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1.
Nat Commun ; 11(1): 4653, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938923

RESUMO

Cancer cells demand excess nutrients to support their proliferation, but how tumours exploit extracellular amino acids during systemic metabolic perturbations remain incompletely understood. Here, we use a Drosophila model of high-sugar diet (HSD)-enhanced tumourigenesis to uncover a systemic host-tumour metabolic circuit that supports tumour growth. We demonstrate coordinate induction of systemic muscle wasting with tumour-autonomous Yorkie-mediated SLC36-family amino acid transporter expression as a proline-scavenging programme to drive tumourigenesis. We identify Indole-3-propionic acid as an optimal amino acid derivative to rationally target the proline-dependency of tumour growth. Insights from this whole-animal Drosophila model provide a powerful approach towards the identification and therapeutic exploitation of the amino acid vulnerabilities of tumourigenesis in the context of a perturbed systemic metabolic network.


Assuntos
Açúcares da Dieta/efeitos adversos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Neoplasias Experimentais/fisiopatologia , Prolina/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Animais Geneticamente Modificados , Carcinogênese , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Hemolinfa/efeitos dos fármacos , Hemolinfa/metabolismo , Larva , Debilidade Muscular/induzido quimicamente , Debilidade Muscular/patologia , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/patologia , Neoplasias Experimentais/etiologia , Proteínas Nucleares/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transativadores/genética , Proteínas ras/genética
2.
Nat Commun ; 11(1): 4866, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978391

RESUMO

Mitochondria house evolutionarily conserved pathways of carbon and nitrogen metabolism that drive cellular energy production. Mitochondrial bioenergetics is regulated by calcium uptake through the mitochondrial calcium uniporter (MCU), a multi-protein complex whose assembly in the inner mitochondrial membrane is facilitated by the scaffold factor MCUR1. Intriguingly, many fungi that lack MCU contain MCUR1 homologs, suggesting alternate functions. Herein, we characterize Saccharomyces cerevisiae homologs Put6 and Put7 of MCUR1 as regulators of mitochondrial proline metabolism. Put6 and Put7 are tethered to the inner mitochondrial membrane in a large hetero-oligomeric complex, whose abundance is regulated by proline. Loss of this complex perturbs mitochondrial proline homeostasis and cellular redox balance. Yeast cells lacking either Put6 or Put7 exhibit a pronounced defect in proline utilization, which can be corrected by the heterologous expression of human MCUR1. Our work uncovers an unexpected role of MCUR1 homologs in mitochondrial proline metabolism.


Assuntos
Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Prolina/metabolismo , Saccharomyces cerevisiae/metabolismo , Canais de Cálcio , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Homeostase , Humanos , Proteínas de Membrana/genética , Redes e Vias Metabólicas/genética , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transcriptoma
3.
Mol Cell ; 79(3): 376-389.e8, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32640193

RESUMO

Activation of dual-specificity tyrosine-phosphorylation-regulated kinases 1A and 1B (DYRK1A and DYRK1B) requires prolyl hydroxylation by PHD1 prolyl hydroxylase. Prolyl hydroxylation of DYRK1 initiates a cascade of events leading to the release of molecular constraints on von Hippel-Lindau (VHL) ubiquitin ligase tumor suppressor function. However, the proline residue of DYRK1 targeted by hydroxylation and the role of prolyl hydroxylation in tyrosine autophosphorylation of DYRK1 are unknown. We found that a highly conserved proline in the CMGC insert of the DYRK1 kinase domain is hydroxylated by PHD1, and this event precedes tyrosine autophosphorylation. Mutation of the hydroxylation acceptor proline precludes tyrosine autophosphorylation and folding of DYRK1, resulting in a kinase unable to preserve VHL function and lacking glioma suppression activity. The consensus proline sequence is shared by most CMGC kinases, and prolyl hydroxylation is essential for catalytic activation. Thus, formation of prolyl-hydroxylated intermediates is a novel mechanism of kinase maturation and likely a general mechanism of regulation of CMGC kinases in eukaryotes.


Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Isoenzimas/genética , Prolina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Glioma/patologia , Células HEK293 , Xenoenxertos , Humanos , Hidroxilação , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Camundongos , Camundongos Nus , Proteína Quinase 14 Ativada por Mitógeno/química , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Mutação , Neuroglia/metabolismo , Neuroglia/patologia , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
4.
Prostate ; 80(13): 1071-1086, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32687633

RESUMO

BACKGROUND: The emergence of reactive stroma is a hallmark of prostate cancer (PCa) progression and a potential source for prognostic and diagnostic markers of PCa. Collagen is a main component of reactive stroma and changes systematically and quantitatively to reflect the course of PCa, yet has remained undefined due to a lack of tools that can define collagen protein structure. Here we use a novel collagen-targeting proteomics approach to investigate zonal regulation of collagen-type proteins in PCa prostatectomies. METHODS: Prostatectomies from nine patients were divided into zones containing 0%, 5%, 20%, 70% to 80% glandular tissue and 0%, 5%, 25%, 70% by mass of PCa tumor following the McNeal model. Tissue sections from zones were graded by a pathologist for Gleason score, percent tumor present, percent prostatic intraepithelial neoplasia and/or inflammation (INF). High-resolution accurate mass collagen targeting proteomics was done on a select subset of tissue sections from patient-matched tumor or nontumor zones. Imaging mass spectrometry was used to investigate collagen-type regulation corresponding to pathologist-defined regions. RESULTS: Complex collagen proteomes were detected from all zones. COL17A and COL27A increased in zones of INF compared with zones with tumor present. COL3A1, COL4A5, and COL8A2 consistently increased in zones with tumor content, independent of tumor size. Collagen hydroxylation of proline (HYP) was altered in tumor zones compared with zones with INF and no tumor. COL3A1 and COL5A1 showed significant changes in HYP peptide ratios within tumor compared with zones of INF (2.59 ± 0.29, P value: .015; 3.75 ± 0.96 P value .036, respectively). By imaging mass spectrometry COL3A1 showed defined localization and regulation to tumor pathology. COL1A1 and COL1A2 showed gradient regulation corresponding to PCa pathology across zones. Pathologist-defined tumor regions showed significant increases in COL1A1 HYP modifications compared with COL1A2 HYP modifications. Certain COL1A1 and COL1A2 peptides could discriminate between pathologist-defined tumor and inflammatory regions. CONCLUSIONS: Site-specific posttranslational regulation of collagen structure by proline hydroxylation may be involved in reactive stroma associated with PCa progression. Translational and posttranslational regulation of collagen protein structure has potential for new markers to understand PCa progression and outcomes.


Assuntos
Colágeno/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Processamento de Proteína Pós-Traducional , Idoso , Sequência de Aminoácidos , Autoantígenos , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Colágeno Tipo IV/metabolismo , Colágeno Tipo VIII/metabolismo , Progressão da Doença , Colágenos Fibrilares/metabolismo , Humanos , Hidroxilação , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Colágenos não Fibrilares , Prolina/metabolismo , Próstata/metabolismo , Prostatectomia , Neoplasias da Próstata/diagnóstico por imagem , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos
6.
PLoS One ; 15(7): e0235556, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614916

RESUMO

To gain a better insight into the selenium nanoparticle (nSe) benefits/toxicity, this experiment was carried out to address the behavior of bitter melon seedlings to nSe (0, 1, 4, 10, 30, and 50 mgL-1) or bulk form (selenate). Low doses of nSe increased biomass accumulation, while concentrations of 10 mgL-1 and above were associated with stem bending, impaired root meristem, and severe toxicity. Responses to nSe were distinct from that of bulk in that the nano-type exhibited a higher efficiency to stimulate growth and organogenesis than the bulk. The bulk form displayed higher phytotoxicity than the nano-type counterpart. According to the MSAP-based analysis, nSe mediated substantial variation in DNA cytosine methylation, reflecting the epigenetic modification. By increasing the concentration of nSe, the expression of the WRKY1 transcription factor linearly up-regulated (mean = 7.9-fold). Transcriptions of phenylalanine ammonia-lyase (PAL) and 4-Coumarate: CoA-ligase (4CL) genes were also induced. The nSe treatments at low concentrations enhanced the activity of leaf nitrate reductase (mean = 52%) in contrast with the treatment at toxic concentrations. The toxic concentration of nSe increased leaf proline concentration by 80%. The nSe supplement also stimulated the activities of peroxidase (mean = 35%) and catalase (mean = 10%) enzymes. The nSe-treated seedlings exhibited higher PAL activity (mean = 39%) and soluble phenols (mean = 50%). The nSe toxicity was associated with a disrupted differentiation of xylem conducting tissue. The callus formation and performance of the explants originated from the nSe-treated seedlings had a different trend than that of the control. This experiment provides new insights into the nSe-associated advantage/ cytotoxicity and further highlights the necessity of designing convincing studies to introduce novel methods for plant cell/tissue cultures and agriculture.


Assuntos
Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Momordica charantia/metabolismo , Nanopartículas/toxicidade , Selênio/química , Citosina/metabolismo , Momordica charantia/efeitos dos fármacos , Momordica charantia/crescimento & desenvolvimento , Nanopartículas/química , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , Fenóis/metabolismo , Fenilalanina Amônia-Liase/genética , Fenilalanina Amônia-Liase/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Prolina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacos
7.
Ecotoxicol Environ Saf ; 201: 110869, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32585490

RESUMO

Chromium (Cr) contamination is a potential threat to the agricultural soil. Arbuscular mycorrhizal (AM) fungi have potential to remediate the heavy metal polluted soils. It was hypothesized that Cr phytoremediation potentiality of AM fungi could be enhanced in combination with saprophytic filamentous fungi and soil amendment. Tomato plants were raised in Cr polluted technosol amended with compost, inoculated with mixed-culture of AM fungi and Aspergillus terreus. It was found that, triple treatment (soil amendment with compost along with AM fungi and A. terreus inoculation) enhanced biomass production (up to 315%), fruit setting (up to 49%), photosynthetic pigments (up to 214%) and carbohydrate content (up to 400%) whereas reduced the proline (up to 76.5%), catalase (up to 34.2%), peroxidase (up to 58.9%) and root membrane permeability (up to 74.2%). The effect of AM fungi with compost amendment was additive, while it was synergistic with A. terreus. AM fungi enhanced the extraction of Cr from the substrate, but retained in the mycorrhizal root, thereby reduced the translocation into shoot and in fruit, Cr translocation was undetectable. At the end of experiment Cr content in the substrate was significantly decreased (up to 37.9%). Soil amendment with compost along with AM fungi and A. terreus inoculation can be used for reclamation of Cr polluted soils at field scale.


Assuntos
Aspergillus/fisiologia , Cromo/farmacocinética , Lycopersicon esculentum/metabolismo , Micorrizas/fisiologia , Poluentes do Solo/farmacocinética , Biodegradação Ambiental , Biomassa , Metabolismo dos Carboidratos , Catalase/metabolismo , Compostagem , Lycopersicon esculentum/enzimologia , Peroxidase/metabolismo , Raízes de Plantas/metabolismo , Prolina/metabolismo
8.
PLoS Biol ; 18(6): e3000741, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32520929

RESUMO

Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.


Assuntos
Metabolômica , Mitocôndrias/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismo , Trifosfato de Adenosina/biossíntese , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Elétrons , Glucose/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Oxirredução , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Transcriptoma/genética , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/genética
9.
Ecotoxicol Environ Saf ; 202: 110890, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32593096

RESUMO

The presence of tetracycline is ubiquitous and has adverse effects on aquatic systems. A hydroponic experiment was conducted to investigate the ecological sensitivity of Hydrocharis dubia (Bl.) Backer and Trapa bispinosa Roxb. Exposed to different concentrations of tetracycline (0, 0.1, 1, 10, 30 and 50 mg/L) for one day (1D) and 14 days (14D). The results showed that after 1D of tetracycline exposure, the physiological indices of H. dubia had no remarkable change except for proline which was significantly stimulated under 0.1 mg/L tetracycline. For T. bispinosa, guaiacol peroxidase (POD), polyphenol oxidase (PPO) and ascorbate peroxidase (APX) activity and protein and proline content were notably promoted under different concentrations of tetracycline, but PPO activity was significantly decreased in 50 mg/L. After 14D, tetracycline caused no harm to the growth and protein content of H. dubia, but negatively influenced lipid peroxidation product and chlorophyll content in H. dubia under high tetracycline concentrations. Superoxide dismutase (SOD) and POD activity of H. dubia significantly increased at high tetracycline concentrations, while catalase (CAT) and PPO activity significantly decreased. APX activity in H. dubia increased with tetracycline concentrations at low tetracycline concentrations. For T. bispinosa, high concentrations of tetracycline application significantly inhibited its growth and the content of protein and chlorophyll. SOD, POD, CAT, and PPO activity of T. bispinosa were induced under different concentrations of tetracycline and no lipid peroxidation was observed. APX activity in T. bispinosa was significantly inhibited at high tetracycline concentrations. The results suggest that tetracycline can cause oxidative damage in H. dubia but harm the metabolism process of T. bispinosa without inducing oxidative damage. Overall, the sensitivity of T. bispinosa exposed to tetracycline exposure is higher than that of H. dubia.


Assuntos
Antibacterianos/toxicidade , Hydrocharitaceae/fisiologia , Tetraciclina/toxicidade , Antioxidantes/metabolismo , Ascorbato Peroxidases/metabolismo , Catalase/metabolismo , Clorofila/metabolismo , Hydrocharitaceae/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Lythraceae , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Peroxidase , Prolina/metabolismo , Superóxido Dismutase/metabolismo
10.
Proc Natl Acad Sci U S A ; 117(25): 14158-14167, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513738

RESUMO

Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a K d of 16 µM, whereas the otherwise identical Nt-Pro-bearing sequence PGLW binds to GID4 more tightly, with a K d of 1.9 µM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW-bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.


Assuntos
Prolina/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/química , Proteínas de Transporte Vesicular/química , Humanos , Modelos Moleculares , Prolina/metabolismo , Complexo de Endopeptidases do Proteassoma , Conformação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
11.
Ecotoxicol Environ Saf ; 200: 110721, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32464438

RESUMO

Glutaredoxins (Grxs) are small (10-15 kDa) glutathione (GSH) - dependent redox proteins. The role of Grxs are well documented in tolerance to heavy metal stress in prokaryotic and mammalian systems and a few plant genera, but is poorly understood in plants against drought. In the present study, two rice glutaredoxin (Osgrx) genes (LOC_Os02g40500 and LOC_Os01g27140) responsible for tolerance against heavy metal stress have been studied for investigating their role against drought. Each glutaredoxin gene was over-expressed in Arabidopsis thaliana to reveal their role in drought stress. The relative expression of both Osgrx genes was higher in the transgenic lines. Transgenic lines of both Osgrxs showed longer roots, higher seed germination, and survival efficiency during drought stress. The physiological parameters (PN, gs, E, WUE, qP, NPQ and ETR), antioxidant enzymes (GRX, GR, GPX, GST, APX, POD, SOD, CAT, DHAR, and MDHAR), antioxidant molecules (ascorbate and GSH) and stress-responsive amino acids (cysteine and proline) levels were additionally increased in transgenic lines of both Osgrxs to provide drought tolerance. The outcomes from this study strongly determined that each Osgrx gene participated in the moderation of drought and might be utilized in biological engineering strategies to overcome drought conditions in different crops.


Assuntos
Glutarredoxinas/genética , Oryza/enzimologia , Estresse Fisiológico , Antioxidantes/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Ácido Ascórbico/metabolismo , Cisteína/metabolismo , Secas , Genes de Plantas , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Prolina/metabolismo , Estresse Fisiológico/genética
12.
J Anim Sci ; 98(6)2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32415842

RESUMO

This study determined whether extracellular citrulline is degraded by ruminal bacteria of sheep. In the first experiment, whole rumen fluid (3 mL) from six adult Suffolk sheep was incubated at 37 °C with 5 mM l-glutamine (Gln), l-glutamate (Glu), l-arginine (Arg), or l-citrulline (Cit) for 0, 0.5, 1, and 2 h or with 0, 0.5, 2, or 5 mM Gln, Glu, Arg, or Cit for 2 h. An aliquot (50 µL) of the incubation solution was collected at the predetermined time points for amino acids (AA) analyses. Results showed extensive hydrolysis of Gln into Glu and ammonia, of Arg into l-ornithine and l-proline, but little or no degradation of extracellular Cit or Glu by ruminal microbes. In the second experiment, six adult Suffolk sheep were individually fed each of three separate supplements (8 g Gln , Cit, or urea) on three separate days along with regular feed (800 g/animal). Blood (2 mL) was sampled from the jugular vein prior to feeding (time 0) and at 0.5, 1, 2, and 4 h after consuming the supplement. Plasma was analyzed for AA, glucose, ammonia, and urea. The concentrations of Cit in the plasma of sheep consuming this AA increased (P < 0.001) by 117% at 4 h and those of Arg increased by 23% at 4 h, compared with the baseline values. Urea or Gln feeding did not affect (P > 0.05) the concentrations of Cit or Arg in plasma. These results indicate that Cit is not metabolized by ruminal microbes of sheep and is, therefore, absorbed as such by the small intestine and used for the synthesis of Arg by extrahepatic tissues.


Assuntos
Bactérias/metabolismo , Citrulina/metabolismo , Rúmen/microbiologia , Ovinos/microbiologia , Amônia/metabolismo , Ração Animal/análise , Animais , Arginina/metabolismo , Dieta/veterinária , Suplementos Nutricionais , Feminino , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Ornitina/sangue , Prolina/metabolismo , Ureia
13.
PLoS Pathog ; 16(5): e1008244, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365082

RESUMO

Viral escape from CD8+ cytotoxic T lymphocyte responses correlates with disease progression and represents a significant challenge for vaccination. Here, we demonstrate that CD8+ T cell recognition of the naturally occurring MHC-I-restricted LCMV-associated immune escape variant Y4F is restored following vaccination with a proline-altered peptide ligand (APL). The APL increases MHC/peptide (pMHC) complex stability, rigidifies the peptide and facilitates T cell receptor (TCR) recognition through reduced entropy costs. Structural analyses of pMHC complexes before and after TCR binding, combined with biophysical analyses, revealed that although the TCR binds similarly to all complexes, the p3P modification alters the conformations of a very limited amount of specific MHC and peptide residues, facilitating efficient TCR recognition. This approach can be easily introduced in peptides restricted to other MHC alleles, and can be combined with currently available and future vaccination protocols in order to prevent viral immune escape.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vírus da Coriomeningite Linfocítica/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Antivirais/metabolismo , Linfócitos T CD8-Positivos/fisiologia , Proteínas de Ligação a DNA/imunologia , Epitopos/imunologia , Epitopos de Linfócito T/imunologia , Genes RAG-1/imunologia , Ligantes , Ativação Linfocitária/imunologia , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/patogenicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptídeos/metabolismo , Prolina/metabolismo , Ligação Proteica , Linfócitos T Citotóxicos/imunologia , Vacinação/métodos
14.
Ecotoxicol Environ Saf ; 200: 110732, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32460049

RESUMO

This paper reports the role of exogenous glycine betaine (25 and 50 mM GB at a rate of 50 mL per plant) in enhancing NaCl-stress tolerance in common bean (Phaseolus vulgaris L.). Irrigating plants by simulated saline water, containing 0, 50 and 100 mM sodium chloride (NaCl), significantly reduced the growth dynamics, photosynthetic pigments (i.e., Chl a, Chl b, and carotenoids), membrane stability index (MSI), relative water content (RWC), and pod yield. While, malondialdehyde (MDA), endogenous proline, and glutathione contents, electrolyte leakage (EL), antioxidant defense system, and Na+ accumulation markedly increased upon exposure to NaCl-stress. However, the application of exogenous GB significantly improved salt tolerance of common bean as it increased the antioxidant defense including both enzymatic (i.e., peroxidase, superoxide dismutase, and catalase) and nonenzymatic (i.e., proline and glutathione) agents. Consequently, MSI, RWC, EL, and photosynthetic pigments have been improved recording significantly higher values than the control. Moreover, the pod yield increased by 29.8 and 59.4% when plants grown under 50 and 100 mM NaCl, respectively, were sprayed with 25 mM GB. Our results show that GB-induced slat tolerance in common bean plants mainly depends on the osmoregulation effect of GB and to a lesser extent on its antioxidant capacity. Foliar application of GB significantly reduced the accumulation of Na+ and at the same time induced K+ uptake maintaining a higher K+/Na+ ratio. Despite some changes in the activities of antioxidant enzymes induced by the application of GB, no consistent contribution in the salt tolerance could be cited in this study. Therefore, we suggest that salt tolerance is largely unrelated to the antioxidant defense ability of GB in common bean. While the potential role of GB in ameliorating salt tolerance is mainly due to the adjustment of ions uptake through limiting Na+ uptake and alternatively increasing K+ accumulation in plant tissues.


Assuntos
Betaína/farmacologia , Phaseolus/efeitos dos fármacos , Potássio/metabolismo , Tolerância ao Sal , Sódio/metabolismo , Antioxidantes/metabolismo , Transporte Biológico/efeitos dos fármacos , Catalase/metabolismo , Cátions , Glutationa/metabolismo , Malondialdeído/análise , Osmorregulação/efeitos dos fármacos , Peroxidase/metabolismo , Phaseolus/química , Phaseolus/enzimologia , Phaseolus/metabolismo , Fotossíntese/efeitos dos fármacos , Potássio/análise , Prolina/metabolismo , Sódio/análise , Superóxido Dismutase/metabolismo
15.
J Biol Chem ; 295(20): 6926-6935, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32249210

RESUMO

Mouse hepatitis virus (MHV; murine coronavirus) causes meningoencephalitis, myelitis, and optic neuritis followed by axonal loss and demyelination. This murine virus is used as a common model to study acute and chronic virus-induced demyelination in the central nervous system. Studies with recombinant MHV strains that differ in the gene encoding the spike protein have demonstrated that the spike has a role in MHV pathogenesis and retrograde axonal transport. Fusion peptides (FPs) in the spike protein play a key role in MHV pathogenesis. In a previous study of the effect of deleting a single proline residue in the FP of a demyelinating MHV strain, we found that two central, consecutive prolines are important for cell-cell fusion and pathogenesis. The dihedral fluctuation of the FP was shown to be repressed whenever two consecutive prolines were present, in contrast to the presence of a single proline in the chain. Using this proline-deleted MHV strain, here we investigated whether intracranial injection of this strain can induce optic neuritis by retrograde axonal transport from the brain to the retina through the optic nerve. We observed that the proline-deleted recombinant MHV strain is restricted to the optic nerve, is unable to translocate to the retina, and causes only minimal demyelination and no neuronal death. We conclude that an intact proline dyad in the FP of the recombinant demyelinating MHV strain plays a crucial role in translocation of the virus through axons and subsequent neurodegeneration.


Assuntos
Transporte Axonal/genética , Vírus da Hepatite Murina/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Transporte Axonal/fisiologia , Axônios/metabolismo , Axônios/virologia , Encéfalo/metabolismo , Infecções por Coronavirus/patologia , Doenças Desmielinizantes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vírus da Hepatite Murina/metabolismo , Nervo Óptico/metabolismo , Nervo Óptico/virologia , Peptídeos/metabolismo , Prolina/metabolismo , Deleção de Sequência/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas do Envelope Viral/metabolismo
16.
PLoS One ; 15(4): e0230615, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32251430

RESUMO

Availability of good quality irrigation water is a big challenge in arid and semi arid regions of the world. Drought stress results in poor plant growth and low yield; however, the rhizobacteria, capable of producing 1-aminocyclopropane-1-carboxylate (ACC)-deaminase are likely to improve crop growth and productivity under drought stress. Similarly, biochar could also ameliorate the negative impacts of drought stress. Therefore, this pot experiment was conducted to evaluate the role of ACC-deaminase producing plant growth promoting rhizobacteria (PGPR) alone and in combinations with timber-waste biochar in improving maize growth under drought stress. The ACC-deaminase producing rhizobacteria, Pseudomonas aeruginosa, Enterobacter cloacae, Achromobacter xylosoxidans and Leclercia adecarboxylata were studied along with two rates (0.75 and 1.50% of the soil weight) of biochar under three moisture levels i.e., normal moisture, mild drought stress and severe drought stress. The E. cloacae in conjunction with higher rate of biochar produced a significant improvement i.e., up to 60, 73, 43, 69, 76 and 42% respectively, in grain yield plant-1, photosynthetic rate, stomatal conductance, chlorophyll a, total chlorophyll and carotenoids contents of maize as compared to the control under mild drought stress. Similarly, A. xylosoxidans with higher rate of biochar also enhanced grain yield plant-1, photosynthetic rate, stomatal conductance, chlorophyll a, total chlorophyll and carotenoids contents of maize up to 200, 213, 113, 152, 148 and 284%, respectively over control under severe drought stress. In conclusion, combination of ACC-deaminase containing PGPR, A. xylosoxidans and biochar (0.75%) proved an effective technique to improve maize growth and productivity under drought stress.


Assuntos
Bactérias/enzimologia , Carbono-Carbono Liases/biossíntese , Carvão Vegetal/farmacologia , Secas , Rizosfera , Zea mays/efeitos dos fármacos , Zea mays/microbiologia , Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Carotenoides/metabolismo , Clorofila/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Prolina/metabolismo , Estresse Fisiológico , Zea mays/crescimento & desenvolvimento , Zea mays/fisiologia
17.
PLoS One ; 15(4): e0231555, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32315321

RESUMO

The proline-specific enzymes dipeptidyl peptidase 4 (DPP4), prolylcarboxypeptidase (PRCP), fibroblast activation protein α (FAP) and prolyl oligopeptidase (PREP) are known for their involvement in the immune system and blood pressure regulation. Only very limited information is currently available on their enzymatic activity and possible involvement in patients with sepsis and septic-shock. The activity of the enzymes was measured in EDTA-plasma of patients admitted to the intensive care unit (ICU): 40 septic shock patients (sepsis-2) and 22 ICU control patients after major intracranial surgery. These data were used to generate receiver operating characteristic (ROC) curves. A survival analysis (at 90 days) and an association study with other parameters was performed. PRCP (day 1) and PREP (all days) enzymatic activities were higher in septic shock patients compared to controls. In contrast, FAP and DPP4 were lower in these patients on all studied time points. Since large differences were found, ROC curves were generated and these yielded area under the curve (AUC) values for PREP, FAP and DPP4 of 0.88 (CI: 0.80-0.96), 0.94 (CI: 0.89-0.99) and 0.86 (CI: 0.77-0.95), respectively. PRCP had a lower predicting value with an AUC of 0.71 (CI: 0.58-0.83). A nominally significant association was observed between survival and the DPP4 enzymatic activity at day 1 (p<0.05), with a higher DPP4 activity being associated with an increase in survival. All four enzymes were dysregulated in septic shock patients. DPP4, FAP and PREP are good in discriminating between septic shock patients and ICU controls and should be further explored to see whether they are already dysregulated in earlier stages, opening perspectives for their further investigation as biomarkers in sepsis. DPP4 also shows potential as a prognostic biomarker. Additionally, the associations found warrant further research.


Assuntos
Carboxipeptidases/sangue , Dipeptidil Peptidase 4/sangue , Gelatinases/sangue , Proteínas de Membrana/sangue , Serina Endopeptidases/sangue , Choque Séptico/sangue , Choque Séptico/enzimologia , Área Sob a Curva , Biomarcadores/sangue , Cuidados Críticos , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Prolina/metabolismo , Estudos Prospectivos , Curva ROC , Choque Séptico/mortalidade , Choque Séptico/terapia , Análise de Sobrevida
18.
PLoS Pathog ; 16(4): e1008441, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32294136

RESUMO

Gut symbiotic bacteria have a substantial impact on host physiology and ecology. However, the contribution of gut microbes to host fitness during long-term low-temperature stress is still unclear. This study examined the role of gut microbiota in host low-temperature stress resistance at molecular and biochemical levels in the oriental fruit fly Bactrocera dorsalis. The results showed that after the gut bacteria of flies were removed via antibiotic treatment, the median survival time was significantly decreased to approximately 68% of that in conventional flies following exposure to a temperature stress of 10°C. Furthermore, we found that Klebsiella michiganensis BD177 is a key symbiotic bacterium, whose recolonization in antibiotic treated (ABX) flies significantly extended the median survival time to 160% of that in the ABX control, and restored their lifespan to the level of conventional flies. Notably, the relative levels of proline and arginine metabolites were significantly downregulated by 34- and 10-fold, respectively, in ABX flies compared with those in the hemolymph of conventional flies after exposure to a temperature stress of 10°C whereas recolonization of ABX flies by K. michiganensis BD177 significantly upregulated the levels of proline and arginine by 13- and 10- fold, respectively, compared with those found in the hemolymph of ABX flies. qPCR analysis also confirmed that K. michiganensis-recolonized flies significantly stimulated the expression of transcripts from the arginine and proline metabolism pathway compared with the ABX controls, and RNAi mediated silencing of two key genes Pro-C and ASS significantly reduced the survival time of conventional flies, postexposure low-temperature stress. We show that microinjection of L-arginine and L-proline into ABX flies significantly increased their survival time following exposure to temperature stress of 10°C. Transmission electron microscopy (TEM) analysis further revealed that low-temperature stress caused severe destruction in cristae structures and thus resulted in abnormal circular shapes of mitochondria in ABX flies gut, while the recolonization of live K. michiganensis helped the ABX flies to maintain mitochondrial functionality to a normal status, which is important for the arginine and proline induction. Our results suggest that gut microbiota plays a vital role in promoting the host resistance to low-temperature stress in B. dorsalis by stimulating its arginine and proline metabolism pathway.


Assuntos
Arginina/metabolismo , Microbioma Gastrointestinal , Prolina/metabolismo , Tephritidae/microbiologia , Animais , Temperatura Baixa , Klebsiella/genética , Klebsiella/crescimento & desenvolvimento , Klebsiella/isolamento & purificação , Klebsiella/fisiologia , Masculino , Estresse Fisiológico , Simbiose , Tephritidae/fisiologia
19.
PLoS One ; 15(4): e0231348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298338

RESUMO

Salt stress is one of the devastating factors that hampers growth and productivity of soybean. Use of Pseudomonas pseudoalcaligenes to improve salt tolerance in soybean has not been thoroughly explored yet. Therefore, we observed the response of hydroponically grown soybean plants, inoculated with halotolerant P. pseudoalcaligenes (SRM-16) and Bacillus subtilis (SRM-3) under salt stress. In vitro testing of 44 bacterial isolates revealed that four isolates showed high salt tolerance. Among them, B. subtilis and P. pseudoalcaligenes showed ACC deaminase activity, siderophore and indole acetic acid (IAA) production and were selected for the current study. We determined that 106 cells/mL of B. subtilis and P. pseudoalcaligenes was sufficient to induce tolerance in soybean against salinity stress (100 mM NaCl) in hydroponics by enhancing plant biomass, relative water content and osmolytes. Upon exposure of salinity stress, P. pseudoalcaligenes inoculated soybean plants showed tolerance by the increased activities of defense related system such as ion transport, antioxidant enzymes, proline and MDA content in shoots and roots. The Na+ concentration in the soybean plants was increased in the salt stress; while, bacterial priming significantly reduced the Na+ concentration in the salt stressed soybean plants. However, the antagonistic results were observed for K+ concentration. Additionally, soybean primed with P. pseudoalcaligenes and exposed to 100 mM NaCl showed a new protein band of 28 kDa suggesting that P. pseudoalcaligenes effectively reduced salt stress. Our results showed that salinity tolerance was more pronounced in P. pseudoalcaligenes as compared to B. subtilis. However, a detailed study at molecular level to interpret the mechanism by which P. pseudoalcaligenes alleviates salt stress in soybean plants need to be explored.


Assuntos
Bacillus subtilis/patogenicidade , Pseudomonas pseudoalcaligenes/patogenicidade , Tolerância ao Sal , Soja/metabolismo , Bacillus subtilis/metabolismo , Transporte de Íons , Malondialdeído/metabolismo , Peroxidase/genética , Peroxidase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Pseudomonas pseudoalcaligenes/metabolismo , Espécies Reativas de Oxigênio , Soja/microbiologia
20.
Artigo em Inglês | MEDLINE | ID: mdl-32297058

RESUMO

Proline is one of the most important compatible osmolyte in cells, which accumulates in response to various stresses, including salt, water deficit, heavy metal, pathogen infection and extreme temperature. In this study, a growth chamber was employed to simulate heat environment for Avicennia marina seedlings. We detected some physiological indices in the leaves of A. marina at 40 °C, including the activity of delta-1-pyrroline-5-carboxylate synthase (P5CS), the content of free proline and soluble protein, transpiration rate and membrane permeability, and discussed the relationship between these five indices and heat resistant ability. And then a P5CS gene was cloned from A. marina using homologous cloning and rapid amplification of cDNA ends methods. It was designated as AmP5CS, encoding a protein that contained a feedback inhibition site of proline, proA, proB, conserved Leu zipper, GSA-DH domain and other functional domains of P5CS protein in high plants. Expression analysis of AmP5CS gene indicated it was involved in heat stress response. It is the first time that P5CS from A. marina has been cloned and the findings laid the foundation of figuring out heat resistant mechanisms and relieving heat damage, which is significant during global warming.


Assuntos
Avicennia/fisiologia , Genes de Plantas , Resposta ao Choque Térmico/fisiologia , Áreas Alagadas , Sequência de Aminoácidos , Avicennia/genética , Clonagem Molecular , Prolina/metabolismo
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