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1.
J Agric Food Chem ; 67(33): 9265-9276, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31361479

RESUMO

Fungal infections significantly alter the emissions of volatile organic compounds (VOCs) by plants, but the mechanisms for VOCs affecting fungal infections of plants remain largely unknown. Here, we found that infection by Botrytis cinerea upregulated linalool production by strawberries and fumigation with linalool was able to inhibit the infection of fruits by the fungus. Linalool treatment downregulated the expression of rate-limiting enzymes in the ergosterol biosynthesis pathway, and this reduced the ergosterol content in the fungi cell membrane and impaired membrane integrity. Linalool treatment also caused damage to mitochondrial membranes by collapsing mitochondrial membrane potential and also downregulated genes involved in adenosine triphosphate (ATP) production, resulting in a significant decrease in the ATP content. Linalool treatment increased the levels of reactive oxygen species (ROS), in response to which the treated fungal cells produced more of the ROS scavenger pyruvate. RNA-Seq and proteomic analysis data showed that linalool treatment slowed the rates of transcription and translation.


Assuntos
Botrytis/efeitos dos fármacos , Fragaria/metabolismo , Frutas/microbiologia , Monoterpenos/metabolismo , Doenças das Plantas/microbiologia , Compostos Orgânicos Voláteis/metabolismo , Trifosfato de Adenosina/metabolismo , Botrytis/crescimento & desenvolvimento , Fragaria/química , Fragaria/microbiologia , Frutas/química , Frutas/metabolismo , Interações Hospedeiro-Patógeno , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Monoterpenos/farmacologia , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteômica , Espécies Reativas de Oxigênio/metabolismo , Compostos Orgânicos Voláteis/farmacologia
2.
Nat Commun ; 10(1): 2928, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266959

RESUMO

Stochastic formation of Mycobacterium tuberculosis (Mtb) persisters achieves a high level of antibiotic-tolerance and serves as a source of multidrug-resistant (MDR) mutations. As conventional treatment is not effective against infections by persisters and MDR-Mtb, novel therapeutics are needed. Several approaches were proposed to kill persisters by altering their metabolism, obviating the need to target active processes. Here, we adapted a biofilm culture to model Mtb persister-like bacilli (PLB) and demonstrated that PLB underwent trehalose metabolism remodeling. PLB use trehalose as an internal carbon to biosynthesize central carbon metabolism intermediates instead of cell surface glycolipids, thus maintaining levels of ATP and antioxidants. Similar changes were identified in Mtb following antibiotic-treatment, and MDR-Mtb as mechanisms to circumvent antibiotic effects. This suggests that trehalose metabolism is associated not only with transient drug-tolerance but also permanent drug-resistance, and serves as a source of adjunctive therapeutic options, potentiating antibiotic efficacy by interfering with adaptive strategies.


Assuntos
Farmacorresistência Bacteriana Múltipla , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Trealose/metabolismo , Trifosfato de Adenosina/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Humanos , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia
3.
J Agric Food Chem ; 67(32): 8773-8782, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31283205

RESUMO

Conquering rapid postripeness and deterioration of Agaricus bisporus is quite challenging. We previously observed that methyl jasmonate (MeJA) pretreatment postponed the deterioration of A. bisporus, but the mechanism is unknown. Here, a nontargeted metabolomics analysis by ultrahigh-pressure liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) revealed that MeJA increased the synthesis of malate by inhibiting the decomposition of fumarate and cis-aconitate. MeJA maintained energy supply by enhancing ATP content and energy charge level and improving hexokinase and glucose-6-phosphate dehydrogenase activities as well. These results promoted ATP supply by maintaining glycolysis, the TCA cycle, and the pentose phosphate pathway. In addition, we revealed that the delayed deterioration was attributed to MeJA treatment which stimulated the energy status of A. bisporus by reducing the respiration rate and nutrient decomposition, thus maintaining energy production. Our results provide a new insight into the role of MeJA treatment in delaying deterioration of A. bisporus through ATP production and supply.


Assuntos
Acetatos/farmacologia , Agaricus/efeitos dos fármacos , Agaricus/metabolismo , Ciclopentanos/farmacologia , Oxilipinas/farmacologia , Ácido Aconítico/metabolismo , Trifosfato de Adenosina/metabolismo , Agaricus/química , Agaricus/crescimento & desenvolvimento , Cromatografia Líquida de Alta Pressão/métodos , Metabolismo Energético/efeitos dos fármacos , Fumaratos/metabolismo , Malatos/metabolismo , Metabolômica , Espectrometria de Massas em Tandem/métodos
4.
Nature ; 571(7766): 515-520, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31341297

RESUMO

The mitochondrial ADP/ATP carrier (AAC) is a major transport protein of the inner mitochondrial membrane. It exchanges mitochondrial ATP for cytosolic ADP and controls cellular production of ATP. In addition, it has been proposed that AAC mediates mitochondrial uncoupling, but it has proven difficult to demonstrate this function or to elucidate its mechanisms. Here we record AAC currents directly from inner mitochondrial membranes from various mouse tissues and identify two distinct transport modes: ADP/ATP exchange and H+ transport. The AAC-mediated H+ current requires free fatty acids and resembles the H+ leak via the thermogenic uncoupling protein 1 found in brown fat. The ADP/ATP exchange via AAC negatively regulates the H+ leak, but does not completely inhibit it. This suggests that the H+ leak and mitochondrial uncoupling could be dynamically controlled by cellular ATP demand and the rate of ADP/ATP exchange. By mediating two distinct transport modes, ADP/ATP exchange and H+ leak, AAC connects coupled (ATP production) and uncoupled (thermogenesis) energy conversion in mitochondria.


Assuntos
Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Prótons , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Coenzimas/metabolismo , Ácidos Graxos/metabolismo , Transporte de Íons , Masculino , Camundongos , Consumo de Oxigênio
5.
Nat Commun ; 10(1): 2393, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160557

RESUMO

Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains (NBDs) power polypeptide translocation through a central channel comprised of a hexameric spiral of protomers that contact substrate via conserved pore-loop interactions. Here we report cryo-EM structures of a hyperactive ClpB variant bound to the model substrate, casein in the presence of slowly hydrolysable ATPγS, which reveal the translocation mechanism. Distinct substrate-gripping interactions are identified for NBD1 and NBD2 pore loops. A trimer of N-terminal domains define a channel entrance that binds the polypeptide substrate adjacent to the topmost NBD1 contact. NBD conformations at the seam interface reveal how ATP hydrolysis-driven substrate disengagement and re-binding are precisely tuned to drive a directional, stepwise translocation cycle.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Caseínas/metabolismo , Endopeptidase Clp/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Escherichia coli/metabolismo , Proteínas de Choque Térmico/ultraestrutura , Transporte Proteico , Domínio AAA , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Hidrólise , Modelos Moleculares , Peptídeos/metabolismo , Agregados Proteicos , Subunidades Proteicas/metabolismo
6.
Biochemistry (Mosc) ; 84(4): 407-415, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228932

RESUMO

Proton-translocating FOF1-ATP synthase (F-type ATPase, F-ATPase or FOF1) performs ATP synthesis/hydrolysis coupled to proton transport across the membrane in mitochondria, chloroplasts, and most eubacteria. The ATPase activity of the enzyme is suppressed in the absence of protonmotive force by several regulatory mechanisms. The most conserved of these mechanisms is noncompetitive inhibition of ATP hydrolysis by the MgADP complex (ADP-inhibition) which has been found in all the enzymes studied. When MgADP binds without phosphate in the catalytic site, the enzyme enters an inactive state, and MgADP gets locked in the catalytic site and does not exchange with the medium. The degree of ADP-inhibition varies in FOF1 enzymes from different organisms. In the Escherichia coli enzyme, ADP-inhibition is relatively weak and, in contrast to other organisms, is enhanced rather than suppressed by phosphate. In this study, we used site-directed mutagenesis to investigate the role of amino acid residues ß139, ß158, ß189, and ß319 of E. coli FOF1-ATP synthase in the mechanism of ADP-inhibition and its modulation by the protonmotive force. The amino acid residues in these positions differ in the enzymes from beta- and gammaproteobacteria (including E. coli) and FOF1-ATP synthases from other eubacteria, mitochondria, and chloroplasts. The ßN158L substitution produced no effect on the enzyme activity, while substitutions ßF139Y, ßF189L, and ßV319T only slightly affected ATP (1 mM) hydrolysis. However, in a mixture of ATP and ADP, the activity of the mutants was less suppressed than that of the wild-type enzyme. In addition, mutations ßF189L and ßV319T weakened the ATPase activity inhibition by phosphate in the presence of ADP. We suggest that residues ß139, ß189, and ß319 are involved in the mechanism of ADP-inhibition and its modulation by phosphate.


Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Difosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Cinética , Mutagênese Sítio-Dirigida , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Força Próton-Motriz , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/genética , Alinhamento de Sequência
7.
Nat Commun ; 10(1): 2474, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31171772

RESUMO

Diabetes is a global health problem caused primarily by the inability of pancreatic ß-cells to secrete adequate levels of insulin. The molecular mechanisms underlying the progressive failure of ß-cells to respond to glucose in type-2 diabetes remain unresolved. Using a combination of transcriptomics and proteomics, we find significant dysregulation of major metabolic pathways in islets of diabetic ßV59M mice, a non-obese, eulipidaemic diabetes model. Multiple genes/proteins involved in glycolysis/gluconeogenesis are upregulated, whereas those involved in oxidative phosphorylation are downregulated. In isolated islets, glucose-induced increases in NADH and ATP are impaired and both oxidative and glycolytic glucose metabolism are reduced. INS-1 ß-cells cultured chronically at high glucose show similar changes in protein expression and reduced glucose-stimulated oxygen consumption: targeted metabolomics reveals impaired metabolism. These data indicate hyperglycaemia induces metabolic changes in ß-cells that markedly reduce mitochondrial metabolism and ATP synthesis. We propose this underlies the progressive failure of ß-cells in diabetes.


Assuntos
Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/genética , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Perfilação da Expressão Gênica , Gluconeogênese , Glicólise , Secreção de Insulina , Metabolômica , Camundongos , Camundongos Transgênicos , NAD/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio , Canais de Potássio Corretores do Fluxo de Internalização/genética , Proteômica
8.
Chem Commun (Camb) ; 55(52): 7482-7485, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31184653

RESUMO

Protein arginine (Arg) phosphorylation regulates stress responses and virulence in bacteria. With fluorescent activity probes, we show that McsB, a protein Arg kinase, can dephosphorylate phosphoarginine (pArg) residues to produce ATP from ADP, implicating the dynamic control of protein pArg levels by the kinase even without a phosphatase.


Assuntos
Arginina Quinase/metabolismo , Corantes Fluorescentes/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Arginina/análogos & derivados , Arginina/análise , Arginina/química , Arginina/metabolismo , Cromatografia Líquida de Alta Pressão , Compostos Organofosforados/análise , Fosforilação
9.
Ann Hematol ; 98(8): 1845-1854, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31154474

RESUMO

Primary immune thrombocytopenia is an autoimmune disease, characterized with decreased platelet and increased risk of bleeding. Recent studies have shown the reduction and dysfunction of regulatory T (Treg) cells in ITP patients. CD39 is highly expressed on the surface of Treg cells. It degrades ATP to AMP and CD73 dephosphorylates AMP into adenosine. Then adenosine binds with adenosine receptor and suppresses immune response by activating Treg cells and inhibiting the release of inflammatory cytokines from effector T (Teff) cells. Adenosine receptor has several subtypes and adenosine A2A receptor (A2AR) plays a crucial role especially within lymphocytes. The CD39+ Treg cells and the expression of A2AR showed abnormality in some autoimmune disease. But knowledge of CD39+ Treg cells and A2AR which are crucial in the adenosine immunosuppressive pathway is still limited in ITP. Thirty-one adult patients with newly diagnosed ITP were enrolled in this study. CD39 and A2AR expression was measured by flow cytometry and RT-PCR. The function of CD39 was reflected by the change of ATP concentration detected by CellTiter-Glo Luminescent Cell Viability Assay. CD39 expression within CD4+CD25+ Treg cells in ITP patients was decreased compared to normal controls. After high-dose dexamethasone therapy, response (R) group showed increased CD39 expression within Treg cells while non-response (NR) group did not show any difference in contrast to those before treatment. The expression of A2AR in CD4+CD25- Teff and CD4+CD25+ Treg cells was both lower in ITP patients than that of normal controls. After therapy, CD4+CD25- Teff cells had higher A2AR expression while CD4+CD25+ Treg cells did not show any difference in comparison to that before treatment. The enzymatic activity of CD39 was damaged in ITP patients and improved after high-dose dexamethasone therapy. In ITP, there was not only numerical decrease but also impaired enzymatic activity in CD39+ Treg cells. After high-dose dexamethasone treatment, these two defects could be reversed. Our results also suggested that ITP patients had reduced A2AR expression in both CD4+CD25+ Treg cells and CD4+CD25- Teff cells. CD4+CD25- Teff cells had increased A2AR expression after treatment.


Assuntos
Apirase/genética , Dexametasona/uso terapêutico , Imunossupressores/uso terapêutico , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Receptor A2A de Adenosina/genética , Linfócitos T Reguladores/efeitos dos fármacos , Adenosina/imunologia , Adenosina/metabolismo , Trifosfato de Adenosina/imunologia , Trifosfato de Adenosina/metabolismo , Adulto , Idoso , Apirase/imunologia , Estudos de Casos e Controles , Feminino , Expressão Gênica , Humanos , Imunofenotipagem , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Púrpura Trombocitopênica Idiopática/enzimologia , Púrpura Trombocitopênica Idiopática/genética , Púrpura Trombocitopênica Idiopática/imunologia , Receptor A2A de Adenosina/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/enzimologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/enzimologia , Linfócitos T Reguladores/imunologia
10.
Nat Commun ; 10(1): 2420, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160570

RESUMO

Replication-Factor-C (RFC) and RFC-like complexes (RLCs) mediate chromatin engagement of the proliferating cell nuclear antigen (PCNA). It remains controversial how RFC and RLCs cooperate to regulate PCNA loading and unloading. Here, we show the distinct PCNA loading or unloading activity of each clamp loader. ATAD5-RLC possesses the potent PCNA unloading activity. ATPase motif and collar domain of ATAD5 are crucial for the unloading activity. DNA structures did not affect PCNA unloading activity of ATAD5-RLC. ATAD5-RLC could unload ubiquitinated PCNA. Through single molecule measurements, we reveal that ATAD5-RLC unloaded PCNA through one intermediate state before ATP hydrolysis. RFC loaded PCNA through two intermediate states on DNA, separated by ATP hydrolysis. Replication proteins such as Fen1 could inhibit the PCNA unloading activity of Elg1-RLC, a yeast homolog of ATAD5-RLC in vitro. Our findings provide molecular insights into how PCNA is released from chromatin to finalize DNA replication/repair.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína de Replicação C/metabolismo , Adenosina Trifosfatases , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Endonucleases Flap/metabolismo , Humanos , Hidrólise , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo
11.
Sheng Li Xue Bao ; 71(3): 388-394, 2019 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-31218329

RESUMO

The aim of this study was to determine the effects of extremely low frequency electromagnetic field (ELF-EMF) on energy metabolism and oxidative stress in Caenorhabditis elegans (C. elegans). Worms in three adult stages (young adult stage, egg-laying stage and peak egg-laying stage) were investigated under 50 Hz, 3 mT ELF-EMF exposure. ATP levels, ATP synthase activity in vivo, reactive oxygen species (ROS) content, and changes of total antioxidant capacity (TAC) were detected, and worms' oxidative stress responses were also evaluated under ELF-EMF exposure. The results showed that ATP levels were significantly increased under this ELF-EMF exposure, and mitochondrial ATP synthase activity was upregulated simultaneously. In young adult stage, worms' ROS level was significantly elevated, together with upregulated TAC but with a decreased ROS-TAC score indicated by principal component analysis. ROS level and TAC of worms had no significant changes in egg-laying and peak egg-laying stages. Based on these results, we concluded that ELF-EMF can enhance worm energy metabolism and elicit oxidative stress, mainly manifesting as ATP and ROS level elevation together with ATP synthase upregulation and ROS-TAC score decrease in young adult C. elegans.


Assuntos
Caenorhabditis elegans/efeitos da radiação , Radiação Eletromagnética , Metabolismo Energético , Estresse Oxidativo , Trifosfato de Adenosina/metabolismo , Animais , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Espécies Reativas de Oxigênio/análise
12.
Cancer Sci ; 110(8): 2456-2470, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31148343

RESUMO

Extracellular ATP has been shown to play an important role in invasion and the epithelial-mesenchymal transition (EMT) process in breast cancer; however, the mechanism is unclear. Here, by using a cDNA microarray, we demonstrated that extracellular ATP could stimulate hypoxia-inducible factor (HIF) signaling and upregulate hypoxia-inducible factor 1/2α (HIF-1/2α) expression. After knocking down HIF-1/2α using siRNA, we found that ATP-driven invasion and EMT were significantly attenuated via HIF2A-siRNA in breast cancer cells. By using ChIP assays, we revealed that the biological function of extracellular ATP in invasion and EMT process depended on HIF-2α direct targets, among which lysyl oxidase-like 2 (LOXL2) and matrix metalloproteinase-9 (MMP-9) mediated ATP-driven invasion, and E-cadherin and Snail mediated ATP-driven EMT, respectively. In addition, using silver staining and mass spectrometry, we found that phosphoglycerate kinase 1 (PGK1) could interact with HIF-2α and mediate ATP-driven HIF-2α upregulation. Furthermore, we demonstrated that expressions of HIF-2α and its target proteins could be regulated via ATP by AKT-PGK1 pathway. Using a Balb/c mice model, we illustrated the function of HIF-2α in promoting tumor growth and metastasis in vivo. Moreover, by exploring online databases, we found that molecules involved in ATP-HIF-2α signaling were highly expressed in human breast carcinoma tissues and were associated with poor prognosis. Altogether, these findings suggest that extracellular ATP could promote breast carcinoma invasion and EMT via HIF-2α signaling, which may be a potential target for future anti-metastasis therapy.


Assuntos
Trifosfato de Adenosina/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal/fisiologia , Hipóxia/patologia , Invasividade Neoplásica/patologia , Aminoácido Oxirredutases/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Células MCF-7 , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia
13.
J Photochem Photobiol B ; 196: 111512, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31129505

RESUMO

Cancer is a leading cause of death worldwide, and doxorubicin (DOX) has become one of the most commonly prescribed drugs. Stem cell (SC) therapy is proving to be a promising strategy to alleviate DOX adverse effects on non-cancerous cells. However, the drug also has a toxic action on SCs, reducing the efficiency of cell therapy from a preventive view. The present study shows that the DOX toxicity in mesenchymal SCs (MSCs) can be partially overcome by low-level laser irradiation (LLLI). To achieve this, we applied the low-level red laser (wavelength: 660 nm; output power: 30 mW; laser beam: 0.028 cm2; irradiation: 1.07 mW/cm2; Ga-Al-As Photon Laser III, DMC, São Paulo, Brazil) in rat adipose tissue-derived MSCs before their exposure to different DOX concentrations. Results revealed that the DOX reduced the viability and adenosine triphosphate level of MSCs. These findings were followed by significantly increased apoptosis as well as oxidative stress in the MSCs. Interestingly, LLLI at the dose of 0.2 J alleviated the effects of DOX on cell viability and apoptosis, and inhibited oxidative stress in the MSCs. In summary, this study provides a crucial step toward the future application of LLLI as a protective approach against DOX-induced toxicity in MSCs, particularly cell death. This study also lays the groundwork for further investigation into the role of oxidative stress and inflammation as an instructive milieu for cell protection.


Assuntos
Apoptose/efeitos da radiação , Doxorrubicina/farmacologia , Lasers , Trifosfato de Adenosina/metabolismo , Tecido Adiposo/citologia , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Citocinas/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Ratos
14.
Croat Med J ; 60(2): 127-140, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31044584

RESUMO

AIM: To propose potential mechanisms of action of electromagnetic fields (EMF) on astrocytes and microglia and to elucidate the role of heat shock proteins (HSP), adenosine triphosphate (ATP), calcium ions (Ca2+), and hypoxia-inducible factor 1α (HIF1α) in neurorestoration following the application of EMF. METHODS: We reviewed the existing studies within the public domain and cross-evaluated their results in order to conclude on the molecular mechanisms of microglia-astrocyte crosstalk at work during EMF treatment. RESULTS: The existing studies suggest that EMF induces the increase of HSP70 expression and inhibition of HIF1α, thus decreasing inflammation and allowing the microglia-astrocyte crosstalk to initiate the formation of a glial scar within the central nervous system. Furthermore, by potentially up-regulating A2A and A3 adenosine receptors, EMF increases cAMP accumulation from astrocytes and reduces the expression of inflammatory cytokines TNF α and IL-8, thus initiating neurorestoration. CONCLUSION: The microglia-astrocyte crosstalk during EMF treatment is crucial for the initiation of neurorestoration. Elucidating the exact mechanisms of EMF actions upon microglia and astrocytes, and its role in neurorestoration could be a key step in further research of the therapeutic potential of EMFs in various neurological disorders.


Assuntos
Astrócitos/fisiologia , Terapia de Campo Magnético , Microglia/fisiologia , Doenças Neurodegenerativas/terapia , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Citocinas , Campos Eletromagnéticos , Proteínas de Choque Térmico/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/metabolismo , Inflamação/terapia , Doenças Neurodegenerativas/imunologia , Receptor Cross-Talk , Fator de Necrose Tumoral alfa
15.
J Enzyme Inhib Med Chem ; 34(1): 1010-1017, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31072165

RESUMO

The Mur ligases form a series of consecutive enzymes that participate in the intracellular steps of bacterial peptidoglycan biosynthesis. They therefore represent interesting targets for antibacterial drug discovery. MurC, D, E and F are all ATP-dependent ligases. Accordingly, with the aim being to find multiple inhibitors of these enzymes, we screened a collection of ATP-competitive kinase inhibitors, on Escherichia coli MurC, D and F, and identified five promising scaffolds that inhibited at least two of these ligases. Compounds 1, 2, 4 and 5 are multiple inhibitors of the whole MurC to MurF cascade that act in the micromolar range (IC50, 32-368 µM). NMR-assisted binding studies and steady-state kinetics studies performed on aza-stilbene derivative 1 showed, surprisingly, that it acts as a competitive inhibitor of MurD activity towards D-glutamic acid, and additionally, that its binding to the D-glutamic acid binding site is independent of the enzyme closure promoted by ATP.


Assuntos
Trifosfato de Adenosina/antagonistas & inibidores , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Ligases/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Cinética , Ligases/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade
16.
Nat Chem Biol ; 15(6): 556-559, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086327

RESUMO

Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and specific small-molecule inhibitor of the NLRP3 pathway, but its molecular target is not defined. Here, we show that MCC950 directly interacts with the Walker B motif within the NLRP3 NACHT domain, thereby blocking ATP hydrolysis and inhibiting NLRP3 activation and inflammasome formation.


Assuntos
Trifosfato de Adenosina/antagonistas & inibidores , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Inflamassomos/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Sulfonas/farmacologia , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/química , Humanos , Hidrólise/efeitos dos fármacos , Inflamassomos/biossíntese , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sulfonas/química
17.
J Phys Chem Lett ; 10(11): 3071-3079, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31117686

RESUMO

Vesicle transport conducted by motor protein multiplexes (MPMs), which is ubiquitous among eukaryotes, shows anomalous and stochastic dynamics qualitatively different from the dynamics of thermal motion and artificial active matter; the relationship between in vivo vesicle-delivery dynamics and the underlying physicochemical processes is not yet quantitatively understood. Addressing this issue, we perform accurate tracking of individual vesicles, containing upconverting nanoparticles, transported by kinesin-dynein-multiplexes along axonal microtubules. The mean-square-displacement of vesicles along the microtubule exhibits unusual dynamic phase transitions that are seemingly inconsistent with the scaling behavior of the mean-first-passage time over the travel length. These paradoxical results and the vesicle displacement distribution are quantitatively explained and predicted by a multimode MPM model, developed in the current work, where ATP-hydrolysis-coupled motion of MPM has both unidirectional and bidirectional modes.


Assuntos
Dineínas/metabolismo , Cinesina/metabolismo , Corpos Multivesiculares/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte Axonal , Transporte Biológico Ativo , Linhagem Celular , Humanos , Hidrólise , Cinética , Microtúbulos/metabolismo , Modelos Biológicos , Nanopartículas/metabolismo
18.
Eur J Med Chem ; 175: 330-348, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31096154

RESUMO

Human DNA topoisomerases represent one of the key targets of modern chemotherapy. An emerging group of catalytic inhibitors of human DNA topoisomerase IIα comprises a new paradigm directed to circumvent the known limitations of topoisomerase II poisons such as cardiotoxicity and induction of secondary tumors. In our previous studies, 4,6-substituted-1,3,5-triazin-2(1H)-ones were discovered as catalytic inhibitors of topo IIα. Here, we report the results of our efforts to optimize several properties of the initial chemical series that did not exhibit cytotoxicity on cancer cell lines. Using an optimized synthetic route, a focused chemical library was designed aimed at further functionalizing substituents at the position 4 of the 1,3,5-triazin-2(1H)-one scaffold to enable additional interactions with the topo IIα ATP binding site. After virtual screening, selected 36 analogues were synthesized and experimentally evaluated for human topo IIα inhibition. The optimized series displayed improved inhibition of topo IIα over the initial series and the catalytic mode of inhibition was confirmed for the selected active compounds. The optimized series also showed cytotoxicity against HepG2 and MCF-7 cell lines and did not induce double-strand breaks, thus displaying a mechanism of action that differs from the topo II poisons on the cellular level. The new series represents a new step in the development of the 4,6-substituted-1,3,5-triazin-2(1H)-one class towards novel efficient anticancer therapies utilizing the catalytic topo IIα inhibition paradigm.


Assuntos
DNA Topoisomerases Tipo II/efeitos dos fármacos , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Triazinas/química , Triazinas/farmacologia , Trifosfato de Adenosina/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Catálise , Quebras de DNA de Cadeia Dupla , Células Hep G2 , Histonas/metabolismo , Humanos , Células MCF-7 , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/metabolismo
19.
Enzyme Microb Technol ; 127: 58-64, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31088617

RESUMO

Cadaverine, also known as 1,5-pentanediamine, is an important platform chemical with a wide range of applications and can be produced either by fermentation or bioconversion. Bioconversion of cadaverine from l-lysine is the preferred method because of its many benefits, including rapid reaction time and an easy downstream process. In our previous study, we replaced pyridoxal-5-phosphate (PLP) with pyridoxal kinase (PdxY) along with pyridoxal (PL) because it could achieve 80% conversion with 0.4 M of l-lysine in 6 h. However, conversion was sharply decreased in the presence of high concentrations of l-lysine (i.e., 1 M), resulting in less than 40% conversion after several hours. In this study, we introduced an ATP regeneration system using polyphosphate kinase (ppk) into systems containing cadaverine decarboxylase (CadA) and PdxY for a sufficient supply of PLP, which resulted in enhanced cadaverine production. In addition, to improve transport efficiency, the use of surfactants was tested. We found that membrane permeabilization via hexadecyltrimethylammonium bromide (CTAB) increased the yield of cadaverine in the presence of high concentrations of l-lysine. By combining these two strategies, the ppk system and addition of CTAB, we enhanced cadaverine production up to 100% with 1 M of l-lysine over the course of 6 h.


Assuntos
Trifosfato de Adenosina/metabolismo , Cadaverina/metabolismo , Cetrimônio/metabolismo , Escherichia coli/metabolismo , Fosfato de Piridoxal/metabolismo , Biotransformação , Escherichia coli/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo
20.
Int J Mol Sci ; 20(9)2019 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-31083504

RESUMO

Hsp70s use ATP to generate forces that disassemble protein complexes and aggregates, and that translocate proteins into organelles. Entropic pulling has been proposed as a novel mechanism, distinct from the more familiar power-stroke and Brownian ratchet models, for how Hsp70s generate these forces. Experimental evidence supports entropic pulling, but this model may not be well understood among scientists studying these systems. In this review we address persistent misconceptions regarding the dynamics of proteins in solution that contribute to this lack of understanding, and we clarify the basic physics of entropic pulling with some simple analogies. We hope that increased understanding of the entropic pulling mechanism will inform future efforts to characterize how Hsp70s function as motors, and how they coordinate with their regulatory cochaperones in mechanochemical cycles that transduce the energy of ATP hydrolysis into physical changes in their protein substrates.


Assuntos
Entropia , Proteínas de Choque Térmico HSP70/metabolismo , Modelos Biológicos , Proteínas Motores Moleculares/metabolismo , Trifosfato de Adenosina/metabolismo
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