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1.
Zhonghua Jie He He Hu Xi Za Zhi ; 42(10): 760-764, 2019 Oct 12.
Artigo em Chinês | MEDLINE | ID: mdl-31594110

RESUMO

Objective: To explore the difference of mRNA, protein expression levels and the indexes of peripheral blood antioxidant capacity in peripheral blood lymphocytes of different EPHX1 genotypes in chronic obstructive pulmonary disease(COPD). Methods: A case-control study was conducted to collect peripheral blood samples of 220 stable chronic COPD patients with smoking history and 230 healthy smokers (control group) from October 2016 to February 2018 in the First Affiliated Hospital of Kunming Medical University, and the genetic testing was carried out according to the operation instructions of BigDye Terminator v1.1 DNA Sequencing Kit. Based on their EPHX1 exon 3 and exon 4 polymorphism status, the EPHX1 was classified into 4 groups, i. e., normal activity, slow activity, extremely slow activity and fast activity. Then COPD patients were allocated to either a slow activity group (slow and very slow activity) or a fast activity group (normal and fast activity) according to EPHX1 genotype and gene activity. The expression of EPHX1 mRNA and protein in peripheral blood lymphocytes were detected by qRT-PCR and Western blot, and indexes of serum antioxidant capacity was detected by corresponding kits. Results: (1)The 2(-ΔΔCt) of the control group was 1.000, and the 2(-ΔΔCt) of the COPD group was 1.052±0.023. There was no significant difference in the level of EPHX1 mRNA expression between the two groups (t=1.992 P=0.865). The level of EPHX1 mRNA expression in the slow activity group was not different significantly compared to that in the fast-active group (1.053±0.023 vs 1.048±0.021, t=1.133, P=0.260). (2)The level of EPHX1 protein expression by Western blot analysis showed that the EHPX1/GAPDH gray ratio was not different significantly between the COPD group and the control group (0.613±0.089 vs 0.602±0.075, t=0.805, P=0.422). The level of EPHX1 protein expression in the slow activity group was not significantly different compared to that in the fast activity group (0.606±0.088 vs 0.622±0.092, t=-0.786 P=0.434). (3)There were significant differences in indexes of antioxidant capacity between the control group and the COPD group (P<0.05). There were significant differences in indexes of antioxidant capacity between the slow activity group and the fast activity group of COPD patients (P<0.05). Conclusions: The different antioxidant capacity of COPD patients with different EPHX1 genotypes may be related to the polymorphism of EPHX1 gene affecting the activity of microsomal epoxidase, but not to the level of EPHX1 mRNA and protein expression.


Assuntos
Antioxidantes/metabolismo , Grupo com Ancestrais do Continente Asiático/genética , Epóxido Hidrolases/genética , Doença Pulmonar Obstrutiva Crônica/genética , Fumar/efeitos adversos , Adulto , Western Blotting , Estudos de Casos e Controles , China/epidemiologia , Epóxido Hidrolases/metabolismo , Predisposição Genética para Doença/genética , Genótipo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Humanos , Pessoa de Meia-Idade , Polimorfismo Genético , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Fumar/epidemiologia
2.
PLoS Biol ; 17(10): e3000433, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31613873

RESUMO

Cell-to-cell heterogeneity within an isogenic population has been observed in prokaryotic and eukaryotic cells. Such heterogeneity often manifests at the level of individual protein abundance and may have evolutionary benefits, especially for organisms in fluctuating environments. Although general features and the origins of cellular noise have been revealed, details of the molecular pathways underlying noise regulation remain elusive. Here, we used experimental evolution of Saccharomyces cerevisiae to select for mutations that increase reporter protein noise. By combining bulk segregant analysis and CRISPR/Cas9-based reconstitution, we identified the methyltransferase Hmt1 as a general regulator of noise buffering. Hmt1 methylation activity is critical for the evolved phenotype, and we also show that two of the Hmt1 methylation targets can suppress noise. Hmt1 functions as an environmental sensor to adjust noise levels in response to environmental cues. Moreover, Hmt1-mediated noise buffering is conserved in an evolutionarily distant yeast species, suggesting broad significance of noise regulation.


Assuntos
Regulação Fúngica da Expressão Gênica , Heterogeneidade Genética , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sistemas CRISPR-Cas , Evolução Molecular Direcionada , Metanossulfonato de Etila/farmacologia , Edição de Genes , Genes Reporter , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Metilação , Mutação , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
BMC Plant Biol ; 19(1): 366, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31426752

RESUMO

BACKGROUND: Drought stress is one of the major abiotic stresses that affects plant growth and productivity. The GAPCp genes play important roles in drought stress tolerance in multiple species. The aim of this experiment was to identify the core cis-regulatory elements that may respond to drought stress in the GAPCp2 and GAPCp3 promoter sequences. RESULTS: In this study, the promoters of GAPCp2 and GAPCp3 were cloned. The promoter activities were significantly improved under abiotic stress via regulation of Rluc reporter gene expression, while promoter sequence analysis indicated that these fragments were not almost identical. In transgenic Arabidopsis with the expression of the GUS reporter gene under the control of one of these promoters, the activities of GUS were strong in almost all tissues except the seeds, and the activities were induced after abiotic stress. The yeast one-hybrid system and EMSA demonstrated that TaMYB bound TaGAPCp2P/3P. By analyzing different 5' deletion mutants of these promoters, it was determined that TaGAPCp2P (- 1312~ - 528) and TaGAPCp3P (- 2049~ - 610), including the MYB binding site, contained enhancer elements that increased gene expression levels under drought stress. We used an effector and a reporter to co-transform tobacco and found that TaMYB interacted with the specific MYB binding sites of TaGAPCp2P (- 1197~ - 635) and TaGAPCp3P (- 1456~ - 1144 and - 718~ - 610) in plant cells. Then, the Y1H system and EMSA assay demonstrated that these MYB binding sites in TaGAPCp2P (- 1135 and - 985) and TaGAPCp3P (- 1414 and - 665) were the target cis-elements of TaMYB. The deletion of the specific MYB binding sites in the promoter fragments significantly restrained the drought response, and these results confirmed that these MYB binding sites (AACTAAA/C) play vital roles in improving the transcription levels under drought stress. The results of qRT-PCR in wheat protoplasts transiently overexpressing TaMYB indicated that the expression of TaGAPCp2/3 induced by abiotic stress was upregulated by TaMYB. CONCLUSION: The MYB binding sites (AACTAAA/C) in TaGAPCp2P/3P were identified as the key cis-elements for responding to drought stress and were bound by the transcription factor TaMYB.


Assuntos
Secas , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Proteínas de Plantas/genética , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Triticum/fisiologia , Sítios de Ligação , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Regiões Promotoras Genéticas/genética , Tabaco/genética , Tabaco/fisiologia , Fatores de Transcrição/metabolismo , Triticum/genética
4.
Environ Monit Assess ; 191(8): 490, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31297613

RESUMO

Eukaryotes employ various mechanisms to survive environmental stress conditions. Multicellular organisms eliminate permanently damaged cells by apoptosis, while unicellular eukaryotes like yeast react by decelerating cell aging. In the present study, transcriptomic and proteomic approaches were employed to elucidate the underlying mechanism of delayed apoptosis. Our findings suggest that Candida tropicalis 3Aer has a set of tightly controlled genes that are activated under Cd+2 exposition. Acute exposure to Cd+2 halts the cell cycle at the G2/M phase checkpoint and activates multiple cytoplasmic proteins that overcome effects of Cd+2-induced reactive oxygen species. Prolonged Cd+2 stress damages DNA and initiates GAPDH amyloid formation. This is the first report that Cd+2 challenge initiates dynamic redistribution of GAPDH and MDH and alters various metabolic pathways including the pentose phosphate pathway. In conclusion, the intracellular redistribution of GAPDH and MDH induced by prolonged cadmium stress modulates various cellular reactions, which facilitate delayed aging in the yeast cell.


Assuntos
Apoptose/efeitos dos fármacos , Cádmio/toxicidade , Candida tropicalis/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Malato Desidrogenase/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Candida tropicalis/metabolismo , Ciclo Celular/efeitos dos fármacos , Proteômica , Fatores de Tempo
5.
Diagn Microbiol Infect Dis ; 95(3): 114860, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31353066

RESUMO

Chagas disease is caused by Trypanosoma cruzi and affects about 7 million people worldwide. Benznidazole and nifurtimox have low efficacy and high toxicity. The present study was designed to identify the trypanocidal effect of (-)-α-Bisabolol (BIS) and investigate its mechanism. Epimastigotes and trypomastigotes were cultured with BIS and the viable cells were counted. BIS antiamastigote effect was evaluated using infected LLC-MK2 cells. MTT assay was performed to evaluate BIS cytotoxicity. Growth recovery was assessed to evaluate BIS effect after short times of exposure. BIS mechanism was investigated through flow cytometry, with 7-AAD and Annexin V-PE. DCFH-DA, rhodamine 123 (Rho123) and acridine orange (AO). Finally, enzymatic and computational assays were performed to identify BIS interaction with T. cruzi GAPDH (tcGAPDH). BIS showed an inhibitory effect on epimastigotes after all tested periods, as well on trypomastigotes. It caused cytotoxicity on LLC-MK2 cells at higher concentrations, with selectivity index (SeI) = 26.5. After treatment, infected cells showed a decrease in infected cells, the number of amastigotes per infected cell and the survival index (SuI). Growth recovery demonstrated that BIS effect causes rapid death of T. cruzi. Flow cytometry showed that BIS biological effect is associated with apoptosis induction, increase in cytoplasmic ROS and mitochondrial transmembrane potential, while reservosome swelling was observed at a late stage. Also, BIS action mechanism may be associated to tcGAPDH inhibition. Altogether, the results demonstrate that BIS causes cell death in Trypanosoma cruzi Y strain forms, with the involvement of apoptosis and oxidative stress and enzymatic inhibition.


Assuntos
/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/antagonistas & inibidores , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Concentração Inibidora 50 , Macaca mulatta , Simulação de Acoplamento Molecular , Estrutura Molecular , Estresse Oxidativo/efeitos dos fármacos , Trypanosoma cruzi/fisiologia
6.
BMC Immunol ; 20(1): 24, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286875

RESUMO

BACKGROUND: Multiple sclerosis is a demyelinating and autoimmune disease and its immune response is not fully elucidated. This study was conducted to examine the pathological changes and B cell subsets in experimental autoimmune encephalomyelitis (EAE) mice, and analyze the expression of triosephosphate isomerase (TPI) and GADPH to define the role of B cell subsets in the disease. RESULTS: Female C57BL/6 mice were randomly divided into EAE group (n = 18) and control (n = 18). During the experiments, the weight and nerve function scores were determined. The proportions of B cell subsets in the peripheral blood were measured by flow cytometry. Seven, 18 and 30 days after immunization, the brain and spinal cord tissues were examined for the infiltration of inflammatory cells using hematoxylin-eosin (HE) HE staining and the demyelination using Luxol fast blue staining. The expression of B cell-related proteins was detected immunohistochemistrially and the expression of antigenic TPI and GADPH was analyzed using enzyme-linked immunosorbent assay (ELISA). HE staining showed that mice had more severe EAE 18 d than 7 d after modelling, while the symptoms were significantly relieved at 30 d. The results were consistent with the weight measurements and neural function scores. Immunohistochemistry studies showed that B cells aggregated in the spinal cord, but not much in the brain. Flow cytometry studies showed that there were more B cells in control than in EAE models from day 7 and the difference was narrowed at day 30. The level of plasma cells increased continuously, reached the top at day 21 and obviously declined at day 30. On other hand, the numbers of memory B cells increased gradually over the experimental period. The numbers of plasma and memory B cells were similar between the control and EAE mice. ELISA data revealed that the brain contents of TPI and GAPDH were higher in EAE mice than in control at day 7, while at day 18, the levels were reversed. CONCLUSIONS: In the central pathological process of EAE mice, B cells exert role through the mechanism other than producing antibodies and the levels of brain TPI and GADPH are related to the severity of autoimmune induced-damage.


Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Encéfalo/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Esclerose Múltipla/imunologia , Medula Espinal/metabolismo , Triose-Fosfato Isomerase/metabolismo , Animais , Encéfalo/patologia , Separação Celular , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Humanos , Memória Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Medula Espinal/patologia
7.
J Microbiol Biotechnol ; 29(4): 538-547, 2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-30939634

RESUMO

The aim of the present study was to evaluate the effects of two well-known natural antioxidants vitamin C (VC) and vitamin E (VE) on the antifungal activity of honokiol against Candida albicans. The broth microdilution method was employed to test the antifungal activities of honokiol with or without antioxidants in the medium against C. albicans strain. Intracellular reactive oxygen species (ROS) and lipid peroxidation were determined by fluorescence staining assay. Mitochondrial dysfunction was assessed by detecting the mitochondrial DNA and the mitochondrial membrane potential. We observed that VC could significantly potentiate the antifungal activities of honokiol while VE reduced the effectiveness of honokiol against C. albicans. In addition, VC accelerated honokiol-induced mitochondrial dysfunction and inhibited glycolysis leading to a decrease in cellular ATP. However, VE could protect against mitochondrial membrane lipid peroxidation and rescue mitochondrial function after honokiol treatment. Our research provides new insight into the understanding of the action mechanism of honokiol and VC combination against C. albicans.


Assuntos
Antifúngicos/farmacologia , Ácido Ascórbico/antagonistas & inibidores , Compostos de Bifenilo/farmacologia , Candida albicans/efeitos dos fármacos , Antagonismo de Drogas , Lignanas/farmacologia , Vitamina E/antagonistas & inibidores , Antioxidantes/farmacologia , Candida albicans/citologia , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/isolamento & purificação , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Glicólise/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
8.
Int J Legal Med ; 133(3): 899-908, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30864069

RESUMO

The assessment of postmortem degradation of skeletal muscle proteins has emerged as a novel approach to estimate the time since death in the early to mid-postmortem phase (approximately 24 h postmortem (hpm) to 120 hpm). Current protein-based methods are limited to a small number of skeletal muscle proteins, shown to undergo proteolysis after death. In this study, we investigated the usability of a target-based and unbiased system-wide protein analysis to gain further insights into systemic postmortem protein alterations and to identify additional markers for postmortem interval (PMI) delimitation. We performed proteomic profiling to globally analyze postmortem alterations of the rat and mouse skeletal muscle proteome at defined time points (0, 24, 48, 72, and 96 hpm), harnessing a mass spectrometry-based quantitative proteomics approach. Hierarchical clustering analysis for a total of 579 (rat) and 896 (mouse) quantified proteins revealed differentially expressed proteins during the investigated postmortem period. We further focused on two selected proteins (eEF1A2 and GAPDH), which were shown to consistently degrade postmortem in both rat and mouse, suggesting conserved intra- and interspecies degradation behavior, and thus preserved association with the PMI and possible transferability to humans. In turn, we validated the usefulness of these new markers by classical Western blot experiments in a rat model and in human autopsy cases. Our results demonstrate the feasibility of mass spectrometry-based analysis to discover novel protein markers for PMI estimation and show that the proteins eEF1A2 and GAPDH appear to be valuable markers for PMI estimation in humans.


Assuntos
Biomarcadores/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Mudanças Depois da Morte , Proteômica , Idoso , Animais , Cromatografia Líquida , Análise por Conglomerados , Feminino , Patologia Legal/métodos , Humanos , Masculino , Espectrometria de Massas , Camundongos Endogâmicos ICR , Modelos Animais , Ratos Sprague-Dawley
9.
Microb Pathog ; 129: 74-77, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30710674

RESUMO

Pasteurella multocida is the causative agent of a wide range of disease (pasteurellosis) and a zoonotic pathogen in humans. Some pathogenic bacteria are able to exploit host plasminogen for migration across tissue barriers or evade from host innate immunity. However, there is no study on host plasminogen exploitation of P. multocida. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has been reported to be a plasminogen receptor in many pathogenic bacteria, but its role in P. multocida exploiting plasminogen has not yet been characterized. The aim of this study was to detect the activity of P. multocida to exploit host plasminogen and evaluate the ability of GAPDH to act as a receptor in the recruitment process. P. multocida could recruit host plasminogen and exhibited plasmin activity when stimulated by urokinase. GAPDH exhibited binding activity to plasminogen. GAPDH Antiserum significantly decrease the plasminogen recruitment activity of P. multocida. In conclusion, P. multocida is able to exploit host plasminogen via GAPDH. To our knowledge, this is the first report on host plasminogen exploitation of P. multocida.


Assuntos
Proteínas de Bactérias/metabolismo , Fibrinolíticos/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Pasteurella multocida/metabolismo , Pasteurella multocida/patogenicidade , Plasminogênio/metabolismo , Animais , Feminino , Humanos , Camundongos Endogâmicos BALB C , Ligação Proteica
10.
Food Res Int ; 116: 354-361, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30716956

RESUMO

The objective of this study was to unravelling the possible mechanism underlying drip loss and identify the protein markers associated with water holding capacity (WHC) of meat. Pectoralis major muscles from geese were assigned to high and low drip loss groups. The physio-chemical properties and proteome profiles were compared between these two groups. Label-free quantitative mass spectrometry was applied to investigate the differentially expressed proteins, and the results were confirmed with parallel reaction monitoring (PRM). 21 differential proteins were identified in abundance between high and low drip loss groups, and they fall generally into the structural proteins, metabolic enzymes, antioxidant enzymes and stress response proteins. The results may provide a perspective to the discovery of new biomarkers for drip loss.


Assuntos
Gansos , Proteínas Musculares/metabolismo , Músculos Peitorais/metabolismo , Proteômica , Água/análise , Animais , Culinária , Desmina/metabolismo , Glutationa Redutase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Proteínas de Choque Térmico/metabolismo , Concentração de Íons de Hidrogênio , Queratinas/metabolismo , Cadeias Leves de Miosina/metabolismo , Superóxido Dismutase/metabolismo , Espectrometria de Massas em Tandem
11.
J Thorac Cardiovasc Surg ; 157(2): 494-503.e1, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30195603

RESUMO

OBJECTIVE: The aim of the study was to examine whether the cannabinoid agonist WIN55212-2 could attenuate ischemic spinal cord injury (SCI) in rats through inhibition of GAPDH/Siah1 signaling. METHODS: Male Sprague-Dawley rats were distributed randomly into 5 groups: (1) sham group that received no aortic occlusion and injected intraperitoneally (i.p.) with vehicle control after reperfusion; (2) control group that received a 12-minute aortic occlusion and injected i.p. with vehicle control after reperfusion; (3) WIN55212-2 group (WIN) that received the aortic occlusion and injected i.p. with 1 mg/kg of WIN55212-2 after reperfusion; and (4) WIN55212-2 plus AM251 group and (5) WIN55212-2 plus AM630 group that received the same surgical operation as the WIN group, except that 1 mg/kg of AM251 or AM630 was injected i.p. 30 min before each dose of WIN55212-2 injection, respectively. Neurologic function was assessed 48 hours after reperfusion. Histopathologic examination was performed to determine the number of normal neurons in anterior spinal cord. Protein expression of active caspase-3, total caspase-3, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), inducible nitric oxide synthase (iNOS), nuclear factor kappa light chain enhancer of activated B cells (NF-κB), Siah1, tumor necrosis factor α, and interleukin 1ß were determined with Western blot and enzyme-linked immunosorbent assay; coimmunoprecipitation assays were also used to determine GAPDH/Siah1 complexing. Finally, terminal deoxynucleotidyl transferase dUTP nick end labeling staining was used to determine neuronal apoptosis in the lumbar spinal cord. RESULTS: The nuclear translocation of GAPDH and Siah1 in the spinal cord was initiated after ischemic spinal cord injury (SCI) along with the increased formation of GAPDH/Siah1 complexes. However, the activation of GAPDH/Siah1 was blocked by WIN. In addition, the treatment of WIN55212-2 promoted neuronal survival in the spinal cord, reduced apoptosis and inflammation, and improved neurologic scores. Furthermore, these beneficial effects of WIN55212-2 were abolished by the combined treatment of the CB2 antagonist AM630, but not the CB1 antagonist AM251. CONCLUSIONS: Our findings reveal GAPDH/Siah1 signaling cascades as a novel therapeutic target for ischemic SCI and identify WIN55212-2 with the potential to treat ischemic SCI by targeting this pathway.


Assuntos
Benzoxazinas/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Morfolinas/farmacologia , Naftalenos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Isquemia do Cordão Espinal , Medula Espinal , Animais , Apoptose/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Inflamação/metabolismo , Masculino , Neurônios/citologia , Neurônios/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Ratos , Ratos Sprague-Dawley , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Isquemia do Cordão Espinal/tratamento farmacológico , Isquemia do Cordão Espinal/prevenção & controle , Ubiquitina-Proteína Ligases/metabolismo
12.
Free Radic Biol Med ; 131: 144-153, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30500420

RESUMO

A single clove of edible garlic (Allium sativum L.) of about 10 g produces up to 5 mg of allicin (diallylthiosulfinate), a thiol-reactive sulfur-containing defence substance that gives injured garlic tissue its characteristic smell. Allicin induces apoptosis or necrosis in a dose-dependent manner but biocompatible doses influence cellular metabolism and signalling cascades. Oxidation of protein thiols and depletion of the glutathione pool are thought to be responsible for allicin's physiological effects. Here, we studied the effect of allicin on post-translational thiol-modification in human Jurkat T-cells using shotgun LC-MS/MS analyses. We identified 332 proteins that were modified by S-thioallylation in the Jurkat cell proteome which causes a mass shift of 72 Da on cysteines. Many S-thioallylated proteins are highly abundant proteins, including cytoskeletal proteins tubulin, actin, cofilin, filamin and plastin-2, the heat shock chaperones HSP90 and HSPA4, the glycolytic enzymes GAPDH, ALDOA, PKM as well the protein translation factor EEF2. Allicin disrupted the actin cytoskeleton in murine L929 fibroblasts. Allicin stimulated the immune response by causing Zn2+ release from proteins and increasing the Zn2+-dependent IL-1-triggered production of IL-2 in murine EL-4 T-cells. Furthermore, allicin caused inhibition of enolase activity, an enzyme considered a cancer therapy target. In conclusion, our study revealed the widespread extent of S-thioallylation in the human Jurkat cell proteome and showed effects of allicin exposure on essential cellular functions of selected targets, many of which are targets for cancer therapy.


Assuntos
Alho/química , Processamento de Proteína Pós-Traducional , Ácidos Sulfínicos/farmacologia , Fatores de Despolimerização de Actina/genética , Fatores de Despolimerização de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Filaminas/genética , Filaminas/metabolismo , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Proteínas de Choque Térmico HSP110/genética , Proteínas de Choque Térmico HSP110/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Células Jurkat , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteoma/genética , Proteoma/metabolismo , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Compostos de Sulfidrila/metabolismo , Ácidos Sulfínicos/isolamento & purificação , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Zinco/metabolismo
13.
Methods ; 155: 116-123, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30521847

RESUMO

Both RNA synthesis and decay must be balanced within a cell to achieve proper gene expression. Additionally, modulation of RNA decay specifically offers the cell an opportunity to rapidly reshape the transcriptome in response to specific stimuli or cues. Therefore, it is critical to understand the underlying mechanisms through which RNA decay contribute to gene expression homeostasis. Cell-free reconstitution approaches have been used successfully to reveal mechanisms associated with numerous post-transcriptional RNA processes. Historically, it has been difficult to examine all aspects of RNA decay in such an in vitro setting due, in part, to limitations on the ability to resolve larger RNAs through denaturing polyacrylamide gels. Thus, in vitro systems to study RNA decay rely on smaller, less biologically relevant RNA fragments. Herein, we present an approach to more confidently examine RNA decay parameters of large mRNA size transcripts through the inclusion of an engineered XRN1-resistant reporter RNA (xrRNA). By placing a 67 nucleotide xrRNA near the 3' end of any in vitro transcribed RNA with variable size or sequence context, investigators can observe the accumulation of the xrRNA as a readout of exoribonuclease-mediated 5'-3' decay. This approach may allow in vitro RNA decay assays to include full biologically relevant mRNA/mRNPs, extending their utility and allow improved experimental design considerations to promote biologically relevant outcomes.


Assuntos
Engenharia Genética/métodos , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Estabilidade de RNA , RNA Mensageiro/genética , RNA Viral/genética , Sistema Livre de Células , Eletroforese em Gel de Gradiente Desnaturante , Vírus da Dengue/química , Vírus da Dengue/genética , Exorribonucleases/genética , Exorribonucleases/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Transcrição Genética
14.
J Exp Bot ; 70(2): 653-670, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30395279

RESUMO

Non-enzymatic post-translational modifications of proteins can occur when the nucleophilic amino acid side chains of lysine and arginine encounter a reactive metabolite to form advanced glycation end products (AGEs). Glycation arises predominantly from the degradation of reducing sugars, and glycation has been observed during metabolic stress from glucose metabolism in both animals and plants. The implications of glycating proteins on plant proteins and biology has received little attention, and here we describe a robust assessment of global glycation profiles. We identified 112 glycated proteins that were common under a range of growth conditions and abiotic stress treatments, but also showed rosette age, diurnal, and drought stress-specific targets. Among 18 drought stress-specific glycation targets included several thioredoxin and thioredoxin-like proteins. In vitro glycation of two carbohydrate metabolism enzymes led either to a reduction or to a complete inhibition of activity, demonstrating the impact of glycation on protein function. Taken together, our results suggest that stress-specific glycation patterns of a small number of regulatory proteins may have a much broader impact on downstream target proteins that are, for example, associated with primary metabolism.


Assuntos
Arabidopsis/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Estresse Fisiológico , Proteínas de Arabidopsis/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Tiorredoxinas/metabolismo , Triose-Fosfato Isomerase/metabolismo
15.
Microb Pathog ; 127: 359-367, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30553015

RESUMO

GAPDH being a key enzyme in the glycolytic pathway is one of the surface adhesins of many Gram-positive bacteria including Streptococcus agalactiae. This anchorless adhesin is known to bind to host plasminogen (PLG) and fibrinogen (Fg), which enhances the virulence and modulates the host immune system. The crystal structure of the recombinant GAPDH from S. agalactiae (SagGAPDH) was determined at 2.6 Šresolution by molecular replacement. The structure was found to be highly conserved with a typical NAD binding domain and a catalytic domain. In this paper, using biolayer interferometry studies, we report that the multifunctional SagGAPDH enzyme binds to a variety of host molecules such as PLG, Fg, laminin, transferrin and mucin with a KD value of 4.4 × 10-7 M, 9.8 × 10-7 M, 1 × 10-5 M, 9.7 × 10-12 M and 1.4 × 10-7 M respectively. The ligand affinity blots reveal that SagGAPDH binds specifically to α and ß subunits of Fg and the competitive binding ELISA assay reveals that the Fg and PLG binding sites on GAPDH does not overlap each other. The PLG binding motif of GAPDH varies with organisms, however positively charged residues in the hydrophobic surroundings is essential for PLG binding. The lysine analogue competitive binding assay and lysine succinylation experiments deciphered the role of SagGAPDH lysines in PLG binding. On structural comparison with S. pneumoniae GAPDH, K171 of SagGAPDH is being predicted to be involved in PLG binding. Further SagGAPDH exhibited enzymatic activity in the presence of Fg, PLG and transferrin. This suggests that these host molecules does not mask the active site and bind at some other region of GAPDH.


Assuntos
Adesinas Bacterianas/química , Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/química , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Streptococcus agalactiae/enzimologia
16.
J Neuroinflammation ; 15(1): 308, 2018 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-30400801

RESUMO

BACKGROUND: Inflammation plays a critical role in accelerating the progression of neurodegenerative diseases, such as Alzheimer's disease (AD) and ataxia telangiectasia (A-T). In A-T mouse models, LPS-induced neuroinflammation advances the degenerative changes found in cerebellar Purkinje neurons both in vivo and in vitro. In the current study, we ask whether ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), can have the opposite effect and delay the symptoms of the disease. METHODS: We tested the beneficial effects of ibuprofen in both in vitro and in vivo models. Conditioned medium from LPS stimulated primary microglia (LM) applied to cultures of dissociated cortical neurons leads to numerous degenerative changes. Pretreatment of the neurons with ibuprofen, however, blocked this damage. Systemic injection of LPS into either adult wild-type or adult Atm-/- mice produced an immune challenge that triggered profound behavioral, biochemical, and histological effects. We used a 2-week ibuprofen pretreatment regimen to investigate whether these LPS effects could be blocked. We also treated young presymptomatic Atm-/- mice to determine if ibuprofen could delay the appearance of symptoms. RESULTS: Adding ibuprofen directly to neuronal cultures significantly reduced LM-induced degeneration. Curiously, adding ibuprofen to the microglia cultures before the LPS challenge had little effect, thus implying a direct effect of the NSAID on the neuronal cultures. In vivo administration of ibuprofen to Atm-/- animals before a systemic LPS immune challenge suppressed cytological damage. The ibuprofen effects were widespread as microglial activation, p38 phosphorylation, DNA damage, and neuronal cell cycle reentry were all reduced. Unfortunately, ibuprofen only slightly improved the LPS-induced behavioral deficits. Yet, while the behavioral symptoms could not be reversed once they were established in adult Atm-/- animals, administration of ibuprofen to young mutant pups prevented their symptoms from appearing. CONCLUSION: Inflammatory processes impact the normal progression of A-T implying that modulation of the immune system can have therapeutic benefit for both the behavioral and cellular symptoms of this neurodegenerative disease.


Assuntos
Ataxia Telangiectasia/prevenção & controle , Ibuprofeno/farmacologia , Animais , Animais Recém-Nascidos , Anti-Inflamatórios não Esteroides/farmacologia , Ataxia Telangiectasia/induzido quimicamente , Ataxia Telangiectasia/fisiopatologia , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/genética , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Comportamento Exploratório/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
17.
J Biol Chem ; 293(51): 19886-19898, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30385504

RESUMO

Urate is often viewed as an antioxidant. Here, we present an alternative perspective by showing that, when oxidized, urate propagates oxidative stress. Oxidation converts urate to the urate radical and the electrophilic products dehydrourate, 5-hydroxyisourate, and urate hydroperoxide, which eventually break down to allantoin. We investigated whether urate-derived electrophiles are intercepted by nucleophilic amino acid residues to form stable adducts on proteins. When urate was oxidized in the presence of various peptides and proteins, two adducts derived from urate (M r 167 Da) were detected and had mass additions of 140 and 166 Da, occurring mainly on lysine residues and N-terminal amines. The adduct with a 140-Da mass addition was detected more frequently and was stable. Dehydrourate (M r 166 Da) also formed transient adducts with cysteine residues. Urate-derived adducts were detected on human serum albumin in plasma of healthy donors. Basal adduct levels increased when neutrophils were added to plasma and stimulated, and relied on the NADPH oxidase, myeloperoxidase, hydrogen peroxide, and superoxide. Adducts of oxidized urate on serum albumin were elevated in plasma and synovial fluid from individuals with gout and rheumatoid arthritis. We propose that rather than acting as an antioxidant, urate's conversion to electrophiles contributes to oxidative stress. The addition of urate-derived electrophiles to nucleophilic amino acid residues, a process we call oxidative uratylation, will leave a footprint on proteins that could alter their function when critical sites are modified.


Assuntos
Ácido Úrico/química , Aminas/química , Sequência de Aminoácidos , Ativação Enzimática/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Humanos , Inflamação/metabolismo , Modelos Moleculares , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Conformação Proteica , Albumina Sérica/química , Albumina Sérica/metabolismo , Ácido Úrico/metabolismo , Ácido Úrico/farmacologia
18.
Nature ; 561(7722): 263-267, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30209366

RESUMO

Starvation poses a fundamental challenge to cell survival. Whereas the role of autophagy in promoting energy homeostasis in this setting has been extensively characterized1, other mechanisms are less well understood. Here we reveal that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibits coat protein I (COPI) transport by targeting a GTPase-activating protein (GAP) towards ADP-ribosylation factor 1 (ARF1) to suppress COPI vesicle fission. GAPDH inhibits multiple other transport pathways, also by targeting ARF GAPs. Further characterization suggests that this broad inhibition is activated by the cell during starvation to reduce energy consumption. These findings reveal a remarkable level of coordination among the intracellular transport pathways that underlies a critical mechanism of cellular energy homeostasis.


Assuntos
Metabolismo Energético , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Homeostase , Adenilato Quinase/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Animais , Autofagia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Linhagem Celular , Cricetulus , Fibroblastos , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/química , Humanos , Camundongos , Fosforilação , Ribonucleotídeos/metabolismo , Inanição
19.
BMC Plant Biol ; 18(1): 184, 2018 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-30189844

RESUMO

BACKGROUND: Plant cytosolic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GapC) displays redox-dependent changes in its subcellular localizations and activity. Apart from its fundamental role in glycolysis, it also exhibits moonlighting properties. Since the exceptional redox-sensitivity of GapC has been suggested to play a crucial role in its various functions, we here studied its redox-dependent subcellular localization and the influence of the redox-state on GapC protein interactions. RESULTS: In mesophyll protoplasts from Arabidopsis thaliana, colocalization of GapC with mitochondria was more pronounced under reducing conditions than upon oxidative stress. In accordance, reduced GapC showed an increased affinity to the mitochondrial voltage-dependent anion-selective channel (VDAC) compared to the oxidized one. On the other hand, nuclear localization of GapC was increased under oxidizing conditions. The essential role of the catalytic cysteine for nuclear translocation was shown by using the corresponding cysteine mutants. Furthermore, interaction of GapC with the thioredoxin Trx-h3 as a candidate to revert the redox-modifications, occurred in the nucleus of oxidized protoplasts. In a yeast complementation assay, we could demonstrate that the plant-specific non-phosphorylating glyceraldehyde 3-P dehydrogenase (GapN) can substitute for glucose 6-P dehydrogenase to generate NADPH for re-reduction of the Trx system and ROS defense. CONCLUSIONS: The preferred association of reduced, glycolytically active GapC with VDAC suggests a substrate-channeling metabolon at the mitochondrial surface for efficient energy generation. Increased occurrence of oxidized GapC in the nucleus points to a function in signal transduction and gene expression. Furthermore, the interaction of GapC with Trx-h3 in the nucleus indicates reversal of the oxidative cysteine modification after re-establishment of cellular homeostasis. Both, energy metabolism and signal transfer for long-term adjustment and protection from redox-imbalances are mediated by the various functions of GapC. The molecular properties of GapC as a redox-switch are key to its multiple roles in orchestrating energy metabolism.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citosol/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cisteína/metabolismo , Metabolismo Energético , Teste de Complementação Genética , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Mitocôndrias/metabolismo , Mutação , Oxirredução , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Tiorredoxinas/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo
20.
Sci Rep ; 8(1): 12856, 2018 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-30150703

RESUMO

There is strong evidence indicating neuroinflammation is an important mediator in multiple sclerosis (MS), with astrogliosis playing a significant role in this process. Surprisingly, astrocytes exert paradoxical roles during disease development, but the mechanisms remain unknown. Previously, we have reported that administering an interfering peptide (GluA2-G-Gpep) which specifically disrupts the GluA2-GAPDH interaction rescued neurological symptoms in the EAE mouse model of MS. In this study, we validated that the GluA2-GAPDH complex was elevated in LPS-induced primary reactive astrocytes, and GluA2-G-Gpep treatment significantly reduced GFAP expression levels in both EAE mice and reactive astrocytes. Further in vivo and in vitro analyses revealed that GluA2-G-Gpep administration normalized EAAT1 and EAAT2 expression, rescued compromised blood-brain barrier integrity via AQP4, promoted actin reorganization and changed mitochondrial dynamics. These alterations may partially be explained by changes in the nuclear GAPDH and p53 transcription pathways. Our findings provide critical implications for understanding the astrocyte properties regulated by GluA2-GAPDH associated with MS, and insights for novel treatment options targeting at astrocytes.


Assuntos
Astrócitos/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Esclerose Múltipla/metabolismo , Receptores de AMPA/metabolismo , Animais , Aquaporina 4/genética , Aquaporina 4/metabolismo , Biomarcadores , Barreira Hematoencefálica/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/metabolismo , Transportador 1 de Aminoácido Excitatório/genética , Transportador 1 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Imunofluorescência , Camundongos , Mitocôndrias/metabolismo , Permeabilidade , Ligação Proteica
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