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1.
Medicine (Baltimore) ; 99(28): e20934, 2020 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-32664090

RESUMO

This study aimed to investigate the myocardial protective effect of liquid sodium phosphocreatine cardiac arrest in extracorporeal circulation surgery treating infants with atrial septal defects.Eighty-four infants with atrial septal defects who required extracorporeal circulation surgery treatment at our hospital from January 2016 to June 2018 were divided into an observation group and a control group through a digitally randomized method, with 42 cases in each group. The control group adopted the conventional modified St Thomas II high potassium cold liquid crystal cardiac arrest, while the observation group adopted the liquid sodium phosphocreatine cardiac arrest.The myocardial enzyme indexes of the 2 groups 3, 6, 12, and 24 hours postoperatively were higher than before establishing the cardiopulmonary bypass and the enzyme indexes of the control group at the same time were higher than that of the observation group; adenosine triphosphate, adenosine diphosphate, and other energy levels and the postoperative recovery rate energy levels of the observation group were higher than those in the control group, the difference was statistically significant (P < .05).Liquid sodium phosphocreatine cardiac arrest used in extracorporeal circulation surgery treating infants with atrial septal defects can reduce myocardial ischemia-reperfusion injury, maintain energy supply during ischemia, strengthen the St Thomas II effect, and aid postoperative cardiac function recovery of high potassium cold liquid crystal cardiac arrest used in infants with atrial septal defects and treated with extracorporeal circulation surgery.


Assuntos
Ponte Cardiopulmonar/métodos , Cardiotônicos/farmacologia , Parada Cardíaca Induzida/métodos , Comunicação Interatrial/cirurgia , Fosfocreatina/farmacologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Cardiotônicos/administração & dosagem , Estudos de Casos e Controles , Pré-Escolar , Circulação Extracorpórea/métodos , Feminino , Parada Cardíaca/induzido quimicamente , Comunicação Interatrial/diagnóstico , Comunicação Interatrial/tratamento farmacológico , Humanos , Lactente , Masculino , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/química , Miocárdio/enzimologia , Preservação de Órgãos/métodos , Fosfocreatina/administração & dosagem , Período Pós-Operatório , Cloreto de Potássio/administração & dosagem , Cloreto de Potássio/farmacologia , Substâncias Protetoras/administração & dosagem , Recuperação de Função Fisiológica/efeitos dos fármacos
3.
Metabolism ; 108: 154257, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32370945

RESUMO

BACKGROUND: Protein degradation is an energy-dependent process, requiring ATP at multiple steps. However, reports conflict as to the relationship between intracellular energetics and the rate of proteasome-mediated protein degradation. METHODS: To determine whether the concentration of the adenine nucleotide pool (ATP + ADP + AMP) affects protein degradation in muscle cells, we overexpressed an AMP degrading enzyme, AMP deaminase 3 (AMPD3), via adenovirus in C2C12 myotubes. RESULTS: Overexpression of AMPD3 resulted in a dose- and time-dependent reduction of total adenine nucleotides (ATP, ADP and AMP) without increasing the ADP/ATP or AMP/ATP ratios. In agreement, the reduction of total adenine nucleotide concentration did not result in increased Thr172 phosphorylation of AMP-activated protein kinase (AMPK), a common indicator of intracellular energetic state. Furthermore, LC3 protein accumulation and ULK1 (Ser 555) phosphorylation were not induced. However, overall protein degradation and ubiquitin-dependent proteolysis were slowed by overexpression of AMPD3, despite unchanged content of several proteasome subunit proteins and proteasome activity in vitro under standard conditions. CONCLUSIONS: Altogether, these findings indicate that a physiologically relevant decrease in ATP content, without a concomitant increase in ADP or AMP, is sufficient to decrease the rate of protein degradation and activity of the ubiquitin-proteasome system in muscle cells. This suggests that adenine nucleotide degrading enzymes, such as AMPD3, may be a viable target to control muscle protein degradation and perhaps muscle mass.


Assuntos
AMP Desaminase/metabolismo , Trifosfato de Adenosina/metabolismo , Músculo Esquelético/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Fosforilação/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Proteólise , Ubiquitina/metabolismo
4.
Nat Commun ; 11(1): 1916, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317635

RESUMO

mHsp60-mHsp10 assists the folding of mitochondrial matrix proteins without the negative ATP binding inter-ring cooperativity of GroEL-GroES. Here we report the crystal structure of an ATP (ADP:BeF3-bound) ground-state mimic double-ring mHsp6014-(mHsp107)2 football complex, and the cryo-EM structures of the ADP-bound successor mHsp6014-(mHsp107)2 complex, and a single-ring mHsp607-mHsp107 half-football. The structures explain the nucleotide dependence of mHsp60 ring formation, and reveal an inter-ring nucleotide symmetry consistent with the absence of negative cooperativity. In the ground-state a two-fold symmetric H-bond and a salt bridge stitch the double-rings together, whereas only the H-bond remains as the equatorial gap increases in an ADP football poised to split into half-footballs. Refolding assays demonstrate obligate single- and double-ring mHsp60 variants are active, and complementation analysis in bacteria shows the single-ring variant is as efficient as wild-type mHsp60. Our work provides a structural basis for active single- and double-ring complexes coexisting in the mHsp60-mHsp10 chaperonin reaction cycle.


Assuntos
Chaperonina 10/química , Chaperonina 60/química , Mitocôndrias/química , Proteínas Mitocondriais/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Citosol/química , Humanos , Ligação de Hidrogênio , Hidrólise , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Dobramento de Proteína
5.
Am J Physiol Regul Integr Comp Physiol ; 318(5): R972-R980, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32233925

RESUMO

Mitochondria utilize the majority of oxygen (O2) consumed by aerobic organisms as the final electron acceptor for oxidative phosphorylation (OXPHOS) but also to generate reactive oxygen species (mtROS) that participate in cell signaling, physiological hormesis, and disease pathogenesis. Simultaneous monitoring of mtROS production and oxygen consumption (Jo2) from tissue mitochondrial preparations is an attractive investigative approach, but it introduces dynamic changes in media O2 concentration ([O2]) that can confound experimental results and interpretation. We utilized high-resolution fluorespirometry to evaluate Jo2 and hydrogen peroxide release (Jh2o2) from isolated mitochondria (Mt), permeabilized fibers (Pf), and tissue homogenates (Hm) prepared from murine heart and skeletal muscle across a range of experimental [O2]s typically encountered during respirometry protocols (400-50 µM). Results demonstrate notable variations in Jh2o2 across tissues and sample preparations during nonphosphorylating (LEAK) and OXPHOS-linked respiration states at 250 µM [O2] but a linear decline in Jh2o2 of 5-15% per 50-µM decrease in chamber [O2] in all samples. Jo2 was generally stable in Mt and Hm across [O2]s above 50 µM but tended to decline below 250 µM in Pf, leading to wide variations in assayed rates of Jh2o2/O2 across chamber [O2]s and sample preparations. Development of chemical background fluorescence from the H2O2 probe (Amplex Red) was also O2 sensitive, emphasizing relevant calibration considerations. This study highlights the importance of monitoring and reporting the chamber [O2] at which Jo2 and Jh2o2 are recorded during fluorespirometry experiments and provides a basis for selecting sample preparations for studies addressing the role of mtROS in physiology and disease.


Assuntos
Peróxido de Hidrogênio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Consumo de Oxigênio , Oxigênio/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Respiração Celular , Fluorometria , Cinética , Masculino , Camundongos , Modelos Biológicos , Fosforilação Oxidativa
6.
Mol Biotechnol ; 62(4): 252-259, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32146690

RESUMO

Classic toxicology studies often utilize in vivo animal models. Newer approaches employing in vitro organ-specific cellular models have been developed in recent years to help accelerate the speed and reduce the cost of traditional toxicology testing. Toward the goal of supporting in vitro cellular model research with a regulatory application in mind, we have developed a 'designer' human kidney cell line called HK2-Vi that can fluorescently measure the cytotoxicity of potential toxins on proximal tubule cell viability in a direct exposure in vitro model. HK2-Vi was designed to be a reagent-less kinetic assay that can yield data on short- or long-term cell viability after toxin exposure. To generate HK2-Vi, we used monocistronic lentiviral transduction methods to genetically engineer a human kidney cell line called HK-2 to stably co-express two transgenes. The first is Perceval HR, which encodes a fluorescent biosensor of both cytosolic ATP and ADP and the second is pHRed, which encodes a biosensor of cytosolic pH. Relative levels of cellular ATP and ADP effectively serve as a reliable and robust indicator of cell viability. Because the fluorescence Perceval HR is pH-dependent, we co-expressed the pHRed genetic biosensor to correct for variations in pH if necessary. Heterogenous populations of transduced renal cells were enriched by flow cytometry before monoclonal cellular populations were isolated by cell culture methods. A single clonal population of co-transduced cells expressing both Perceval HR and pHRed was selected to be HK2-Vi. This established cell line can now serve as a tool for in vitro toxicology testing and the methods described herein serve as a model for developing designer cell lines derived from other organs.


Assuntos
Linhagem Celular , Túbulos Renais Proximais/efeitos dos fármacos , Testes de Toxicidade , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Técnicas Biossensoriais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fluorescência , Engenharia Genética , Humanos , Concentração de Íons de Hidrogênio , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Transgenes
7.
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
8.
Adv Exp Med Biol ; 1202: 35-65, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32034708

RESUMO

The chapter is focused on the mechanism of action of metabotropic P2Y nucleotide receptors: P2Y1, P2Y2, P2Y12, P2Y14 and the ionotropic P2X7 receptor in glioma C6 cells. P2Y1 and P2Y12 both respond to ADP, but while P2Y1 links to PLC and elevates cytosolic Ca2+ concentration, P2Y12 negatively couples to adenylate cyclase, maintaining cAMP at low level. In glioma C6, these two P2Y receptors modulate activities of ERK1/2 and PI3K/Akt signaling and the effects depend on physiological conditions of the cells. During prolonged serum deprivation, cell growth is arrested, the expression of the P2Y1 receptor strongly decreases and P2Y12 becomes a major player responsible for ADP-evoked signal transduction. The P2Y12 receptor activates ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulates Akt activity, contributing to glioma invasiveness. In contrast, P2Y1 has an inhibitory effect on Akt pathway signaling. Furthermore, the P2X7 receptor, often responsible for apoptotic fate, is not involved in Ca2+elevation in C6 cells. The shift in nucleotide receptor expression from P2Y1 to P2Y12 during serum withdrawal, the cross talk between both receptors and the lack of P2X7 activity shows the precise self-regulating mechanism, enhancing survival and preserving the neoplastic features of C6 cells.


Assuntos
Glioma/metabolismo , Nucleotídeos/metabolismo , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais , Difosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glioma/patologia , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
9.
Genes Dev ; 34(5-6): 263-284, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32029451

RESUMO

ADP-ribosylation is an intricate and versatile posttranslational modification involved in the regulation of a vast variety of cellular processes in all kingdoms of life. Its complexity derives from the varied range of different chemical linkages, including to several amino acid side chains as well as nucleic acids termini and bases, it can adopt. In this review, we provide an overview of the different families of (ADP-ribosyl)hydrolases. We discuss their molecular functions, physiological roles, and influence on human health and disease. Together, the accumulated data support the increasingly compelling view that (ADP-ribosyl)hydrolases are a vital element within ADP-ribosyl signaling pathways and they hold the potential for novel therapeutic approaches as well as a deeper understanding of ADP-ribosylation as a whole.


Assuntos
ADP-Ribosilação/fisiologia , Difosfato de Adenosina/metabolismo , Hidrolases/química , Hidrolases/metabolismo , Humanos , Hidrolases/classificação , Domínios Proteicos , Relação Estrutura-Atividade
10.
Nat Struct Mol Biol ; 27(2): 202-209, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32042153

RESUMO

The mitochondrial membrane-bound AAA protein Bcs1 translocate substrates across the mitochondrial inner membrane without previous unfolding. One substrate of Bcs1 is the iron-sulfur protein (ISP), a subunit of the respiratory Complex III. How Bcs1 translocates ISP across the membrane is unknown. Here we report structures of mouse Bcs1 in two different conformations, representing three nucleotide states. The apo and ADP-bound structures reveal a homo-heptamer and show a large putative substrate-binding cavity accessible to the matrix space. ATP binding drives a contraction of the cavity by concerted motion of the ATPase domains, which could push substrate across the membrane. Our findings shed light on the potential mechanism of translocating folded proteins across a membrane, offer insights into the assembly process of Complex III and allow mapping of human disease-associated mutations onto the Bcs1 structure.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/química , Chaperonas Moleculares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cristalografia por Raios X , Camundongos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Transporte Proteico
11.
Exp Parasitol ; 210: 107842, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31978393

RESUMO

Free-living amoebae of the genus Acanthamoeba have been associated with keratitis and encephalitis. Some factors related to their pathogenic potential have been described, including the release of hydrolytic enzymes, and the adhesion and phagocytosis processes. However, other factors such as their effect over the hemodynamics and microcirculation elements have not been fully investigated. This work determines the in vitro activity of potentially pathogenic environmental isolates of Acanthamoeba genotype T4 and T5 over erythrocytes and platelets. The hemolytic activity (dependent and independent of contact), as well as the production of ADP of ten environmental isolates of Acanthamoeba obtained from dental units, combined emergency showers, dust, and hospital water, were measured. Tests were carried out over erythrocytes in suspension and blood agar plates, incubated at 4 °C, room temperature and 37 °C. Erythrophagocytosis and platelet aggregation assays were also performed. Live trophozoites of all of the isolates tested showed a hemolytic activity that was temperature-dependent. Over erythrocytes in suspension, variable hemolysis percentages were obtained: a maximum of 41% and a minimum of 15%. Regarding hemolysis over agar plates, two patterns of hemolysis were observed: double and simple halos. Conditioned medium and crude extracts of trophozoites did not show hemolytic activity. Erythrophagocytosis by Acanthamoeba was also observed; however, no production of ADP was determined by the employed methodology.


Assuntos
Acanthamoeba/fisiologia , Plaquetas/parasitologia , Meio Ambiente , Eritrócitos/parasitologia , Acanthamoeba/classificação , Acanthamoeba/genética , Acanthamoeba/patogenicidade , Difosfato de Adenosina/metabolismo , Doenças Transmissíveis Emergentes/parasitologia , Meios de Cultivo Condicionados , Eritrócitos/fisiologia , Genótipo , Hemólise , Humanos , Fagocitose , Agregação Plaquetária , Temperatura , Trofozoítos/classificação , Trofozoítos/genética , Trofozoítos/patogenicidade , Trofozoítos/fisiologia
12.
Plant Mol Biol ; 102(3): 323-337, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31900819

RESUMO

KEY MESSAGE: There is a link between PAP/SAL retrograde pathway, ethylene signaling and Fe metabolism in Arabidopsis. Nuclear gene expression is regulated by a diversity of retrograde signals that travel from organelles to the nucleus in a lineal or classical model. One such signal molecule is 3'-phosphoadenisine-5'-phosphate (PAP) and it's in vivo levels are regulated by SAL1/FRY1, a phosphatase enzyme located in chloroplast and mitochondria. This metabolite inhibits the action of a group of exorribonucleases which participate in post-transcriptional gene expression regulation. Transcriptome analysis of Arabidopsis thaliana mutant plants in PAP-SAL1 pathway revealed that the ferritin genes AtFER1, AtFER3, and AtFER4 are up-regulated. In this work we studied Fe metabolism in three different mutants of the PAP/SAL1 retrograde pathway. Mutant plants showed increased Fe accumulation in roots, shoots and seeds when grown in Fe-sufficient condition, and a constitutive activation of the Strategy I Fe uptake genes. As a consequence, they grew more vigorously than wild type plants in Fe-deficient medium. However, when mutant plants grown in Fe-deficient conditions were sprayed with Fe in their leaves, they were unable to deactivate root Fe uptake. Ethylene synthesis inhibition revert the constitutive Fe uptake phenotype. We propose that there is a link between PAP/SAL pathway, ethylene signaling and Fe metabolism.


Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ferro/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais , Difosfato de Adenosina/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Clorofila , Cloroplastos/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Homeostase , Mitocôndrias/metabolismo , Mutação , Monoéster Fosfórico Hidrolases/genética , Folhas de Planta/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo
13.
Nat Commun ; 11(1): 547, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992706

RESUMO

TrkH is a bacterial ion channel implicated in K+ uptake and pH regulation. TrkH assembles with its regulatory protein, TrkA, which closes the channel when bound to ADP and opens it when bound to ATP. However, it is unknown how nucleotides control the gating of TrkH through TrkA. Here we report the structures of the TrkH-TrkA complex in the presence of ADP or ATP. TrkA forms a tetrameric ring when bound to ADP and constrains TrkH to a closed conformation. The TrkA ring splits into two TrkA dimers in the presence of ATP and releases the constraints on TrkH, resulting in an open channel conformation. Functional studies show that both the tetramer-to-dimer conversion of TrkA and the loss of constraints on TrkH are required for channel gating. In addition, deletion of TrkA in Escherichia coli depolarizes the cell, suggesting that the TrkH-TrkA complex couples changes in intracellular nucleotides to membrane potential.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Potenciais da Membrana/fisiologia , Canais de Potássio/química , Canais de Potássio/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Difosfato de Adenosina , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transporte Biológico/fisiologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutagênese , Potássio/metabolismo , Canais de Potássio/genética , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência , Vibrio parahaemolyticus/genética , Difração de Raios X
14.
Commun Biol ; 3(1): 46, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992852

RESUMO

The hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI form a 3.3 MDa cage, proposed to assist assembly of specific respiratory complexes in E. coli. Here, we show that inside the LdcI-RavA cage, RavA hexamers adopt an asymmetric spiral conformation in which the nucleotide-free seam is constrained to two opposite orientations. Cryo-EM reconstructions of free RavA reveal two co-existing structural states: an asymmetric spiral, and a flat C2-symmetric closed ring characterised by two nucleotide-free seams. The closed ring RavA state bears close structural similarity to the pseudo two-fold symmetric crystal structure of the AAA+ unfoldase ClpX, suggesting a common ATPase mechanism. Based on these structures, and in light of the current knowledge regarding AAA+ ATPases, we propose different scenarios for the ATP hydrolysis cycle of free RavA and the LdcI-RavA cage-like complex, and extend the comparison to other AAA+ ATPases of clade 7.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Carboxiliases/química , Carboxiliases/metabolismo , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Difosfato de Adenosina/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Cristalização , Cristalografia por Raios X , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Ligação Proteica , Conformação Proteica em alfa-Hélice
15.
Nucleic Acids Res ; 48(1): 200-211, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31665475

RESUMO

Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution. Whereas ADP-DnaA was predominantly monomeric, AMP-PNP-DnaA (a non-hydrolysable ATP-analog bound-DnaA) was oligomeric, primarily dimeric. Functional studies using DnaA mutants revealed that DnaA(H136Q) is defective in initiating replication in vivo. The mutant retains high-affinity ATP binding, but was defective in producing replication-competent initiation complexes. Docking of ATP on a structure of E. coli DnaA, modeled upon the crystallographic structure of Aquifex aeolicus DnaA, predicts a hydrogen bond between ATP and imidazole ring of His136, which is disrupted when Gln is present at position 136. SAXS performed on AMP-PNP-DnaA (H136Q) indicates that the protein has lost its ability to form oligomers. These results show the importance of high ATP in DnaA oligomerization and its dependence on the His136 residue.


Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Replicação do DNA , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cromossomos Bacterianos/química , Cromossomos Bacterianos/metabolismo , Cristalografia por Raios X , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Escherichia coli/metabolismo , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Mutação , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Origem de Replicação , Termodinâmica
16.
Arch Biochem Biophys ; 680: 108226, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31843644

RESUMO

SIRT7, an epigenetic modulator is related to several important cellular processes like aging, genome stability, and metabolism. The mechanistic and regulatory aspect of this enzyme needs to be explored. SIRT7 contains a conserved catalytic core with long flanking N- and C-terminal extensions. We find that the N terminus is involved in substrate binding, thus also in its dual enzyme activity i.e. deacetylation and ADP ribosylation. The C-terminus is not essential for its catalysis. Mutation of certain residues at the active site suggests that mono ADP-ribosylation and deacetylation are two distinct activities of SIRT7. In this study, we also find that the SIRT7 enzyme can specifically transfer a single moiety of ADP ribose on other nuclear proteins, with a preference for NAD+. For this, the ADPr transfer follows the enzymatic reaction mechanism. Nicotinamide and certain metal ions have a significant negative effect on this mono ADP ribosylation process. A comparison of these dual activities suggests SIRT7's preference for the mono ADPr transfer over its deacetylation of H3K18Ac. Mono ADP ribosylation in cells is often linked to different metabolic disease conditions. This kind of modification of transcription factors, p53 and ELK4 by SIRT7 may play a key role in maintaining the tumor phenotype. Thus, SIRT7 becomes an important therapeutic hotspot for drug designing against several diseases. Finally, we can also relate SIRT7 to the DNA repair process through ADP ribosylation of one of its key players, PARP1. Here, SIRT7 positively regulates the PARP1 activity.


Assuntos
ADP-Ribosilação , Sirtuínas/metabolismo , ADP Ribose Transferases/química , ADP Ribose Transferases/metabolismo , Difosfato de Adenosina/metabolismo , Domínio Catalítico , Histonas/metabolismo , Humanos , NAD/metabolismo , Mapas de Interação de Proteínas , Sirtuínas/química
17.
Proc Natl Acad Sci U S A ; 117(2): 1167-1173, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31879356

RESUMO

Chemiosmosis and substrate-level phosphorylation are the 2 mechanisms employed to form the biological energy currency adenosine triphosphate (ATP). During chemiosmosis, a transmembrane electrochemical ion gradient is harnessed by a rotary ATP synthase to phosphorylate adenosine diphosphate to ATP. In microorganisms, this ion gradient is usually composed of [Formula: see text], but it can also be composed of Na+ Here, we show that the strictly anaerobic rumen bacterium Pseudobutyrivibrio ruminis possesses 2 ATP synthases and 2 distinct respiratory enzymes, the ferredoxin:[Formula: see text] oxidoreductase (Rnf complex) and the energy-converting hydrogenase (Ech complex). In silico analyses revealed that 1 ATP synthase is [Formula: see text]-dependent and the other Na+-dependent, which was validated by biochemical analyses. Rnf and Ech activity was also biochemically identified and investigated in membranes of P. ruminis Furthermore, the physiology of the rumen bacterium and the role of the energy-conserving systems was investigated in dependence of 2 different catabolic pathways (the Embden-Meyerhof-Parnas or the pentose-phosphate pathway) and in dependence of Na+ availability. Growth of P. ruminis was greatly stimulated by Na+, and a combination of physiological, biochemical, and transcriptional analyses revealed the role of the energy conserving systems in P. ruminis under different metabolic scenarios. These data demonstrate the use of a 2-component ion circuit for [Formula: see text] bioenergetics and a 2nd 2-component ion circuit for Na+ bioenergetics in a strictly anaerobic rumen bacterium. In silico analyses infer that these 2 circuits are prevalent in a number of other strictly anaerobic microorganisms.


Assuntos
Complexos de ATP Sintetase/metabolismo , Trifosfato de Adenosina/metabolismo , Clostridiales/metabolismo , Metabolismo Energético/fisiologia , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Clostridiales/enzimologia , Clostridiales/genética , Clostridiales/crescimento & desenvolvimento , Metabolismo Energético/genética , Ferredoxinas/metabolismo , Hidrogenase/metabolismo , Transporte de Íons , Oxirredução , Oxirredutases/metabolismo , Sódio/metabolismo
18.
Proc Natl Acad Sci U S A ; 117(1): 395-404, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31862713

RESUMO

Hsp90 plays a central role in cell homeostasis by assisting folding and maturation of a large variety of clients. It is a homo-dimer, which functions via hydrolysis of ATP-coupled to conformational changes. Hsp90's conformational cycle in the absence of cochaperones is currently postulated as apo-Hsp90 being an ensemble of "open"/"closed" conformations. Upon ATP binding, Hsp90 adopts an active ATP-bound closed conformation where the N-terminal domains, which comprise the ATP binding site, are in close contact. However, there is no consensus regarding the conformation of the ADP-bound Hsp90, which is considered important for client release. In this work, we tracked the conformational states of yeast Hsp90 at various stages of ATP hydrolysis in frozen solutions employing electron paramagnetic resonance (EPR) techniques, particularly double electron-electron resonance (DEER) distance measurements. Using rigid Gd(III) spin labels, we found the C domains to be dimerized with same distance distribution at all hydrolysis states. Then, we substituted the ATPase Mg(II) cofactor with paramagnetic Mn(II) and followed the hydrolysis state using hyperfine spectroscopy and measured the inter-N-domain distance distributions via Mn(II)-Mn(II) DEER. The point character of the Mn(II) spin label allowed us resolve 2 different closed states: The ATP-bound (prehydrolysis) characterized by a distance distribution having a maximum of 4.3 nm, which broadened and shortened, shifting the mean to 3.8 nm at the ADP-bound state (posthydrolysis). This provides experimental evidence to a second closed conformational state of Hsp90 in solution, referred to as "compact." Finally, the so-called high-energy state, trapped by addition of vanadate, was found structurally similar to the posthydrolysis state.


Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Domínios Proteicos/genética , Leveduras/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Manganês/química , Modelos Moleculares , Mutação , Marcadores de Spin , Leveduras/genética
19.
Food Chem ; 311: 126008, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31869639

RESUMO

The effects of hydrogen peroxide (H2O2) on the contents of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP), the level of energy charge, and the activity of adenosine triphosphatase (ATPase) in pulp of harvested longan fruit, and its association with longan pulp breakdown occurrence were studied. The results showed that, compared to the control longans, H2O2-treated longans exhibited a higher index of pulp breakdown, a higher amount of AMP, but lower levels of ATP, ADP and energy charge. H2O2-treated longans also exhibited lower activities of Mg2+-ATPase, Ca2+-ATPase, and H+-ATPase in mitochondrial membrane, vacuolar membrane, and plasma membrane as compared to the control longans. Above findings demonstrated that H2O2 caused longan pulp breakdown by depleting energy and lowering the ATPase activity, indicating H2O2-induced pulp breakdown in harvested longan fruit was due to energy deficit.


Assuntos
Adenosina Trifosfatases/metabolismo , Peróxido de Hidrogênio/metabolismo , Sapindaceae/metabolismo , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Metabolismo Energético/efeitos dos fármacos , Armazenamento de Alimentos , Frutas/metabolismo , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo
20.
Platelets ; 31(1): 68-78, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30810440

RESUMO

Despite the transient hyporeactivity of neonatal platelets, full-term neonates do not display a bleeding tendency, suggesting potential compensatory mechanisms which allow for balanced and efficient neonatal hemostasis. This study aimed to utilize small-volume, whole blood platelet functional assays to assess the neonatal platelet response downstream of the hemostatic platelet agonists thrombin and adenosine diphosphate (ADP). Thrombin activates platelets via the protease-activated receptors (PARs) 1 and 4, whereas ADP signals via the receptors P2Y1 and P2Y12 as a positive feedback mediator of platelet activation. We observed that neonatal and cord blood-derived platelets exhibited diminished PAR1-mediated granule secretion and integrin activation relative to adult platelets, correlating to reduced PAR1 expression by neonatal platelets. PAR4-mediated granule secretion was blunted in neonatal platelets, correlating to lower PAR4 expression as compared to adult platelets, while PAR4 mediated GPIIb/IIIa activation was similar between neonatal and adult platelets. Under high shear stress, cord blood-derived platelets yielded similar thrombin generation rates but reduced phosphatidylserine expression as compared to adult platelets. Interestingly, we observed enhanced P2Y1/P2Y12-mediated dense granule trafficking in neonatal platelets relative to adults, although P2Y1/P2Y12 expression in neonatal, cord, and adult platelets were similar, suggesting that neonatal platelets may employ an ADP-mediated positive feedback loop as a potential compensatory mechanism for neonatal platelet hyporeactivity.


Assuntos
Plaquetas/metabolismo , Grânulos Citoplasmáticos/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte Biológico , Biomarcadores , Coagulação Sanguínea , Humanos , Recém-Nascido , Integrinas/metabolismo , Ativação Plaquetária , Agregação Plaquetária , Resistência ao Cisalhamento , Trombina/metabolismo
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