function and significantly decreased the viability of cultured Nnt-/- mouse astrocytes. We conclude that even though the tested compounds indeed presented inhibitory effects on NNT activity, at effective concentrations, they cause important undesirable effects on mitochondrial respiratory function and cell viability.


Assuntos
Inibidores Enzimáticos/farmacologia , Mitocôndrias Hepáticas/enzimologia , NADP Trans-Hidrogenase Específica para A ou B/antagonistas & inibidores , NADP Trans-Hidrogenase Específica para A ou B/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Animais , Inibidores Enzimáticos/química , Feminino , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/genética , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , NADP Trans-Hidrogenase Específica para A ou B/genética , Consumo de Oxigênio/genética
3.
Nat Commun ; 11(1): 3360, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620763

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is considered the next major health epidemic with an estimated 25% worldwide prevalence. No drugs have yet been approved and NAFLD remains a major unmet need. Here, we identify MCJ (Methylation-Controlled J protein) as a target for non-alcoholic steatohepatitis (NASH), an advanced phase of NAFLD. MCJ is an endogenous negative regulator of the respiratory chain Complex I that acts to restrain mitochondrial respiration. We show that therapeutic targeting of MCJ in the liver with nanoparticle- and GalNAc-formulated siRNA efficiently reduces liver lipid accumulation and fibrosis in multiple NASH mouse models. Decreasing MCJ expression enhances the capacity of hepatocytes to mediate ß-oxidation of fatty acids and minimizes lipid accumulation, which results in reduced hepatocyte damage and fibrosis. Moreover, MCJ levels in the liver of NAFLD patients are elevated relative to healthy subjects. Thus, inhibition of MCJ emerges as an alternative approach to treat NAFLD.


Assuntos
Ácidos Graxos/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Fígado/patologia , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Adulto , Idoso , Animais , Conjuntos de Dados como Assunto , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Proteínas de Choque Térmico HSP40/antagonistas & inibidores , Proteínas de Choque Térmico HSP40/genética , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Nanopartículas/administração & dosagem , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Oxirredução/efeitos dos fármacos , Cultura Primária de Células , RNA Interferente Pequeno/administração & dosagem , RNA-Seq
4.
Biochim Biophys Acta Bioenerg ; 1861(10): 148247, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32565080

RESUMO

The alternative oxidase (AOX) is a monotopic di­iron carboxylate protein which acts as a terminal respiratory chain oxidase in a variety of plants, fungi and protists. Of particular importance is the finding that both emerging infectious diseases caused by human and plant fungal pathogens, the majority of which are multi-drug resistant, appear to be dependent upon AOX activity for survival. Since AOX is absent in mammalian cells, AOX is considered a viable therapeutic target for the design of specific fungicidal and anti-parasitic drugs. In this work, we have mutated conserved residues within the hydrophobic channel (R96, D100, R118, L122, L212, E215 and T219), which crystallography has indicated leads to the active site. Our data shows that all mutations result in a drastic reduction in Vmax and catalytic efficiency whilst some also affected the Km for quinol and oxygen. The extent to which mutation effects inhibitor sensitivity was also investigated, with mutation of R118 and T219 leading to a complete loss of inhibitor potency. However, only a slight reduction in IC50 values was observed when R96 was mutated, implying that this residue is less important in inhibitor binding. In silico modelling has been used to provide insight into the reason for such changes, which we suggest is due to disruptions in the proton transfer network, resulting in a reduction in overall reaction kinetics. We discuss our results in terms of the structural features of the ubiquinol binding site and consider the implications of such findings on the nature of the catalytic cycle. SIGNIFICANCE: The alternative oxidase is a ubiquinol oxidoreductase enzyme that catalyses the oxidation of ubiquinol and the reduction of oxygen to water. It is widely distributed amongst the plant, fungal and parasitic kingdoms and plays a central role in metabolism through facilitating the turnover of the TCA cycle whilst reducing ROS production.


Assuntos
Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Oxigênio/metabolismo , Proteínas de Plantas/metabolismo , Trypanosoma brucei brucei/enzimologia , Ubiquinona/análogos & derivados , Sítios de Ligação , Inibidores Enzimáticos/farmacologia , Cinética , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Simulação de Acoplamento Molecular , Mutação , Oxirredução , Oxirredutases/antagonistas & inibidores , Oxirredutases/química , Oxirredutases/genética , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformação Proteica , Ubiquinona/metabolismo
5.
Gene ; 753: 144807, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32461017

RESUMO

Mitochondrial transcription factor A (TFAM), which is required for mitochondrial DNA (mtDNA) transcription, has been linked to metabolic changes that contribute to tumorigenesis and chemoresistance. In this work, we investigated the expression pattern and role of TFAM in hepatocellular carcinoma (HCC). TFAM expression level is similar in 18 out of 20 paired normal liver and HCC tissues with only 2 HCC tissues showing 1.8-fold increase in TFAM. Similar phenomenon was observed in HCC cell lines compared to normal liver lines. Interestingly, TFAM expression is upregulated in resistant HCC cells regardless of the differential TFAM expression level in their parental lines and mechanism of resistance. TFAM depletion led to inhibition of growth and survival but not migration, and sensitization to doxorubicin and sorafenib treatment, through AMPK activation, reduction of nucleoside triphosphates and mitochondrial respiration in HCC cells. In addition, we demonstrated that resistant HCC cell lines were more sensitive to TFAM inhibition than parental lines, and this might be due to the increased mitochondrial biogenesis in resistant HCC cell lines. Our work reveals the preferential role of TFAM in HCC cell response to standard of care drugs, which suggests a potential sensitizing therapeutic target for HCC treatment.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Proteínas de Ligação a DNA/deficiência , Doxorrubicina/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Mitocôndrias/metabolismo , Proteínas Mitocondriais/deficiência , Sorafenibe/farmacologia , Fatores de Transcrição/deficiência , Proteínas Quinases Ativadas por AMP/genética , Adulto , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias/genética , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima
6.
J Dairy Sci ; 103(6): 5561-5574, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32278565

RESUMO

Inflammation is critical in the progression from benign hepatic lipidosis to pathological hepatic steatosis. The objective of this study was to examine the potential role of the outer mitochondrial membrane protein mitofusin 2 (MFN2) in the etiology of hepatic steatosis in dairy cows during early lactation. Using a nested case-control design, we compared blood and liver samples from 10 healthy cows and 10 age-matched cows with moderate fatty liver. Cows with moderate fatty liver had high liver triacylglycerols, elevated plasma concentrations of free fatty acids (FFA) and ß-hydroxybutyrate, and low concentrations of glucose. Cows with moderate fatty liver had overactivated inflammatory pathways in the liver, as indicated by increased abundance of phosphorylated nuclear factor κB (NF-κB) p65, NLR family pyrin domain containing 3 (NLRP3) and caspase-1 inflammasome protein, and elevated plasma concentrations and hepatic mRNA abundance of their molecular targets IL-1ß, IL-6, and tumor necrosis factor α (TNF-α). In the cell culture model, we were able to replicate our findings in cows with moderate fatty liver: 1.2 mM exogenous FFA decreased the abundance of MFN2 and upregulated phosphorylation levels of the inhibitor of NF-κB (IκB) α and NF-κB p65, the IκB kinase ß activity, and the abundance of NLRP3, caspase-1, IL-1ß, IL-6, and TNF-α. Whereas MFN2 knockdown potentiated the FFA-induced activation of these inflammatory pathways, overexpression of MFN2 attenuated the detrimental effect of excess exogenous FFA by improving mitochondrial function and decreasing the release of reactive oxygen species, suggesting that MFN2 may be a potential therapeutic target for FFA-induced hepatic inflammation in dairy cows during early lactation.


Assuntos
Doenças dos Bovinos/prevenção & controle , Ácidos Graxos não Esterificados/efeitos adversos , Fígado Gorduroso/veterinária , GTP Fosfo-Hidrolases/antagonistas & inibidores , Inflamação/veterinária , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Animais , Estudos de Casos e Controles , Bovinos , Ácidos Graxos não Esterificados/sangue , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/prevenção & controle , Feminino , GTP Fosfo-Hidrolases/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Lactação/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismo
7.
Metabolism ; 105: 154182, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32061660

RESUMO

The impairment of podocyte protein filtration function caused by excessive mitochondrial calcium intake is a critical feature of diabetic nephropathy (DN). Ca2+ channel transient receptor potential cation channel subfamily V member 1 (TRPV1) has been reported to protect against ischemia-reperfusion induced acute renal injury, but there is no report about its role in DN. Here, we report that dietary capsaicin potently inhibits and reverses chronic renal structural and functional damages in db/db or streptozotocin (STZ)-induced diabetic mice in a TRPV1-dependent manner. Activation of TRPV1 by capsaicin alleviated hyperglycemia-induced mitochondrial dysfunction in podocytes, accompanied by reduced mitochondria-associated membranes (MAMs) formation and fewer Ca2+ transport from endoplasmic reticulum (ER) to mitochondria. Mechanistically, TRPV1-mediated transient Ca2+ influx activated 5' AMP-activated protein kinase (AMPK) that reduced the transcription of Fundc1, a key molecule participating in MAMs formation. Inhibition of AMPK or overexpression of Fundc1 obviously blocked the inhibitory effect of capsaicin on MAMs formation and functional decline in podocytes. These findings emphasize the critical role of mitochondrial Ca2+ homeostasis in the maintenance of normal renal function and suggest an effective intervention method to counteract DN.


Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Podócitos/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Canais de Cálcio/metabolismo , Capsaicina/uso terapêutico , Dieta , Inibidores Enzimáticos/farmacologia , Hiperglicemia/tratamento farmacológico , Hiperglicemia/microbiologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Membranas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/biossíntese
8.
Appl Environ Microbiol ; 86(7)2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32005728

RESUMO

Ergosterol plays an important role in maintaining cell membrane sterol homeostasis in fungi, and as such, it is considered an effective target in antifungal chemotherapy. In yeast, the enzyme acetyl-coenzyme A (CoA) acetyltransferase (ERG10) catalyzes the Claisen condensation of two acetyl-CoA molecules to acetoacetyl-CoA in the ergosterol biosynthesis pathway and is reported as being critical for cell viability. Using yeast ERG10 for alignment, two orthologues, AfERG10A (AFUB_000550) and AfERG10B (AFUB_083570), were discovered in the opportunistic fungal pathogen Aspergillus fumigatus Despite the essentiality of AfERG10B having been previously validated, the biological function of AfERG10A remains unclear. In this study, we have characterized recombinant AfERG10A as a functional acetyl-CoA acetyltransferase catalyzing both synthetic and degradative reactions. Unexpectedly, AfERG10A localizes to the mitochondria in A. fumigatus, as shown by C-terminal green fluorescent protein (GFP) tag fusion. Both knockout and inducible promoter strategies demonstrate that Aferg10A is essential for the survival of A. fumigatus The reduced expression of Aferg10A leads to severe morphological defects and increased susceptibility to oxidative and cell wall stresses. Although the catalytic mechanism of acetyl-CoA acetyltransferase family is highly conserved, the crystal structure of AfERG10A and its complex with CoA are solved, revealing four substitutions within the CoA binding site that are different from human orthologues. Taken together, our combination of genetic and structural studies demonstrates that mitochondrial AfERG10A is essential for A. fumigatus cell viability and could be a potential drug target to feed the antifungal drug development pipeline.IMPORTANCE A growing number of people worldwide are suffering from invasive aspergillosis caused by the human opportunistic fungal pathogen A. fumigatus Current therapeutic options rely on a limited repertoire of antifungals. Ergosterol is an essential component of the fungal cell membrane as well as a target of current antifungals. Approximately 20 enzymes are involved in ergosterol biosynthesis, of which acetyl-CoA acetyltransferase (ACAT) is the first enzyme. Two ACATs in A. fumigatus are AfErg10A and AfErg10B. However, the biological function of AfErg10A is yet to be investigated. In this study, we showed that AfErg10A is localized in the mitochondria and is essential for A. fumigatus survival and morphological development. In combination with structural studies, we validated AfErg10A as a potential drug target that will facilitate the development of novel antifungals and improve the efficiency of existing drugs.


Assuntos
Acetil-CoA C-Acetiltransferase/antagonistas & inibidores , Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas Mitocondriais/antagonistas & inibidores , Acetil-CoA C-Acetiltransferase/genética , Acetil-CoA C-Acetiltransferase/metabolismo , Aspergillus fumigatus/enzimologia , Ergosterol/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
9.
Biochem Pharmacol ; 174: 113832, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32006470

RESUMO

Cardiorenal syndrome type-3 (CRS-3) is characterized by acute cardiac injury induced by acute kidney injury. Here, we investigated the causes of CRS-3 by analyzing cardiac function after renal ischemia-reperfusion injury (IRI) using echocardiography and evaluation of pro-inflammatory markers, calcium balance, mitochondrial function, and cardiomyocyte death. Our results show that renal IRI reduces cardiac diastolic function associated with cardiomyocyte death and inflammatory responses. Renal IRI also disrupts cardiomyocyte energy metabolism, induces calcium overload, and impairs mitochondrial function, as evidenced by reduced mitochondrial membrane potential and increased mitochondrial fission. Further, renal IRI induces phosphorylation of inositol 1,4,5-trisphosphate receptor (IP3R) and expression of mitochondrial calcium uniporter (MCU), resulting in cytoplasmic calcium overload and mitochondrial calcium accumulation. Pretreatment with melatonin attenuates renal IRI-mediated cardiac damage by maintaining myocardial diastolic function and reducing cardiomyocyte death. Melatonin also inhibits IP3R phosphorylation and MCU expression, thereby alleviating cytoplasmic and mitochondrial calcium overload. Blockade of IP3R has similar cardioprotective effects, whereas MCU activation abrogates the melatonin-mediated cardioprotection. These results show that the negative effects of renal IRI on myocardial viability and cardiac function are caused by induced IP3R phosphorylation, MCU upregulation, and calcium overload. Melatonin protects cardiac function against CRS-3 by suppressing IP3R-MCU signaling.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Síndrome Cardiorrenal/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Melatonina/farmacologia , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Síndrome Cardiorrenal/tratamento farmacológico , Síndrome Cardiorrenal/patologia , Células Cultivadas , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Masculino , Melatonina/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/antagonistas & inibidores , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
10.
Eur J Pharmacol ; 874: 173029, 2020 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-32084419

RESUMO

Central post-stroke pain (CPSP) is a type of neuropathic pain for which the mechanism and relevant drug pathways remain unknown. Recently, it was reported that intracerebroventricular (ICV) administration of orexin-A suppresses pain and ischemia. In this study, we tested the role of orexin-A in CPSP induction in mice. Male ddY mice were subjected to 30 min of bilateral carotid artery occlusion (BCAO). CPSP was assessed by von Frey test. Colocalization of orexin 1 receptor (OX1R) with various neuron markers were determined by double-immunofluorescence. The hindpaw withdrawal responses to mechanical stimuli were significantly increased 3 days post-BCAO compared with those of sham groups. ICV injection of orexin-A dose-dependently suppressed BCAO-induced mechanical allodynia. These effects were inhibited by pre-treatment with SB334867 (an OX1R antagonist; ICV injection), yohimbine (a noradrenaline α2 receptor antagonist; intrathecal (IT) injection), and WAY100635 (a serotonin 5-HT1A receptor antagonist; IT injection), but not TCS OX2 29 (an OX2R antagonist; ICV injection). OX1R colocalized with TH (a noradrenergic neuron marker) and TPH (a serotonergic neuron marker) in the locus ceruleus (LC) and the rostral ventromedial medulla (RVM), respectively. The number of c-Fos positive cells in the LC and the RVM of BCAO mice was increased at 90 min after ICV injection of orexin-A compared to saline group. These results indicate that orexin-A/OX1R signaling plays an important role through activation of the descending pain control system in the induction of CPSP in mice.


Assuntos
Isquemia Encefálica/metabolismo , Hiperalgesia/metabolismo , Proteínas Mitocondriais/metabolismo , Neuralgia/metabolismo , Antagonistas de Receptores Adrenérgicos alfa 2/farmacologia , Animais , Benzoxazóis/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Isoquinolinas/farmacologia , Masculino , Camundongos , Proteínas Mitocondriais/antagonistas & inibidores , Naftiridinas/farmacologia , Antagonistas dos Receptores de Orexina/farmacologia , Receptores de Orexina/metabolismo , Orexinas/farmacologia , Piperazinas/farmacologia , Piridinas/farmacologia , Antagonistas da Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ureia/análogos & derivados , Ureia/farmacologia , Ioimbina/farmacologia
11.
J Biol Chem ; 295(51): 17684-17697, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33454007

RESUMO

Tuberculosis (TB), caused by the infection of Mycobacterium tuberculosis (MTB), is one of the leading causes of death worldwide, especially in children. However, the mechanisms by which MTB infects its cellular host, activates an immune response, and triggers inflammation remain unknown. Mitochondria play important roles in the initiation and activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, where mitochondria-associated endoplasmic reticulum membranes (MAMs) may serve as the platform for inflammasome assembly and activation. Additionally, mitofusin 2 (MFN2) is implicated in the formation of MAMs, but, the roles of mitochondria and MFN2 in MTB infection have not been elucidated. Using mircroarry profiling of TB patients and in vitro MTB stimulation of macrophages, we observed an up-regulation of MFN2 in the peripheral blood mononuclear cells of active TB patients. Furthermore, we found that MTB stimulation by MTB-specific antigen ESAT-6 or lysate of MTB promoted MFN2 interaction with NLRP3 inflammasomes, resulting in the assembly and activation of the inflammasome and, subsequently, IL-1ß secretion. These findings suggest that MFN2 and mitochondria play important role in the pathogen-host interaction during MTB infection.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Inflamassomos/metabolismo , Proteínas Mitocondriais/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Tuberculose/patologia , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Estudos de Casos e Controles , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/genética , Interações Hospedeiro-Patógeno , Humanos , Interleucina-1beta/metabolismo , Leucócitos Mononucleares/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Mycobacterium tuberculosis/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Tuberculose/metabolismo , Regulação para Cima
12.
Dokl Biochem Biophys ; 488(1): 342-345, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31768856

RESUMO

The antioxidant and antiradical properties of the tetra nitrosyl iron complex with thiosulfate ligands (TNIC) were studied in vitro in mouse brain homogenates. It was found for the first time that TNIC is an effective antioxidant. The effect of TNIC on the catalytic activity of mitochondrial enzymes cytochrome c oxidase and monoamine oxidase A was studied. It was shown for the first time that TNIC is an inhibitor of the catalytic activity of cytochrome c oxidase and monoamine oxidase A in animal brain mitochondria in vitro.


Assuntos
Encéfalo/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons , Ferro , Mitocôndrias/enzimologia , Proteínas Mitocondriais , Inibidores da Monoaminoxidase , Óxidos de Nitrogênio , Tiossulfatos , Animais , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ferro/química , Ferro/farmacologia , Camundongos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/metabolismo , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/síntese química , Inibidores da Monoaminoxidase/química , Inibidores da Monoaminoxidase/farmacologia , Óxidos de Nitrogênio/síntese química , Óxidos de Nitrogênio/química , Óxidos de Nitrogênio/farmacologia , Tiossulfatos/síntese química , Tiossulfatos/química , Tiossulfatos/farmacologia
13.
ChemMedChem ; 14(23): 1951-1962, 2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31692274

RESUMO

Inhibitors of apoptosis proteins (IAPs) inhibit caspase activity, allowing various cancers to reduce programmed cell death (apoptosis) and resist drug treatment. The second mitochondrial-derived activator of caspases (SMAC) protein is an endogenous IAP antagonist, which can be considered as a potential anticancer therapy. Small-molecule SMAC mimetics based on the Ala-Val-Pro-Ile motif have been validated as potent IAP antagonists. In particular, most bivalent SMAC mimetics, which target both the baculovirus IAP repeat 2 (BIR2) and BIR3 domains in X-linked IAP (XIAP), antagonize IAPs better than the corresponding monovalent mimetics. Here we focus on strategies for designing bivalent small-molecule SMAC mimetics and progress in using them to antagonize IAPs. We also consider their clinical potential. Our discussion will hopefully help guide further study of these interesting mimetics.


Assuntos
Antineoplásicos/metabolismo , Materiais Biomiméticos/química , Inibidores de Caspase/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Sequência de Aminoácidos , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Apoptose , Inibidores de Caspase/administração & dosagem , Inibidores de Caspase/química , Caspases/metabolismo , Desenho de Fármacos , Humanos , Proteínas Inibidoras de Apoptose/administração & dosagem , Proteínas Inibidoras de Apoptose/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Conformação Molecular , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Engenharia de Proteínas , Multimerização Proteica , Transdução de Sinais , Relação Estrutura-Atividade , Resultado do Tratamento
15.
Cell Commun Signal ; 17(1): 150, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31744505
16.
Cancer Res ; 79(24): 6215-6226, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31582380

RESUMO

The regulators of mitochondrial cell death in cancer have remained elusive, hampering the development of new therapies. Here, we showed that protein isoforms of mitochondrial fission factor (MFF1 and MFF2), a molecule that controls mitochondrial size and shape, that is, mitochondrial dynamics, were overexpressed in patients with non-small cell lung cancer and formed homo- and heterodimeric complexes with the voltage-dependent anion channel-1 (VDAC1), a key regulator of mitochondrial outer membrane permeability. MFF inserted into the interior hole of the VDAC1 ring using Arg225, Arg236, and Gln241 as key contact sites. A cell-permeable MFF Ser223-Leu243 d-enantiomeric peptidomimetic disrupted the MFF-VDAC1 complex, acutely depolarized mitochondria, and triggered cell death in heterogeneous tumor types, including drug-resistant melanoma, but had no effect on normal cells. In preclinical models, treatment with the MFF peptidomimetic was well-tolerated and demonstrated anticancer activity in patient-derived xenografts, primary breast and lung adenocarcinoma 3D organoids, and glioblastoma neurospheres. These data identify the MFF-VDAC1 complex as a novel regulator of mitochondrial cell death and an actionable therapeutic target in cancer. SIGNIFICANCE: These findings describe mitochondrial fission regulation using a peptidomimetic agent that disturbs the MFF-VDAC complex and displays anticancer activity in multiple tumor models.See related commentary by Rao, p. 6074.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Proteínas de Membrana/metabolismo , Mitocôndrias/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/patologia , Proteínas Mitocondriais/antagonistas & inibidores , Permeabilidade/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Canal de Ânion 1 Dependente de Voltagem/antagonistas & inibidores , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Cell Metab ; 30(6): 1131-1140.e7, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31564441

RESUMO

The identification of molecular targets and pharmacodynamic markers for Parkinson's disease (PD) will empower more effective clinical management and experimental therapies. Miro1 is localized on the mitochondrial surface and mediates mitochondrial motility. Miro1 is removed from depolarized mitochondria to facilitate their clearance via mitophagy. Here, we explore the clinical utility of Miro1 for detecting PD and for gauging potential treatments. We measure the Miro1 response to mitochondrial depolarization using biochemical assays in skin fibroblasts from a broad spectrum of PD patients and discover that more than 94% of the patients' fibroblast cell lines fail to remove Miro1 following depolarization. We identify a small molecule that can repair this defect of Miro1 in PD fibroblasts. Treating patient-derived neurons and fly models with this compound rescues the locomotor deficits and dopaminergic neurodegeneration. Our results indicate that tracking this Miro1 marker and engaging in Miro1-based therapies could open new avenues to personalized medicine.


Assuntos
Antiparkinsonianos/farmacologia , Proteínas de Drosophila , Proteínas Mitocondriais , Degeneração Neural/tratamento farmacológico , Neurônios/efeitos dos fármacos , Doença de Parkinson , Proteínas rho de Ligação ao GTP , Adulto , Idoso , Animais , Antiparkinsonianos/uso terapêutico , Biomarcadores/metabolismo , Drosophila , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Feminino , Fibroblastos , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Pessoa de Meia-Idade , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/metabolismo , Neurônios/patologia , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/metabolismo
18.
Sci Adv ; 5(9): eaax1978, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31535025

RESUMO

How lipid metabolism is regulated at the outer mitochondrial membrane (OMM) for transducing stress signaling remains largely unknown. We show here that this process is controlled by trafficking of ceramide synthase 1 (CerS1) from the endoplasmic reticulum (ER) to the OMM by a previously uncharacterized p17, which is now renamed protein that mediates ER-mitochondria trafficking (PERMIT). Data revealed that p17/PERMIT associates with newly translated CerS1 on the ER surface to mediate its trafficking to the OMM. Cellular stress induces Drp1 nitrosylation/activation, releasing p17/PERMIT to retrieve CerS1 for its OMM trafficking, resulting in mitochondrial ceramide generation, mitophagy and cell death. In vivo, CRISPR-Cas9-dependent genetic ablation of p17/PERMIT prevents acute stress-mediated CerS1 trafficking to OMM, attenuating mitophagy in p17/PERMIT-/- mice, compared to controls, in various metabolically active tissues, including brain, muscle, and pancreas. Thus, these data have implications in diseases associated with accumulation of damaged mitochondria such as cancer and/or neurodegeneration.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/fisiologia , Mitocôndrias/patologia , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/fisiologia , Mitofagia , Esfingosina N-Aciltransferase/fisiologia , Estresse Fisiológico , Animais , Sistemas CRISPR-Cas , Ceramidas/metabolismo , Retículo Endoplasmático/patologia , Humanos , Metabolismo dos Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Transporte Proteico
19.
FASEB J ; 33(11): 13002-13013, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31525300

RESUMO

African trypanosomiasis, sleeping sickness in humans or nagana in animals, is a potentially fatal neglected tropical disease and a threat to 65 million human lives and 100 million small and large livestock animals in sub-Saharan Africa. Available treatments for this devastating disease are few and have limited efficacy, prompting the search for new drug candidates. Simultaneous inhibition of the trypanosomal glycerol kinase (TGK) and trypanosomal alternative oxidase (TAO) is considered a validated strategy toward the development of new drugs. Our goal is to develop a TGK-specific inhibitor for coadministration with ascofuranone (AF), the most potent TAO inhibitor. Here, we report on the identification of novel compounds with inhibitory potency against TGK. Importantly, one of these compounds (compound 17) and its derivatives (17a and 17b) killed trypanosomes even in the absence of AF. Inhibition kinetics revealed that derivative 17b is a mixed-type and competitive inhibitor for TGK and TAO, respectively. Structural data revealed the molecular basis of this dual inhibitory action, which, in our opinion, will aid in the successful development of a promising drug to treat trypanosomiasis. Although the EC50 of compound 17b against trypanosome cells was 1.77 µM, it had no effect on cultured human cells, even at 50 µM.-Balogun, E. O., Inaoka, D. K., Shiba, T., Tsuge, C., May, B., Sato, T., Kido, Y., Nara, T., Aoki, T., Honma, T., Tanaka, A., Inoue, M., Matsuoka, S., Michels, P. A. M., Watanabe, Y.-I., Moore, A. L., Harada, S., Kita, K. Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei.


Assuntos
Cumarínicos/farmacologia , Descoberta de Drogas , Glicerol Quinase/antagonistas & inibidores , Proteínas Mitocondriais/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Proteínas de Plantas/antagonistas & inibidores , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Animais , Cumarínicos/química , Glicerol Quinase/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Trypanosoma brucei brucei/enzimologia
20.
PLoS Pathog ; 15(9): e1008065, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31557263

RESUMO

Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27°C and are unable to survive prolonged exposure to 37°C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes.


Assuntos
Proteínas de Protozoários/metabolismo , Tiorredoxinas/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Técnicas de Silenciamento de Genes , Genes de Protozoários , Humanos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Oxirredução , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/patogenicidade
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