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1.
Int J Mol Sci ; 22(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34502162

RESUMO

Sepsis in the young population, which is particularly at risk, is rarely studied. O-GlcNAcylation is a post-translational modification involved in cell survival, stress response and metabolic regulation. O-GlcNAc stimulation is beneficial in adult septic rats. This modification is physiologically higher in the young rat, potentially limiting the therapeutic potential of O-GlcNAc stimulation in young septic rats. The aim is to evaluate whether O-GlcNAc stimulation can improve sepsis outcome in young rats. Endotoxemic challenge was induced in 28-day-old rats by lipopolysaccharide injection (E. Coli O111:B4, 20 mg·kg-1) and compared to control rats (NaCl 0.9%). One hour after lipopolysaccharide injection, rats were randomly assigned to no therapy, fluidotherapy (NaCl 0.9%, 10 mL·kg-1) ± NButGT (10 mg·kg-1) to increase O-GlcNAcylation levels. Physiological parameters and plasmatic markers were evaluated 2h later. Finally, untargeted mass spectrometry was performed to map cardiac O-GlcNAcylated proteins. Lipopolysaccharide injection induced shock with a decrease in mean arterial pressure and alteration of biological parameters (p < 0.05). NButGT, contrary to fluidotherapy, was associated with an improvement of arterial pressure (p < 0.05). ATP citrate lyase was identified among the O-GlcNAcylated proteins. In conclusion, O-GlcNAc stimulation improves outcomes in young septic rats. Interestingly, identified O-GlcNAcylated proteins are mainly involved in cellular metabolism.


Assuntos
ATP Citrato (pro-S)-Liase/metabolismo , Acetilglucosamina/metabolismo , Processamento de Proteína Pós-Traducional , Choque Séptico/metabolismo , Acetilação , Animais , Hidratação/métodos , Lipopolissacarídeos/toxicidade , Ratos , Choque Séptico/etiologia , Choque Séptico/terapia
2.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445285

RESUMO

N-glycosylation is essential for many biological processes in mammals. A variety of N-glycan structures exist, of which, the formation of bisecting N-acetylglucosamine (GlcNAc) is catalyzed by N-acetylglucosaminyltransferase-III (GnT-III, encoded by the Mgat3 gene). We previously identified various bisecting GlcNAc-modified proteins involved in Alzheimer's disease and cancer. However, the mechanisms by which GnT-III acts on the target proteins are unknown. Here, we performed comparative glycoproteomic analyses using brain membranes of wild type (WT) and Mgat3-deficient mice. Target glycoproteins of GnT-III were enriched with E4-phytohemagglutinin (PHA) lectin, which recognizes bisecting GlcNAc, and analyzed by liquid chromatograph-mass spectrometry. We identified 32 N-glycosylation sites (Asn-Xaa-Ser/Thr, Xaa ≠ Pro) that were modified with bisecting GlcNAc. Sequence alignment of identified N-glycosylation sites that displayed bisecting GlcNAc suggested that GnT-III does not recognize a specific primary amino acid sequence. The molecular modeling of GluA1 as one of the good cell surface substrates for GnT-III in the brain, indicated that GnT-III acts on N-glycosylation sites located in a highly flexible and mobile loop of GluA1. These results suggest that the action of GnT-III is partially affected by the tertiary structure of target proteins, which can accommodate bisecting GlcNAc that generates a bulky flipped-back conformation of the modified glycans.


Assuntos
Acetilglucosamina/metabolismo , Encéfalo/metabolismo , Membrana Celular/metabolismo , Peptídeos/metabolismo , Receptores de AMPA/metabolismo , Análise de Sequência de Proteína , Acetilglucosamina/genética , Animais , Membrana Celular/genética , Glicosilação , Camundongos , Camundongos Knockout , N-Acetilglucosaminiltransferases/deficiência , N-Acetilglucosaminiltransferases/metabolismo , Mapeamento de Peptídeos , Peptídeos/genética , Receptores de AMPA/genética
3.
Nat Commun ; 12(1): 5068, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417460

RESUMO

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis.


Assuntos
Acetilglucosamina/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Restrição Calórica , Linhagem Celular , Colforsina/farmacologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Epinefrina/metabolismo , Glucagon/metabolismo , Glucocorticoides/metabolismo , Gluconeogênese/efeitos dos fármacos , Glicosilação , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrocortisona/metabolismo , Hiperglicemia/complicações , Hiperglicemia/metabolismo , Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/complicações , Obesidade/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Ácido Pirúvico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética
4.
Biomolecules ; 11(7)2021 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-34356680

RESUMO

Proteins of the major histocompatibility complex (MHC) class I, or human leukocyte antigen (HLA) in humans interact with endogenous peptides and present them to T cell receptors (TCR), which in turn tune the immune system to recognize and discriminate between self and foreign (non-self) peptides. Of especial importance are peptides derived from tumor-associated antigens. T cells recognizing these peptides are found in cancer patients, but not in cancer-free individuals. What stimulates this recognition, which is vital for the success of checkpoint based therapy? A peptide derived from the protein p53 (residues 161-169 or p161) was reported to show this behavior. T cells recognizing this unmodified peptide could be further stimulated in vitro to create effective cancer killing CTLs (cytotoxic T lymphocytes). We hypothesize that the underlying difference may arise from post-translational glycosylation of p161 in normal individuals, likely masking it against recognition by TCR. Defects in glycosylation in cancer cells may allow the presentation of the native peptide. We investigate the structural consequences of such peptide glycosylation by investigating the associated structural dynamics.


Assuntos
Antígeno HLA-A24/química , Antígeno HLA-A24/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilglucosamina/metabolismo , Glicosilação , Proteínas do Vírus da Imunodeficiência Humana/química , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Receptores de Antígenos de Linfócitos T/química , Proteína Supressora de Tumor p53/química
5.
Appl Environ Microbiol ; 87(19): e0052921, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34319813

RESUMO

The fish pathogen Aliivibrio (Vibrio) salmonicida LFI1238 is thought to be incapable of utilizing chitin as a nutrient source, since approximately half of the genes representing the chitinolytic pathway are disrupted by insertion sequences. In the present study, we combined a broad set of analytical methods to investigate this hypothesis. Cultivation studies revealed that A. salmonicida grew efficiently on N-acetylglucosamine (GlcNAc) and chitobiose [(GlcNAc)2], the primary soluble products resulting from enzymatic chitin hydrolysis. The bacterium was also able to grow on chitin particles, albeit at a lower rate than on the soluble substrates. The genome of the bacterium contains five disrupted chitinase genes (pseudogenes) and three intact genes encoding a glycoside hydrolase family 18 (GH18) chitinase and two auxiliary activity family 10 (AA10) lytic polysaccharide monooxygenases (LPMOs). Biochemical characterization showed that the chitinase and LPMOs were able to depolymerize both α- and ß-chitin to (GlcNAc)2 and oxidized chitooligosaccharides, respectively. Notably, the chitinase displayed up to 50-fold lower activity than other well-studied chitinases. Deletion of the genes encoding the intact chitinolytic enzymes showed that the chitinase was important for growth on ß-chitin, whereas the LPMO gene deletion variants only showed minor growth defects on this substrate. Finally, proteomic analysis of A. salmonicida LFI1238 growth on ß-chitin showed expression of all three chitinolytic enzymes and, intriguingly, also three of the disrupted chitinases. In conclusion, our results show that A. salmonicida LFI1238 can utilize chitin as a nutrient source and that the GH18 chitinase and the two LPMOs are needed for this ability. IMPORTANCE The ability to utilize chitin as a source of nutrients is important for the survival and spread of marine microbial pathogens in the environment. One such pathogen is Aliivibrio (Vibrio) salmonicida, the causative agent of cold water vibriosis. Due to extensive gene decay, many key enzymes in the chitinolytic pathway have been disrupted, putatively rendering this bacterium incapable of chitin degradation and utilization. In the present study, we demonstrate that A. salmonicida can degrade and metabolize chitin, the most abundant biopolymer in the ocean. Our findings shed new light on the environmental adaption of this fish pathogen.


Assuntos
Aliivibrio salmonicida/metabolismo , Quitina/metabolismo , Acetilglucosamina/metabolismo , Aliivibrio salmonicida/genética , Animais , Quitinases/genética , Quitinases/metabolismo , Dissacarídeos/metabolismo , Peixes , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Transdução de Sinais
6.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203166

RESUMO

Placental dysfunction can lead to fetal growth restriction which is associated with perinatal morbidity and mortality. Fetal growth restriction increases the risk of obesity and diabetes later in life. Placental O-GlcNAc transferase (OGT) has been identified as a marker and a mediator of placental insufficiency in the setting of prenatal stress, however, its role in the fetal programming of metabolism and glucose homeostasis remains unknown. We aim to determine the long-term metabolic outcomes of offspring with a reduction in placental OGT. Mice with a partial reduction and a full knockout of placenta-specific OGT were generated utilizing the Cre-Lox system. Glucose homeostasis and metabolic parameters were assessed on a normal chow and a high-fat diet in both male and female adult offspring. A reduction in placental OGT did not demonstrate differences in the metabolic parameters or glucose homeostasis compared to the controls on a standard chow. The high-fat diet provided a metabolic challenge that revealed a decrease in body weight gain (p = 0.02) and an improved insulin tolerance (p = 0.03) for offspring with a partially reduced placental OGT but not when OGT was fully knocked out. Changes in body weight were not associated with changes in energy homeostasis. Offspring with a partial reduction in placental OGT demonstrated increased hepatic Akt phosphorylation in response to insulin treatment (p = 0.02). A partial reduction in placental OGT was protective from weight gain and insulin intolerance when faced with the metabolic challenge of a high-fat diet. This appears to be, in part, due to increased hepatic insulin signaling. The findings of this study contribute to the greater understanding of fetal metabolic programming and the effect of placental OGT on peripheral insulin sensitivity and provides a target for future investigation and clinical applications.


Assuntos
Acetilglucosamina/metabolismo , Peso Corporal/fisiologia , Resistência à Insulina/fisiologia , Insulina/farmacologia , N-Acetilglucosaminiltransferases/metabolismo , Placenta/efeitos dos fármacos , Placenta/metabolismo , Animais , Peso Corporal/genética , Feminino , Resistência à Insulina/genética , Masculino , Camundongos , N-Acetilglucosaminiltransferases/genética , Gravidez
7.
Nat Commun ; 12(1): 4173, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234137

RESUMO

The integration of circadian and metabolic signals is essential for maintaining robust circadian rhythms and ensuring efficient metabolism and energy use. Using Drosophila as an animal model, we show that cellular protein O-GlcNAcylation exhibits robust 24-hour rhythm and represents a key post-translational mechanism that regulates circadian physiology. We observe strong correlation between protein O-GlcNAcylation rhythms and clock-controlled feeding-fasting cycles, suggesting that O-GlcNAcylation rhythms are primarily driven by nutrient input. Interestingly, daily O-GlcNAcylation rhythms are severely dampened when we subject flies to time-restricted feeding at unnatural feeding time. This suggests the presence of clock-regulated buffering mechanisms that prevent excessive O-GlcNAcylation at non-optimal times of the day-night cycle. We show that this buffering mechanism is mediated by the expression and activity of GFAT, OGT, and OGA, which are regulated through integration of circadian and metabolic signals. Finally, we generate a mathematical model to describe the key factors that regulate daily O-GlcNAcylation rhythm.


Assuntos
Ritmo Circadiano/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Hexosaminas/biossíntese , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Acetilglucosamina/metabolismo , Animais , Animais Geneticamente Modificados , Vias Biossintéticas/genética , Relógios Circadianos/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Comportamento Alimentar/fisiologia , Feminino , Perfilação da Expressão Gênica , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Masculino , Modelos Animais , N-Acetilglucosaminiltransferases/genética , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo
8.
ACS Chem Biol ; 16(6): 1040-1049, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34105348

RESUMO

O-GlcNAcylation is an O-linked ß-N-acetyl-glucosamine (O-GlcNAc)-monosaccharide modification of serine or threonine in proteins that plays a vital role in many critical cellular processes. Owing to its low molecular weight, uncharged property, and difficulty in distinguishing from ß-N-acetyl-galactosamine (GalNAc), the lack of high specificity and avidity tools and sophisticated quantification methods have always been the bottleneck in analyzing O-GlcNAc functions. Here, we compared glycan array data of the mutant of Clostridium perfringen OGA (CpOGAD298N), O-GlcNAc antibody CTD110.6, and several lectins. We found that CpOGAD298N can effectively distinguish GlcNAc from GalNAc. Glycan array analysis and isothermal titration calorimetry (ITC) show that CpOGAD298N has a GlcNAc specific binding characteristic. CpOGAD298N could be used in far-western, flow cytometry analysis, and confocal imaging to demonstrate the existence of O-GlcNAc proteins. Using the CpOGAD298N affinity column, we identified 84 highly confident O-GlcNAc modified peptides from 82 proteins in the MCF-7 cell line and 33 highly confident peptides in 33 proteins from mouse liver tissue; most of them are novel O-GlcNAc proteins and could not bind with wheat germ agglutinin (WGA). Besides being used as a facile enrichment tool, a combination of CpOGAD298N with the proximity ligation assay (PLA) is successfully used to quantify O-GlcNAc modified histone H2B, which is as low as femtomoles in MCF-7 cell lysate. These results suggest that CpOGAD298N is a specific tool for detection (far-western, flow cytometry analysis, and confocal imaging) and enrichment of O-GlcNAcylated proteins and peptides, and the CpOGAD298N-PLA method is useful for quantifying certain O-GlcNAc protein.


Assuntos
Acetilglucosamina/metabolismo , Proteínas de Bactérias/metabolismo , Clostridium perfringens/metabolismo , Acetilglucosamina/análise , Acilação , Proteínas de Bactérias/química , Clostridium perfringens/química , Glicosilação , Polissacarídeos/análise , Polissacarídeos/metabolismo
9.
Curr Protoc ; 1(5): e129, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34004049

RESUMO

O-GlcNAc is a common post-translational modification of nuclear, mitochondrial, and cytoplasmic proteins that regulates normal physiology and the cell stress response. Dysregulation of O-GlcNAc cycling is implicated in the etiology of type II diabetes, heart failure, hypertension, and Alzheimer's disease, as well as cardioprotection. These protocols cover simple and comprehensive techniques for detecting proteins modified by O-GlcNAc and studying the enzymes that add or remove O-GlcNAc. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Increasing the stoichiometry of O-GlcNAc on proteins before analysis Basic Protocol 2: Detection of proteins modified by O-GlcNAc using antibodies Basic Protocol 3: Detection of proteins modified by O-GlcNAc using the lectin sWGA Support Protocol 1: Control for O-linked glycosylation Basic Protocol 4: Detection and enrichment of proteins using WGA-agarose Support Protocol 2: Digestion of proteins with hexosaminidase Alternate Protocol: Detection of proteins modified by O-GlcNAc using galactosyltransferase Support Protocol 3: Autogalactosylation of galactosyltransferase Support Protocol 4: Assay of galactosyltransferase activity Basic Protocol 5: Characterization of labeled glycans by ß-elimination and chromatography Basic Protocol 6: Detection of O-GlcNAc in 96-well plates Basic Protocol 7: Assay for OGT activity Support Protocol 5: Desalting of O-GlcNAc transferase Basic Protocol 8: Assay for O-GlcNAcase activity.


Assuntos
Acetilglucosamina , Diabetes Mellitus Tipo 2 , Acetilglucosamina/metabolismo , Núcleo Celular/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glicosilação , Humanos , Processamento de Proteína Pós-Traducional
10.
J Biol Chem ; 296: 100800, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019870

RESUMO

Thousands of proteins have been found to be modified by O-GlcNAc, a common glycosylation modification of serine and threonine residues throughout the cytosol and nucleus. O-GlcNAc is enzymatically added and removed from proteins, making it a potential dynamic regulator of cell signaling. However, compared with other posttranslational modifications like phosphorylation, relatively few O-GlcNAc-regulated pathways have been discovered and biochemically characterized. We previously discovered one such pathway, where O-GlcNAc controls the contraction of fibroblasts initiated by the signaling lipid sphingosine-1-phosphate. Specifically, we found that O-GlcNAc modification of the phosphatase MYPT1 maintains its activity, resulting in dephosphorylation and deactivation of the myosin light chain of the actinomyosin complex. Another signaling lipid that leads to contraction of fibroblasts is lysophosphatidic acid, and this signaling pathway also converges on MYPT1 and actinomyosin. We therefore rationalized that O-GlcNAc would also control this pathway. Here, we used a combination of small molecule inhibitors, 2D and 3D cell cultures, and biochemistry to confirm our hypothesis. Specifically, we found that O-GlcNAc levels control the sensitivity of mouse and primary human dermal fibroblasts to lysophosphatidic acid-induced contraction in culture and the phosphorylation of MLC and that MYPT1 O-GlcNAc modification is responsible. These findings further solidify the importance of O-GlcNAc in regulating the biology of fibroblasts in response to procontractile stimuli.


Assuntos
Fibroblastos/citologia , Lisofosfolipídeos/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Acetilglucosamina/metabolismo , Animais , Linhagem Celular , Fibroblastos/metabolismo , Glicosilação , Humanos , Camundongos , Células NIH 3T3
11.
Biochim Biophys Acta Gen Subj ; 1865(8): 129930, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34019948

RESUMO

BACKGROUND: Chromatin modifier metastasis-associated protein 1 (MTA1), closely associated with tumor angiogenesis in breast cancer, plays an important role in gene expression and cancer cell behavior. Recently, an association between O-GlcNAc transferase (OGT) and MTA1 was identified by mass spectroscopy. However, the potential relationship between MTA1 and O-GlcNAc modification has not yet explored. METHODS: In the current study, the role of MTA1 and its O-GlcNAc modification in breast cancer cell genotoxic adaptation was investigated through quantitative proteomics, chromatin immunoprecipitation followed by sequencing (ChIP-seq), transcriptome analysis, and loss- and gain-of-function experiments. RESULTS: We demonstrate that the O-GlcNAc modification promotes MTA1 to interaction with chromatin and thus changes the expression of target genes, contributing to breast cancer cell genotoxic adaptation. MTA1 is modified with O-GlcNAc residues at serine (S) residues S237/S241/S246 in adriamycin-adaptive breast cancer cells, and this modification improves the genome-wide interactions of MTA1 with gene promotor regions by enhancing its association with nucleosome remodeling and histone deacetylation (NuRD) complex. Further, O-GlcNAc modification modulates MTA1 chromatin binding, influencing the specific transcriptional regulation of genes involved in the adaptation of breast cancer cells to genotoxic stress. CONCLUSIONS: Our findings reveal a previously unrecognized role for O-GlcNAc-modified MTA1 in transcriptional regulation and suggest that the O-GlcNAc modification is a key to the molecular regulation of chemoresistance in breast cancers.


Assuntos
Acetilglucosamina/química , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Dano ao DNA , Regulação Neoplásica da Expressão Gênica , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Acetilglucosamina/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Perfilação da Expressão Gênica , Humanos , N-Acetilglucosaminiltransferases/metabolismo , Prognóstico , Proteínas Repressoras/química , Proteínas Repressoras/genética , Taxa de Sobrevida , Transativadores/química , Transativadores/genética , Células Tumorais Cultivadas
12.
Biochem Biophys Res Commun ; 558: 126-133, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33915326

RESUMO

Myocardial ischemia/reperfusion (I/R) injury is a major determinant of morbidity and mortality in patients undergoing treatment for cardiac disease. A variety of treatments are reported to have benefits against reperfusion injury, yet their cardioprotective effects seem to be diminished in obesity, and the underlying mechanism remains elusive. In this study, we found that db/db mice exhibit cardiac hyper-O-GlcNAcylation. In parallel, palmitate treatment (200 mM; 12 h) in H9c2 cells showed an increase in global protein O-GlcNAcylation, along with an impaired insulin response against reperfusion injury. To investigate whether O-GlcNAcylation underlies this phenomenon, glucosamine was used to increase global protein O-GlcNAc levels. Interestingly, histological staining, electrophysiological studies, serum cardiac markers and oxidative stress biomarker assays showed that preischemic treatment with glucosamine attenuated insulin cardioprotection against myocardial infarction, arrhythmia and oxidative stress. Mechanistically, glucosamine treatment decreased insulin-stimulated Akt phosphorylation, a key modulator of cell survival. Furthermore, inhibition of O-GlcNAcylation via 6-diazo-5-oxo-l-norleucine (DON) apparently increased insulin-induced Akt phosphorylation and restored its cardioprotective response against reperfusion injury in palmitate-induced insulin-resistant H9c2 cells. Our findings demonstrated that obesity-induced hyper-O-GlcNAcylation might contribute to the attenuation of insulin cardioprotection against I/R injury.


Assuntos
Acetilglucosamina/metabolismo , Arritmias Cardíacas/complicações , Arritmias Cardíacas/metabolismo , Insulina/metabolismo , Traumatismo por Reperfusão Miocárdica/complicações , Traumatismo por Reperfusão Miocárdica/metabolismo , Obesidade/complicações , Obesidade/metabolismo , Animais , Cardiotônicos/metabolismo , Hipóxia Celular , Linhagem Celular , Diazo-Oxo-Norleucina/farmacologia , Modelos Animais de Doenças , Glicosilação/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos
13.
Biosci Biotechnol Biochem ; 85(6): 1433-1440, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-33836055

RESUMO

Regulation of hyaluronan (HA) is important for the maintenance of epidermal homeostasis. Here, we examined the mechanism by which 1-ethyl-ß-N-acetylglucosaminide (ß-NAG2), a newly developed N-acetylglucosamine (NAG) derivative, increases HA production in cultured human epidermal keratinocytes. When keratinocytes were treated with ß-NAG2, mRNA expression of HA synthase 3, which is responsible for HA production in human keratinocytes, was not influenced, but the intracellular level of UDP-NAG, a substrate used for HA synthesis, was increased. By using a synthetic substrate for ß-N-acetylglucosaminidase (ß-NAGase), keratinocytes were found to possess ß-NAGase activity, and treatment of o-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino N-phenyl carbamate (PUGNAc), an inhibitor of ß-NAGase, abolished the release of NAG from ß-NAG2 in keratinocytes. Furthermore, PUGNAc attenuated the ß-NAG2-induced intracellular UDP-NAG and HA production in keratinocytes. These results suggest that ß-NAG2 is converted to NAG by endogenous ß-NAGase in keratinocytes, and the resulting NAG is further metabolized to UDP-NAG and utilized for HA production.


Assuntos
Acetilglucosamina/metabolismo , Acetilglucosaminidase/metabolismo , Ácido Hialurônico/biossíntese , Queratinócitos/metabolismo , Glicosilação , Humanos
14.
Mar Drugs ; 19(3)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799701

RESUMO

Many corals establish symbiosis with Symbiodiniaceae cells from surrounding environments, but very few Symbiodiniaceae cells exist in the water column. Given that the N-acetyl-d-glucosamine-binding lectin ActL attracts Symbiodiniaceae cells, we hypothesized that corals must attract Symbiodiniaceae cells using ActL to acquire them. Anti-ActL antibody inhibited acquisition of Symbiodiniaceae cells, and rearing seawater for juvenile Acropora tenuis contained ActL, suggesting that juvenile A. tenuis discharge ActL to attract these cells. Among eight Symbiodiniaceae cultured strains, ActL attracted NBRC102920 (Symbiodinium tridacnidorum) most strongly followed by CS-161 (Symbiodinium tridacnidorum), CCMP2556 (Durusdinium trenchii), and CCMP1633 (Breviolum sp.); however, it did not attract GTP-A6-Sy (Symbiodinium natans), CCMP421 (Effrenium voratum), FKM0207 (Fugacium sp.), and CS-156 (Fugacium sp.). Juvenile polyps of A. tenuis acquired limited Symbiodiniaceae cell strains, and the number of acquired Symbiodiniaceae cells in a polyp also differed from each other. The number of Symbiodiniaceae cells acquired by juvenile polyps of A. tenuis was correlated with the ActL chemotactic activity. Thus, ActL could be used to attract select Symbiodiniaceae cells and help Symbiodiniaceae cell acquisition in juvenile polyps of A. tenuis, facilitating establishment of symbiosis between A. tenuis and Symbiodiniaceae cells.


Assuntos
Acetilglucosamina/metabolismo , Antozoários/metabolismo , Dinoflagelados/metabolismo , Lectinas/metabolismo , Animais , Técnicas de Cultura de Células , Dinoflagelados/citologia , Simbiose
15.
Biochim Biophys Acta Mol Basis Dis ; 1867(7): 166129, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33744394

RESUMO

Hexosamine biosynthetic (HBP) and PI3K/AKT/mTOR pathways are found to predominate the proliferation and survival of prostate cancer cells. Both these pathways have their own specific intermediates to propagate the secondary signals in down-stream cascades and besides having their own structured network, also have shared interconnecting branches. These interconnections are either competitive or co-operative in nature depending on the microenvironmental conditions. Specifically, in prostate cancer HBP and mTOR pathways increases the expression and protein level of androgen receptor in order to support cancer cell proliferation, advancement and metastasis. Pharmacological inhibition of a single pathway is therefore insufficient to stop disease progression as the cancer cells manage to alter the signalling channel. This is one of the primary reasons for the therapeutic failure in prostate cancer and emergence of chemoresistance. Inhibition of these multiple pathways at their common junctures might prove to be of benefit in men suffering from an advanced disease state. Hence, a thorough understanding of these cellular intersecting points and their significance with respect to signal transduction mechanisms might assist in the rational designing of combinations for effective management of prostate cancer.


Assuntos
Acetilglucosamina/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/patologia , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Humanos , Masculino , Fosfatidilinositol 3-Quinases/química , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/química , Transdução de Sinais , Serina-Treonina Quinases TOR/química
16.
Arch Microbiol ; 203(5): 1953-1969, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33710379

RESUMO

One of the most abundant natural polymers on earth, chitin is a fibrous and structural polysaccharide, composed of N-acetyl-D-glucosamine. The biopolymer is the major structural constituent of fungi, arthropods, mollusks, nematodes, and some algae. The biodegradation of chitin is largely manifested by chitinolytic enzyme secreting organisms including bacteria, insects, and plants. Among them, bacterial chitinases represent the most promising, inexpensive, and sustainable source of proteins that can be employed for industrial-scale applications. To this end, the presented review comes at a timely moment to highlight the major sources of chitinolytic bacteria. It also discusses the potential pros and cons of prospecting bacterial chitinases that can be easily manipulated through genetic engineering. Additionally, we have elaborated the recent applications of the chitin thereby branding chitinases as potential candidates for biorefinery and biomedical research for eco-friendly and sustainable management of chitin waste in the environment.


Assuntos
Bactérias/metabolismo , Bioprospecção , Quitina/metabolismo , Quitinases/metabolismo , Acetilglucosamina/metabolismo , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Quitina/química , Quitinases/genética , Engenharia Genética
17.
J Biol Chem ; 296: 100519, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33684445

RESUMO

Endo-ß-N-acetylmuramidases, commonly known as lysozymes, are well-characterized antimicrobial enzymes that catalyze an endo-lytic cleavage of peptidoglycan; i.e., they hydrolyze the ß-1,4-glycosidic bonds connecting N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc). In contrast, little is known about exo-ß-N-acetylmuramidases, which catalyze an exo-lytic cleavage of ß-1,4-MurNAc entities from the non-reducing ends of peptidoglycan chains. Such an enzyme was identified earlier in the bacterium Bacillus subtilis, but the corresponding gene has remained unknown so far. We now report that ybbC of B. subtilis, renamed namZ, encodes the reported exo-ß-N-acetylmuramidase. A ΔnamZ mutant accumulated specific cell wall fragments and showed growth defects under starvation conditions, indicating a role of NamZ in cell wall turnover and recycling. Recombinant NamZ protein specifically hydrolyzed the artificial substrate para-nitrophenyl ß-MurNAc and the peptidoglycan-derived disaccharide MurNAc-ß-1,4-GlcNAc. Together with the exo-ß-N-acetylglucosaminidase NagZ and the exo-muramoyl-l-alanine amidase AmiE, NamZ degraded intact peptidoglycan by sequential hydrolysis from the non-reducing ends. A structure model of NamZ, built on the basis of two crystal structures of putative orthologs from Bacteroides fragilis, revealed a two-domain structure including a Rossmann-fold-like domain that constitutes a unique glycosidase fold. Thus, NamZ, a member of the DUF1343 protein family of unknown function, is now classified as the founding member of a new family of glycosidases (CAZy GH171; www.cazy.org/GH171.html). NamZ-like peptidoglycan hexosaminidases are mainly present in the phylum Bacteroidetes and less frequently found in individual genomes within Firmicutes (Bacilli, Clostridia), Actinobacteria, and γ-proteobacteria.


Assuntos
Acetilglucosamina/metabolismo , Bacillus subtilis/enzimologia , Glicosídeo Hidrolases/metabolismo , Ácidos Murâmicos/metabolismo , Peptidoglicano/metabolismo , Cristalografia por Raios X , Glicosídeo Hidrolases/química , Hidrólise , Conformação Proteica
18.
Molecules ; 26(4)2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669256

RESUMO

O-GlcNAcylation is a posttranslational modification that occurs at serine and threonine residues of protein substrates by the addition of O-linked ß-d-N-acetylglucosamine (GlcNAc) moiety. Two enzymes are involved in this modification: O-GlcNac transferase (OGT), which attaches the GlcNAc residue to the protein substrate, and O-GlcNAcase (OGA), which removes it. This biological balance is important for many biological processes, such as protein expression, cell apoptosis, and regulation of enzyme activity. The extent of this modification has sparked interest in the medical community to explore OGA and OGT as therapeutic targets, particularly in degenerative diseases. While some OGA inhibitors are already in phase 1 clinical trials for the treatment of Alzheimer's disease, OGT inhibitors still have a long way to go. Due to complex expression and instability, the discovery of potent OGT inhibitors is challenging. Over the years, the field has grappled with this problem, and scientists have developed a number of techniques and assays. In this review, we aim to highlight assays and techniques for OGT inhibitor discovery, evaluate their strength for the field, and give us direction for future bioassay methods.


Assuntos
Bioensaio/métodos , N-Acetilglucosaminiltransferases/metabolismo , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Fenômenos Biofísicos , Química Click , Ligação Proteica
19.
Chem Rev ; 121(3): 1513-1581, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33416322

RESUMO

Protein O-linked ß-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) is a unique monosaccharide modification discovered in the early 1980s. With the technological advances in the past several decades, great progress has been made to reveal the biochemistry of O-GlcNAcylation, the substrates of O-GlcNAcylation, and the functional importance of protein O-GlcNAcylation. As a nutrient sensor, protein O-GlcNAcylation plays important roles in almost all biochemical processes examined. Although the functional importance of O-GlcNAcylation of proteins has been extensively reviewed previously, the chemical and biochemical aspects have not been fully addressed. In this review, by critically evaluating key publications in the past 35 years, we aim to provide a comprehensive understanding of this important post-translational modification (PTM) from analytical and biochemical perspectives. Specifically, we will cover (1) multiple analytical advances in the characterization of O-GlcNAc cycling components (i.e., the substrate donor UDP-GlcNAc, the two key enzymes O-GlcNAc transferase and O-GlcNAcase, and O-GlcNAc substrate proteins), (2) the biochemical characterization of the enzymes with a variety of chemical tools, and (3) exploration of O-GlcNAc cycling and its modulating chemicals as potential biomarkers and therapeutic drugs for diseases. Last but not least, we will discuss the challenges and possible solutions for basic and translational research of protein O-GlcNAcylation in the future.


Assuntos
Acetilglucosamina/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Acetilglucosamina/química , Humanos , N-Acetilglucosaminiltransferases/química , beta-N-Acetil-Hexosaminidases/química
20.
Nutrients ; 13(1)2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33430126

RESUMO

Micronutrient sensing is critical for cellular growth and differentiation. Deficiencies in essential nutrients such as iron strongly affect neuronal cell development and may lead to defects in neuronal function that cannot be remedied by subsequent iron supplementation. To understand the adaptive intracellular responses to iron deficiency in neuronal cells, we developed and utilized a Stable Isotopic Labeling of Amino acids in Cell culture (SILAC)-based quantitative phosphoproteomics workflow. Our integrated approach was designed to comprehensively elucidate the changes in phosphorylation signaling under both acute and chronic iron-deficient cell models. In addition, we analyzed the differential cellular responses between iron deficiency and hypoxia (oxygen-deprived) in neuronal cells. Our analysis identified nearly 16,000 phosphorylation sites in HT-22 cells, a hippocampal-derived neuronal cell line, more than ten percent of which showed at least 2-fold changes in response to either hypoxia or acute/chronic iron deficiency. Bioinformatic analysis revealed that iron deficiency altered key metabolic and epigenetic pathways including the phosphorylation of proteins involved in iron sequestration, glutamate metabolism, and histone methylation. In particular, iron deficiency increased glutamine-fructose-6-phosphate transaminase (GFPT1) phosphorylation, which is a key enzyme in the glucosamine biosynthesis pathway and a target of 5' AMP-activated protein kinase (AMPK), leading to reduced GFPT1 enzymatic activity and consequently lower global O-GlcNAc modification in neuronal cells. Taken together, our analysis of the phosphoproteome dynamics in response to iron and oxygen deprivation demonstrated an adaptive cellular response by mounting post-translational modifications that are critical for intracellular signaling and epigenetic programming in neuronal cells.


Assuntos
Acetilglucosamina/metabolismo , Ferro/deficiência , Neurônios/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Hipóxia Celular , Linhagem Celular , Dano ao DNA , Reparo do DNA , Epigênese Genética , Homeostase , Inflamação/metabolismo , Metais/metabolismo , Camundongos , Fosforilação , Proteínas Quinases/metabolismo , Proteoma/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais
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