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1.
Chem Commun (Camb) ; 55(88): 13291-13294, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31626249

RESUMO

O-GlcNAc transferase (OGT) glycosylates numerous proteins and is implicated in many diseases. To date, most OGT inhibitors lack either sufficient potency or characterized specificity in cells. We report the first targeted covalent inhibitor that predominantly reacts with OGT but does not affect other functionally similar enzymes. This study provides a new strategy to interrogate cellular OGT functions and to investigate other glycosyltransferases.


Assuntos
Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Inibidores Enzimáticos/química , Células HEK293 , Humanos , Células MCF-7 , Modelos Moleculares , N-Acetilglucosaminiltransferases/metabolismo
2.
Med Sci Monit ; 25: 6491-6503, 2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31465316

RESUMO

BACKGROUND EXT1 is an endoplasmic reticulum-resident glycosyl transferase whose intracellular expression alters the biosynthesis and distribution of heparan sulfate. EXT1 is regarded as a classic tumor suppressor. MiR-665 can act as either an oncogene or tumor-suppressing gene in different tumors. The aim of the current study was to determine the function and molecular mechanisms of EXT1 and miR-665 in acute lymphoblastic leukemia (ALL). MATERIAL AND METHODS EXT1 expression in ALL was evaluated by real-time polymerase chain reaction (RT-PCR) and western blotting. The effects of EXT1 in ALL were explored by Cell Counting Kit-8 (CCK-8)/EdU assays, western blotting, flow cytometry, and in vivo tumorigenesis assays. Label-free quantification was used to detect differentially expressed proteins in EXT1-overexpressing Reh cells. RESULTS EXT1 expression is downregulated in ALL and negatively correlated with miR-665 expression. Moreover, low EXT1 and high miR-665 expression levels in adult ALL bone marrow tissues are correlated with poor patient survival. Our study showed that EXT1 modulates the proliferation and apoptosis of ALL cells in vitro and in vivo and that miR-665 promotes cell growth and inhibits apoptosis by suppressing EXT1. EXT1 promotes cell apoptosis via deactivating the ERK1/2 pathway. CONCLUSIONS In conclusion, this study is the first to confirm the association between low EXT1 levels and several clinical features of ALL. Low bone marrow EXT1 levels independently predict poor prognoses in adult ALL patients. Thus, our study suggests that EXT1- or miR-665-targeted strategies can confer the therapeutic effect of promoting apoptosis by deactivating the ERK1/2 pathway.


Assuntos
Apoptose/genética , Sistema de Sinalização das MAP Quinases , MicroRNAs/metabolismo , N-Acetilglucosaminiltransferases/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Adolescente , Adulto , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Masculino , Camundongos , MicroRNAs/genética , Análise Multivariada , N-Acetilglucosaminiltransferases/metabolismo , Análise de Sobrevida , Adulto Jovem
3.
Basic Res Cardiol ; 114(4): 28, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-31152247

RESUMO

Several post-translational modifications figure prominently in ventricular remodeling. The beta-O-linkage of N-acetylglucosamine (O-GlcNAc) to proteins has emerged as an important signal in the cardiovascular system. Although there are limited insights about the regulation of the biosynthetic pathway that gives rise to the O-GlcNAc post-translational modification, much remains to be elucidated regarding the enzymes, such as O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which regulate the presence/absence of O-GlcNAcylation. Recently, we showed that the transcription factor, E2F1, could negatively regulate OGT and OGA expression in vitro. The present study sought to determine whether E2f1 deletion would improve post-infarct ventricular function by de-repressing expression of OGT and OGA. Male and female mice were subjected to non-reperfused myocardial infarction (MI) and followed for 1 or 4 week. MI significantly increased E2F1 expression. Deletion of E2f1 alone was not sufficient to alter OGT or OGA expression in a naïve setting. Cardiac dysfunction was significantly attenuated at 1-week post-MI in E2f1-ablated mice. During chronic heart failure, E2f1 deletion also attenuated cardiac dysfunction. Despite the improvement in function, OGT and OGA expression was not normalized and protein O-GlcNAcyltion was not changed at 1-week post-MI. OGA expression was significantly upregulated at 4-week post-MI but overall protein O-GlcNAcylation was not changed. As an alternative explanation, we also performed guided transcriptional profiling of predicted targets of E2F1, which indicated potential differences in cardiac metabolism, angiogenesis, and apoptosis. E2f1 ablation increased heart size and preserved remote zone capillary density at 1-week post-MI. During chronic heart failure, cardiomyocytes in the remote zone of E2f1-deleted hearts were larger than wildtype. These data indicate that, overall, E2f1 exerts a deleterious effect on ventricular remodeling. Thus, E2f1 deletion improves ventricular remodeling with limited impact on enzymes regulating O-GlcNAcylation.


Assuntos
Fator de Transcrição E2F1/deficiência , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Função Ventricular Esquerda , Remodelação Ventricular , Animais , Capilares/metabolismo , Capilares/patologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Modelos Animais de Doenças , Fator de Transcrição E2F1/genética , Feminino , Deleção de Genes , Glicosilação , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , N-Acetilglucosaminiltransferases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo
4.
Glycobiology ; 29(8): 582-592, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31094413

RESUMO

Binding and uptake of triglyceride-rich lipoproteins (TRLs) in mice depend on heparan sulfate and the hepatic proteoglycan, syndecan-1 (SDC1). Alteration of glucosamine N-sulfation by deletion of glucosamine N-deacetylase-N-sulfotransferase 1 (Ndst1) and 2-O-sulfation of uronic acids by deletion of uronyl 2-O-sulfotransferase (Hs2st) led to diminished lipoprotein metabolism, whereas inactivation of glucosaminyl 6-O-sulfotransferase 1 (Hs6st1), which encodes one of the three 6-O-sulfotransferases, had little effect on lipoprotein binding. However, other studies have suggested that 6-O-sulfation may be important for TRL binding and uptake. In order to explain these discrepant findings, we used CRISPR/Cas9 gene editing to create a library of mutants in the human hepatoma cell line, Hep3B. Inactivation of EXT1 encoding the heparan sulfate copolymerase, NDST1 and HS2ST dramatically reduced binding of TRLs. Inactivation of HS6ST1 had no effect, but deletion of HS6ST2 reduced TRL binding. Compounding mutations in HS6ST1 and HS6ST2 did not exacerbate this effect indicating that HS6ST2 is the dominant 6-O-sulfotransferase and that binding of TRLs indeed depends on 6-O-sulfation of glucosamine residues. Uptake studies showed that TRL internalization was also affected in 6-O-sulfation deficient cells. Interestingly, genetic deletion of SDC1 only marginally impacted binding of TRLs but reduced TRL uptake to the same extent as treating the cells with heparin lyases. These findings confirm that SDC1 is the dominant endocytic proteoglycan receptor for TRLs in human Hep3B cells and that binding and uptake of TRLs depend on SDC1 and N- and 2-O-sulfation as well as 6-O-sulfation of heparan sulfate chains catalyzed by HS6ST2.


Assuntos
Lipoproteínas/metabolismo , Mutação com Perda de Função , N-Acetilglucosaminiltransferases/metabolismo , Sulfotransferases/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Células Cultivadas , Hepatócitos/metabolismo , Humanos , Lipoproteínas/química , N-Acetilglucosaminiltransferases/genética , Ligação Proteica , Sulfotransferases/genética , Sindecana-1/genética , Sindecana-1/metabolismo , Triglicerídeos/química , Triglicerídeos/metabolismo
5.
J Zhejiang Univ Sci B ; 20(5): 437-448, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31090269

RESUMO

O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic post-translational modification occurring on myriad proteins in the cell nucleus, cytoplasm, and mitochondria. The donor sugar for O-GlcNAcylation, uridine-diphosphate N-acetylglucosamine (UDP-GlcNAc), is synthesized from glucose through the hexosamine biosynthetic pathway (HBP). The recycling of O-GlcNAc on proteins is mediated by two enzymes in cells-O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which catalyze the addition and removal of O-GlcNAc, respectively. O-GlcNAcylation is involved in a number of important cell processes including transcription, translation, metabolism, signal transduction, and apoptosis. Deregulation of O-GlcNAcylation has been reported to be associated with various human diseases such as cancer, diabetes, neurodegenerative diseases, and cardiovascular diseases. A better understanding of the roles of O-GlcNAcylation in physiopathological processes would help to uncover novel avenues for therapeutic intervention. The aim of this review is to discuss the recent updates on the mechanisms and impacts of O-GlcNAcylation on these diseases, and its potential as a new clinical target.


Assuntos
Acetilglucosamina/química , Doenças Cardiovasculares/metabolismo , Doenças Neurodegenerativas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Apoptose , Catálise , Núcleo Celular/metabolismo , Proliferação de Células , Citoplasma/metabolismo , Complicações do Diabetes/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hexosaminas/química , Humanos , Insulina , Mitocôndrias/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Neoplasias/metabolismo , Fosforilação , Transdução de Sinais , beta-N-Acetil-Hexosaminidases/metabolismo
6.
Org Biomol Chem ; 17(17): 4326-4334, 2019 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-30976765

RESUMO

An unnatural monosaccharide with a C6-azide, Ac36AzGalNAc, has been developed as a potent and selective probe for O-GlcNAc-modified proteins. Combined with click chemistry, we demonstrate that Ac36AzGalNAc can robustly label O-GlcNAc glycosylation in a wide range of cell lines. Meanwhile, cell imaging and LC-MS/MS proteomics verify its selective activity on O-GlcNAc. More importantly, the protocol presented here provides a general methodology for tracking, capturing and identifying unnatural monosaccharide modified proteins in cells or cell lysates.


Assuntos
Galactosamina/química , Sondas Moleculares/química , N-Acetilglucosaminiltransferases/análise , beta-N-Acetil-Hexosaminidases/análise , Animais , Células Cultivadas , Galactosamina/análogos & derivados , Galactosamina/síntese química , Humanos , Camundongos , Sondas Moleculares/síntese química , Estrutura Molecular , N-Acetilglucosaminiltransferases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo
7.
Neuroscience ; 409: 204-221, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-30872163

RESUMO

ALG13 (asparagine-linked glycosylation 13 homolog) encodes a crucial protein involved in the process of N-linked glycosylation, and abnormal N-linked glycosylation is considered an important risk factor that leads to neurological deficits and disorders. However, the causal relationship between ALG13 and epilepsy remains unknown. This study applied a kainic acid (KA)-induced epileptic mouse model to determine whether ALG13 deficiency resulted in increased susceptibility to and severity of epileptic seizures. This report found that the expression of ALG13 in the central nervous system (CNS) had histologically and cellular specificity, mainly in the neurons in the cortex and hippocampus, epilepsy commonly occurs. In addition, KA-induced seizures significantly affected the expression levels of ALG13 mRNA and protein in the forebrain of wild-type (WT) mice. KA-induced epileptic progressions were dramatically increased in Alg13 knockout (KO) mice, including prolonged electrographic seizures, strikingly increased mortality rates, and the severity of responses to epileptic seizures. Furthermore, KA-induced epilepsy-related pathological changes of the brain were predominantly exacerbated in Alg13 KO mice. This study also preliminarily explored the possible mechanisms of ALG13-involved epilepsy by showing hyperactive mTOR signaling pathways in the cortex and hippocampus of Alg13 KO mice. To the best of our knowledge, this report is the first evidence of the association between ALG13 and epilepsy in experimental animals.


Assuntos
Epilepsia/diagnóstico , Hipocampo/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Neurônios/metabolismo , Convulsões/diagnóstico , Animais , Modelos Animais de Doenças , Epilepsia/induzido quimicamente , Epilepsia/genética , Epilepsia/metabolismo , Ácido Caínico , Camundongos , Camundongos Knockout , N-Acetilglucosaminiltransferases/genética , Convulsões/induzido quimicamente , Convulsões/genética , Convulsões/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo
8.
J Biol Chem ; 294(17): 6843-6856, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-30858176

RESUMO

Inhibitory GABAergic transmission is required for proper circuit function in the nervous system. However, our understanding of molecular mechanisms that preferentially influence GABAergic transmission, particularly presynaptic mechanisms, remains limited. We previously reported that the ubiquitin ligase EEL-1 preferentially regulates GABAergic presynaptic transmission. To further explore how EEL-1 functions, here we performed affinity purification proteomics using Caenorhabditis elegans and identified the O-GlcNAc transferase OGT-1 as an EEL-1 binding protein. This observation was intriguing, as we know little about how OGT-1 affects neuron function. Using C. elegans biochemistry, we confirmed that the OGT-1/EEL-1 complex forms in neurons in vivo and showed that the human orthologs, OGT and HUWE1, also bind in cell culture. We observed that, like EEL-1, OGT-1 is expressed in GABAergic motor neurons, localizes to GABAergic presynaptic terminals, and functions cell-autonomously to regulate GABA neuron function. Results with catalytically inactive point mutants indicated that OGT-1 glycosyltransferase activity is dispensable for GABA neuron function. Consistent with OGT-1 and EEL-1 forming a complex, genetic results using automated, behavioral pharmacology assays showed that ogt-1 and eel-1 act in parallel to regulate GABA neuron function. These findings demonstrate that OGT-1 and EEL-1 form a conserved signaling complex and function together to affect GABA neuron function.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Neurônios GABAérgicos/fisiologia , N-Acetilglucosaminiltransferases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Aldicarb/farmacologia , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/isolamento & purificação , Cromatografia de Afinidade , Neurônios GABAérgicos/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Ligação Proteica , Proteômica , Transdução de Sinais , Transmissão Sináptica/efeitos dos fármacos , Ubiquitina-Proteína Ligases/isolamento & purificação
9.
Amino Acids ; 51(4): 739-743, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30725225

RESUMO

O-GlcNAcylation, like phosphorylation, is a dynamic and rapid posttranslational modification which regulates many cellular processes. Phosphorylation on tyrosine and O-GlcNAcylation on nearby serine or threonine residues may modulate each other. Indeed, by using a microarray with a peptide model system based on the ZO-3 protein, extensive cross talk between O-GlcNAcylation by OGT and phosphorylation by kinases was observed. However, studying the effects of kinases and OGT without the reverse processes catalyzed by phosphatases and O-GlcNAcase (OGA) does not provide a complete picture of the cross talk. The study of the missing part showed that nearby phosphorylation affects the de-O-GlcNAcylation by OGA, but not to the same extent as it affects the O-GlcNAcylation by OGT. Both the phosphorylation and de-phosphorylation processes were only slightly affected by the presence of an O-GlcNAc residue on a nearby serine.


Assuntos
N-Acetilglucosaminiltransferases/metabolismo , Fragmentos de Peptídeos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Tirosina/metabolismo , Proteínas da Zônula de Oclusão/metabolismo , Humanos , Fosforilação , Análise Serial de Proteínas
10.
Cell Mol Life Sci ; 76(9): 1821-1832, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30737517

RESUMO

Branching and processing of N-glycans in the medial-Golgi rely both on the transport of the donor UDP-N-acetylglucosamine (UDP-GlcNAc) to the Golgi lumen by the SLC35A3 nucleotide sugar transporter (NST) as well as on the addition of the GlcNAc residue to terminal mannoses in nascent N-glycans by several linkage-specific N-acetyl-glucosaminyltransferases (MGAT1-MGAT5). Previous data indicate that the MGATs and NSTs both form higher order assemblies in the Golgi membranes. Here, we investigate their specific and mutual interactions using high-throughput FRET- and BiFC-based interaction screens. We show that MGAT1, MGAT2, MGAT3, MGAT4B (but not MGAT5) and Golgi alpha-mannosidase IIX (MAN2A2) form several distinct molecular assemblies with each other and that the MAN2A2 acts as a central hub for the interactions. Similar assemblies were also detected between the NSTs SLC35A2, SLC35A3, and SLC35A4. Using in vivo BiFC-based FRET interaction screens, we also identified novel ternary complexes between the MGATs themselves or between the MGATs and the NSTs. These findings suggest that the MGATs and the NSTs self-assemble into multi-enzyme/multi-transporter complexes in the Golgi membranes in vivo to facilitate efficient synthesis of complex N-glycans.


Assuntos
Complexo de Golgi/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Complexos Multienzimáticos/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Animais , Células COS , Linhagem Celular , Polissacarídeos/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismo , alfa-Manosidase/metabolismo
11.
mBio ; 10(1)2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30782656

RESUMO

Cardiolipin (CL) is an anionic phospholipid that plays an important role in regulating protein biochemistry in bacteria and mitochondria. Deleting the CL synthase gene (Δcls) in Rhodobacter sphaeroides depletes CL and decreases cell length by 20%. Using a chemical biology approach, we found that a CL deficiency does not impair the function of the cell wall elongasome in R. sphaeroides; instead, biosynthesis of the peptidoglycan (PG) precursor lipid II is decreased. Treating R. sphaeroides cells with fosfomycin and d-cycloserine inhibits lipid II biosynthesis and creates phenotypes in cell shape, PG composition, and spatial PG assembly that are strikingly similar to those seen with R. sphaeroides Δcls cells, suggesting that CL deficiency alters the elongation of R. sphaeroides cells by reducing lipid II biosynthesis. We found that MurG-a glycosyltransferase that performs the last step of lipid II biosynthesis-interacts with anionic phospholipids in native (i.e., R. sphaeroides) and artificial membranes. Lipid II production decreases 25% in R. sphaeroides Δcls cells compared to wild-type cells, and overexpression of MurG in R. sphaeroides Δcls cells restores their rod shape, indicating that CL deficiency decreases MurG activity and alters cell shape. The R. sphaeroides Δcls mutant is more sensitive than the wild-type strain to antibiotics targeting PG synthesis, including fosfomycin, d-cycloserine, S-(3,4-dichlorobenzyl)isothiourea (A22), mecillinam, and ampicillin, suggesting that CL biosynthesis may be a potential target for combination chemotherapies that block the bacterial cell wall.IMPORTANCE The phospholipid composition of the cell membrane influences the spatial and temporal biochemistry of cells. We studied molecular mechanisms connecting membrane composition to cell morphology in the model bacterium Rhodobacter sphaeroides The peptidoglycan (PG) layer of the cell wall is a dominant component of cell mechanical properties; consequently, it has been an important antibiotic target. We found that the anionic phospholipid cardiolipin (CL) plays a role in determination of the shape of R. sphaeroides cells by affecting PG precursor biosynthesis. Removing CL in R. sphaeroides alters cell morphology and increases its sensitivity to antibiotics targeting proteins synthesizing PG. These studies provide a connection to spatial biochemical control in mitochondria, which contain an inner membrane with topological features in common with R. sphaeroides.


Assuntos
Cardiolipinas/metabolismo , Parede Celular/metabolismo , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/metabolismo , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Proteínas da Membrana Bacteriana Externa/metabolismo , Vias Biossintéticas , Deleção de Genes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Uridina Difosfato Ácido N-Acetilmurâmico/biossíntese
12.
Immunity ; 50(3): 576-590.e6, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30770249

RESUMO

Elevated glucose metabolism in immune cells represents a hallmark feature of many inflammatory diseases, such as sepsis. However, the role of individual glucose metabolic pathways during immune cell activation and inflammation remains incompletely understood. Here, we demonstrate a previously unrecognized anti-inflammatory function of the O-linked ß-N-acetylglucosamine (O-GlcNAc) signaling associated with the hexosamine biosynthesis pathway (HBP). Despite elevated activities of glycolysis and the pentose phosphate pathway, activation of macrophages with lipopolysaccharide (LPS) resulted in attenuated HBP activity and protein O-GlcNAcylation. Deletion of O-GlcNAc transferase (OGT), a key enzyme for protein O-GlcNAcylation, led to enhanced innate immune activation and exacerbated septic inflammation. Mechanistically, OGT-mediated O-GlcNAcylation of the serine-threonine kinase RIPK3 on threonine 467 (T467) prevented RIPK3-RIPK1 hetero- and RIPK3-RIPK3 homo-interaction and inhibited downstream innate immunity and necroptosis signaling. Thus, our study identifies an immuno-metabolic crosstalk essential for fine-tuning innate immune cell activation and highlights the importance of glucose metabolism in septic inflammation.


Assuntos
Apoptose/fisiologia , Inflamação/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Necrose/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Linhagem Celular , Glucose/metabolismo , Humanos , Imunidade Inata/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Serina/metabolismo , Transdução de Sinais/fisiologia , Treonina/metabolismo
13.
Bioorg Med Chem Lett ; 29(6): 802-805, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30713024

RESUMO

O-Linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslationalmonosaccaride-modification found on Ser or Thr residues of intracellular proteins in most eukaryotes. The dynamic nature of O-GlcNAc has enabled researchers to modulate the stoichiometry of O-GlcNAc on proteins in order to investigate its function. Cell permeable small moleculars have proven invaluable tools to increase O-GlcNAc levels. Herein, using in vitro substrate screening, we identified GlcNAcF3 as an OGT-accepted but OGA-resistant sugar mimic. Cellular experiments with cell-permeable peracetylated-GlcNAcF3 (Ac4GlcNAcF3) displayed that Ac4GlcNAcF3 was a potent tool to increase O-GlcNAc levels in several cell lines. Further, NIH3T3 cells interfered with OGT (siOGT) showed significant decreasing of O-GlcNAc levels with Ac4GlcNAcF3 treatment, indicating O-GlcNAcF3 was an OGT-dependent modification. In addition, cellular toxic assay confirmed O-GlcNAcF3 production has no significant effect on cell proliferation or viability. Thus, Ac4GlcNAcF3 represents a safe and dual regulator for both OGT and OGA, which will benefit the study of O-GlcNAc.


Assuntos
Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Acetilglucosamina/toxicidade , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/toxicidade , Glicosilação/efeitos dos fármacos , Humanos , Camundongos , Células NIH 3T3 , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores
14.
Int J Oncol ; 54(3): 859-868, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30664192

RESUMO

The molecular mechanism of hereditary multiple exostoses (HME) remains ambiguous and a limited number of studies have investigated the pathogenic mechanism of mutations in patients with HME. In the present study, a novel heterozygous splice mutation (c.1284+2del) in exostosin glycosyltransferase 1 (EXT1) gene was identified in a three­generation family with HME. Bioinformatics and TA clone­sequencing indicated that the splice site mutation would result in exon 4 skipping. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) revealed that the expression levels of wild­type EXT1/EXT2 mRNA in patients with HME were significantly decreased, compared with normal control participants (P<0.05). Abnormal EXT1 transcript lacking exon 4 (EXT1­DEL) and full­length EXT1 mRNA (EXT1­FL) were overexpressed in 293­T cells and Cos­7 cells using lentivirus infection. RT­qPCR demonstrated that the expression level of EXT1­DEL was significantly increased, compared with EXT1­FL (17.032 vs. 6.309, respectively; P<0.05). The protein encoded by EXT1­DEL was detected by western blot analysis, and the level was increased, compared with EXT1­FL protein expression. Immunofluorescence indicated that the protein encoded by EXT1­DEL was located in the cytoplasm of Cos­7 cells, which was consistent with the localization of the EXT1­FL protein. In conclusion, the present study identified a novel splice mutation that causes exon 4 skipping during mRNA splicing and causes reduced expression of EXT1/EXT2. The mutation in EXT1­DEL generated a unique peptide that is located in the cytoplasm in vitro, and it expands the mutation spectrum and provides molecular genetic evidence for a novel pathogenic mechanism of HME.


Assuntos
Exostose Múltipla Hereditária/genética , N-Acetilglucosaminiltransferases/genética , Processamento de RNA/genética , Adulto , Idoso , Linhagem Celular Transformada , Citoplasma/metabolismo , Exostose Múltipla Hereditária/metabolismo , Exostose Múltipla Hereditária/patologia , Feminino , Expressão Gênica , Estudos de Associação Genética , Testes Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , N-Acetilglucosaminiltransferases/metabolismo , Sítios de Splice de RNA/genética , RNA Mensageiro/metabolismo
15.
Pathol Oncol Res ; 25(2): 759-768, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30689164

RESUMO

Mammary carcinoma is the most common malignant tumor in women, and it is the leading cause of mortality. In tumor context, glycosylation promotes post translational modifications necessary for cell progression, emerging as a relevant tumor hallmarker. This study aimed to analyze the association between polypeptide N-acetylgalactosaminyltransferase-6 (ppGalNAc-T6), -T8, N-acetylglucosaminyltransferase III (GnT-III) expression, Phaseolus vulgaris-leucoagglutinin (PHA-L), wheat germ agglutinin (WGA) and peanut agglutinin (PNA) staining with clinic-histopathological factors from patients with pure ductal carcinoma in situ (DCIS) and DCIS with invasive ductal carcinoma (DCIS-IDC) of breast. Formalin-fixed and paraffin-embedded samples (n = 109) were analyzed. In pure DCIS samples GnT-III was over-expressed in comedo lesions (p = 0.007). In DCIS-IDC, GnT-III expression was associated with high nuclear grade tumors (p = 0.039) while the presence of PHA-L and WGA were inversely related to HER-2 expression (p = 0.001; p = 0.036, respectively). These findings pointed to possible involvement of GnT-III, ppGalNAc-T8, L-PHA and WGA as probes in prognostic evaluation of DCIS.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , N-Acetilglucosaminiltransferases/metabolismo , Adulto , Neoplasias da Mama/enzimologia , Carcinoma Ductal de Mama/enzimologia , Carcinoma Intraductal não Infiltrante/enzimologia , Feminino , Humanos , Pessoa de Meia-Idade , N-Acetilglucosaminiltransferases/análise
16.
Org Lett ; 21(4): 876-879, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30698984

RESUMO

We have explored a method to convert a muraymycin biosynthetic intermediate 3 to an anticancer drug lead 2 for in vivo and thorough preclinical studies. Cu(OAc)2 forms a stable complex with the amide 4 and prevents electrophilic reactions at the 2-((3-aminopropyl)amino)acetamide moiety. Under the present conditions, the desired 5″-primary amine was selectively protected with (Boc)2O to yield 6. The intermediate 6 was converted to 2 in two steps with 90% yield.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Nucleosídeos/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Conformação Molecular , N-Acetilglucosaminiltransferases/metabolismo , Nucleosídeos/metabolismo , Relação Estrutura-Atividade
17.
Neurobiol Dis ; 124: 531-543, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30625365

RESUMO

Temporal Lobe Epilepsy (TLE) is frequently associated with changes in protein composition and post-translational modifications (PTM) that exacerbate the disorder. O-linked-ß-N-acetyl glucosamine (O-GlcNAc) is a PTM occurring at serine/threonine residues that is derived from and closely associated with metabolic substrates. The enzymes O-GlcNActransferase (OGT) and O-GlcNAcase (OGA) mediate the addition and removal, respectively, of the O-GlcNAc modification. The goal of this study was to characterize OGT/OGA and protein O-GlcNAcylation in the epileptic hippocampus and to determine and whether direct manipulation of these proteins and PTM's alter epileptiform activity. We observed reduced global and protein specific O-GlcNAcylation and OGT expression in the kainate rat model of TLE and in human TLE hippocampal tissue. Inhibiting OGA with Thiamet-G elevated protein O-GlcNAcylation, and decreased both seizure duration and epileptic spike events, suggesting that OGA may be a therapeutic target for seizure control. These findings suggest that loss of O-GlcNAc homeostasis in the kainate model and in human TLE can be reversed via targeting of O-GlcNAc related pathways.


Assuntos
Epilepsia do Lobo Temporal/metabolismo , Glucosamina/metabolismo , Hipocampo/metabolismo , Homeostase/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Histona Acetiltransferases/metabolismo , Humanos , Masculino , N-Acetilglucosaminiltransferases/metabolismo , Ratos , Ratos Sprague-Dawley
18.
J Biol Chem ; 294(7): 2211-2231, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30626734

RESUMO

In the early 1980s, while using purified glycosyltransferases to probe glycan structures on surfaces of living cells in the murine immune system, we discovered a novel form of serine/threonine protein glycosylation (O-linked ß-GlcNAc; O-GlcNAc) that occurs on thousands of proteins within the nucleus, cytoplasm, and mitochondria. Prior to this discovery, it was dogma that protein glycosylation was restricted to the luminal compartments of the secretory pathway and on extracellular domains of membrane and secretory proteins. Work in the last 3 decades from several laboratories has shown that O-GlcNAc cycling serves as a nutrient sensor to regulate signaling, transcription, mitochondrial activity, and cytoskeletal functions. O-GlcNAc also has extensive cross-talk with phosphorylation, not only at the same or proximal sites on polypeptides, but also by regulating each other's enzymes that catalyze cycling of the modifications. O-GlcNAc is generally not elongated or modified. It cycles on and off polypeptides in a time scale similar to phosphorylation, and both the enzyme that adds O-GlcNAc, the O-GlcNAc transferase (OGT), and the enzyme that removes O-GlcNAc, O-GlcNAcase (OGA), are highly conserved from C. elegans to humans. Both O-GlcNAc cycling enzymes are essential in mammals and plants. Due to O-GlcNAc's fundamental roles as a nutrient and stress sensor, it plays an important role in the etiologies of chronic diseases of aging, including diabetes, cancer, and neurodegenerative disease. This review will present an overview of our current understanding of O-GlcNAc's regulation, functions, and roles in chronic diseases of aging.


Assuntos
Envelhecimento/metabolismo , Diabetes Mellitus/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Nutrientes/metabolismo , Transdução de Sinais , Transcrição Genética , Envelhecimento/patologia , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Doença Crônica , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Diabetes Mellitus/patologia , Glicosilação , Humanos , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , N-Acetilglucosaminiltransferases/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/patologia , Fosforilação
19.
Nat Chem ; 11(2): 161-169, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30532014

RESUMO

Contemporary chemoenzymatic approaches can provide highly complex multi-antennary N-linked glycans. These procedures are, however, very demanding and typically involve as many as 100 chemical steps to prepare advanced intermediates that can be diversified by glycosyltransferases in a branch-selective manner to give asymmetrical structures commonly found in nature. Only highly specialized laboratories can perform such syntheses, which greatly hampers progress in glycoscience. Here we describe a biomimetic approach in which a readily available bi-antennary glycopeptide can be converted in ten or fewer chemical and enzymatic steps into multi-antennary N-glycans that at each arm can be uniquely extended by glycosyltransferases to give access to highly complex asymmetrically branched N-glycans. A key feature of our approach is the installation of additional branching points using recombinant MGAT4 and MGAT5 in combination with unnatural sugar donors. At an appropriate point in the enzymatic synthesis, the unnatural monosaccharides can be converted into their natural counterpart, allowing each arm to be elaborated into a unique appendage.


Assuntos
Materiais Biomiméticos/metabolismo , Polissacarídeos/metabolismo , Asparagina/metabolismo , Sequência de Carboidratos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Galactosidases/metabolismo , Glicopeptídeos/metabolismo , Glicosilação , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/química , Sialiltransferases/metabolismo
20.
J Biol Chem ; 294(4): 1363-1379, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30523150

RESUMO

The addition of a single ß-d-GlcNAc sugar (O-GlcNAc) by O-GlcNAc-transferase (OGT) and O-GlcNAc removal by O-GlcNAcase (OGA) maintain homeostatic O-GlcNAc levels on cellular proteins. Changes in protein O-GlcNAcylation regulate cellular differentiation and cell fate decisions, but how these changes affect erythropoiesis, an essential process in blood cell formation, remains unclear. Here, we investigated the role of O-GlcNAcylation in erythropoiesis by using G1E-ER4 cells, which carry the erythroid-specific transcription factor GATA-binding protein 1 (GATA-1) fused to the estrogen receptor (GATA-1-ER) and therefore undergo erythropoiesis after ß-estradiol (E2) addition. We observed that during G1E-ER4 differentiation, overall O-GlcNAc levels decrease, and physical interactions of GATA-1 with both OGT and OGA increase. RNA-Seq-based transcriptome analysis of G1E-ER4 cells differentiated in the presence of the OGA inhibitor Thiamet-G (TMG) revealed changes in expression of 433 GATA-1 target genes. ChIP results indicated that the TMG treatment decreases the occupancy of GATA-1, OGT, and OGA at the GATA-binding site of the lysosomal protein transmembrane 5 (Laptm5) gene promoter. TMG also reduced the expression of genes involved in differentiation of NB4 and HL60 human myeloid leukemia cells, suggesting that O-GlcNAcylation is involved in the regulation of hematopoietic differentiation. Sustained treatment of G1E-ER4 cells with TMG before differentiation reduced hemoglobin-positive cells and increased stem/progenitor cell surface markers. Our results show that alterations in O-GlcNAcylation disrupt transcriptional programs controlling erythropoietic lineage commitment, suggesting a role for O-GlcNAcylation in regulating hematopoietic cell fate.


Assuntos
Acetilglucosamina/metabolismo , Diferenciação Celular , Células Eritroides/citologia , Hematopoese , Homeostase , Células Mieloides/citologia , N-Acetilglucosaminiltransferases/metabolismo , Células Cultivadas , Células Eritroides/metabolismo , Fator de Transcrição GATA1/metabolismo , Humanos , Células Mieloides/fisiologia
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