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1.
Adv Neurobiol ; 23: 125-145, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31667807

RESUMO

Glycogen constitutes the main store of glucose in animal cells. Being present at much lower concentrations in the brain than in liver and muscles, brain glycogen has long been considered as an emergency source of glucose, mobilized under stress conditions (including hypoglyceamia). Nevertheless, over the past decade, multiple studies have shed a new light on the roles of brain glycogen, being notably an energy supply critical for high-cognitive processes such as learning and memory consolidation. Glycogen phosphorylase (GP) is the key enzyme regulating the mobilization of glycogen in cells. It is found in humans as three isozymes: muscle (mGP), liver (lGP) and brain GP (bGP). In the brain, astrocytes express both mGP and bGP while neurons only express the brain isoform. Although GP isozymes are very similar, their distinct regulatory features confer them distinct metabolic functions that are strongly related to the roles of glycogen in different tissues. Here, we provide an overview of the functions, the regulations and the structures of GPs in the brain and their relation to the specific roles of glycogen in astrocytes and neurons. We also discuss novel findings concerning the specific regulations of bGP by oxidative stress, and the potential of these enzymes as therapeutic targets in the brain.


Assuntos
Encéfalo/enzimologia , Glicogênio Fosforilase/química , Glicogênio Fosforilase/metabolismo , Glicogênio , Animais , Encéfalo/metabolismo , Glicogênio/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Músculos/enzimologia , Músculos/metabolismo
2.
ACS Chem Biol ; 14(7): 1460-1470, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31243960

RESUMO

Several C-ß-d-glucopyranosyl azoles have recently been uncovered as among the most potent glycogen phosphorylase (GP) catalytic site inhibitors discovered to date. Toward further exploring their translational potential, ex vivo experiments have been performed for their effectiveness in reduction of glycogenolysis in hepatocytes. New compounds for these experiments were predicted in silico where, for the first time, effective ranking of GP catalytic site inhibitor potencies using the molecular mechanics-generalized Born surface area (MM-GBSA) method has been demonstrated. For a congeneric training set of 27 ligands, excellent statistics in terms of Pearson (RP) and Spearman (RS) correlations (both 0.98), predictive index (PI = 0.99), and area under the receiver operating characteristic curve (AU-ROC = 0.99) for predicted versus experimental binding affinities were obtained, with ligand tautomeric/ionization states additionally considered using density functional theory (DFT). Seven 2-aryl-4(5)-(ß-d-glucopyranosyl)-imidazoles and 2-aryl-4-(ß-d-glucopyranosyl)-thiazoles were subsequently synthesized, and kinetics experiments against rabbit muscle GPb revealed new potent inhibitors with best Ki values in the low micromolar range (5c = 1.97 µM; 13b = 4.58 µM). Ten C-ß-d-glucopyranosyl azoles were then tested ex vivo in mouse primary hepatocytes. Four of these (5a-c and 9d) demonstrated significant reduction of glucagon stimulated glycogenolysis (IC50 = 30-60 µM). Structural and predicted physicochemical properties associated with their effectiveness were analyzed with permeability related parameters identified as crucial factors. The most effective ligand series 5 contained an imidazole ring, and the calculated pKa (Epik: 6.2; Jaguar 5.5) for protonated imidazole suggests that cellular permeation through the neutral state is favored, while within the cell, there is predicted more favorable binding to GP in the protonated form.


Assuntos
Azóis/farmacologia , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Glicogenólise/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Animais , Azóis/química , Células CACO-2 , Desenho de Drogas , Inibidores Enzimáticos/química , Glicogênio Fosforilase/metabolismo , Hepatócitos/metabolismo , Humanos , Modelos Moleculares , Coelhos , Relação Estrutura-Atividade
3.
Top Curr Chem (Cham) ; 377(4): 19, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31165274

RESUMO

This review is an effort to summarize recent developments in synthesis of O-glycosides and N-, C-glycosyl molecules with promising antidiabetic potential. Articles published after 2000 are included. First, the O-glycosides used in the treatment of diabetes are presented, followed by the N-glycosides and finally the C-glycosides constituting the largest group of antidiabetic drugs are described. Within each group of glycosides, we presented how the structure of compounds representing potential drugs changes and when discussing chemical compounds of a similar structure, achievements are presented in the chronological order. C-Glycosyl compounds mimicking O-glycosides structure, exhibit the best features in terms of pharmacodynamics and pharmacokinetics. Therefore, the largest part of the article is concerned with the description of the synthesis and biological studies of various C-glycosides. Also N-glycosides such as N-(ß-D-glucopyranosyl)-amides, N-(ß-D-glucopyranosyl)-ureas, and 1,2,3-triazolyl derivatives belong to the most potent classes of antidiabetic agents. In order to indicate which of the compounds presented in the given sections have the best inhibitory properties, a list of the best inhibitors is presented at the end of each section. In summary, the best inhibitors were selected from each of the summarizing figures and the results of the ranking were placed. In this way, the reader can learn about the structure of the compounds having the best antidiabetic activity. The compounds, whose synthesis was described in the article but did not appear on the figures presenting the structures of the most active inhibitors, did not show proper activity as inhibitors. Thus, the article also presents studies that have not yielded the desired results and show directions of research that should not be followed. In order to show the directions of the latest research, articles from 2018 to 2019 are described in a separate Sect. 5. In Sect. 6, biological mechanisms of action of the glycosides and patents of marketed drugs are described.


Assuntos
Diabetes Mellitus/tratamento farmacológico , Descoberta de Drogas/métodos , Glicosídeos/química , Glicosídeos/farmacologia , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Animais , Diabetes Mellitus/enzimologia , Diabetes Mellitus/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Glicogênio Fosforilase/antagonistas & inibidores , Glicogênio Fosforilase/metabolismo , Glicosídeos/farmacocinética , Glicosídeos/uso terapêutico , Humanos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/uso terapêutico , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/química , Inibidores do Transportador 2 de Sódio-Glicose/farmacocinética , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Relação Estrutura-Atividade
4.
Food Chem ; 293: 537-544, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151645

RESUMO

To verify the effect of protein phosphorylation on glycolysis and elucidate the regulatory mechanism from the perspective of enzyme activity, ovine muscle was treated with a kinase inhibitor, dimethyl sulfoxide, or a phosphatase inhibitor and the activities of glycogen phosphorylase, pyruvate kinase and phosphofructokinase were determined. The protein phosphorylation level was significantly different after incubation of muscle with kinase or phosphatase inhibitors. The pH value and lactate content revealed that a high phosphorylation level was the reason for the fast glycolysis. The glycogen phosphorylase, pyruvate kinase and phosphofructokinase activities were significantly higher in the phosphatase inhibitor group than in the other two groups (p < 0.05). Therefore, protein phosphorylation is involved in activating these three enzymes. In summary, protein phosphorylation plays a role in post-mortem glycolysis through the regulation of enzyme activity in ovine muscle.


Assuntos
Glicogênio Fosforilase/metabolismo , Músculos/enzimologia , Fosfofrutoquinases/metabolismo , Piruvato Quinase/metabolismo , Animais , Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Glicólise/efeitos dos fármacos , Fosfofrutoquinases/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piruvato Quinase/antagonistas & inibidores , Ovinos
5.
Biomed Pharmacother ; 112: 108715, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30970519

RESUMO

BACKGROUND: Dysregulation of glucose and glycogen metabolism are crucial mechanisms implicated in type 2 diabetes mellitus (T2DM). Centella asiatica (L.) Urban (Apiaceae) has been utilized as a traditional medicine in Africa and Asia for centuries and is commercially available as a dietary supplement. AIM: We explored for the first time, the possible efficacy of Centella asiatica (CA) extract in ameliorating T2DM-induced changes in key enzymes involved in glucose and glycogen metabolism in the rat skeletal muscle. METHODS: Diabetic rats were orally treated with vehicle, CA (500 and 1000 mg/kg) or metformin (300 mg/kg) daily for 14 days. Skeletal muscle activities of hexokinase (HK), phosphofructokinase (PFK) and fructose 1,6-bisphosphatase (FBPase) were determined by spectrophotometric assays while those of glycogen synthase (GS) and glycogen phosphorylase (GP) were assayed radio-chemically. Histological examination of skeletal muscle was also performed. RESULTS: Rats with induced T2DM had reduced activities of HK (25%), PFK (88%), and GS (38%) when compared to non-diabetic rats. Treatment of diabetic rats with CA500 increased the activities of PFK (7-fold), and FBPase (23%). Further, treatment of diabetic rats with CA1000 also increased the activities of GS (27%) and GP (50%) with little change in these parameters for diabetic rats treated with CA500. These effects probably led to the reduced blood glucose level and elevated skeletal muscle glycogen content observed in CA-treated rats relative to diabetic controls. Furthermore, CA treated rats had reduced the morphological damage of skeletal muscle fibres compared to the non-treated diabetic control rats. CONCLUSION: Our findings strongly suggest that the anti-diabetic effects of CA in part target muscle glucose and glycogen metabolism and hence supporting its folkloric medical use as an anti-diabetic remedy.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucose/metabolismo , Glicogênio/metabolismo , Hipoglicemiantes/uso terapêutico , Músculo Esquelético/efeitos dos fármacos , Triterpenos/uso terapêutico , Animais , Glicemia/análise , Centella/química , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glicogênio Fosforilase/metabolismo , Glicogênio Sintase/metabolismo , Hipoglicemiantes/farmacologia , Masculino , Medicina Tradicional , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Folhas de Planta/química , Ratos Sprague-Dawley , Estreptozocina , Triterpenos/farmacologia
6.
PLoS Biol ; 17(3): e2006146, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30860988

RESUMO

Stress responses are crucial processes that require activation of genetic programs that protect from the stressor. Stress responses are also energy consuming and can thus be deleterious to the organism. The mechanisms coordinating energy consumption during stress response in multicellular organisms are not well understood. Here, we show that loss of the epigenetic regulator G9a in Drosophila causes a shift in the transcriptional and metabolic responses to oxidative stress (OS) that leads to decreased survival time upon feeding the xenobiotic paraquat. During OS exposure, G9a mutants show overactivation of stress response genes, rapid depletion of glycogen, and inability to access lipid energy stores. The OS survival deficiency of G9a mutants can be rescued by a high-sugar diet. Control flies also show improved OS survival when fed a high-sugar diet, suggesting that energy availability is generally a limiting factor for OS tolerance. Directly limiting access to glycogen stores by knocking down glycogen phosphorylase recapitulates the OS-induced survival defects of G9a mutants. We propose that G9a mutants are sensitive to stress because they experience a net reduction in available energy due to (1) rapid glycogen use, (2) an inability to access lipid energy stores, and (3) an overinduced transcriptional response to stress that further exacerbates energy demands. This suggests that G9a acts as a critical regulatory hub between the transcriptional and metabolic responses to OS. Our findings, together with recent studies that established a role for G9a in hypoxia resistance in cancer cell lines, suggest that G9a is of wide importance in controlling the cellular and organismal response to multiple types of stress.


Assuntos
Histona Metiltransferases/metabolismo , Animais , Antioxidantes/metabolismo , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Epigênese Genética/genética , Glicogênio Fosforilase/genética , Glicogênio Fosforilase/metabolismo , Histona Metiltransferases/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Masculino , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Filogenia , Análise de Sequência de RNA
7.
Phys Chem Chem Phys ; 21(14): 7685-7696, 2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30912774

RESUMO

A fluorescence study of N1-(ß-d-glucopyranosyl)-N4-[2-acridin-9(10H)-onyl]-cytosine (GLAC), the first fluorescent potent inhibitor of glycogen phosphorylase (GP), in neutral aqueous solution, is presented herein. Quantum chemistry (TD-DFT) calculations show the existence of several conformers both in the ground and first excited states. They result from rotations of the acridone and cytosine moieties around an NH bridge which may lead to the formation of non-emitting charge-transfer states. The fingerprints of various conformers have been detected by time-resolved fluorescence spectroscopy (fluorescence upconversion and time-correlated single photon counting) and identified using as criteria their energy, polarization and relative population resulting from computations. Such an analysis should contribute to the design of new GP inhibitors with better fluorescence properties, suitable for imaging applications.


Assuntos
Inibidores Enzimáticos/metabolismo , Glicogênio Fosforilase/metabolismo , Teoria Quântica , Acridonas/síntese química , Acridonas/química , Acridonas/metabolismo , Benzoatos/síntese química , Benzoatos/química , Benzoatos/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glicogênio Fosforilase/antagonistas & inibidores , Espectrometria de Fluorescência , Termodinâmica
8.
Artigo em Inglês | MEDLINE | ID: mdl-30710892

RESUMO

Vertebrate freeze tolerance requires multiple adaptations underpinned by specialized biochemistry. Freezing of extracellular water leads to intracellular dehydration as pure water is incorporated into growing ice crystals and also results in the cessation of blood supply to tissues, creating an anoxic cellular environment. Hence, the freeze tolerant wood frog, Rana sylvatica, must endure both dehydration and anoxia stresses in addition to freezing. The metabolic responses to freezing, dehydration and anoxia involve both protein/enzyme adaptations and the production of metabolites with metabolic or osmotic functions, particularly glucose and urea. The present study uses a phosphoproteome analysis to examine the differential phosphorylation of metabolic enzymes involved in the production of these two metabolites in liver in response to freezing, anoxia, or dehydration exposures. Our results show stress-specific responses in the abundance of phosphopeptides retrieved from nine glycolytic enzymes and three urea cycle enzymes in liver of wood frogs exposed to 24 h freezing, 24 h anoxia, or dehydration to 40% of total body water loss, as compared with 5 °C acclimated controls. Data show changes in the abundance of phosphopeptides belonging to glycogen phosphorylase (GP) and phosphofructokinase 2 (PFK2) that were consistent with differential phosphorylation control of glycogenolysis and a metabolic block at PFK1 that can facilitate glucose synthesis as the cryoprotectant during freezing. Anoxia-exposed animals showed similar changes in GP phosphorylation but no changes to PFK2; changes that would facilitate mobilization of glycogen as a fermentative fuel for anaerobic glycolysis. Urea is commonly produced as a compatible osmolyte in response to amphibian dehydration. Selected urea cycle enzymes showed small changes in phosphopeptide abundance in response to dehydration, but during freezing differential phosphorylation occurred that may facilitate this ATP expensive process when energy resources are sparse. These results add to the growing body of literature demonstrating the importance and efficiency of reversible protein phosphorylation as a regulatory mechanism allowing animals to rapidly respond to environmental stress.


Assuntos
Aclimatação , Resposta ao Choque Frio , Glucose/metabolismo , Oxigênio/metabolismo , Ranidae/fisiologia , Ureia/metabolismo , Proteínas de Anfíbios/metabolismo , Animais , Congelamento , Glicogênio Fosforilase/metabolismo , Fosfofrutoquinase-2/metabolismo , Fosforilação , Água/metabolismo
9.
J Microbiol Biotechnol ; 29(3): 357-366, 2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30691252

RESUMO

We first confirmed the involvement of MalQ (4-α-glucanotransferase) in Escherichia coli glycogen breakdown by both in vitro and in vivo assays. In vivo tests of the knock-out mutant, ΔmalQ, showed that glycogen slowly decreased after the stationary phase compared to the wild-type strain, indicating the involvement of MalQ in glycogen degradation. In vitro assays incubated glycogen-mimic substrate, branched cyclodextrin (maltotetraosyl-ß-CD: G4- ß-CD) and glycogen phosphorylase (GlgP)-limit dextrin with a set of variable combinations of E. coli enzymes, including GlgX (debranching enzyme), MalP (maltodextrin phosphorylase), GlgP and MalQ. In the absence of GlgP, the reaction of MalP, GlgX and MalQ on substrates produced glucose-1-P (glc-1-P) 3-fold faster than without MalQ. The results revealed that MalQ led to disproportionate G4 released from GlgP-limit dextrin to another acceptor, G4, which is phosphorylated by MalP. In contrast, in the absence of MalP, the reaction of GlgX, GlgP and MalQ resulted in a 1.6-fold increased production of glc-1-P than without MalQ. The result indicated that the G4-branch chains of GlgP-limit dextrin are released by GlgX hydrolysis, and then MalQ transfers the resultant G4 either to another branch chain or another G4 that can immediately be phosphorylated into glc-1-P by GlgP. Thus, we propose a model of two possible MalQ-involved pathways in glycogen degradation. The operon structure of MalP-defecting enterobacteria strongly supports the involvement of MalQ and GlgP as alternative pathways in glycogen degradation.


Assuntos
Escherichia coli/enzimologia , Escherichia coli/metabolismo , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Glicogênio/metabolismo , Ciclodextrinas/metabolismo , Dextrinas/antagonistas & inibidores , Dextrinas/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Glucanos/metabolismo , Glucose/metabolismo , Glucofosfatos/metabolismo , Glucosiltransferases/metabolismo , Glicogênio/genética , Sistema da Enzima Desramificadora do Glicogênio/genética , Glicogênio Fosforilase/metabolismo , Glicosilação , Redes e Vias Metabólicas , Família Multigênica
10.
J Biol Chem ; 294(12): 4345-4358, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30647127

RESUMO

We recently uncovered a regulatory pathway of the muscle isoform of glycogen phosphorylase (PYGM) that plays an important role in regulating immune function in T cells. Here, using various enzymatic, pulldown, and immunoprecipitation assays, we describe signaling cross-talk between the small GTPases RAS and RAP1A, member of RAS oncogene family (RAP1) in human Kit 225 lymphoid cells, which, in turn, is regulated by the epidermal growth factor receptor (EGFR). We found that this communication bridge is essential for glycogen phosphorylase (PYG) activation through the canonical pathway because this enzyme is inactive in the absence of adenylyl cyclase type 6 (ADCY6). PYG activation required stimulation of both exchange protein directly activated by cAMP 2 (EPAC2) and RAP1 via RAS and ADCY6 phosphorylation, with the latter being mediated by Raf-1 proto-oncogene, Ser/Thr kinase (RAF1). Consistent with this model, PYG activation was EGFR-dependent and may be initiated by the constitutively active form of RAS. Consequently, PYG activation in Kit 225 T cells could be blocked with specific inhibitors of RAS, EPAC, RAP1, RAF1, ADCY6, and cAMP-dependent protein kinase. Our results establish a new paradigm for the mechanism of PYG activation, which depends on the type of receptor involved.


Assuntos
Receptores ErbB/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Glicogênio Fosforilase/metabolismo , Linfócitos T/enzimologia , Animais , Linhagem Celular , Ativação Enzimática , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Fosforilação , Transdução de Sinais , Proteínas rap1 de Ligação ao GTP/metabolismo
11.
Colloids Surf B Biointerfaces ; 173: 725-732, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30384269

RESUMO

Acanthamoeba keratitis is an ophthalmic disease with no specific treatment that specially affects contact lens users. The silencing of serine phosphatase (SP) and glycogen phosphorylase (GP) proteins produced by Acanthamoeba has been shown to significantly reduce the cytopathic effect, although no vehicle was proposed yet to deliver the siRNA sequences to the trophozoites. In this study, PEGylated cationic liposomes were proposed and optimized using Box-Behnken design. The influence of DOTAP:DOPE ratio, DSPE-PEG concentration, and siRNA/DOTAP charge ratio were evaluated over both biological response and physicochemical properties of liposomes. The ratio of DOTAP:DOPE had an effect in the trophozoite activity whereas the charge ratio influenced both size and protease activity. The predicted values were very close to the observed values, yielding a formulation with good activity and toxicity profile, which was used in the following experiments. A murine model of ocular keratitis was treated with siGP + siSP-loaded liposomes, as well as their respective controls, and combined treatment of liposomes and chlorhexidine. After 15 days of eight daily administrations, the liposomal complex combined with chlorhexidine was the only treatment able to reverse the more severe lesions associated with keratitis. There was 60% complete regression in corneal damage, with histological sections demonstrating the presence of an integral epithelium, without lymphocytic infiltrate. The set of results demonstrate the efficacy of a combined therapy based on siRNA with classical drugs for a better prognosis of keratitis caused by Acanthamoeba.


Assuntos
Ceratite por Acanthamoeba/terapia , Acanthamoeba/efeitos dos fármacos , Clorexidina/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Lipossomos/química , Proteínas de Protozoários/antagonistas & inibidores , Trofozoítos/efeitos dos fármacos , Acanthamoeba/enzimologia , Acanthamoeba/patogenicidade , Ceratite por Acanthamoeba/parasitologia , Ceratite por Acanthamoeba/patologia , Animais , Córnea/efeitos dos fármacos , Córnea/parasitologia , Córnea/patologia , Modelos Animais de Doenças , Esquema de Medicação , Composição de Medicamentos/métodos , Quimioterapia Combinada , Análise Fatorial , Ácidos Graxos Monoinsaturados/química , Regulação da Expressão Gênica , Glicogênio Fosforilase/antagonistas & inibidores , Glicogênio Fosforilase/genética , Glicogênio Fosforilase/metabolismo , Humanos , Lipossomos/metabolismo , Fosfatidiletanolaminas/química , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Polietilenoglicóis/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Compostos de Amônio Quaternário/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Trofozoítos/enzimologia , Trofozoítos/patogenicidade
12.
Biochem Biophys Res Commun ; 508(4): 1101-1105, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30551876

RESUMO

Small heat shock proteins (sHsps) are molecular chaperones preventing protein aggregation. Dynamics of quaternary structure plays an important role in the chaperone-like activity of sHsps. However, an interrelation between the oligomeric state and chaperone-like activity of sHsps remains insufficiently characterized. Most of the accumulated data were obtained in dilute protein solutions, leaving the question of the oligomeric state of sHsps in crowded intracellular media largely unanswered. Here, we analyzed the effect of crowding on the oligomeric state of αB-crystallin (αB-Cr) using analytical ultracentrifugation. Marked increase in the sedimentation coefficient of αB-Cr was observed in the presence of polyethylene glycol (PEG), polyvinylpyrrolidone (PVP) and trimethylamine N-oxide (TMAO) at 48 °C. An especially pronounced effect was detected for the PEG and TMAO mixture, where the sedimentation coefficient (s20,w) of αB-Cr increased from 10.7 S in dilute solution up to 40.7 S in the presence of crowding agents. In the PEG + TMAO mixture, addition of model protein substrate (muscle glycogen phosphorylase b) induced dissociation of large αB-Cr oligomers and formation of complexes with smaller sedimentation coefficients, supporting the idea that, under crowding conditions, protein substrates can promote dissociation of large αB-Cr oligomers.


Assuntos
Multimerização Proteica , Cadeia B de alfa-Cristalina/química , Área Sob a Curva , Difusão Dinâmica da Luz , Glicogênio Fosforilase/metabolismo , Humanos , Estrutura Quaternária de Proteína , Temperatura Ambiente
13.
Food Chem ; 272: 613-618, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30309589

RESUMO

The aim of this study was to investigate the effects of protein S-nitrosylation on the glycogen metabolism in postmortem pork. The pork samples were incubated with control (0.9% NaCl), nitric oxide synthase (NOS) inhibitor, or NO donor for 4 and 12 h at 4 °C. Results indicate that NOS inhibitor treatment led to significantly lower level of glycogen and higher lactate content at 24 h compared those of control and NO donor treatments (P < 0.05). The pH of NOS inhibitor treatment was significantly lower than other treatments, which indicates the fast glycolysis during postmortem aging (P < 0.05). In addition, the activities of glycolytic enzymes including GP, GAPDH and PK were significantly different among three treatments (P < 0.05) possibly due to the different modification of protein S-nitrosylation. These results suggest that NO could regulate the glycogen metabolism through modulating the activities of glycolytic enzymes by protein S-nitrosylation.


Assuntos
Glicogênio/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , S-Nitrosotióis/análise , Animais , Glicogênio Fosforilase/metabolismo , Glicólise , Concentração de Íons de Hidrogênio , Proteínas Musculares/química , Doadores de Óxido Nítrico/química , Doadores de Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Suínos
14.
Chem Commun (Camb) ; 54(91): 12863-12866, 2018 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-30375590

RESUMO

The interactome of arzanol was investigated by MS-based chemical proteomics, a pioneering technology for small molecule target discovery. Brain glycogen phosphorylase (bGP), a key regulator of glucose metabolism so far refractory to small molecule modulation, was identified as the main high-affinity target of arzanol. Competitive affinity-based proteomics, DARTS, molecular docking, surface plasmon resonance and in vitro biological assays provided molecular mechanistic insights into the arzanol-enzyme interaction, qualifying this positive modulator of bGP for further studies in the realm of neurodegeneration and cancer.


Assuntos
Encéfalo/enzimologia , Glicogênio Fosforilase/metabolismo , Floroglucinol/análogos & derivados , Pironas/metabolismo , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Sítios de Ligação , Glicogênio Fosforilase/química , Células HeLa , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Espectrometria de Massas , Simulação de Acoplamento Molecular , Floroglucinol/química , Floroglucinol/metabolismo , Estrutura Terciária de Proteína , Proteômica , Pironas/química , Ressonância de Plasmônio de Superfície
15.
Hum Reprod ; 33(10): 1898-1906, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30169642

RESUMO

STUDY QUESTION: Is there any difference in the protein composition of the endometrial fluid aspirate (EFA) obtained the day of embryo transfer in in vitro fertilization (IVF) cycles achieving and not achieving pregnancy? SUMMARY ANSWER: Comparative analysis identified a differential protein expression pattern in 'implantative' and 'non-implantative' IVF cycles. WHAT IS KNOWN ALREADY: EFA allows non-invasive characterization of the endometrium, and may contain important information on its receptivity when performing (IVF) cycles. Endometrial side of implantation has usually been studied with endometrial biopsy in an IVF cycle prior to embryo transfer, focusing on 'receptive/non-receptive' endometria and with low-throughput proteomic techniques. STUDY DESIGN, SIZE, DURATION: We have compared the protein expression patterns in EFA from a total of 110 women undergoing IVF, corresponding to 50 implantative and 60 non-implantative IVF cycles. Discovery (38 patients) and Validation (42 patients) sample cohorts were analyzed using a high-throughput differential proteomic approach. Then, the differential expression of glycogen phosphorylase B (PYGB) was validated by western blotting in an additional cohort (30 patients). The study period was 18 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: The population under study consisted of 110 women aged 18-40 years old, undergoing their first or second IVF/ intracytoplasmic sperm injection cycle, with normal uterus and endometrium, and 1-2 good quality embryos, and embryo transfer being performed on Day 3. Endometrial fluid aspiration was performed immediately before the embryo transfer. Samples (80) were initially distributed in two independent cohorts and analyzed by liquid chromatography-mass spectrometry. The first cohort was used for the discovery and the second for the validation of the results. Filter-aided sample preparation was used for the in-solution tryptic digestion of the proteins present in the samples, followed by label-free mass spectrometry analysis. In order to unravel the molecular features of receptivity, the lists of differential proteins were thoroughly analyzed using different bioinformatic tools, including GSEA, IPA and GO analysis. MAIN RESULTS AND THE ROLE OF CHANCE: A false discovery rate-based correction of the t-test P-values was carried out in order to strengthen the reliability of the results. Functional analyses denoted the deregulation of important processes governing receptivity, such as antimicrobial response, cell-cell interaction, immune response and inflammatory signaling, among others. Overall eight proteins were commonly deregulated in both studied datasets and brain form glycogen phosphorylase (PYGB) was selected for confirmatory analysis. LIMITATIONS, REASONS FOR CAUTION: Our results were obtained from patients with normal uterus and endometrium and with good quality embryos, who had fresh Day-3 embryo transfer, in stimulated cycles. Therefore, our observations may not be applicable to poor prognosis cases or non-stimulated cycles. WIDER IMPLICATIONS OF THE FINDINGS: This work provides insights into the molecular features of implantative IVF cycles using non-invasive methods. It reveals that EFA may reflect an increased inflammatory state in non-implantative endometrium. Additionally, it proposes PYGB as a potential biomarker for endometrial receptivity or implantation success. This knowledge opens a new avenue for developing embryo transfer strategies, through the improvement of embryo culture media or modifying endometrial fluid composition to increase pregnancy rates. STUDY FUNDING/COMPETING INTEREST(S): This study was partially funded by a Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). Authors declare no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.


Assuntos
Glicemia/metabolismo , Transferência Embrionária/métodos , Endométrio/metabolismo , Glicogênio Fosforilase/metabolismo , Adulto , Biomarcadores/metabolismo , Estudos de Casos e Controles , Feminino , Fertilização In Vitro , Glicogênio Fosforilase/análise , Humanos , Gravidez , Proteômica , Reprodutibilidade dos Testes , Adulto Jovem
16.
Artigo em Inglês | MEDLINE | ID: mdl-30017911

RESUMO

Glycogen, as an intracellular deposit of polysaccharide, takes important roles in energy balance of many animals. In fish, however, the role of glycogen during development is poorly understood. In the present study, we assessed changes in glycogen concentration and gene expression patterns of glycogen-metabolizing enzymes in developing masu salmon (Oncorhynchus masou masou), a salmonid species inhabiting west side of North Pacific Ocean. As we measured glycogen levels in the bodies and yolk sacs containing the liver separately, the glycogen concentration increased in both parts as the fish developed, whereas it transiently decreased in the yolk sac after hatching, implying glycogen synthesis and breakdown in these tissues. Immunofluorescence staining using anti-glycogen monoclonal antibody revealed localization of glycogen in the liver, muscle and yolk syncytial layer of the pre-hatching embryos and hatched larvae. In order to estimate glycogen metabolism in the fish, the genes encoding homologs of glycogen synthase (gys1 and gys2) and glycogen phosphorylase (pygma, pygmb and pygl) were cloned, and their expression patterns were assessed by quantitative PCR and in situ hybridization. In the fish, gys1 and gys2 were robustly expressed in the muscle and liver, respectively. Also, expression of pyg isoforms was found in muscle, liver and yolk syncytial layer during hatching. With changes in glycogen concentration and expression patterns of relevant genes, our results suggest, for the first time, possible involvement of glycogen in energy balance of fish embryos, especially during hatching.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Glicogênio/metabolismo , Fígado/enzimologia , Músculos/enzimologia , Salmão/metabolismo , Animais , Clonagem Molecular , Feminino , Imunofluorescência , Glicogênio Fosforilase/metabolismo , Fígado/crescimento & desenvolvimento , Masculino , Desenvolvimento Muscular , Filogenia , RNA Mensageiro/genética , Salmão/genética , Salmão/crescimento & desenvolvimento
17.
ChemMedChem ; 13(15): 1608-1616, 2018 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-29905983

RESUMO

Liver glycogen phosphorylase (GP) is a key enzyme for human health, as its increased activity is associated with type 2 diabetes. The GP catalytic mechanism has been explored by quantum mechanics/molecular mechanics (QM/MM) methods. Herein, we propose a mechanism that proceeds by three steps: 1) it begins with transfer of a hydrogen atom from the phosphate group of the pyridoxal 5'-phosphate (HPO42- -PLP) cofactor to the phosphate substrate; 2) the glycosidic linkage is then cleaved through protonation of the glycosidic oxygen atom by a hydroxy group of the inorganic phosphate group; and 3) an oxygen atom of the phosphate performs a nucleophilic attack on the anomeric carbon atom of glucose, concomitant with the return of a proton from phosphate to PO43- -PLP, which finally leads to formation of the glucose-1-phosphate product and recovers the initial state of the PLP cofactor. The glycosidic bond cleavage and nucleophilic attack from the phosphate group to the glycosyl molecule have the highest activation free energies. The structural properties of the hereby characterized transition states could be very useful in structure-based drug design studies against liver GP.


Assuntos
Glicogênio Fosforilase/metabolismo , Glicogenólise , Teoria Quântica , Catálise , Humanos , Cinética
18.
PLoS One ; 13(6): e0198322, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29927967

RESUMO

The honey bee has been extensively studied as a model for neuronal circuit and memory function and more recently has emerged as an unconventional model in biogerontology. Yet, the detailed knowledge of neuronal processing in the honey bee brain contrasts with the very sparse information available on glial cells. In other systems glial cells are involved in nutritional homeostasis, detoxification, and aging. These glial functions have been linked to metabolic enzymes, such as glutamine synthetase and glycogen phosphorylase. As a step in identifying functional roles and potential differences among honey bee glial types, we examined the spatial distribution of these enzymes and asked if enzyme abundance is associated with aging and other processes essential for survival. Using immunohistochemistry and confocal laser microscopy we demonstrate that glutamine synthetase and glycogen phosphorylase are abundant in glia but appear to co-localize with different glial sub-types. The overall spatial distribution of both enzymes was not homogenous and differed markedly between different neuropiles and also within each neuropil. Using semi-quantitative Western blotting we found that rapid aging, typically observed in shortest-lived worker bees (foragers), was associated with declining enzyme levels. Further, we found enzyme abundance changes after severe starvation stress, and that glutamine synthetase is associated with food response. Together, our data indicate that aging and nutritional physiology in bees are linked to glial specific metabolic enzymes. Enzyme specific localization patterns suggest a functional differentiation among identified glial types.


Assuntos
Envelhecimento/fisiologia , Abelhas/enzimologia , Glutamato-Amônia Ligase/metabolismo , Glicogênio Fosforilase/metabolismo , Inanição/enzimologia , Animais , Abelhas/fisiologia , Encéfalo/citologia , Encéfalo/enzimologia , Regulação Enzimológica da Expressão Gênica , Proteínas de Insetos/metabolismo , Microscopia Confocal , Neuroglia/enzimologia , Neurópilo/enzimologia
19.
Meat Sci ; 143: 129-136, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29751219

RESUMO

High pressure processing (HPP) of pre-rigor longissimus thoracis (strip loin) from prime and bull animals substantially decreased the shear force and improved consumer eating attributes of the final meat product. The improved tenderness in both prime and bull meat was associated with a lower myofibrillar fragmentation index and reduced calpain 1 activity which indicated the mechanism of tenderisation was different from that which occurred in chill aged meat. Light microscopy showed disruption to the fibre packing within the muscle and electron microscopy confirmed significant disruption of the Z discs and M lines and disappearance of the A lines. Thus, HPP is associated with a reduction in the structural integrity and strength of the sarcomeres. These effects were consistent in strip loins sourced from prime and bull stock. HPP also led to the movement of glycogen phosphorylase from the sarcoplasmic fraction to the insoluble myofibrillar fraction in all animals and this was associated with a higher pH at 24 h.


Assuntos
Músculos do Dorso/química , Qualidade dos Alimentos , Produtos da Carne/análise , Indústria de Embalagem de Carne/métodos , Carne/análise , Estresse Mecânico , Animais , Músculos do Dorso/metabolismo , Músculos do Dorso/ultraestrutura , Calpaína/metabolismo , Bovinos , Culinária , Feminino , Preferências Alimentares , Glicogênio Fosforilase/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Masculino , Mastigação , Microscopia Eletrônica de Transmissão , Miofibrilas/química , Miofibrilas/metabolismo , Miofibrilas/ultraestrutura , Nova Zelândia , Sarcômeros/química , Sarcômeros/metabolismo , Sarcômeros/ultraestrutura , Espectrometria de Massas em Tandem
20.
Molecules ; 23(6)2018 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-29844263

RESUMO

A few new anthranilate diamide derivatives, 3a⁻e, 5a⁻c and 7a⁻d, were designed, synthesized, and evaluated for their inhibitory activity against two interesting antidiabetic targets, α-glucosidase and glycogen phosphorylase enzymes. Different instrumental analytical tools were applied in identification and conformation of their structures like; 13C NMR, ¹H NMR and elemental analysis. The screening of the novel compounds showed potent inhibitory activity with nanomolar concentration values. The most active compounds (5c) and (7b) showed the highest inhibitory activity against α-glucosidase and glycogen phosphorylase enzymes IC50 = 0.01247 ± 0.01 µM and IC50 = 0.01372 ± 0.03 µM, respectively. In addition, in vivo testing of the highly potent α-glucosidase inhibitor (7b) on rats with DTZ-induced diabetes was done and showed significant reduction of blood glucose levels compared to the reference drug. Furthermore, a molecular docking study was performed to help understand the binding interactions of the most active analogs with these two enzymes. The data obtained from the molecular modeling were correlated with those obtained from the biological screening. These data showed considerable antidiabetic activity for these newly synthesized compounds.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Glicogênio Fosforilase/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases/farmacologia , Hipoglicemiantes/farmacologia , alfa-Glucosidases/química , ortoaminobenzoatos/farmacologia , Animais , Sítios de Ligação , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/fisiopatologia , Ensaios Enzimáticos , Glicogênio Fosforilase/química , Glicogênio Fosforilase/metabolismo , Inibidores de Glicosídeo Hidrolases/síntese química , Hipoglicemiantes/síntese química , Masculino , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Coelhos , Ratos , Ratos Sprague-Dawley , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Estreptozocina , Relação Estrutura-Atividade , alfa-Glucosidases/metabolismo , ortoaminobenzoatos/síntese química
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