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1.
Nat Commun ; 12(1): 120, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33402698

RESUMO

Mitochondrial ATP synthase plays a key role in inducing membrane curvature to establish cristae. In Apicomplexa causing diseases such as malaria and toxoplasmosis, an unusual cristae morphology has been observed, but its structural basis is unknown. Here, we report that the apicomplexan ATP synthase assembles into cyclic hexamers, essential to shape their distinct cristae. Cryo-EM was used to determine the structure of the hexamer, which is held together by interactions between parasite-specific subunits in the lumenal region. Overall, we identified 17 apicomplexan-specific subunits, and a minimal and nuclear-encoded subunit-a. The hexamer consists of three dimers with an extensive dimer interface that includes bound cardiolipins and the inhibitor IF1. Cryo-ET and subtomogram averaging revealed that hexamers arrange into ~20-megadalton pentagonal pyramids in the curved apical membrane regions. Knockout of the linker protein ATPTG11 resulted in the loss of pentagonal pyramids with concomitant aberrantly shaped cristae. Together, this demonstrates that the unique macromolecular arrangement is critical for the maintenance of cristae morphology in Apicomplexa.


Assuntos
Mitocôndrias/ultraestrutura , Membranas Mitocondriais/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/química , Subunidades Proteicas/química , Proteínas de Protozoários/química , Toxoplasma/ultraestrutura , Sítios de Ligação , Cardiolipinas/química , Cardiolipinas/metabolismo , Microscopia Crioeletrônica , Expressão Gênica , Mitocôndrias/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Especificidade por Substrato , Termodinâmica , Toxoplasma/genética , Toxoplasma/metabolismo
2.
Int J Mol Sci ; 22(3)2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33498298

RESUMO

Several genetic variants in the mitochondrial genome (mtDNA), including ancient polymorphisms, are associated with chronic inflammatory conditions, but investigating the functional consequences of such mtDNA polymorphisms in humans is challenging due to the influence of many other polymorphisms in both mtDNA and the nuclear genome (nDNA). Here, using the conplastic mouse strain B6-mtFVB, we show that in mice, a maternally inherited natural mutation (m.7778G > T) in the mitochondrially encoded gene ATP synthase 8 (mt-Atp8) of complex V impacts on the cellular metabolic profile and effector functions of CD4+ T cells and induces mild changes in oxidative phosphorylation (OXPHOS) complex activities. These changes culminated in significantly lower disease susceptibility in two models of inflammatory skin disease. Our findings provide experimental evidence that a natural variation in mtDNA influences chronic inflammatory conditions through alterations in cellular metabolism and the systemic metabolic profile without causing major dysfunction in the OXPHOS system.


Assuntos
DNA Mitocondrial/genética , Epidermólise Bolhosa Adquirida/genética , Linfócitos/metabolismo , Polimorfismo de Nucleotídeo Único , Animais , Células Cultivadas , Citocinas/metabolismo , Epidermólise Bolhosa Adquirida/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/genética , Mitocôndrias Hepáticas/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética
4.
Nat Commun ; 11(1): 3606, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681016

RESUMO

Mitochondrial metabolism has emerged as a promising target against the mechanisms of tumor growth. Herein, we have screened an FDA-approved library to identify drugs that inhibit mitochondrial respiration. The ß1-blocker nebivolol specifically hinders oxidative phosphorylation in cancer cells by concertedly inhibiting Complex I and ATP synthase activities. Complex I inhibition is mediated by interfering the phosphorylation of NDUFS7. Inhibition of the ATP synthase is exerted by the overexpression and binding of the ATPase Inhibitory Factor 1 (IF1) to the enzyme. Remarkably, nebivolol also arrests tumor angiogenesis by arresting endothelial cell proliferation. Altogether, targeting mitochondria and angiogenesis triggers a metabolic and oxidative stress crisis that restricts the growth of colon and breast carcinomas. Nebivolol holds great promise to be repurposed for the treatment of cancer patients.


Assuntos
Antagonistas Adrenérgicos/farmacologia , Indutores da Angiogênese/farmacologia , Neoplasias da Mama/fisiopatologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/fisiopatologia , Mitocôndrias/efeitos dos fármacos , Nebivolol/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Feminino , Humanos , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Proteínas/genética , Proteínas/metabolismo
5.
PLoS One ; 15(5): e0233177, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32413073

RESUMO

Mitochondrial oxidative phosphorylation (oxphos) is the process by which the ATP synthase conserves the energy released during the oxidation of different nutrients as ATP. The yeast ATP synthase consists of three assembly modules, one of which is a ring consisting of 10 copies of the Atp9 subunit. We previously reported the existence in yeast mitochondria of high molecular weight complexes composed of mitochondrially encoded Atp9 and of Cox6, an imported structural subunit of cytochrome oxidase (COX). Pulse-chase experiments indicated a correlation between the loss of newly translated Atp9 complexed to Cox6 and an increase of newly formed Atp9 ring, but did not exclude the possibility of an alternate source of Atp9 for ring formation. Here we have extended studies on the functions and structure of this complex, referred to as Atco. We show that Atco is the exclusive source of Atp9 for the ATP synthase assembly. Pulse-chase experiments show that newly translated Atp9, present in Atco, is converted to a ring, which is incorporated into the ATP synthase with kinetics characteristic of a precursor-product relationship. Even though Atco does not contain the ring form of Atp9, cross-linking experiments indicate that it is oligomeric and that the inter-subunit interactions are similar to those of the bona fide ring. We propose that, by providing Atp9 for biogenesis of ATP synthase, Atco complexes free Cox6 for assembly of COX. This suggests that Atco complexes may play a role in coordinating assembly and maintaining proper stoichiometry of the two oxphos enzymes.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA Mitocondrial/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação Oxidativa , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
6.
Biochem Pharmacol ; 177: 113995, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32339494

RESUMO

Few discoveries have influenced drug discovery programs more than the finding that mitochondrial membranes undergo swings in permeability in response to cellular perturbations. The conductor of these permeability changes is the aptly named mitochondrial permeability transition pore which, although not yet precisely defined, is comprised of several integral proteins that differentially act to regulate the flux of ions, proteins and metabolic byproducts during the course of cellular physiological functions but also pathophysiological insults. Pursuit of the pore's exact identity remains a topic of keen interest, but decades of research have unearthed provocative functions for the integral proteins leading to their evaluation to develop novel therapeutics for a wide range of clinical indications. Chief amongst these targeted, integral proteins have been the Voltage Dependent Anion Channel (VDAC) and the F1FO ATP synthase. Research associated with the roles and ligands of VDAC has been extensive and we will expand upon 3 examples of ligand:VDAC interactions for consideration of drug discovery projects: Tubulin:VDAC1, Hexokinase I/II:VDAC1 and olesoxime:VDAC1. The discoveries that cyclosporine blocks mitochondrial permeability transition via binding to cyclophilin D, and that cyclophilin D is an important component of F1FO ATP synthase, has heightened interest in the F1FO ATP synthase as a focal point for drug discovery, and we will discuss 2 plausible campaigns associated with disease indications. To date no drug has emerged from prospective targeting these integral proteins; however, continued exploration such as the approaches suggested in this Commentary will increase the likelihood of providing important therapeutics for severely unmet medical needs.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Colestenonas/uso terapêutico , Ciclosporina/uso terapêutico , Mitocôndrias/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/genética , Canal de Ânion 1 Dependente de Voltagem/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Ciclofilinas/genética , Ciclofilinas/metabolismo , Regulação da Expressão Gênica , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/patologia , Permeabilidade/efeitos dos fármacos , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Canal de Ânion 1 Dependente de Voltagem/antagonistas & inibidores , Canal de Ânion 1 Dependente de Voltagem/metabolismo
7.
Food Chem ; 322: 126759, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32283374

RESUMO

Detection of animal species in meat product is crucial to prevent adulterated and unnecessary contamination during processing. Gold standard is the real-time PCR assays, which can be conducted at highly equipped laboratories. Toward the development of point-of-need test, two rapid molecular assays based on recombinase polymerase amplification (RPA) for the detection of pork and horse DNA were established. Target genes are the porcine mitochondrial ND2 and equine ATP 6-8 genes. The pork and horse_RPA assays detected 16 and one DNA molecules/µl in eleven to six minutes, respectively. The myoglobin in the meat did not influence the assays performances, while the presence of high background-DNA induced a one log decrease in the sensitivity. Both assays are highly specific and identify down to 0.1% of their target DNA in meat mixtures. Both RPA assays could be used on-site as a rapid and mobile detection system to determine contamination of meat products.


Assuntos
DNA/análise , Cavalos/genética , Carne/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Suínos/genética , Animais , DNA/metabolismo , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , NADH Desidrogenase/genética , Sistemas Automatizados de Assistência Junto ao Leito , Recombinases/metabolismo
8.
Cell Physiol Biochem ; 54(2): 211-229, 2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32100973

RESUMO

BACKGROUND/AIMS: Mitochondrial ATP synthase, in addition to being involved in ATP synthesis, is involved in permeability transition pore (PTP) formation, which precedes apoptosis in mammalian cells and programmed cell death in yeast. Mutations in genes encoding ATP synthase subunits cause neuromuscular disorders and have been identified in cancer samples. PTP is also involved in pathology. We previously found that in Saccharomyces cerevisiae, two mutations in ATP synthase subunit a (atp6-P163S and atp6-K90E, equivalent to those detected in prostate and thyroid cancer samples, respectively) in the OM45-GFP background affected ROS and calcium homeostasis and delayed yeast PTP (yPTP) induction upon calcium treatment by modulating the dynamics of ATP synthase dimer/oligomer formation. The Om45 protein is a component of the porin complex, which is equivalent to mammalian VDAC. We aimed to investigate yPTP function in atp6-P163S and atp6-K90E mutants lacking the e and g dimerization subunits of ATP synthase. METHODS: Triple mutants with the atp6-P163S or atp6-K90E mutation, the OM45-GFP gene and deletion of the TIM11 gene encoding subunit e were constructed by crossing and tetrad dissection. In spores capable of growing, the original atp6 mutations reverted to wild type, and two compensatory mutations, namely, atp6-C33S-T215C, were selected. The effects of these mutations on cellular physiology, mitochondrial morphology, bioenergetics and permeability transition (PT) were analyzed by fluorescence and electron microscopy, mitochondrial respiration, ATP synthase activity, calcium retention capacity and swelling assays. RESULTS: The atp6-C33S-T215C mutations in the OM45-GFP background led to delayed growth at elevated temperature on both fermentative and respiratory media and increased sensitivity to high calcium ions concentration or hydrogen peroxide in the medium. The ATP synthase activity was reduced by approximately 50% and mitochondrial network was hyperfused in these cells grown at elevated temperature. The atp6-C33S-T215C stabilized ATP synthase dimers and restored the yPTP properties in Tim11∆ cells. In OM45-GFP cells, in which Tim11 is present, these mutations increased the fraction of swollen mitochondria by up to 85% vs 60% in the wild type, although the time required for calcium release doubled. CONCLUSION: ATP synthase subunit e is essential in the S. cerevisiae atp6-P163S and atp6-K90E mutants. In addition to subunits e and g, subunit a is critical for yPTP induction and conduction. The increased yPTP conduction decrease the S. cerevisiae cell fitness.


Assuntos
Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cálcio/metabolismo , Cobre/farmacologia , DNA Mitocondrial/metabolismo , Dimerização , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/genética , Mutagênese , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Temperatura
9.
Proc Natl Acad Sci U S A ; 117(5): 2412-2421, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31964824

RESUMO

Mitochondria have a characteristic ultrastructure with invaginations of the inner membrane called cristae that contain the protein complexes of the oxidative phosphorylation system. How this particular morphology of the respiratory membrane impacts energy conversion is currently unknown. One proposed role of cristae formation is to facilitate the establishment of local proton gradients to fuel ATP synthesis. Here, we determined the local pH values at defined sublocations within mitochondria of respiring yeast cells by fusing a pH-sensitive GFP to proteins residing in different mitochondrial subcompartments. Only a small proton gradient was detected over the inner membrane in wild type or cristae-lacking cells. Conversely, the obtained pH values did barely permit ATP synthesis in a reconstituted system containing purified yeast F1F0 ATP synthase, although, thermodynamically, a sufficiently high driving force was applied. At higher driving forces, where robust ATP synthesis was observed, a P-side pH value of 6 increased the ATP synthesis rate 3-fold compared to pH 7. In contrast, when ATP synthase was coreconstituted with an active proton-translocating cytochrome oxidase, ATP synthesis readily occurred at the measured, physiological pH values. Our study thus reveals that the morphology of the inner membrane does not influence the subcompartmental pH values and is not necessary for robust oxidative phosphorylation in mitochondria. Instead, it is likely that the dense packing of the oxidative phosphorylation complexes in the cristae membranes assists kinetic coupling between proton pumping and ATP synthesis.


Assuntos
Trifosfato de Adenosina/biossíntese , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Prótons , Transporte de Elétrons , Concentração de Íons de Hidrogênio , Cinética , Mitocôndrias/química , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/química , Membranas Mitocondriais/enzimologia , ATPases Mitocondriais Próton-Translocadoras/genética , Fosforilação Oxidativa , Proteolipídeos/metabolismo , Bombas de Próton/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
10.
Brain Dev ; 42(1): 69-72, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31500933

RESUMO

BACKGROUND: The mitochondrial DNA MT-ATP6 gene encodes the ATP6 subunit of the mitochondrial ATP synthase. The m.9185 T > C variant in MT-ATP6 has been reported to cause various neurological disorders including late-onset Leigh syndrome (LS). To our knowledge, there has been no reported case of infantile-onset LS associated with the m.9185 T > C variant. Herein, we report a patient with early-onset LS complicated with infantile spasms who exhibited profound developmental delay. CASE REPORT: A 3-month-old Japanese girl presented with focal seizures. Brain magnetic resonance imaging (MRI) revealed bilateral lesions in the basal ganglia and cerebral peduncle. Laboratory evaluation demonstrated marked elevations of lactate and pyruvate in both venous blood and cerebrospinal fluid. At 6 months, she developed infantile spasms, which were ceased by adrenocorticotropic hormone therapy. At 2 years of age, she was bedridden due to hypotonic quadriplegia and was unable to make eye contact. Whole-exome sequencing identified apparently de novo homoplasmic m.9185 T > C variant in her blood. CONCLUSION: This is the first case report describing early infantile-onset LS associated with the m.9185 T > C variant, and thereby broadens the phenotypic spectrum of m.9185 T > C-related disorders.


Assuntos
Doença de Leigh/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Espasmos Infantis/genética , Feminino , Humanos , Lactente , Mutação
11.
Mol Cell Proteomics ; 19(1): 142-154, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31723016

RESUMO

We previously reported that tumor inflammasomes play a key role in tumor control and act as favorable prognostic markers in nasopharyngeal carcinoma (NPC). Activated inflammasomes frequently form distinguishable specks and govern the cellular secretion of IL-1ß. However, we know little about the biological and biochemical differences between cells with and without apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) speck formation. In this study, we used proteomic iTRAQ analysis to analyze the proteomes of NPC cells that differ in their ASC speck formation upon cisplatin treatment. We identified proteins that were differentially over-expressed in cells with specks, and found that they fell into two Gene ontology (GO) pathways: mitochondrial oxidative phosphorylation (OxPhos) and ubiquinone metabolism. We observed up-regulation of various components of the OxPhos machinery (including NDUFB3, NDUFB8 and ATP5B), and subsequently found that these changes lead to mitochondrial ROS (mtROS) production, which promotes the formation and activation of NLRP3 inflammasomes and subsequent pyroptosis. In NPC patients, better local recurrence-free survival was significantly associated with high-level expression of NDUFB8 (p = 0.037) and ATP5B (p = 0.029), as examined using immunohistochemistry. However, there were no significant associations between the expression of NDUFB8 and ATP5B with overall survival of NPC patients. Together, our results demonstrate that up-regulated mitochondrial OxPhos components are strongly associated with NLRP3 inflammasome activation in NPC. Our findings further suggest that high-level expression of OxPhos components could be markers for local recurrence and/or promising therapeutic targets in patients with NPC.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Inflamassomos/metabolismo , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Intervalo Livre de Doença , Complexo I de Transporte de Elétrons/genética , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , ATPases Mitocondriais Próton-Translocadoras/genética , Carcinoma Nasofaríngeo/mortalidade , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/mortalidade , Neoplasias Nasofaríngeas/patologia , Fosforilação Oxidativa , Proteômica/métodos , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/genética
12.
Biochim Biophys Acta Bioenerg ; 1861(1): 148091, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31669489

RESUMO

F1FO ATP synthase, also known as complex V, is a key enzyme of mitochondrial energy metabolism that can synthesize and hydrolyze ATP. It is not known whether the ATP synthase and ATPase function are correlated with a different spatio-temporal organisation of the enzyme. In order to analyze this, we tracked and localized single ATP synthase molecules in situ using live cell microscopy. Under normal conditions, complex V was mainly restricted to cristae indicated by orthogonal trajectories along the cristae membranes. In addition confined trajectories that are quasi immobile exist. By inhibiting glycolysis with 2-DG, the activity and mobility of complex V was altered. The distinct cristae-related orthogonal trajectories of complex V were obliterated. Moreover, a mobile subpopulation of complex V was found in the inner boundary membrane. The observed changes in the ratio of dimeric/monomeric complex V, respectively less mobile/more mobile complex V and its activity changes were reversible. In IF1-KO cells, in which ATP hydrolysis is not inhibited by IF1, complex V was more mobile, while inhibition of ATP hydrolysis by BMS-199264 reduced the mobility of complex V. Taken together, these data support the existence of different subpopulations of complex V, ATP synthase and ATP hydrolase, the latter with higher mobility and probably not prevailing at the cristae edges. Obviously, complex V reacts quickly and reversibly to metabolic conditions, not only by functional, but also by spatial and structural reorganization.


Assuntos
Trifosfato de Adenosina/metabolismo , Mitocôndrias/enzimologia , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Trifosfato de Adenosina/genética , Células HeLa , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Translocadoras de Prótons/genética
13.
Cell Death Dis ; 10(11): 857, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31719530

RESUMO

Parkinson's disease (PD), the second most common neurodegenerative disorder, is characterized by dopaminergic neuronal loss that initiates in the substantia nigra pars compacta and by the formation of intracellular inclusions mainly constituted by aberrant α-synuclein (α-syn) deposits known as Lewy bodies. Most cases of PD are sporadic, but about 10% are familial, among them those caused by mutations in SNCA gene have an autosomal dominant transmission. SNCA encodes α-syn, a small 140-amino acids protein that, under physiological conditions, is mainly localized at the presynaptic terminals. It is prevalently cytosolic, but its presence has been reported in the nucleus, in the mitochondria and, more recently, in the mitochondria-associated ER membranes (MAMs). Whether different cellular localizations may reflect specific α-syn activities is presently unclear and its action at mitochondrial level is still a matter of debate. Mounting evidence supports a role for α-syn in several mitochondria-derived activities, among which maintenance of mitochondrial morphology and modulation of complex I and ATP synthase activity. α-syn has been proposed to localize at the outer membrane (OMM), in the intermembrane space (IMS), at the inner membrane (IMM) and in the mitochondrial matrix, but a clear and comparative analysis of the sub-mitochondrial localization of WT and mutant α-syn is missing. Furthermore, the reasons for this spread sub-mitochondrial localization under physiological and pathological circumstances remain elusive. In this context, we decided to selectively monitor the sub-mitochondrial distribution of the WT and PD-related α-syn mutants A53T and A30P by taking advantage from a bimolecular fluorescence complementation (BiFC) approach. We also investigated whether cell stress could trigger α-syn translocation within the different mitochondrial sub-compartments and whether PD-related mutations could impinge on it. Interestingly, the artificial targeting of α-syn WT (but not of the mutants) to the mitochondrial matrix impacts on ATP production, suggesting a potential role within this compartment.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Mitocôndrias/genética , Doença de Parkinson/metabolismo , alfa-Sinucleína/genética , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/genética , Citosol/metabolismo , Citosol/patologia , Dopamina/genética , Dopamina/metabolismo , Neurônios Dopaminérgicos/patologia , Expressão Gênica/genética , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas Mutantes/genética , Doença de Parkinson/patologia , Parte Compacta da Substância Negra/metabolismo , Parte Compacta da Substância Negra/patologia , Terminações Pré-Sinápticas/metabolismo
14.
Int J Mol Sci ; 20(23)2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31757021

RESUMO

Mucopolysaccharidosis type II (MPS II) is a rare lysosomal storage disease (LSD) involving a genetic error in iduronic acid-2-sulfatase (IDS) metabolism that leads to accumulation of glycosaminoglycans within intracellular lysosomes. The primary treatment for MPS II, enzyme replacement therapy, is not effective for central nervous system (CNS) symptoms, such as intellectual disability, because the drugs do not cross the blood-brain barrier. Recently, autophagy has been associated with LSDs. In this study, we examined the morphologic relationship between neuronal damage and autophagy in IDS knockout mice using antibodies against subunit c of mitochondrial adenosine triphosphate (ATP) synthetase and p62. Immunohistological changes suggesting autophagy, such as vacuolation, were observed in neurons, microglia, and pericytes throughout the CNS, and the numbers increased over postnatal development. Oral administration of chloroquine, which inhibits autophagy, did not suppress damage to microglia and pericytes, but greatly reduced neuronal vacuolation and eliminated neuronal cells with abnormal inclusions. Thus, decreasing autophagy appears to prevent neuronal degeneration. These results suggest that an autophagy modulator could be used in addition to conventional enzyme replacement therapy to preserve the CNS in patients with MPS II.


Assuntos
Autofagia , Mucopolissacaridose II/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Cloroquina/farmacologia , Iduronato Sulfatase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mucopolissacaridose II/patologia , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo
16.
FASEB J ; 33(12): 14103-14117, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31652072

RESUMO

Biogenesis of F1Fo ATP synthase, the key enzyme of mitochondrial energy provision, depends on transmembrane protein 70 (TMEM70), localized in the inner mitochondrial membrane of higher eukaryotes. TMEM70 absence causes severe ATP-synthase deficiency and leads to a neonatal mitochondrial encephalocardiomyopathy in humans. However, the exact biochemical function of TMEM70 remains unknown. Using TMEM70 conditional knockout in mice, we show that absence of TMEM70 impairs the early stage of enzyme biogenesis by preventing incorporation of hydrophobic subunit c into rotor structure of the enzyme. This results in the formation of an incomplete, pathologic enzyme complex consisting of F1 domain and peripheral stalk but lacking Fo proton channel composed of subunits c and a. We demonstrated direct interaction between TMEM70 and subunit c and showed that overexpression of subunit c in TMEM70-/- cells partially rescued TMEM70 defect. Accordingly, TMEM70 knockdown prevented subunit c accumulation otherwise observed in F1-deficient cells. Altogether, we identified TMEM70 as specific ancillary factor for subunit c. The biologic role of TMEM70 is to increase the low efficacy of spontaneous assembly of subunit c oligomer, the key and rate-limiting step of ATP-synthase biogenesis, and thus to reach an adequately high physiologic level of ATP synthase in mammalian tissues.-Kovalcíková, J., Vrbacký, M., Pecina, P., Tauchmannová, K., Nusková, H., Kaplanová, V., Brázdová, A., Alán, L., Eliás, J., Cunátová, K., Korínek, V., Sedlacek, R., Mrácek, T., Houstek, J. TMEM70 facilitates biogenesis of mammalian ATP synthase by promoting subunit c incorporation into the rotor structure of the enzyme.


Assuntos
Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica , Técnicas de Inativação de Genes/métodos , Genótipo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Proteolipídeos/metabolismo , Tamoxifeno/farmacologia
17.
J Genet ; 982019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31544786

RESUMO

Cytoplasmic male sterility (CMS) is an important trait for large-scale hybrid seed production which avoids manual emasculation and undesired horizontal spread of pollen. Rearrangements in mitochondrial genome in terms of deletions and insertions are frequent causes leading to CMS. Mitochondrial ATP synthase is a multisubunit molecular machine which is involved in synthesis of ATP. In this study, three mutations in ATPase subunit 6 were identified and their cosegregation with male sterility was established using tobacco male sterile hybrids and Nicotiana suvaolensis. A breeder friendly Kompetitive allele specific polymerase chain reaction (KASP) SNP marker was developed for high throughput and quick genotyping. Introgression of this trait into selected germplasm lines (n = 9) was achieved based on foreground for CMS and background selection for recurrent parent using KASP marker and 50K custom tobacco SNP array, respectively. Analysis of genotyping data from SNP array revealed the presence of 88-99% of recurrent parent genome in BC3F1 plants. The selected BC3F1 plants exhibit CMS and are indistinguishable from the fertile recurrent parent (germplasm) in terms of plant morphology.


Assuntos
ATPases Mitocondriais Próton-Translocadoras/genética , Infertilidade das Plantas/genética , Pólen/genética , Tabaco/genética , Alelos , DNA Mitocondrial/genética , Marcadores Genéticos , Genótipo , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único
19.
Mikrochim Acta ; 186(9): 634, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31428871

RESUMO

A PCR method is described to identify the species origin of various animal and human tissue-derived biochemical drugs. Four commercialized drugs, including spermary tablets, compound embryonic bovine liver extract tablets, spleen aminopeptide solution, and placenta polypeptide injection, were used as a proof-of-principle in this study. Primers were designed to amplify conservative regions of mitochondrial cytochrome b and ATPase 8 genes from beef, pork, lamb and human DNA, respectively. The specificity of primers for ATPase 8 gene is found to be higher than those for cytochrome b under the given experimental conditions. The amplicon sizes of ATPase 8 were 212, 271, 293 and 405 bp for pork, beef, lamb and human tissue, respectively. The minimum detectable concentration of DNA sample for species identification is 0.05-0.5 pg·µL-1. The species origin can be distinguished by this method in extremely low concentrations of template DNAs extracted. Conceivably, this PCR method for meat authentication may be extended to quality control of other biochemical drugs and raw materials. Graphical abstract A specific PCR method was developed for the detection of species origin in biochemical drugs via species-specific primers targeting mitochondrial ATPase 8 genes. The PCR products were separated by gel electrophoresis and species origins were indicated by comparison to references.


Assuntos
Primers do DNA/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Preparações Farmacêuticas/análise , Reação em Cadeia da Polimerase , Animais , Bovinos , Citocromos b/genética , DNA Mitocondrial/genética , Eletroforese em Gel de Ágar , Humanos , Carne/análise , Tamanho da Partícula , Ovinos , Propriedades de Superfície , Suínos , Comprimidos
20.
J Bacteriol ; 201(19)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31285242

RESUMO

Mycobacterium tuberculosis, a bacterium that causes tuberculosis, poses a serious threat, especially due to the emergence of drug-resistant strains. M. tuberculosis and other mycobacterial species, such as M. smegmatis, are known to generate an inadequate amount of energy by substrate-level phosphorylation and mandatorily require oxidative phosphorylation (OXPHOS) for their growth and metabolism. Hence, antibacterial drugs, such as bedaquiline, targeting the multisubunit ATP synthase complex, which is required for OXPHOS, have been developed with the aim of eliminating pathogenic mycobacteria. Here, we explored the influence of suboptimal OXPHOS on the physiology and metabolism of M. smegmatis M. smegmatis harbors two identical copies of atpD, which codes for the ß subunit of ATP synthase. We show that upon deletion of one copy of atpD (M. smegmatis ΔatpD), M. smegmatis synthesizes smaller amounts of ATP and enters into an energy-compromised state. The mutant displays remarkable phenotypic and physiological differences from the wild type, such as respiratory slowdown, reduced biofilm formation, lesser amounts of cell envelope polar lipids, and increased antibiotic sensitivity compared to the wild type. Additionally, M. smegmatis ΔatpD overexpresses genes belonging to the dormancy operon, the ß-oxidation pathway, and the glyoxylate shunt, suggesting that the mutant adapts to a low energy state by switching to alternative pathways to produce energy. Interestingly, M. smegmatis ΔatpD shows significant phenotypic, metabolic, and physiological similarities with bedaquiline-treated wild-type M. smegmatis We believe that the identification and characterization of key metabolic pathways functioning during an energy-compromised state will enhance our understanding of bacterial adaptation and survival and will open newer avenues in the form of drug targets that may be used in the treatment of mycobacterial infections.IMPORTANCE M. smegmatis generates an inadequate amount of energy by substrate-level phosphorylation and mandatorily requires oxidative phosphorylation (OXPHOS) for its growth and metabolism. Here, we explored the influence of suboptimal OXPHOS on M. smegmatis physiology and metabolism. M. smegmatis harbors two identical copies of the atpD gene, which codes for the ATP synthase ß subunit. Here, we carried out the deletion of only one copy of atpD in M. smegmatis to understand the bacterial survival response in an energy-deprived state. M. smegmatis ΔatpD shows remarkable phenotypic, metabolic, and physiological differences from the wild type. Our study thus establishes M. smegmatis ΔatpD as an energy-compromised mycobacterial strain, highlights the importance of ATP synthase in mycobacterial physiology, and further paves the way for the identification of novel antimycobacterial drug targets.


Assuntos
Deleção de Genes , ATPases Mitocondriais Próton-Translocadoras/genética , Mycobacterium smegmatis/fisiologia , Fosforilação Oxidativa , Trifosfato de Adenosina/metabolismo , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Diarilquinolinas/farmacologia , Metabolismo Energético/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Óperon , Fosforilação Oxidativa/efeitos dos fármacos , Fenótipo
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