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1.
Eur J Med Chem ; 182: 111632, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31499363

RESUMO

ß-amino acids and their analogues are gathering increased attention not only because of their antibacterial and antifungal activity, but also for their use in designing peptidomimetics with increased oral bioavailability and resistance to metabolic degradation. In this study, a series of α-phenyl substituted chalcones, α-phenyl, ß-amino substituted dihydrochalcones and ß-amino acid derivatives were synthesized and evaluated for their antileishmanial efficacy against experimental visceral leishmaniasis (VL). Among all synthesized derivatives, 10c showed promising antileishmanial efficacy against both extracellular promastigote and intracellular amastigote (IC50 8.2 µM and 20.5 µM respectively) of L. donovani with negligible cytotoxic effect towards J774 macrophages and Vero cells. 10c effectively reduced spleen and liver parasite burden (>90%) in both hamster and Balb/c model of VL without any hepatotoxicity. In vitro pharmacokinetic analysis showed that 10c was stable in gastric fluid and plasma of Balb/c mice at 10 µg/ml. Further analysis of the molecular mechanism revealed that 10c entered into the parasite by depolarizing the plasma membrane rather than forming nonspecific pores and induced molecular events like loss in mitochondrial membrane potential with a gradual decline in ATP production. This, in turn, did not induce programmed cell death of the parasite; rather 10c induced bioenergetic collapse of the parasite by decreasing ATP synthesis through specific inhibition of mitochondrial complex III activity. Altogether, our results allude to the therapeutic potential of ß-amino acid derivatives as novel antileishmanials, identifying them as lead compounds for further exploration in the design of potent candidates for the treatment of visceral leishmaniasis.


Assuntos
Aminoácidos/farmacologia , Antiprotozoários/farmacologia , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Aminoácidos/química , Animais , Antiprotozoários/química , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Relação Dose-Resposta a Droga , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Leishmania donovani/metabolismo , Leishmaniose Visceral/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Células Vero
2.
Molecules ; 24(16)2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426298

RESUMO

Amongst the various approaches to contain aflatoxin contamination of feed and food commodities, the use of inhibitors of fungal growth and/or toxin biosynthesis is showing great promise for the implementation or the replacement of conventional pesticide-based strategies. Several inhibition mechanisms were found taking place at different levels in the biology of the aflatoxin-producing fungal species such as Aspergillus flavus: compounds that influence aflatoxin production may block the biosynthetic pathway through the direct control of genes belonging to the aflatoxin gene cluster, or interfere with one or more of the several steps involved in the aflatoxin metabolism upstream. Recent findings pointed to mitochondrial functionality as one of the potential targets of some aflatoxin inhibitors. Additionally, we have recently reported that the effect of a compound belonging to the class of thiosemicarbazones might be related to the energy generation/carbon flow and redox homeostasis control by the fungal cell. Here, we report our investigation about a putative molecular target of the 3-isopropylbenzaldehyde thiosemicarbazone (mHtcum), using the yeast Saccharomyces cerevisiae as model system, to demonstrate how the compound can actually interfere with the mitochondrial respiratory chain.


Assuntos
Aflatoxinas/antagonistas & inibidores , Antifúngicos/farmacologia , Regulação Fúngica da Expressão Gênica , Mitocôndrias/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Tiossemicarbazonas/farmacologia , Aflatoxinas/biossíntese , Antifúngicos/química , Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/enzimologia , Aspergillus flavus/genética , Sítios de Ligação , Transporte de Elétrons/efeitos dos fármacos , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Simulação de Acoplamento Molecular , Família Multigênica , Ligação Proteica , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Tiossemicarbazonas/química
3.
Med Sci Monit ; 25: 4982-4991, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31276465

RESUMO

BACKGROUND Osteosarcoma (OS) is a common primary malignant bone tumor for which the molecular mechanisms remain unclear. Studies on coding and non-coding RNAs are needed to determine the molecular mechanism. MATERIAL AND METHODS To explore the potential roles of miRNAs and mRNA in OS, we determined the miRNA and mRNA expression profile of 3 pairs of OS and paracancerous tissues from patients with OS by sequencing and bioinformatics analysis. The expression levels of critical miRNAs and mRNAs were verified in 10 pairs of OS and paracancerous tissues. An miRNA inhibitor and mimics were used to investigate the interactions between miRNAs and target genes. The cell counting kit-8 assay was performed to evaluate OS cell proliferation after miRNA interference. RESULTS A total of 184 miRNAs and 2501 mRNAs were identified (fold-change >2.0 or <2.0, P<0.05), with up-regulation of 82 miRNAs and 1320 mRNAs and down-regulation of 102 miRNAs and 1181 mRNAs in OS tissue. The protein protein interaction network revealed that UQCRC1 (ubiquinol-cytochrome c reductase core protein 1) is a critical gene and a potential target gene of miR-214-3p. Both UQCRC1 and miR-214-3p were significantly differentially expressed in OS tissue and cell lines (down and up-regulated, respectively). Down-regulated miR-214-3p expression increased UQCRC1 expression and suppressed OS cell proliferation. In contrast, overexpression of miR-214-3p decreased UQCRC1 expression and promoted OS cell proliferation. CONCLUSIONS High miR-214-3p expression may promote OS cell proliferation by targeting UQCRC1, providing insight into a potential therapeutic target for preventing and treating OS.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Osteossarcoma/genética , Osteossarcoma/patologia , Análise de Sequência de RNA , Adolescente , Adulto , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/genética , Criança , Regulação para Baixo/genética , Feminino , Ontologia Genética , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Mapas de Interação de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/genética , Adulto Jovem
4.
Biochim Biophys Acta Bioenerg ; 1860(8): 618-627, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31251900

RESUMO

Ustilago maydis is an aerobic basidiomycete that depends on oxidative phosphorylation for its ATP supply, pointing to the mitochondrion as a key player in its energy metabolism. Mitochondrial respiratory complexes I, III2, and IV occur in supramolecular structures named respirasome. In this work, we characterized the subunit composition and the kinetics of NADH:Q oxidoreductase activity of the digitonine-solubilized respirasome (1600 kDa) and the free-complex I (990 kDa). In the presence of 2,6-dimethoxy-1,4-benzoquinone (DBQ) and cytochrome c, both the respirasome NADH:O2 and the NADH:DBQ oxidoreductase activities were inhibited by rotenone, antimycin A or cyanide. A value of 2.4 for the NADH oxidized/oxygen reduced ratio was determined for the respirasome activity, while ROS production was less than 0.001% of the oxygen consumption rate. Analysis of the NADH:DBQ oxidoreductase activity showed that respirasome was 3-times more active and showed higher affinity than free-complex I. The results suggest that the contacts between complexes I, III2 and IV in the respirasome increase the catalytic efficiency of complex I and regulate its activity to prevent ROS production.


Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Mitocôndrias/enzimologia , NADH Desidrogenase/metabolismo , Ustilago/enzimologia , Basidiomycota , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Oxirredução , Fosforilação Oxidativa , Espécies Reativas de Oxigênio/antagonistas & inibidores , Ustilago/metabolismo
5.
G3 (Bethesda) ; 9(7): 2225-2234, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31076384

RESUMO

Drosophila melanogaster, like most animal species, displays considerable genetic variation in both nuclear and mitochondrial DNA (mtDNA). Here we tested whether any of four natural mtDNA variants was able to modify the effect of the phenotypically mild, nuclear tko25t mutation, affecting mitochondrial protein synthesis. When combined with tko25t , the mtDNA from wild strain KSA2 produced pupal lethality, accompanied by the presence of melanotic nodules in L3 larvae. KSA2 mtDNA, which carries a substitution at a conserved residue of cytochrome b that is predicted to be involved in subunit interactions within respiratory complex III, conferred drastically decreased respiratory capacity and complex III activity in the tko25t but not a wild-type nuclear background. The complex III inhibitor antimycin A was able to phenocopy effects of the tko25t mutation in the KSA2 mtDNA background. This is the first report of a lethal, nuclear-mitochondrial interaction within a metazoan species, representing a paradigm for understanding genetic interactions between nuclear and mitochondrial genotype relevant to human health and disease.


Assuntos
Núcleo Celular/genética , Drosophila melanogaster/genética , Genótipo , Mitocôndrias/genética , Mutações Sintéticas Letais/genética , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , DNA Mitocondrial , Drosophila melanogaster/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Dosagem de Genes , Regulação Enzimológica da Expressão Gênica , Mitocôndrias/metabolismo , Modelos Moleculares , Mutação , Fosforilação Oxidativa , Fenótipo , Conformação Proteica , Relação Estrutura-Atividade
6.
Nat Commun ; 10(1): 1432, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926815

RESUMO

Of the four separate PE biosynthetic pathways in eukaryotes, one occurs in the mitochondrial inner membrane (IM) and is executed by phosphatidylserine decarboxylase (Psd1). Deletion of Psd1 is lethal in mice and compromises mitochondrial function. We hypothesize that this reflects inefficient import of non-mitochondrial PE into the IM. Here, we test this by re-wiring PE metabolism in yeast by re-directing Psd1 to the outer mitochondrial membrane or the endomembrane system and show that PE can cross the IMS in both directions. Nonetheless, PE synthesis in the IM is critical for cytochrome bc1 complex (III) function and mutations predicted to disrupt a conserved PE-binding site in the complex III subunit, Qcr7, impair complex III activity similar to PSD1 deletion. Collectively, these data challenge the current dogma of PE trafficking and demonstrate that PE made in the IM by Psd1 support the intrinsic functionality of complex III.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Membranas Mitocondriais/metabolismo , Fosfatidiletanolaminas/metabolismo , Saccharomyces cerevisiae/metabolismo , Aerobiose , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Membranas Mitocondriais/ultraestrutura , Mutação/genética , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
Redox Biol ; 22: 101152, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30825773

RESUMO

Mitochondria have originated in eukaryotic cells by endosymbiosis of a specialized prokaryote approximately 2 billion years ago. They are essential for normal cell function by providing energy through their role in oxidizing carbon substrates. Glutathione (GSH) is a major thiol-disulfide redox buffer of the cell including the mitochondrial matrix and intermembrane space. We have generated cardiomyocyte-specific Grx1-roGFP2 GSH redox potential (EGSH) biosensor mice in the past, in which the sensor is targeted to the mitochondrial matrix. Using this mouse model a distinct EGSH of the mitochondrial matrix (-278.9 ±â€¯0.4 mV) in isolated cardiomyocytes is observed. When analyzing the EGSH in isolated mitochondria from the transgenic hearts, however, the EGSH in the mitochondrial matrix is significantly oxidized (-247.7 ±â€¯8.7 mV). This is prevented by adding N-Ethylmaleimide during the mitochondria isolation procedure, which precludes disulfide bond formation. A similar reducing effect is observed by isolating mitochondria in hypoxic (0.1-3% O2) conditions that mimics mitochondrial pO2 levels in cellulo. The reduced EGSH is accompanied by lower ROS production, reduced complex III activity but increased ATP levels produced at baseline and after stimulation with succinate/ADP. Altogether, we demonstrate that oxygenation is an essential factor that needs to be considered when analyzing mitochondrial function ex vivo.


Assuntos
Mitocôndrias/metabolismo , Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Peróxido de Hidrogênio/metabolismo , Hiperóxia/metabolismo , Camundongos , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Oxirredução , Superóxidos/metabolismo
8.
Nat Immunol ; 20(4): 420-432, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30858618

RESUMO

The adoption of Warburg metabolism is critical for the activation of macrophages in response to lipopolysaccharide. Macrophages stimulated with lipopolysaccharide increase their expression of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in NAD+ salvage, and loss of NAMPT activity alters their inflammatory potential. However, the events that lead to the cells' becoming dependent on NAD+ salvage remain poorly defined. We found that depletion of NAD+ and increased expression of NAMPT occurred rapidly after inflammatory activation and coincided with DNA damage caused by reactive oxygen species (ROS). ROS produced by complex III of the mitochondrial electron-transport chain were required for macrophage activation. DNA damage was associated with activation of poly(ADP-ribose) polymerase, which led to consumption of NAD+. In this setting, increased NAMPT expression allowed the maintenance of NAD+ pools sufficient for glyceraldehyde-3-phosphate dehydrogenase activity and Warburg metabolism. Our findings provide an integrated explanation for the dependence of inflammatory macrophages on the NAD+ salvage pathway.


Assuntos
Dano ao DNA , Macrófagos/metabolismo , NAD/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acrilamidas/farmacologia , Animais , Células Cultivadas , Citocinas/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Células HEK293 , Humanos , Inflamação/metabolismo , Ativação de Macrófagos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Piperidinas/farmacologia
9.
Arch Biochem Biophys ; 664: 68-75, 2019 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-30710505

RESUMO

Rieske iron-sulfur protein (RISP) is a catalytic subunit of the complex III in the mitochondrial electron transport chain. Studies for years have revealed that RISP is essential for the generation of intracellular reactive oxygen species (ROS) via delicate signaling pathways associated with many important molecules such as protein kinase C-ε, NADPH oxidase, and ryanodine receptors. More significantly, mitochondrial RISP-mediated ROS production has been implicated in the development of hypoxic pulmonary vasoconstriction, leading to pulmonary hypertension, right heart failure, and death. Investigations have also shown the involvement of RISP in ROS-dependent cardiac ischemic/reperfusion injuries. Further research may provide novel and valuable information that can not only enhance our understanding of the functional roles of RISP and the underlying molecular mechanisms in the pulmonary vasculature and other systems, but also elucidate whether RISP targeting can act as preventative and restorative therapies against pulmonary hypertension, cardiac diseases, and other disorders.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Mitocôndrias/metabolismo , Músculo Liso Vascular/patologia , Artéria Pulmonar/patologia , Transdução de Sinais , Animais , Humanos
10.
Nature ; 565(7740): 495-499, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30626970

RESUMO

Regulatory T cells (Treg cells), a distinct subset of CD4+ T cells, are necessary for the maintenance of immune self-tolerance and homeostasis1,2. Recent studies have demonstrated that Treg cells exhibit a unique metabolic profile, characterized by an increase in mitochondrial metabolism relative to other CD4+ effector subsets3,4. Furthermore, the Treg cell lineage-defining transcription factor, Foxp3, has been shown to promote respiration5,6; however, it remains unknown whether the mitochondrial respiratory chain is required for the T cell-suppression capacity, stability and survival of Treg cells. Here we report that Treg cell-specific ablation of mitochondrial respiratory chain complex III in mice results in the development of fatal inflammatory disease early in life, without affecting Treg cell number. Mice that lack mitochondrial complex III specifically in Treg cells displayed a loss of T cell-suppression capacity without altering Treg cell proliferation and survival. Treg cells deficient in complex III showed decreased expression of genes associated with Treg function, whereas Foxp3 expression remained stable. Loss of complex III in Treg cells increased DNA methylation as well as the metabolites 2-hydroxyglutarate (2-HG) and succinate that inhibit the ten-eleven translocation (TET) family of DNA demethylases7. Thus, Treg cells require mitochondrial complex III to maintain immune regulatory gene expression and suppressive function.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Tolerância a Antígenos Próprios/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Desmetilação do DNA , Metilação de DNA , Transporte de Elétrons , Feminino , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Glutaratos/metabolismo , Inflamação/genética , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Tolerância a Antígenos Próprios/genética , Ácido Succínico/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/enzimologia
12.
EBioMedicine ; 40: 92-105, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30674441

RESUMO

BACKGROUND: Ubiquinol-cytochrome C reductase core protein II (QCR2) is essential for mitochondrial functions, yet, its role in cancer development has remained elusive. METHODS: The expression of QCR2 in cancer patients was assessed by immunohistochemistry. The proliferation of cancer cells was assessed by CCK-8 assay, EdU staining and Flow cytometry analysis. The biological function of QCR2 and PHB were determined using western blotting, RT-qPCR, microarray analysis and xenografts. The interactions between proteins and the ubiquitination of p53 were assessed by immunoprecipitation, mass spectrometry analysis and GST pull down. The subcellular location of PHB and QCR2 was assessed by immunoblotting and immunofluorescence. FINDING: The expression of QCR2 is upregulated in multiple human tumors. Suppression of QCR2 inhibits cancer cell growth by activating p53 signaling and inducing p21-dependent cell cycle arrest and senescence. QCR2 directly interacts with PHB in the mitochondria. Overexpression of QCR2 inhibits PHB binding to p53 in the nucleus, and facilitates p53 ubiquitination and degradation, consequently leading to tumorigenesis. Also, increased QCR2 and decreased PHB protein levels are well correlated with decreased expression of p21 in cervical cancer tissues. INTERPRETATION: These results identify a novel role for QCR2, together with PHB, in negative regulation of p53 stability and activity, thus promote cervical carcinogenesis. FUND: "973" Program of China, the National Science-technology Supporting Plan Projects, the National Natural Science Foundation of China, National Science and Technology Major Sub-Project and Technical Innovation Special Project of Hubei Province.


Assuntos
Transformação Celular Neoplásica/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Senescência Celular/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Imuno-Histoquímica , Camundongos , Modelos Biológicos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Proteólise , Ubiquitinação
13.
Cell Mol Life Sci ; 76(7): 1381-1396, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30666338

RESUMO

Ubiquinol cytochrome c reductase core protein I (UQCRC1) is a component of the complex III in the respiratory chain. Its biological functions are unknown. Here, we showed that knockout of UQCRC1 led to embryonic lethality. Disrupting one UQCRC1 allele in mice (heterozygous mice) of both sexes did not affect their growth but reduced UQCRC1 mRNA and protein in the brain. These mice had decreased complex III formation, complex III activity and ATP content in the brain at baseline. They developed worsened neurological outcome after brain ischemia/hypoxia or focal brain ischemia compared with wild-type mice. The ischemic cerebral cortex of the heterozygous mice had decreased mitochondrial membrane potential and ATP content as well as increased free radicals. Also, the heterozygous mice performed poorly in the Barnes maze and novel object recognition tests. Finally, UQCRC1 was expressed abundantly in neurons and astrocytes. These results suggest a critical role of UQCRC1 in embryo survival. UQCRC1 may also be important by forming the complex III to maintain normal brain ischemic tolerance, learning and memory.


Assuntos
Isquemia Encefálica/patologia , Cognição/fisiologia , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Astrócitos/metabolismo , Isquemia Encefálica/metabolismo , Resistência à Doença , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/genética , Embrião de Mamíferos/metabolismo , Feminino , Masculino , Aprendizagem em Labirinto , Potencial da Membrana Mitocondrial , Memória/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Espécies Reativas de Oxigênio/metabolismo
14.
Nat Struct Mol Biol ; 26(1): 78-83, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30598554

RESUMO

Cytochrome c oxidase (complex IV, CIV) is known in mammals to exist independently or in association with other respiratory proteins to form supercomplexes (SCs). In Saccharomyces cerevisiae, CIV is found solely in an SC with cytochrome bc1 (complex III, CIII). Here, we present the cryogenic electron microscopy (cryo-EM) structure of S. cerevisiae CIV in a III2IV2 SC at 3.3 Å resolution. While overall similarity to mammalian homologs is high, we found notable differences in the supernumerary subunits Cox26 and Cox13; the latter exhibits a unique arrangement that precludes CIV dimerization as seen in bovine. A conformational shift in the matrix domain of Cox5A-involved in allosteric inhibition by ATP-may arise from its association with CIII. The CIII-CIV arrangement highlights a conserved interaction interface of CIII, albeit one occupied by complex I in mammalian respirasomes. We discuss our findings in the context of the potential impact of SC formation on CIV regulation.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Membranas Mitocondriais , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética
15.
Int J Mol Sci ; 20(1)2019 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-30621095

RESUMO

Cancer stem cells (CSCs) have been shown as a distinct population of cancer cells strongly implicated with resistance to conventional chemotherapy. Metformin, the most widely prescribed drug for diabetes, was reported to target cancer stem cells in various cancers. In this study, we sought to determine the effects of metformin on head and neck squamous cell carcinoma (HNSCC). CSCs and non-stem HNSCC cells were treated with metformin and cisplatin alone, and in combination, and cell proliferation levels were measured through MTS assays. Next, potential targets of metformin were explored through computational small molecule binding analysis. In contrast to the reported effects of metformin on CSCs in other cancers, our data suggests that metformin protects HNSCC CSCs against cisplatin in vitro. Treatment with metformin resulted in a dose-dependent induction of the stem cell genes CD44, BMI-1, OCT-4, and NANOG. On the other hand, we observed that metformin successfully decreased the proliferation of non-stem HNSCC cells. Computational drug⁻protein interaction analysis revealed mitochondrial complex III to be a likely target of metformin. Based on our results, we present the novel hypothesis that metformin targets complex III to reduce reactive oxygen species (ROS) levels, leading to the differential effects observed on non-stem cancer cells and CSCs.


Assuntos
Citoproteção/efeitos dos fármacos , Metformina/farmacologia , Células-Tronco Neoplásicas/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Biomarcadores Tumorais/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Subunidades Proteicas/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética
16.
Methods Mol Biol ; 1880: 655-668, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610729

RESUMO

Mitochondrial selective autophagy (mitophagy) is a critical cellular process for mitochondrial homeostasis and survival both under basal and stress conditions. Distinct cell types display different requirements for mitochondrial turnover depending on their metabolic status, differentiation state, and environmental cues. This points to the necessity of developing novel tools for real-time, tissue-specific assessment of mitophagy. Caenorhabditis elegans is an invaluable model organism for this kind of analysis providing a platform for simultaneous monitoring of mitophagy in vivo in different tissues and cell types, during development, stress conditions, and/or throughout life span. In this chapter we describe three versatile, noninvasive methods, developed for monitoring in vivo early and late mitophagic events in body wall muscles and neuronal cells of C. elegans. These procedures can be readily used and/or provide insights into the generation of novel imaging methods to investigate further the role of mitophagy at the organismal level under normal and pathological conditions.


Assuntos
Bioensaio/métodos , Microscopia Intravital/métodos , /fisiologia , Animais , Animais Geneticamente Modificados , Bioensaio/instrumentação , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Genes Reporter/genética , Processamento de Imagem Assistida por Computador/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Microscopia Intravital/instrumentação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Modelos Animais , Músculos/citologia , Músculos/metabolismo , Neurônios/metabolismo
17.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1298-1312, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30690068

RESUMO

In humans, low brown adipose tissue (BAT) mass and activity have been associated with increased adiposity and fasting glucose levels, suggesting that defective BAT-dependent thermogenesis could contribute to the development of obesity and/or type 2 diabetes. The thermogenic function of BAT relies on a vast network of mitochondria exclusively equipped with UCP1. Mitochondrial biogenesis is exquisitely regulated by a well-defined network of transcription factors that coordinate the expression of nuclear genes required for the formation of functional mitochondria. However, less is known about the mitochondrial factors that control the expression of the genes encoded by the mitochondrial genome. Here, we have studied the role of mitochondrial transcription termination factor-4 (MTERF4) in BAT by using a new mouse model devoid of MTERF4 specifically in adipocytes (MTERF4-FAT-KO mice). Lack of MTERF4 in BAT leads to reduced OxPhos mitochondrial protein levels and impaired assembly of OxPhos complexes I, III and IV due to deficient translation of mtDNA-encoded proteins. As a result, brown adipocytes lacking MTERF4 exhibit impaired respiratory capacity. MTERF4-FAT-KO mice show a blunted thermogenic response and are unable to maintain body temperature when exposed to cold. Despite impaired BAT function, MTERF4-FAT-KO mice do not develop obesity or insulin resistance. Still, MTERF4-FAT-KO mice became resistant to the insulin-sensitizing effects of ß3-specific adrenergic receptor agonists. Our results demonstrate that MTERF4 regulates mitochondrial protein translation and is essential for proper BAT thermogenic activity. Our study also supports the notion that pharmacological activation of BAT is a plausible therapeutic target for the treatment of insulin resistance.


Assuntos
Tecido Adiposo Marrom/metabolismo , Glucose/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Termogênese/genética , Fatores de Transcrição/genética , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/patologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Temperatura Baixa , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica , Homeostase/genética , Humanos , Insulina/metabolismo , Insulina/farmacologia , Resistência à Insulina , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Proteínas Mitocondriais/deficiência , Biogênese de Organelas , Fosforilação Oxidativa/efeitos dos fármacos , Transdução de Sinais , Fatores de Transcrição/deficiência
18.
Neurotox Res ; 35(1): 1-18, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29127580

RESUMO

The sigma1 receptor (σ1R) is a chaperone protein residing at mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), where it modulates Ca2+ exchange between the ER and mitochondria by interacting with inositol-1,4,5 trisphosphate receptors (IP3Rs). The σ1R is highly expressed in the central nervous system and its activation stimulates neuromodulation and neuroprotection, for instance in Alzheimer's disease (AD) models in vitro and in vivo. σ1R effects on mitochondria pathophysiology and the downstream signaling are still not fully understood. We here evaluated the impacts of σ1R ligands in mouse mitochondria preparations on reactive oxygen species (ROS) production, mitochondrial respiration, and complex activities, in physiological condition and after direct application of amyloid Aß1-42 peptide. σ1R agonists (2-(4-morpholinethyl)-1-phenylcyclohexanecarboxylate hydrochloride (PRE-084), tetrahydro-N,N-dimethyl-5,5-diphenyl-3-furanmethanamine (ANAVEX1-41, AN1-41), (S)-1-(2,8-dimethyl-1-thia-3,8-diazaspiro[4.5]dec-3-yl)-3-(1H-indol-3-yl)propan-1-one (ANAVEX3-71, AN3-71), dehydroepiandrosterone-3 sulfate (DHEA), donepezil) increased mitochondrial ROS in a σ1R antagonist-sensitive manner but decreased Aß1-42-induced increase in ROS. σ1R ligands (agonists or antagonists) did not impact respiration but attenuated Aß1-42-induced alteration. σ1R agonists (PRE-084, AN1-41, tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine hydrochloride (ANAVEX2-73, AN2-73), AN3-71) increased complex I activity, in a Ca2+-dependent and σ1R antagonist-sensitive manner. σ1R ligands failed to affect complex II, III, and IV activities. The increase in complex I activity explain the σ1R-induced increase in ROS since ligands failed to affect other sources of ROS accumulation in mitochondria and homogenates, namely NADPH oxidase (NOX) and superoxide dismutase (SOD) activities. Furthermore, Aß1-42 significantly decreased the activity of complexes I and IV and σ1R agonists attenuated the Aß1-42-induced complex I and IV dysfunctions. σ1R activity in mitochondria therefore results in a Ying-Yang effect, by triggering moderate ROS increase acting as a physiological signal and promoting a marked anti-oxidant effect in pathological (Aß) conditions.


Assuntos
Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Receptores sigma/agonistas , Peptídeos beta-Amiloides/toxicidade , Animais , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Masculino , Camundongos , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Estresse Oxidativo/fisiologia , Fragmentos de Peptídeos/toxicidade , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores sigma/antagonistas & inibidores , Receptores sigma/metabolismo , Succinato Desidrogenase/metabolismo , Superóxido Dismutase/metabolismo
19.
FEBS Lett ; 593(1): 3-12, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30428128

RESUMO

Catalytic reactions of quinol oxidoreductases may lead to the generation of superoxide due to electron leaks from unstable semiquinone intermediates (SQ). For cytochrome bc1 , the mechanism of suppression of superoxide generation remains unknown. We analyzed conditions of formation of a spin-spin-coupled state between SQ and the Rieske cluster (SQ-FeS) associated with catalysis of the quinol oxidation site of cytochrome bc1 . We reveal that mutants that preclude direct interaction between SQ and the Rieske cluster do not form SQ-FeS and release enhanced superoxide. In the enzymes generating SQ-FeS, little or no superoxide is detected. We propose that SQ-FeS suppresses superoxide generation, becoming an element modulating superoxide release under physiologically relevant conditions slowing electron flow through the enzyme.


Assuntos
Benzoquinonas/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Rhodobacter capsulatus/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Complexo III da Cadeia de Transporte de Elétrons/genética , Mutação , Ligação Proteica , Rhodobacter capsulatus/química , Rhodobacter capsulatus/genética , Superóxidos/metabolismo
20.
Pest Manag Sci ; 75(4): 1181-1189, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30520257

RESUMO

BACKGROUND: Metyltetraprole is a new fungicide with a unique tetrazolinone-moiety and a similar side chain to a known quinone outside inhibitor (QoI), pyraclostrobin. In this study we describe a unique bioactivity of metyltetraprole on QoI-resistant strains of Zymoseptoria tritici and Pyrenophora teres. RESULTS: Metyltetraprole exhibited potent antifungal activity against Ascomycetes; it was especially effective against Z. tritici and P. teres in seedling pot tests. Metyltetraprole was also effective in field tests with QoI-resistant mutants. Antifungal activity tests using field strains of Z. tritici and P. teres showed that the performance of metyltetraprole was unaltered by QoI, succinate dehydrogenase inhibitor (SDHI), and sterol 14α-demethylation inhibitor (DMI) resistance. However, the mitochondrial activity test indicated that the compound inhibits the respiratory chain via complex III. CONCLUSION: Metyltetraprole is a novel fungicide that is highly effective against a wide range of fungal diseases, including important cereal diseases. Although metyltetraprole most likely inhibits the respiratory chain via complex III, it remains effective against QoI resistant strains. Therefore, metyltetraprole is considered as a novel fungicidal agent for controlling diseases affecting cereal crops and overcoming pathogen resistance to existing fungicides. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.


Assuntos
Ascomicetos/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Proteínas Fúngicas/genética , Fungicidas Industriais/farmacologia , Grão Comestível/microbiologia , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Fúngicas/metabolismo , Doenças das Plantas/prevenção & controle
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