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1.
PLoS One ; 15(8): e0238452, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866191

RESUMO

The filamentous fungus Acremonium chrysogenum is the main industrial producer of cephalosporin C (CPC), one of the major precursors for manufacturing of cephalosporin antibiotics. The plasma membrane H+-ATPase (PMA) plays a key role in numerous fungal physiological processes. Previously we observed a decrease of PMA activity in A. chrysogenum overproducing strain RNCM 408D (HY) as compared to the level the wild-type strain A. chrysogenum ATCC 11550. Here we report the relationship between PMA activity and CPC biosynthesis in A. chrysogenum strains. The elevation of PMA activity in HY strain through overexpression of PMA1 from Saccharomyces cerevisiae, under the control of the constitutive gpdA promoter from Aspergillus nidulans, results in a 1.2 to 10-fold decrease in CPC production, shift in beta-lactam intermediates content, and is accompanied by the decrease in cef genes expression in the fermentation process; the characteristic colony morphology on agar media is also changed. The level of PMA activity in A. chrysogenum HY OE::PMA1 strains has been increased by 50-100%, up to the level observed in WT strain, and was interrelated with ATP consumption; the more PMA activity is elevated, the more ATP level is depleted. The reduced PMA activity in A. chrysogenum HY strain may be one of the selected events during classical strain improvement, aimed at elevating the ATP content available for CPC production.


Assuntos
Acremonium/metabolismo , Membrana Celular/metabolismo , Cefalosporinas/biossíntese , Cefalosporinas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Adenosina Trifosfatases/metabolismo , Meios de Cultura/metabolismo , Fermentação/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , beta-Lactamas/metabolismo
2.
PLoS One ; 15(8): e0237569, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32817667

RESUMO

Several 'super-complexes' of individual hetero-oligomeric membrane protein complexes, whose function is to facilitate intra-membrane electron and proton transfer and harvesting of light energy, have been previously characterized in the mitochondrial cristae and chloroplast thylakoid membranes. We report the presence of an intra-membrane super-complex dominated by the ATP-synthase, photosystem I (PSI) reaction-center complex and the ferredoxin-NADP+ Reductase (FNR) in the thylakoid membrane. The presence of the super-complex has been documented by mass spectrometry, clear-native PAGE and Western Blot analyses. This is the first documented presence of ATP synthase in a super-complex with the PSI reaction-center located in the non-appressed stromal domain of the thylakoid membrane.


Assuntos
Cloroplastos/metabolismo , Ferredoxina-NADP Redutase/metabolismo , Óxido Nítrico Sintase/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Tilacoides/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte de Elétrons , Fotossíntese , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Spinacia oleracea/crescimento & desenvolvimento , Spinacia oleracea/metabolismo
3.
Nat Commun ; 11(1): 2615, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457314

RESUMO

F1Fo ATP synthase functions as a biological rotary generator that makes a major contribution to cellular energy production. It comprises two molecular motors coupled together by a central and a peripheral stalk. Proton flow through the Fo motor generates rotation of the central stalk, inducing conformational changes in the F1 motor that catalyzes ATP production. Here we present nine cryo-EM structures of E. coli ATP synthase to 3.1-3.4 Å resolution, in four discrete rotational sub-states, which provide a comprehensive structural model for this widely studied bacterial molecular machine. We observe torsional flexing of the entire complex and a rotational sub-step of Fo associated with long-range conformational changes that indicates how this flexibility accommodates the mismatch between the 3- and 10-fold symmetries of the F1 and Fo motors. We also identify density likely corresponding to lipid molecules that may contribute to the rotor/stator interaction within the Fo motor.


Assuntos
Proteínas de Escherichia coli/química , ATPases Translocadoras de Prótons/química , Difosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Proteínas de Escherichia coli/metabolismo , Lipídeos/química , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Rotação , Relação Estrutura-Atividade
4.
Nat Commun ; 11(1): 2395, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32409656

RESUMO

Pollen tubes are highly polarized tip-growing cells that depend on cytosolic pH gradients for signaling and growth. Autoinhibited plasma membrane proton (H+) ATPases (AHAs) have been proposed to energize pollen tube growth and underlie cell polarity, however, mechanistic evidence for this is lacking. Here we report that the combined loss of AHA6, AHA8, and AHA9 in Arabidopsis thaliana delays pollen germination and causes pollen tube growth defects, leading to drastically reduced fertility. Pollen tubes of aha mutants had reduced extracellular proton (H+) and anion fluxes, reduced cytosolic pH, reduced tip-to-shank proton gradients, and defects in actin organization. Furthermore, mutant pollen tubes had less negative membrane potentials, substantiating a mechanistic role for AHAs in pollen tube growth through plasma membrane hyperpolarization. Our findings define AHAs as energy transducers that sustain the ionic circuit defining the spatial and temporal profiles of cytosolic pH, thereby controlling downstream pH-dependent mechanisms essential for pollen tube elongation, and thus plant fertility.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Tubo Polínico/crescimento & desenvolvimento , Polinização/fisiologia , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Citosol/metabolismo , Técnicas de Silenciamento de Genes , Germinação/fisiologia , Concentração de Íons de Hidrogênio , Potenciais da Membrana/fisiologia , Mutação , Plantas Geneticamente Modificadas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ATPases Translocadoras de Prótons/genética , Análise Espaço-Temporal
5.
Plant Physiol Biochem ; 151: 429-437, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32289636

RESUMO

Potassium (K+) has been reported to alleviate ammonium (NH4+) toxicity in rice through some underlying mechanisms, but it still not clear. In addition, K+ is an important cation for activation of plasma membrane (PM) H+-ATPase activity. Here, we hypothesized that K+ alleviated NH4+ toxicity by mediating PM H+-ATPase function in rice root. In this study, rice plants were cultivated in hydroponic solution with various concentrations of K+ and NH4+. By concurrently supplying K+ with NH4+ or re-supplying K+ after NH4+ toxicity, we found that high K+ concentration reduced the NH4+ uptake rate, enhanced the H+ extrusion rate by the roots, and alleviated rice NH4+ toxicity. The gene expression levels of PM H+-ATPase members (OsA1, 3, 7, 8, and 9) were upregulated by application of increasing concentrations of K+ under NH4+ toxicity. The PM H+-ATPase activity and protein expression in rice roots were also enhanced. Furthermore, the enhancement of PM H+-ATPase activity by a specific stimulator (fusicoccin) rescued rice seedlings from NH4+ toxicity. Taken together, these results indicate that K+ can alleviate NH4+ toxicity, possibly by activating PM H+-ATPase to extrude more H+ and inhibit NH4+ uptake by root. Our results may enhance understanding of the strategy of applying K+ fertilizer to mitigate crop NH4+ toxicity in agriculture.


Assuntos
Compostos de Amônio , Oryza , Potássio , ATPases Translocadoras de Prótons , Compostos de Amônio/metabolismo , Compostos de Amônio/toxicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Ativação Enzimática/efeitos dos fármacos , Oryza/efeitos dos fármacos , Oryza/enzimologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Potássio/farmacologia , ATPases Translocadoras de Prótons/metabolismo
6.
Biochim Biophys Acta Bioenerg ; 1861(7): 148189, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32194063

RESUMO

ATP synthases are important energy-coupling, rotary motor enzymes in all kingdoms of life. In all F-type ATP synthases, the central rotor of the catalytic F1 complex is composed of the γ subunit and the N-terminal domain (NTD) of the ε subunit. In the enzymes of diverse bacteria, the C-terminal domain of ε (εCTD) can undergo a dramatic conformational change to trap the enzyme in a transiently inactive state. This inhibitory mechanism is absent in the mitochondrial enzyme, so the εCTD could provide a means to selectively target ATP synthases of pathogenic bacteria for antibiotic development. For Escherichia coli and other bacterial model systems, it has been difficult to dissect the relationship between ε inhibition and a MgADP-inhibited state that is ubiquitous for FOF1 from bacteria and eukaryotes. A prior study with the isolated catalytic complex from E. coli, EcF1, showed that these two modes of inhibition are mutually exclusive, but it has long been known that interactions of F1 with the membrane-embedded FO complex modulate inhibition by the εCTD. Here, we study membranes containing EcFOF1 with wild-type ε, ε lacking the full εCTD, or ε with a small deletion at the C-terminus. By using compounds with distinct activating effects on F-ATP-ase activity, we confirm that εCTD inhibition and ubiquitous MgADP inhibition are mutually exclusive for membrane-bound E. coli F-ATP-ase. We determine that most of the enzyme complexes in wild-type membranes are in the ε-inhibited state (>50%) or in the MgADP-inhibited state (30%).


Assuntos
Difosfato de Adenosina/farmacologia , Membrana Celular/enzimologia , Escherichia coli/enzimologia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo , Trifosfato de Adenosina/metabolismo , Dimetilaminas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Hidrólise , Domínios Proteicos , Ácido Selenioso/farmacologia , Solubilidade
7.
Am J Physiol Renal Physiol ; 318(5): F1122-F1135, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32174138

RESUMO

Emerging evidence has demonstrated that (pro)renin receptor (PRR)-mediated activation of intrarenal renin-angiotensin system (RAS) plays an essential role in renal handling of Na+ and water balance and blood pressure. The present study tested the possibility that the intrarenal RAS served as a molecular target for the protective action of ELABELA (ELA), a novel endogenous ligand of apelin receptor, in the distal nephron. By RNAscope and immunofluorescence, mRNA and protein expression of endogenous ELA was consistently localized to the collecting duct (CD). Apelin was also found in the medullary CDs as assessed by immunofluorescence. In cultured CD-derived M1 cells, exogenous ELA induced parallel decreases of full-length PRR (fPRR), soluble PRR (sPRR), and prorenin/renin protein expression as assessed by immunoblotting and medium sPRR and prorenin/renin levels by ELISA, all of which were reversed by 8-bromoadenosine 3',5'-cyclic monophosphate. Conversely, deletion of PRR in the CD or nephron in mice elevated Apela and Apln mRNA levels as well as urinary ELA and apelin excretion, supporting the antagonistic relationship between the two systems. Administration of exogenous ELA-32 infusion (1.5 mg·kg-1·day-1, minipump) to high salt (HS)-loaded Dahl salt-sensitive (SS) rats significantly lowered mean arterial pressure, systolic blood pressure, diastolic blood pressure, and albuminuria, accompanied with a reduction of urinary sPRR, angiotensin II, and prorenin/renin excretion. HS upregulated renal medullary protein expression of fPRR, sPRR, prorenin, and renin in Dahl SS rats, all of which were significantly blunted by exogenous ELA-32 infusion. Additionally, HS-induced upregulation of inflammatory cytokines (IL-1ß, IL-2, IL-6, IL-17A, IFN-γ, VCAM-1, ICAM-1, and MCP-1), fibrosis markers (TGF-ß1, FN, Col1A1, PAI-1, and TIMP-1), and kidney injury markers (NGAL, Kim-1, albuminuria, and urinary NGAL excretion) were markedly blocked by exogenous ELA infusion. Together, these results support the antagonistic interaction between ELA and intrarenal RAS in the distal nephron that appears to exert a major impact on blood pressure regulation.


Assuntos
Pressão Sanguínea , Hipertensão/metabolismo , Nefropatias/metabolismo , Rim/metabolismo , Hormônios Peptídicos/metabolismo , Sistema Renina-Angiotensina , Animais , Apelina/genética , Apelina/metabolismo , Receptores de Apelina/genética , Receptores de Apelina/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Rim/efeitos dos fármacos , Rim/patologia , Rim/fisiopatologia , Nefropatias/patologia , Nefropatias/fisiopatologia , Nefropatias/prevenção & controle , Masculino , Camundongos Knockout , Hormônios Peptídicos/administração & dosagem , Hormônios Peptídicos/genética , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Ratos Endogâmicos Dahl , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Transdução de Sinais
8.
J Pharmacol Sci ; 143(2): 127-131, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32156464

RESUMO

The inhibition of retinal ischemia-induced damage by post-ischemic prothymosin alpha (ProTα) was not affected in toll-like receptor 4 knockout (TLR4-/-) mice but blocked by the pretreatment with antibody against F0/F1 ATPase α- or ß-subunit, novel candidate for ProTα-receptor. In addition to the previous observation of ProTα-induced ATP release from cells, the present study showed a ProTα-induced enhancement of ATP hydrolysis activity of recombinant ATP5A1/5B complex. As the protection of retinal function by post-ischemic ProTα was abolished by anti-P2Y12 antibody, the activation of F0/F1 ATPase and subsequent P2Y12 receptor system may play roles in beneficial actions by post-ischemic ProTα.


Assuntos
Isquemia/metabolismo , Isquemia/prevenção & controle , Precursores de Proteínas/administração & dosagem , Precursores de Proteínas/farmacologia , ATPases Translocadoras de Prótons/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Retina , Timosina/análogos & derivados , Animais , Hidrólise/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas Recombinantes/metabolismo , Timosina/administração & dosagem , Timosina/farmacologia
9.
Photochem Photobiol Sci ; 19(1): 88-98, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31904040

RESUMO

Stomatal pores, which are surrounded by pairs of guard cells in the plant epidermis, regulate gas exchange between plants and the atmosphere, thereby controlling photosynthesis and transpiration. Blue light works as a signal to guard cells, to induce intracellular signaling and open stomata. Blue light receptor phototropins (phots) are activated by blue light; phot-mediated signals promote plasma membrane (PM) H+-ATPase activity via C-terminal Thr phosphorylation, serving as the driving force for stomatal opening in guard cells. However, the details of this signaling process are not fully understood. In this study, through an in vitro screening of phot-interacting protein kinases, we obtained the CBC1 and CBC2 that had been reported as signal transducers in stomatal opening. Promoter activities of CBC1 and CBC2 indicated that both genes were expressed in guard cells. Single and double knockout mutants of CBC1 and CBC2 showed no lesions in the context of phot-mediated phototropism, chloroplast movement, or leaf flattening. In contrast, the cbc1cbc2 double mutant showed larger stomatal opening under both dark and blue light conditions. Interestingly, the level of phosphorylation of C-terminal Thr of PM H+-ATPase was higher in double mutant guard cells. The larger stomatal openings of the double mutant were effectively suppressed by the phytohormone abscisic acid (ABA). CBC1 and CBC2 interacted with BLUS1 and PM H+-ATPase in vitro. From these results, we conclude that CBC1 and CBC2 act as negative regulators of stomatal opening, probably via inhibition of PM H+-ATPase activity.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/enzimologia , Estômatos de Plantas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Fosforilação
10.
Proc Natl Acad Sci U S A ; 117(3): 1447-1456, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31896579

RESUMO

The reaction scheme of rotary catalysis and the torque generation mechanism of bovine mitochondrial F1 (bMF1) were studied in single-molecule experiments. Under ATP-saturated concentrations, high-speed imaging of a single 40-nm gold bead attached to the γ subunit of bMF1 showed 2 types of intervening pauses during the rotation that were discriminated by short dwell and long dwell. Using ATPγS as a slowly hydrolyzing ATP derivative as well as using a functional mutant ßE188D with slowed ATP hydrolysis, the 2 pausing events were distinctively identified. Buffer-exchange experiments with a nonhydrolyzable analog (AMP-PNP) revealed that the long dwell corresponds to the catalytic dwell, that is, the waiting state for hydrolysis, while it remains elusive which catalytic state short pause represents. The angular position of catalytic dwell was determined to be at +80° from the ATP-binding angle, mostly consistent with other F1s. The position of short dwell was found at 50 to 60° from catalytic dwell, that is, +10 to 20° from the ATP-binding angle. This is a distinct difference from human mitochondrial F1, which also shows intervening dwell that probably corresponds to the short dwell of bMF1, at +65° from the binding pause. Furthermore, we conducted "stall-and-release" experiments with magnetic tweezers to reveal how the binding affinity and hydrolysis equilibrium are modulated by the γ rotation. Similar to thermophilic F1, bMF1 showed a strong exponential increase in ATP affinity, while the hydrolysis equilibrium did not change significantly. This indicates that the ATP binding process generates larger torque than the hydrolysis process.


Assuntos
Proteínas Mitocondriais/química , ATPases Translocadoras de Prótons/química , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Bovinos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Ligação Proteica , Domínios Proteicos , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Imagem Individual de Molécula
11.
Nature ; 578(7795): 419-424, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31996848

RESUMO

ATP13A2 (PARK9) is a late endolysosomal transporter that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome-a parkinsonism with dementia1-and early-onset Parkinson's disease2. ATP13A2 offers protection against genetic and environmental risk factors of Parkinson's disease, whereas loss of ATP13A2 compromises lysosomes3. However, the transport function of ATP13A2 in lysosomes remains unclear. Here we establish ATP13A2 as a lysosomal polyamine exporter that shows the highest affinity for spermine among the polyamines examined. Polyamines stimulate the activity of purified ATP13A2, whereas ATP13A2 mutants that are implicated in disease are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes the cellular uptake of polyamines by endocytosis and transports them into the cytosol, highlighting a role for endolysosomes in the uptake of polyamines into cells. At high concentrations polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with impaired expression of ATP13A2 or its orthologues. We present defective lysosomal polyamine export as a mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration, and shed light on the molecular identity of the mammalian polyamine transport system.


Assuntos
Lisossomos/metabolismo , Poliaminas/metabolismo , ATPases Translocadoras de Prótons/deficiência , ATPases Translocadoras de Prótons/genética , Animais , Biocatálise , Transporte Biológico , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Catepsina B/metabolismo , Citosol/metabolismo , Modelos Animais de Doenças , Endocitose , Humanos , Lisossomos/patologia , Camundongos , Mutação , Neurônios/metabolismo , Fenótipo , Poliaminas/toxicidade , ATPases Translocadoras de Prótons/metabolismo , Espermidina/metabolismo , Espermina/metabolismo
12.
Clin Sci (Lond) ; 134(2): 239-259, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-31943002

RESUMO

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


Assuntos
Ciclofilina D/metabolismo , Diabetes Mellitus Experimental/metabolismo , Nefropatias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Albuminúria/genética , Albuminúria/metabolismo , Animais , Ciclofilina D/antagonistas & inibidores , Ciclofilina D/genética , Ciclosporina/farmacologia , Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Peróxido de Hidrogênio/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Nefropatias/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , ATPases Translocadoras de Prótons/metabolismo
13.
Arch Biochem Biophys ; 681: 108258, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31917961

RESUMO

Phenylglyoxal (PGO), known to cause post-translational modifications of Arg residues, was used to highlight the role of arginine residues of the F1FO-ATPase, which may be crucial to yield the mitochondrial permeability transition pore (mPTP). In swine heart mitochondria PGO inhibits ATP hydrolysis by the F1FO-ATPase either sustained by the natural cofactor Mg2+ or by Ca2+ by a similar uncompetitive inhibition mechanism, namely the tertiary complex (ESI) only forms when the ATP substrate is already bound to the enzyme, and with similar strength, as shown by the similar K'i values (0.82 ± 0.07 mM in presence of Mg2+ and 0.64 ± 0.05 mM in the presence of Ca2+). Multiple inhibitor analysis indicates that features of the F1 catalytic sites and/or the FO proton binding sites are apparently unaffected by PGO. However, PGO and F1 or FO inhibitors can bind the enzyme combine simultaneously. However they mutually hinder to bind the Mg2+-activated F1FO-ATPase, whereas they do not mutually exclude to bind the Ca2+-activated F1FO-ATPase. The putative formation of PGO-arginine adducts, and the consequent spatial rearrangement in the enzyme structure, inhibits the F1FO-ATPase activity but, as shown by the calcium retention capacity evaluation in intact mitochondria, apparently favours the mPTP formation.


Assuntos
Glioxilatos/metabolismo , Ácidos Mandélicos/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Animais , Cálcio/metabolismo , Magnésio/metabolismo , Suínos
14.
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
15.
Environ Sci Pollut Res Int ; 27(6): 6389-6400, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31873880

RESUMO

Acid rain is a global environmental issue due to inhibiting severely plant growth and productivity. To discover the tolerant mechanism in plants under acid rain stress, we studied the difference in response of two crops (rice and soybean) to simulated acid rain (pH 5.0 ~ 2.5) at growth and physiological, biochemical and molecular levels during exposure and recovery periods by hydroponics. By analyzing the change in relative growth rate, chlorophyll content and plasma permeability in rice and soybean, we found that rice could tolerate acid rain above pH 3.0 whereas soybean could tolerate acid rain above pH 4.5. By RT-PCR analyses, immunoprecipitation and enzyme kinetics study, we observed that pH 4.5 acid rain promoted the transcriptional expression of H+-ATPase genes and the phosphorylation of H+-ATPase and increased H+-ATPase activity in the two crops for resisting acid stress. The increased degree in soybean was larger than that in rice. Acid rain at pH 3.0 still promoted the transcription regulation to maintain H+-ATPase activity higher in rice for resisting stress but caused irreversible inhibition on express of H+-ATPase and decreased H+-ATPase activity in soybean. All results suggest that the different tolerance in rice and soybean to acid rain stress could be associated with difference in plasma membrane H+-ATPase at transcriptional regulation, post-translational modification and the substrate affinity.


Assuntos
Chuva Ácida , Oryza , ATPases Translocadoras de Prótons/metabolismo , Soja , Membrana Celular , Regulação da Expressão Gênica de Plantas , Estresse Fisiológico
16.
Biochemistry (Mosc) ; 84(11): 1247-1255, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31760915

RESUMO

Fo×F1-ATPases of mitochondria, chloroplasts, and microorganisms catalyze transformation of proton motive force (the difference between the electrochemical potentials of hydrogen ion across a coupling membrane) to the free energy of ATP phosphoryl potential. It is often stated that Fo×F1-ATPases operate as reversible chemo-mechano-electrical molecular machines that provide either ATP synthesis or hydrolysis depending on particular physiological demands of an organism; the microreversibility principle of the enzyme catalysis is usually taken as a dogma. Since 1980, the author has upheld the view that the mechanisms of ATP synthesis and hydrolysis by the Fo×F1 complex are different (Vinogradov, A. D. (2000) J. Exp. Biol., 203, 41-49). In this paper, the author proposes a new model considering the existence in coupling membranes of two non-equilibrium isoforms of Fo×F1 unidirectionally catalyzing synthesis and/or hydrolysis of ATP.


Assuntos
Trifosfato de Adenosina/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Biocatálise , Cloroplastos/enzimologia , Hidrólise , Cinética , Mitocôndrias/enzimologia , Paracoccus denitrificans/enzimologia , Força Próton-Motriz
17.
Proc Natl Acad Sci U S A ; 116(51): 25456-25461, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31776250

RESUMO

A method is proposed for analyzing fast (10 µs) single-molecule rotation trajectories in F1 adenosinetriphosphatase ([Formula: see text]-ATPase). This method is based on the distribution of jumps in the rotation angle that occur in the transitions during the steps between subsequent catalytic dwells. The method is complementary to the "stalling" technique devised by H. Noji et al. [Biophys. Rev. 9, 103-118, 2017], and can reveal multiple states not directly detectable as steps. A bimodal distribution of jumps is observed at certain angles, due to the system being in either of 2 states at the same rotation angle. In this method, a multistate theory is used that takes into account a viscoelastic fluctuation of the imaging probe. Using an established sequence of 3 specific states, a theoretical profile of angular jumps is predicted, without adjustable parameters, that agrees with experiment for most of the angular range. Agreement can be achieved at all angles by assuming a fourth state with an ∼10 µs lifetime and a dwell angle about 40° after the adenosine 5'-triphosphate (ATP) binding dwell. The latter result suggests that the ATP binding in one ß subunit and the adenosine 5'-diphosphate (ADP) release from another ß subunit occur via a transient whose lifetime is ∼10 µs and is about 6 orders of magnitude smaller than the lifetime for ADP release from a singly occupied [Formula: see text]-ATPase. An internal consistency test is given by comparing 2 independent ways of obtaining the relaxation time of the probe. They agree and are ∼15 µs.


Assuntos
ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Imagem Individual de Molécula/métodos , Hidrólise , Modelos Moleculares , Rotação
18.
J Antibiot (Tokyo) ; 72(12): 986-990, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31601980

RESUMO

A new antifungal compound YO-001A was found from the culture broth of Streptomyces sp. YO15-A001, which was isolated from a soil sample collected in Toyama Prefecture. YO-001A was identified through morphological changes-based screening of the rice blast fungus, Pyricularia oryzae (P. oryzae). YO-001A is a new 26-membered macrolide of the oligomycin family, which exhibits potent antifungal activity against P. oryzae with an IC50 of 0.012 µM by disrupting mitochondrial respiration via inhibition of the FOF1-ATPase activity.


Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Streptomyces/metabolismo , Antifúngicos/metabolismo , Antifúngicos/toxicidade , Ascomicetos/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Macrolídeos/química , Macrolídeos/farmacologia , Espectroscopia de Ressonância Magnética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estrutura Molecular , Oryza/microbiologia , Doenças das Plantas/microbiologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo , Microbiologia do Solo , Streptomyces/química , Streptomyces/isolamento & purificação
19.
Aquat Toxicol ; 216: 105315, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31561138

RESUMO

Blooms of cyanobacteria, a common event in eutrophic environments, result in the release of potentially toxic substances into the water. The cyanobacterium Radiocystis fernandoi produces microcystin (MC) and other peptides that may disturb homeostasis. This study evaluated the effect of intraperitoneal injections containing the crude extract (CE) of R. fernandoi strain R28 on the gills and kidneys of neotropical fish, Piaractus mesopotamicus, 3, 6 and 24 h post-injection. CE contained MC-RR, MC-YR and minor other oligopeptides. Plasma ions and the activities of the enzymes PP1 and PP2A, Na+/K+-ATPase (NKA), H+-ATPase (HA) and carbonic anhydrase (CA) were determined and morphological changes in both the gills and kidneys were characterized. Compared to controls, the concentration of Na+ within the plasma of P. mesopotamicus decreased after treatment with CE 3 h post treatment and increased after 24 h; the concentration of K+ decreased after 6 h. The activity of the endogenous PP1 and PP2A was unchanged in the gills and was inhibited in the kidneys 6 h after i.p. injection. In the gills, NKA activity increased after 3 h and decreased 6 h post i.p. exposure. Further, NKA activity did not differ from the controls 24-h post injection. In the kidneys, NKA, HA and CA activities were unaffected by treatment. The mitochondria-rich cell (MRC) density in the gills decreased after 3 h in the filament and 3 and 6 h in the lamellae and was restored to the control levels 24 h post-exposure. Filament epithelial hyperplasia and hypertrophy, lamellar atrophy and rupture of the lamellar epithelium were the most common effects of treatment in the gills. No histopathological changes occurred in the kidneys. This study demonstrates that a single dose of toxic CE from R. fernandoi can cause a transitory ion imbalance in P. mesopotamicus which is related to the changes in MRC levels and NKA activity. Ionic balance was recovered 24 h post i.p. injection, however, morphological changes that occurred in the gills took a longer amount of time to return to normal. To conclude, the effects of components contained within the CE of R. fernandoi may be harmful to P. mesopotamicus. In particular, the recovery of ionic regulation depends on MRC responses and histopathological changes produced by CE may affect gas exchange and other gill functions.


Assuntos
Caraciformes/fisiologia , Misturas Complexas/toxicidade , Cianobactérias/metabolismo , Exposição Ambiental , Osmorregulação , Animais , Caraciformes/sangue , Cloretos/sangue , Creatinina/sangue , Brânquias/efeitos dos fármacos , Brânquias/enzimologia , Brânquias/patologia , Íons/sangue , Rim/efeitos dos fármacos , Rim/enzimologia , Rim/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Osmorregulação/efeitos dos fármacos , Potássio/sangue , ATPases Translocadoras de Prótons/metabolismo , Sódio/sangue , ATPase Trocadora de Sódio-Potássio/metabolismo , Poluentes Químicos da Água/toxicidade
20.
Proc Natl Acad Sci U S A ; 116(40): 20226-20231, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31527254

RESUMO

Electrogenic proton pumps have been implicated in the generation of slow wave potentials (SWPs), damage-induced membrane depolarizations that activate the jasmonate (JA) defense pathway in leaves distal to wounds. However, no defined H+-ATPases have been shown to modulate these electrical signals. Pilot experiments revealed that the proton pump activator fusicoccin attenuated SWP duration in Arabidopsis Using mutant analyses, we identified Arabidopsis H+-ATPase 1 (AHA1) as a SWP regulator. The duration of the repolarization phase was strongly extended in reduced function aha1 mutants. Moreover, the duration of SWP repolarization was shortened in the presence of a gain-of-function AHA1 allele. We employed aphid electrodes to probe the effects of the aha1 mutation on wound-stimulated electrical activity in the phloem. Relative to the wild type, the aha1-7 mutant increased the duration and reduced the amplitudes of electrical signals in sieve tube cells. In addition to affecting electrical signaling, expression of the JA pathway marker gene JAZ10 in leaves distal to wounds was enhanced in aha1-7 Consistent with this, levels of wound-response jasmonoyl-isoleucine were enhanced in the mutant, as was defense against a lepidopteran herbivore. The work identifies a discrete member of the P-type ATPase superfamily with a role in leaf-to-leaf electrical signaling and plant defense.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Ciclopentanos/metabolismo , Potenciais da Membrana/genética , Redes e Vias Metabólicas , Oxilipinas/metabolismo , ATPases Translocadoras de Prótons/genética , Transdução de Sinais , Proteínas de Arabidopsis/metabolismo , Fenômenos Eletrofisiológicos , Herbivoria , Fenótipo , Bombas de Próton/genética , Bombas de Próton/metabolismo , ATPases Translocadoras de Prótons/metabolismo
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