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1.
PLoS Comput Biol ; 16(7): e1007996, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32667909

RESUMO

Cortical spreading depression (CSD) is the propagation of a relatively slow wave in cortical brain tissue that is linked to a number of pathological conditions such as stroke and migraine. Most of the existing literature investigates the dynamics of short term phenomena such as the depolarization and repolarization of membrane potentials or large ion shifts. Here, we focus on the clinically-relevant hour-long state of neurovascular malfunction in the wake of CSDs. This dysfunctional state involves widespread vasoconstriction and a general disruption of neurovascular coupling. We demonstrate, using a mathematical model, that dissolution of calcium that has aggregated within the mitochondria of vascular smooth muscle cells can drive an hour-long disruption. We model the rate of calcium clearance as well as the dynamical implications on overall blood flow. Based on reaction stoichiometry, we quantify a possible impact of calcium phosphate dissolution on the maintenance of F0F1-ATP synthase activity.


Assuntos
Depressão Alastrante da Atividade Elétrica Cortical , Potenciais da Membrana , Mitocôndrias/metabolismo , Vasoconstrição , Trifosfato de Adenosina/química , Cálcio/química , Fosfatos de Cálcio/química , Córtex Cerebral/fisiopatologia , Circulação Cerebrovascular , Citosol/química , Retículo Endoplasmático/química , Substância Cinzenta/fisiopatologia , Humanos , Modelos Teóricos , Acoplamento Neurovascular , Oscilometria , Oxigênio/química , Fosforilação , ATPases Translocadoras de Prótons/química , Acidente Vascular Cerebral/fisiopatologia
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
BMC Med Genet ; 21(1): 23, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019516

RESUMO

BACKGROUND: The prevalence of Parkinson's disease (PD) is increasing in sub-Saharan Africa, but little is known about the genetics of PD in these populations. Due to their unique ancestry and diversity, sub-Saharan African populations have the potential to reveal novel insights into the pathobiology of PD. In this study, we aimed to characterise the genetic variation in known and novel PD genes in a group of Black South African and Nigerian patients. METHODS: We recruited 33 Black South African and 14 Nigerian PD patients, and screened them for sequence variants in 751 genes using an Ion AmpliSeq™ Neurological Research panel. We used bcftools to filter variants and annovar software for the annotation. Rare variants were prioritised using MetaLR and MetaSVM prediction scores. The effect of a variant on ATP13A2's protein structure was investigated by molecular modelling. RESULTS: We identified 14,655 rare variants with a minor allele frequency ≤ 0.01, which included 2448 missense variants. Notably, no common pathogenic mutations were identified in these patients. Also, none of the known PD-associated mutations were found highlighting the need for more studies in African populations. Altogether, 54 rare variants in 42 genes were considered deleterious and were prioritized, based on MetaLR and MetaSVM scores, for follow-up studies. Protein modelling showed that the S1004R variant in ATP13A2 possibly alters the conformation of the protein. CONCLUSIONS: We identified several rare variants predicted to be deleterious in sub-Saharan Africa PD patients; however, further studies are required to determine the biological effects of these variants and their possible role in PD. Studies such as these are important to elucidate the genetic aetiology of this disorder in patients of African ancestry.


Assuntos
Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Doença de Parkinson/genética , ATPases Translocadoras de Prótons/genética , Adulto , Grupo com Ancestrais do Continente Africano/genética , Idoso , Idoso de 80 Anos ou mais , Feminino , Frequência do Gene , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Anotação de Sequência Molecular , Mutação de Sentido Incorreto , Nigéria/epidemiologia , Doença de Parkinson/epidemiologia , Doença de Parkinson/patologia , Mutação Puntual , África do Sul/epidemiologia
9.
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
10.
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
11.
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
12.
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
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.
Aquat Toxicol ; 218: 105363, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31783302

RESUMO

The aquatic environment is continuously under threat because it is the final receptor and sink of waste streams. The development of industry, mining activities and agriculture gave rise to an increase in metal pollution in the aquatic system. Thus a wide occurrence of metal mixtures exists in the aquatic environment. The assessment of mixture stress remains a challenge considering that we can not predict the toxicity of a mixture on the basis of single compounds. Therefore the analysis of the effects of environmentally relevant waterborne mixtures is needed to improve our understanding of the impact of metal pollution in aquatic ecosystems. Our aim was to assess whether 10 % of the concentration of the 96 h LC50 (the concentration that is lethal to 50 % of the population in 96 h) of individual metal exposures can be considered as a "safe" concentration when applied in a trinomial mixture. Therefore, common carp were exposed to a sublethal mixture of Cu 0.07 ±â€¯0.001 µM (4.3 ±â€¯0.6 µg/L), Zn 2.71 ±â€¯0.81 µM (176.9 ±â€¯52.8 µg/L) and Cd 0.03 ±â€¯0.0004 µM (3.0 ±â€¯0.4 µg/L) at 20 °C for a period of one week. Parameters assessed included survival rate, bioaccumulation and physiological biomarkers related to ionoregulation and defensive mechanisms such as MT induction. Our results showed a sharp increase in Cu and Cd concentration in gills within the first day of exposure while Zn levels remained stable. The accumulation of these metals led to a Na drop in gills, liver and muscle as well as a decreased K content in the liver. Biomarkers related to Na uptake were also affected: on the first day gene expression for H+-ATPase was transiently increased while a concomitant decreased gene expression of the Na+/H+ exchanger occurred. A fivefold induction of metallothionein gene expression was reported during the entire duration of the experiment. Despite the adverse effects on ionoregulation all fish survived, indicating that common carp are able to cope with these low metal concentrations, at least during a one week exposure.


Assuntos
Bioacumulação , Cádmio/toxicidade , Carpas/metabolismo , Cobre/toxicidade , Poluentes Químicos da Água/toxicidade , Zinco/toxicidade , Animais , Bioacumulação/genética , Biomarcadores/metabolismo , Cádmio/metabolismo , Carpas/genética , Cobre/metabolismo , Ecossistema , Eletrólitos/metabolismo , Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Dose Letal Mediana , Metalotioneína/genética , ATPases Translocadoras de Prótons/genética , Poluentes Químicos da Água/metabolismo , Zinco/metabolismo
15.
Biochim Biophys Acta Gen Subj ; 1864(2): 129330, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-30926442

RESUMO

Micro-chamber arrays enable highly sensitive and quantitative bioassays at the single-molecule level. Accordingly, they are widely used for ultra-sensitive biomedical applications, e.g., digital PCR and digital ELISA. However, the versatility of micro-chambers is generally limited to reactions in aqueous solutions, although various functions of membrane proteins are extremely important. To address this issue, microsystems using arrayed micro-sized chambers sealed with lipid bilayers, referred to here as a "biomembrane microsystems", have been developed by many research groups for the analysis of membrane proteins. In this review, I would like to introduce recent progress on the single molecule analysis of membrane transport proteins using a biomembrane microsystem, and discuss the future prospects for its use in analytical and pharmacological applications.


Assuntos
Bicamadas Lipídicas/química , Proteínas de Membrana Transportadoras/química , Imagem Individual de Molécula/métodos , Técnicas Citológicas/instrumentação , Técnicas Citológicas/métodos , Ensaio de Imunoadsorção Enzimática , Desenho de Equipamento , Proteínas Hemolisinas/química , Concentração de Íons de Hidrogênio , Potenciais da Membrana , Fosfolipídeos/química , Reação em Cadeia da Polimerase , ATPases Translocadoras de Prótons/química
16.
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
17.
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
18.
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
19.
Science ; 366(6471): 1338-1345, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31753851

RESUMO

Eukaryotic genomes are folded into loops and topologically associating domains, which contribute to chromatin structure, gene regulation, and gene recombination. These structures depend on cohesin, a ring-shaped DNA-entrapping adenosine triphosphatase (ATPase) complex that has been proposed to form loops by extrusion. Such an activity has been observed for condensin, which forms loops in mitosis, but not for cohesin. Using biochemical reconstitution, we found that single human cohesin complexes form DNA loops symmetrically at rates up to 2.1 kilo-base pairs per second. Loop formation and maintenance depend on cohesin's ATPase activity and on NIPBL-MAU2, but not on topological entrapment of DNA by cohesin. During loop formation, cohesin and NIPBL-MAU2 reside at the base of loops, which indicates that they generate loops by extrusion. Our results show that cohesin and NIPBL-MAU2 form an active holoenzyme that interacts with DNA either pseudo-topologically or non-topologically to extrude genomic interphase DNA into loops.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Proteínas de Ligação a DNA/química , DNA/química , Conformação de Ácido Nucleico , ATPases Translocadoras de Prótons/química , Células HeLa , Holoenzimas/química , Humanos
20.
Science ; 366(6471): 1345-1349, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31780627

RESUMO

Cohesin is a chromosome-bound, multisubunit adenosine triphosphatase complex. After loading onto chromosomes, it generates loops to regulate chromosome functions. It has been suggested that cohesin organizes the genome through loop extrusion, but direct evidence is lacking. Here, we used single-molecule imaging to show that the recombinant human cohesin-NIPBL complex compacts both naked and nucleosome-bound DNA by extruding DNA loops. DNA compaction by cohesin requires adenosine triphosphate (ATP) hydrolysis and is force sensitive. This compaction is processive over tens of kilobases at an average rate of 0.5 kilobases per second. Compaction of double-tethered DNA suggests that a cohesin dimer extrudes DNA loops bidirectionally. Our results establish cohesin-NIPBL as an ATP-driven molecular machine capable of loop extrusion.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , DNA/química , Conformação de Ácido Nucleico , ATPases Translocadoras de Prótons/química , Humanos , Nucleossomos/química , Multimerização Proteica , Imagem Individual de Molécula
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