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1.
Cell ; 182(6): 1560-1573.e13, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32783916

RESUMO

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.


Assuntos
Metiltransferases/química , RNA Helicases/química , RNA Replicase/química , Proteínas não Estruturais Virais/química , Replicação Viral , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Betacoronavirus/genética , Betacoronavirus/metabolismo , Betacoronavirus/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Holoenzimas/química , Holoenzimas/metabolismo , Magnésio/metabolismo , Metiltransferases/metabolismo , Ligação Proteica , RNA Helicases/metabolismo , RNA Replicase/metabolismo , RNA Viral/química , Proteínas não Estruturais Virais/metabolismo
2.
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.
PLoS Comput Biol ; 16(7): e1008079, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32730244

RESUMO

Exercise training elicits profound metabolic adaptations in skeletal muscle cells. A key molecule in coordinating these adaptations is AMP-activated protein kinase (AMPK), whose activity increases in response to cellular energy demand. AMPK activity dynamics are primarily controlled by the adenine nucleotides ADP and AMP, but how each contributes to its control in skeletal muscle during exercise is unclear. We developed and validated a mathematical model of AMPK signaling dynamics, and then applied global parameter sensitivity analyses with data-informed constraints to predict that AMPK activity dynamics are determined principally by ADP and not AMP. We then used the model to predict the effects of two additional direct-binding activators of AMPK, ZMP and Compound 991, further validating the model and demonstrating its applicability to understanding AMPK pharmacology. The relative effects of direct-binding activators can be understood in terms of four properties, namely their concentrations, binding affinities for AMPK, abilities to enhance AMPK phosphorylation, and the magnitudes of their allosteric activation of AMPK. Despite AMP's favorable values in three of these four properties, ADP is the dominant controller of AMPK activity dynamics in skeletal muscle during exercise by virtue of its higher concentration compared to that of AMP.


Assuntos
Proteínas Quinases Ativadas por AMP , Difosfato de Adenosina , Exercício Físico/fisiologia , Músculo Esquelético , Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacocinética , Animais , Biologia Computacional , Humanos , Camundongos , Modelos Biológicos , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Transdução de Sinais/fisiologia
4.
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
5.
Life Sci ; 256: 117862, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32473244

RESUMO

Vascular smooth muscle cells (VSMCs) exhibit a high degree of plasticity when they undergo the progression from a normal to a disease condition, which makes them a potential target for evaluating early markers and for the development of new therapies. Purinergic signalling plays a key role in vascular tonus control, ATP being an inductor of vasoconstriction, whereas adenosine mediates a vasodilation effect antagonising the ATP actions. The control of extracellular ATP and adenosine levels is done by ectonucleotidases, which represent a potential target to be evaluated in the progression of cardiovascular diseases. In this study, we analysed the basal activity and expression of the ectonucleotidases in aortic rat VSMCs, and we further performed in silico analysis to determine the expression of those enzymes in conditions that mimicked vascular diseases. Cultured in vitro VSMCs showed a prominent expression of Entpd1 followed by Entpd2 and Nt5e (CD73) and very low levels of Entpd3. Slightly faster AMP hydrolysis was observed when compared to ATP and ADP nucleotides. In silico analysis showed that the ectonucleotidases were modulated after induction of conditions that can lead to vascular diseases such as, hypertensive and hypotensive mice models (Nt5e); exposition to high-fat (Entpd1 and Entpd2) or high-phosphate (Nt5e) diet; mechanical stretch (Entpd1, Entpd2 and Nt5e); and myocardial infarction (Entpd1). Our data show that VSMCs are able to efficiently metabolise the extracellular nucleotides generating adenosine. The modulation of Entpd1, Entdp2 and Nt5e in vascular diseases suggests these ectoenzymes as potential targets or markers to be investigated in future studies.


Assuntos
5'-Nucleotidase/metabolismo , Adenosina Trifosfatases/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Músculo Liso Vascular/patologia , Doenças Vasculares/fisiopatologia , Adenosina/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Aorta/citologia , Simulação por Computador , Proteínas Ligadas por GPI/metabolismo , Camundongos , Músculo Liso Vascular/enzimologia , Nucleotídeos/metabolismo , Ratos , Ratos Wistar , Doenças Vasculares/enzimologia
6.
Proc Natl Acad Sci U S A ; 117(24): 13519-13528, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32461373

RESUMO

Networks of branched actin filaments formed by Arp2/3 complex generate and experience mechanical forces during essential cellular functions, including cell motility and endocytosis. External forces regulate the assembly and architecture of branched actin networks both in vitro and in cells. Considerably less is known about how mechanical forces influence the disassembly of actin filament networks, specifically, the dissociation of branches. We used microfluidics to apply force to branches formed from purified muscle actin and fission yeast Arp2/3 complex and observed debranching events in real time with total internal reflection fluorescence microscopy. Low forces in the range of 0 pN to 2 pN on branches accelerated their dissociation from mother filaments more than two orders of magnitude, from hours to <1 min. Neither force on the mother filament nor thermal fluctuations in mother filament shape influenced debranching. Arp2/3 complex at branch junctions adopts two distinct mechanical states with different sensitivities to force, which we name "young/strong" and "old/weak." The "young/strong" state 1 has adenosine 5'-diphosphate (ADP)-P i bound to Arp2/3 complex. Phosphate release converts Arp2/3 complex into the "old/weak" state 2 with bound ADP, which is 20 times more sensitive to force than state 1. Branches with ADP-Arp2/3 complex are more sensitive to debranching by fission yeast GMF (glia maturation factor) than branches with ADP-P i -Arp2/3 complex. These findings suggest that aging of branch junctions by phosphate release from Arp2/3 complex and mechanical forces contribute to disassembling "old" actin filament branches in cells.


Assuntos
Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Fosfatos/metabolismo , Actinas/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Fator de Maturação da Glia/metabolismo , Microfluídica , Microscopia de Fluorescência , Modelos Biológicos , Ligação Proteica , Coelhos , Schizosaccharomyces/metabolismo , Estresse Mecânico
7.
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
8.
Clin Hemorheol Microcirc ; 75(4): 467-474, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32390610

RESUMO

BACKGROUND: Platelet activation is associated with abdominal obesity and exercise training is an important modulator of body weight. OBJECTIVE: We investigated the effects of two high intensity interval exercise (HIIE) protocols of different intensity and duration on platelet indices and platelet aggregation in overweight men. METHODS: Ten overweight men performed 6 intervals of 30s exercise at 110% of peak power output (PPO) interspersed by 3 : 30 min active recovery (1/7 protocol) at 40% of PPO and 6 intervals of 2 min exercise at 85% of PPO interspersed by 2 min active recovery (1/1 protocol) at 30% of PPO in two separate sessions. Platelet indices and platelet aggregation were measured before and immediately after both HIIEs. RESULTS: Platelet indices increased significantly following HIIE (P < 0.05), though, significant differences between the two protocols were only detected for platelet count, which was markedly increased following 1/1 protocol. Platelet aggregation increased significantly (P < 0.05) in response to the two HIIE protocols, with no significant difference being observed between the two protocols (P > 0.05). CONCLUSIONS: It is concluded that HIIE leads to transient increases in markers of thrombus formation and that work to rest ratio is an important factor when investigating the changes in thrombocytosis following HIIE.


Assuntos
Difosfato de Adenosina/metabolismo , Exercício Físico/fisiologia , Treinamento Intervalado de Alta Intensidade/métodos , Sobrepeso/sangue , Agregação Plaquetária/fisiologia , Contagem de Plaquetas/métodos , Adulto , Humanos , Masculino , Adulto Jovem
9.
Nucleic Acids Res ; 48(10): 5457-5466, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282902

RESUMO

The decision whether to replicate DNA is crucial for cell survival, not only to proliferate in favorable conditions, but also to adopt to environmental changes. When a bacteria encounters stress, e.g. starvation, it launches the stringent response, to arrest cell proliferation and to promote survival. During the stringent response a vast amount of polymer composed of phosphate residues, i.e. inorganic polyphosphate (PolyP) is synthesized from ATP. Despite extensive research on PolyP, we still lack the full understanding of the PolyP role during stress. It is also elusive what is the mechanism of DNA replication initiation arrest in starved Escherichia coli cells. Here, we show that during stringent response PolyP activates Lon protease to degrade selectively the replication initiaton protein DnaA bound to ADP, but not ATP. In contrast to DnaA-ADP, the DnaA-ATP does not interact with PolyP, but binds to dnaA promoter to block dnaA transcription. The systems controlling the ratio of nucleotide states of DnaA continue to convert DnaA-ATP to DnaA-ADP, which is proteolysed by Lon, thereby resulting in the DNA replication initiation arrest. The uncovered regulatory mechanism interlocks the PolyP-dependent protease activation with the ATP/ADP cycle of dual-functioning protein essential for bacterial cell proliferation.


Assuntos
Proteínas de Bactérias/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Polifosfatos/metabolismo , Protease La/metabolismo , Estresse Fisiológico/genética , Difosfato de Adenosina/metabolismo , Proteólise
10.
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
11.
Proc Natl Acad Sci U S A ; 117(19): 10593-10602, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32332165

RESUMO

A physiological role for long-chain acyl-CoA esters to activate ATP-sensitive K+ (KATP) channels is well established. Circulating palmitate is transported into cells and converted to palmitoyl-CoA, which is a substrate for palmitoylation. We found that palmitoyl-CoA, but not palmitic acid, activated the channel when applied acutely. We have altered the palmitoylation state by preincubating cells with micromolar concentrations of palmitic acid or by inhibiting protein thioesterases. With acyl-biotin exchange assays we found that Kir6.2, but not sulfonylurea receptor (SUR)1 or SUR2, was palmitoylated. These interventions increased the KATP channel mean patch current, increased the open time, and decreased the apparent sensitivity to ATP without affecting surface expression. Similar data were obtained in transfected cells, rat insulin-secreting INS-1 cells, and isolated cardiac myocytes. Kir6.2ΔC36, expressed without SUR, was also positively regulated by palmitoylation. Mutagenesis of Kir6.2 Cys166 prevented these effects. Clinical variants in KCNJ11 that affect Cys166 had a similar gain-of-function phenotype, but was more pronounced. Molecular modeling studies suggested that palmitoyl-C166 and selected large hydrophobic mutations make direct hydrophobic contact with Kir6.2-bound PIP2 Patch-clamp studies confirmed that palmitoylation of Kir6.2 at Cys166 enhanced the PIP2 sensitivity of the channel. Physiological relevance is suggested since palmitoylation blunted the regulation of KATP channels by α1-adrenoreceptor stimulation. The Cys166 residue is conserved in some other Kir family members (Kir6.1 and Kir3, but not Kir2), which are also subject to regulated palmitoylation, suggesting a general mechanism to control the open state of certain Kir channels.


Assuntos
Canais KATP/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Acil Coenzima A/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cisteína/metabolismo , Células HEK293 , Humanos , Canais KATP/genética , Lipoilação/fisiologia , Mutagênese/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp/métodos , Canais de Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Cultura Primária de Células , Ratos , Receptores Sulfonilureia/genética
12.
Dalton Trans ; 49(19): 6302-6311, 2020 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-32334418

RESUMO

Albeit arsenic As(iii) is a well-known carcinogenic contaminant, the modalities by which it interacts with living organisms are still elusive. Details pertaining to the binding properties of As(iii) by common nucleotides such as AMP, ADP and ATP are indeed mostly unknown. Here we present an investigation, conducted via experimental and quantum-based computational approaches, on the stability of the complexes formed by arsenic with those nucleotides. By means of potentiometric and calorimetric measurements, the relative stability of AMP, ADP and ATP has been evaluated as a function of the pH. It turns out that ATP forms more stable structures with As(iii) than ADP which, in turn, better chelates arsenic than AMP. Such a stability sequestration capability of arsenic (ATP > ADP > AMP) has been interpreted on a twofold basis via state-of-the-art ab initio molecular dynamics (AIMD) and metadynamics (MetD) simulations performed on aqueous solutions of As(iii) chelated by AMP and ATP. In fact, we demonstrate that ATP offers a larger number of effective binding sites than AMP, thus indicating a higher statistical probability for chelating arsenic. Moreover, an evaluation of the free energy associated with the interactions that As(iii) establishes with the nucleotide atoms responsible for the binding quantitatively proves the greater effectiveness of ATP as a chelating agent.


Assuntos
Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Arsênico/metabolismo , Difosfato de Adenosina/química , Monofosfato de Adenosina/química , Trifosfato de Adenosina/química , Arsênico/química , Sítios de Ligação , Simulação de Dinâmica Molecular , Potenciometria , Teoria Quântica , Termodinâmica
13.
Proc Natl Acad Sci U S A ; 117(12): 6491-6501, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32152094

RESUMO

The mitochondria of various tissues from mice, naked mole rats (NMRs), and bats possess two mechanistically similar systems to prevent the generation of mitochondrial reactive oxygen species (mROS): hexokinases I and II and creatine kinase bound to mitochondrial membranes. Both systems operate in a manner such that one of the kinase substrates (mitochondrial ATP) is electrophoretically transported by the ATP/ADP antiporter to the catalytic site of bound hexokinase or bound creatine kinase without ATP dilution in the cytosol. One of the kinase reaction products, ADP, is transported back to the mitochondrial matrix via the antiporter, again through an electrophoretic process without cytosol dilution. The system in question continuously supports H+-ATP synthase with ADP until glucose or creatine is available. Under these conditions, the membrane potential, ∆ψ, is maintained at a lower than maximal level (i.e., mild depolarization of mitochondria). This ∆ψ decrease is sufficient to completely inhibit mROS generation. In 2.5-y-old mice, mild depolarization disappears in the skeletal muscles, diaphragm, heart, spleen, and brain and partially in the lung and kidney. This age-dependent decrease in the levels of bound kinases is not observed in NMRs and bats for many years. As a result, ROS-mediated protein damage, which is substantial during the aging of short-lived mice, is stabilized at low levels during the aging of long-lived NMRs and bats. It is suggested that this mitochondrial mild depolarization is a crucial component of the mitochondrial anti-aging system.


Assuntos
Envelhecimento , Mitocôndrias/fisiologia , Membranas Mitocondriais/fisiologia , Difosfato de Adenosina/metabolismo , Animais , Quirópteros , Creatina/metabolismo , Transporte de Elétrons , Embrião de Mamíferos , Glucose/metabolismo , Hexoquinase/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/metabolismo , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Ratos-Toupeira , Especificidade de Órgãos , Espécies Reativas de Oxigênio/metabolismo , Especificidade da Espécie
14.
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
15.
Biochemistry ; 59(13): 1309-1313, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32207972

RESUMO

In a radical departure from the classical E1-E2-E3 three-enzyme mediated ubiquitination of eukaryotes, the recently described bacterial enzymes of the SidE family of Legionella pneumophila effectors utilize NAD+ to ligate ubiquitin onto target substrate proteins. This outcome is achieved via a two-step mechanism involving (1) ADP ribosylation of ubiquitin followed by (2) phosphotransfer to a target serine residue. Here, using fluorescent NAD+ analogues as well as synthetic substrate mimics, we have developed continuous assays enabling real-time monitoring of both steps of this mechanism. These assays are amenable to biochemical studies and high-throughput screening of inhibitors of these effectors, and the discovery and characterization of putative enzymes similar to members of the SidE family in other organisms. We also show their utility in studying enzymes that can reverse and inhibit this post-translational modification.


Assuntos
Proteínas de Bactérias/metabolismo , Bioquímica/métodos , Corantes Fluorescentes/química , Legionella pneumophila/metabolismo , Serina/metabolismo , Difosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Corantes Fluorescentes/metabolismo , Legionella pneumophila/química , Legionella pneumophila/genética , NAD/química , NAD/metabolismo , Serina/química , Ubiquitinação
16.
Xenobiotica ; 50(8): 929-938, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32065000

RESUMO

We assessed the contribution of CYP2C19 and CYP3A4 metabolic activity to the ADP-induced platelet aggregation 1h and 24h after a loading dose of 60 mg prasugrel or 180 mg ticagrelor in patients with ST-elevation myocardial infarction (STEMI). Further, we assessed the contribution of CYP2C19 polymorphisms and medication to the CYP enzymatic activity.Patients with STEMI were randomly assigned to the treatment with prasugrel (n = 51) or ticagrelor (n = 46). Metabolic activity of CYP2C19 and CYP3A4 was assessed by the rate of 5-hydroxylation and sulfoxidation of lansoprazole. Further, patients were genotyped for CYP2C19 *2 and *17 alleles.In prasugrel-treated patients, high ADP-induced platelet reactivity 1h after the loading dose positively correlated with 5OH-lansoprazole/lansoprazole ratio (r = 0.44, p = 0.002), a marker of CYP2C19 metabolic activity, and negatively with lansoprazole-sulfone/lansoprazole ratio, which reflects CYP3A4 metabolic activity (r = -0.35, p = 0.018).CYP2C19 poor metabolizers had lower 5OH-lansoprazole/lansoprazole ratio and higher lansoprazole-sulfone/lansoprazole ratio, but without any effect on the ADP-induced platelet reactivity. The treatment with amiodarone, a CYP3A4 inhibitor, influenced neither the metabolic ratios nor the ADP-induced platelet reactivity.The CYP3A4 and CYP2C19 metabolic activity is associated with ADP-induced platelet reactivity in prasugrel-treated, but not ticagrelor-treated patients with STEMI.


Assuntos
Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP3A/metabolismo , Cloridrato de Prasugrel/farmacologia , Ticagrelor/farmacologia , Difosfato de Adenosina/metabolismo , Feminino , Humanos , Masculino , Cloridrato de Prasugrel/uso terapêutico , Infarto do Miocárdio com Supradesnível do Segmento ST/tratamento farmacológico , Ticagrelor/uso terapêutico
17.
J Mol Biol ; 432(7): 2217-2231, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32087197

RESUMO

The DEAH/RHA helicase DHX36 has been linked to cellular RNA and DNA quadruplex structures and to AU-rich RNA elements. In vitro, DHX36 remodels DNA and RNA quadruplex structures and unwinds DNA duplexes in an ATP-dependent manner. DHX36 contains the superfamily 2 helicase core and several auxiliary domains that are conserved in orthologs of the enzyme. The role of these auxiliary domains for the enzymatic function of DHX36 is not well understood. Here, we combine structural and biochemical studies to define the function of three auxiliary domains that contact nucleic acid. We first report the crystal structure of mouse DHX36 bound to ADP. The structure reveals an overall architecture of mouse DHX36 that is similar to previously reported architectures of fly and bovine DHX36. In addition, our structure shows conformational changes that accompany stages of the ATP-binding and hydrolysis cycle. We then examine the roles of the DHX36-specific motif (DSM), the OB-fold, and a conserved ß-hairpin (ß-HP) in mouse DHX36 in the remodeling of RNA structures. We demonstrate and characterize RNA duplex unwinding for DHX36 and examine the remodeling of inter- and intramolecular RNA quadruplex structures. We find that the DSM not only functions as a quadruplex binding adaptor but also promotes the remodeling of RNA duplex and quadruplex structures. The OB-fold and the ß-HP contribute to RNA binding. Both domains are also essential for remodeling RNA quadruplex and duplex structures. Our data reveal roles of auxiliary domains for multiple steps of the nucleic acid remodeling reactions.


Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Quadruplex G , RNA/química , RNA/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Camundongos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Domínios Proteicos
18.
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
19.
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
20.
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
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