RESUMO
Inhibiting O-GlcNAcase and thereby up-regulation of the O-GlcNAc levels of tau was a potential approach for discovering AD treatments. Herein, a series of novel highly potent OGA inhibitors embracing a 4-(arylethynyl)piperidine moiety was achieved by capitalizing on the substrate recognition domain. Extensive structure-activity relationships resulted in compound 81 with significant enzymatic inhibition (IC50 = 4.93 ± 2.05 nM) and cellular potency (EC50 = 7.47 ± 3.96 nM in PC12 cells). It markedly increased the protein O-GlcNAcylation levels and reduced the phosphorylation on Ser199, Thr205, and Ser396 of tau in the OA-injured SH-SY5Y cell model, suggesting its potential role for AD treatment. In fact, an in vivo efficacy of ameliorating cognitive impairment was observed following treatment of APP/PS1 mice with compound 81 (100 mg/kg). Additionally, the appropriate plasma PK and beneficial BBB penetration properties were also observed. Compound 81 deserves to be further explored as an anti-AD agent.
Assuntos
Doença de Alzheimer , Inibidores Enzimáticos , Piperidinas , beta-N-Acetil-Hexosaminidases , Doença de Alzheimer/tratamento farmacológico , Animais , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Piperidinas/síntese química , Piperidinas/química , Piperidinas/farmacocinética , Humanos , Relação Estrutura-Atividade , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/metabolismo , Ratos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Inibidores Enzimáticos/farmacocinética , Camundongos , Células PC12 , Descoberta de Drogas , Camundongos Transgênicos , Proteínas tau/metabolismo , Proteínas tau/antagonistas & inibidores , MasculinoRESUMO
Hexosaminidases are key enzymes in glycoconjugate metabolism and occur in all kingdoms of life. Here, we have investigated the phylogeny of the GH20 glycosyl hydrolase family in nematodes and identified a ß-hexosaminidase subclade present only in the Dorylaimia. We have expressed one of these, HEX-2 from Trichuris suis, a porcine parasite, and shown that it prefers an aryl ß-N-acetylgalactosaminide in vitro. HEX-2 has an almost neutral pH optimum and is best inhibited by GalNAc-isofagomine. Toward N-glycan substrates, it displays a preference for the removal of GalNAc residues from LacdiNAc motifs as well as the GlcNAc attached to the α1,3-linked core mannose. Therefore, it has a broader specificity than insect fused lobe (FDL) hexosaminidases but one narrower than distant homologues from plants. Its X-ray crystal structure, the first of any subfamily 1 GH20 hexosaminidase to be determined, is closest to Streptococcus pneumoniae GH20C and the active site is predicted to be compatible with accommodating both GalNAc and GlcNAc. The new structure extends our knowledge about this large enzyme family, particularly as T. suis HEX-2 also possesses the key glutamate residue found in human hexosaminidases of either GH20 subfamily, including HEXD whose biological function remains elusive.
Assuntos
Biologia Computacional , Trichuris , Animais , Trichuris/enzimologia , Especificidade por Substrato , Biologia Computacional/métodos , Cristalografia por Raios X , Sequência de Aminoácidos , Filogenia , Modelos Moleculares , Hexosaminidases/química , Hexosaminidases/metabolismo , Hexosaminidases/genética , Dados de Sequência Molecular , Domínio Catalítico , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , beta-N-Acetil-Hexosaminidases/metabolismo , beta-N-Acetil-Hexosaminidases/química , beta-N-Acetil-Hexosaminidases/genéticaRESUMO
O-GlcNAcase (OGA) is implicated in several important biological and disease-relevant processes. Here, we synthesized fluorogenic probes for OGA by grafting GlcNAc directly or using a self-immolative linker to the hydroxyl position of 4-hydroxylisoindoline (BHID), a typical excited-state intramolecular proton transfer (ESIPT) probe. The probe was used for a fluorogenic assay to determine the half maximal inhibitory concentration of a known OGA inhibitor and differentiate between OGA and hexosaminidase when GlcNAc is replaced by GlcNPr, where a propionyl group is used instead of an acetyl group.
Assuntos
Corantes Fluorescentes , Isoindóis , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Isoindóis/química , Isoindóis/síntese química , Humanos , beta-N-Acetil-Hexosaminidases/metabolismo , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Estrutura Molecular , Espectrometria de FluorescênciaRESUMO
Diabetic retinopathy (DR) is a very serious diabetes complication. Changes in the O-linked N-acetylglucosamine (O-GlcNAc) modification are associated with many diseases. However, its role in DR is not fully understood. In this research, we explored the effect of O-GlcNAc modification regulation by activating AMP-activated protein kinase (AMPK) in DR, providing some evidence for clinical DR treatment in the future. Bioinformatics was used to make predictions from the database, which were validated using the serum samples of diabetic patients. As an in vivo model, diabetic mice were induced using streptozotocin (STZ) injection with/without an AMPK agonist (metformin) or an AMPK inhibitor (compound C) treatment. Electroretinogram (ERG) and H&E staining were used to evaluate the retinal functional and morphological changes. In vitro, 661 w cells were exposed to high-glucose conditions, with or without metformin treatment. Apoptosis was evaluated using TUNEL staining. The protein expression was detected using Western blot and immunofluorescence staining. The angiogenesis ability was detected using a tube formation assay. The levels of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in the serum changed in the DR patients in the clinic. In the diabetic mice, the ERG wave amplitude and retinal thickness decreased. In vitro, the apoptotic cell percentage and Bax expression were increased, and Bcl2 expression was decreased in the 661 w cells under high-glucose conditions. The O-GlcNAc modification was increased in DR. In addition, the expression of GFAT/TXNIP O-GlcNAc was also increased in the 661 w cells after the high-glucose treatment. Additionally, the Co-immunoprecipitation(CO-IP) results show that TXNIP interacted with the O-GlcNAc modification. However, AMPK activation ameliorated this effect. We also found that silencing the AMPKα1 subunit reversed this process. In addition, the conditioned medium of the 661 w cells may have affected the tube formation in vitro. Taken together, O-GlcNAc modification was increased in DR with photoreceptor cell degeneration and neovascularization; however, it was reversed after activating AMPK. The underlying mechanism is linked to the GFAT/TXNIP-O-GlcNAc modification signaling axis. Therefore, the AMPKα1 subunit plays a vital role in the process.
Assuntos
Proteínas Quinases Ativadas por AMP , Acetilglucosamina , Diabetes Mellitus Experimental , Retinopatia Diabética , N-Acetilglucosaminiltransferases , Retinopatia Diabética/metabolismo , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/patologia , Animais , Camundongos , Acetilglucosamina/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Masculino , Apoptose/efeitos dos fármacos , Metformina/farmacologia , beta-N-Acetil-Hexosaminidases/metabolismo , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , Retina/metabolismo , Retina/patologia , Retina/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Linhagem CelularRESUMO
This study aims to investigate the antiallergic effects of Shiikuwasha (Citrus depressa Hayata) leaf and peel extracts by examining the regulation of degranulation and inflammatory cytokine production from rat basophilic leukemia (RBL-2H3) cells and antigen-specific antibody production in sensitized mouse spleen lymphocytes. In vivo antiallergic activity was evaluated using the passive cutaneous anaphylaxis (PCA) reaction model. Extracts of Shiikuwasha leaves and peel were prepared using 80% methanol and dissolved in dimethylsulfoxide. The dinitrophenyl-human serum albumin-induced ß-hexosaminidase levels in immunoglobulin (Ig) E-sensitized RBL-2H3 cells were assessed using enzymatic assays. Cytokine production was measured by enzyme-linked immunosorbent assay. Antibody production capacity was evaluated using lymphocytes isolated from spleens of type I allergy model mice. Lymphocytes were cultured for 72 h with Shiikuwasha extracts, and ovalbumin-specific IgE, IgG1, and IgG2a levels were measured. Shiikuwasha leaf and peel extract significantly reduced ß-hexosaminidase release and suppressed interleukin-4 and tumor necrosis factor-α production from RBL-2H3 cells. Ovalbumin-specific IgE and IgG1 production decreased in Shiikuwasha extract-treated lymphocytes. These extracts also significantly suppressed the PCA reaction. Shiikuwasha leaf and peel extract reduce degranulation in RBL-2H3 cells and antibody production in spleen-derived lymphocytes and therefore exhibit antiallergic effects.
Assuntos
Antialérgicos , Degranulação Celular , Imunoglobulina E , Extratos Vegetais , Folhas de Planta , Baço , Animais , Extratos Vegetais/farmacologia , Ratos , Baço/efeitos dos fármacos , Baço/imunologia , Baço/citologia , Folhas de Planta/química , Camundongos , Linhagem Celular Tumoral , Degranulação Celular/efeitos dos fármacos , Imunoglobulina E/sangue , Antialérgicos/farmacologia , Antialérgicos/uso terapêutico , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Leucemia Basofílica Aguda/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Imunoglobulina G , Masculino , Interleucina-4/metabolismoRESUMO
Understanding the regulation of human embryonic stem cells (hESCs) pluripotency is critical to advance the field of developmental biology and regenerative medicine. Despite the recent progress, molecular events regulating hESC pluripotency, especially the transition between naive and primed states, still remain unclear. Here we show that naive hESCs display lower levels of O-linked N-acetylglucosamine (O-GlcNAcylation) than primed hESCs. O-GlcNAcase (OGA), the key enzyme catalyzing the removal of O-GlcNAc from proteins, is highly expressed in naive hESCs and is important for naive pluripotency. Depletion of OGA accelerates naive-to-primed pluripotency transition. OGA is transcriptionally regulated by EP300 and acts as a transcription regulator of genes important for maintaining naive pluripotency. Moreover, we profile protein O-GlcNAcylation of the two pluripotency states by quantitative proteomics. Together, this study identifies OGA as an important factor of naive pluripotency in hESCs and suggests that O-GlcNAcylation has a broad effect on hESCs homeostasis.
Assuntos
Células-Tronco Embrionárias Humanas , Humanos , Acetilglucosamina/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Diferenciação Celular , Linhagem Celular , Glicosilação , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologiaRESUMO
BACKGROUND: Vitamin K3 (VK3), a fat-soluble synthetic analog of the vitamin K family, has coagulant, anti-inflammatory, antibacterial, and anticancer properties. Pseudo allergy is a IgE-independent immune response associated with mast cells. This study investigated the role of VK3 in IgE-independent mast cell activation. METHODS: Substance P (SP) was used to induce LAD2-cell activation in order to analyze the effects of VK3 in vitro. Cutaneous allergy and systemic allergy mouse models were used to analyze the anti-pseudo-allergic effects of VK3. Proteome microarray assays were used to analyze VK3-binding protein. Biolayer interferometry and immunoprecipitation were used to verify interaction between VK3 and its key targets. RNA interference was used to determine the role of GAB1 in LAD2cell activation. RESULTS: VK3 inhibited SP-induced LAD2-cell activation, and resulted in the release of ß-hexosaminidase, histamine and cytokines; VK3 inhibited SP-induced pseudo allergic reactions in mice, and serum histamine and TNF-α levels decreased. Degranulation of skin mast cells was reduced; GAB1 in mast cells was stably bound to VK3. GAB1 participated in SP-induced LAD2-cell activation. GAB1 knockdown in LAD2 cells prevented SP-induced ß-hexosaminidase release, calcium mobilization and cell skeletal remodeling. VK3 directly binds to GAB1 and reduces its expression to inhibited SP-induced LAD2 cell activation. CONCLUSION: The anti-pseudo-allergic activity of VK3 was confirmed in vitro and in vivo. VK3 can inhibit SP-induced mast cell activation by directly targeting GAB1. This study provides new insights on the activity of VK3 and the mechanism of pseudoallergic reaction.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Mastócitos , Mastócitos/imunologia , Mastócitos/efeitos dos fármacos , Animais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Camundongos , Humanos , Substância P/metabolismo , Degranulação Celular/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Hipersensibilidade/imunologia , Hipersensibilidade/tratamento farmacológico , Antialérgicos/farmacologia , Antialérgicos/uso terapêutico , Feminino , Linhagem Celular , beta-N-Acetil-Hexosaminidases/metabolismo , Modelos Animais de DoençasRESUMO
O-GlcNAcylation is an essential protein glycosylation governed by two O-GlcNAc cycling enzymes: O-GlcNAc transferase (OGT) installs a single sugar moiety N-acetylglucosamine (GlcNAc) on protein serine and threonine residues, and O-GlcNAcase (OGA) removes them. Aberrant O-GlcNAcylation has been implicated in various diseases. However, the large repertoire of more than 1000 O-GlcNAcylated proteins and the elusive mechanisms of OGT/OGA in substrate recognition present significant challenges in targeting the dysregulated O-GlcNAcylation for therapeutic development. Recently, emerging evidence suggested that the non-catalytic domains play critical roles in regulating the functional specificity of OGT/OGA via modulating their protein interactions and substrate recognition. Here, we discuss recent studies on the structures, mechanisms, and related tools of the OGT/OGA non-catalytic domains, highlighting new opportunities for function-specific control.
Assuntos
Acetilglucosamina , N-Acetilglucosaminiltransferases , beta-N-Acetil-Hexosaminidases , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/química , Humanos , Acetilglucosamina/metabolismo , Acetilglucosamina/química , beta-N-Acetil-Hexosaminidases/metabolismo , beta-N-Acetil-Hexosaminidases/química , Especificidade por Substrato , Animais , Glicosilação , Domínios ProteicosRESUMO
Microglia are natural immune cells in the central nervous system, and the activation of microglia is accompanied by a reprogramming of glucose metabolism. In our study, we investigated the role of long non-coding RNA taurine-upregulated gene 1 (TUG1) in regulating microglial glucose metabolism reprogramming and activation. BV2 cells were treated with Lipopolysaccharides (LPS)/Interferon-γ (IFN-γ) to establish a microglial activation model. The glycolysis inhibitor 2-Deoxy-D-glucose (2-DG) was used as a control. The expression levels of TUG1 mRNA and proinflammatory cytokines such as Interleukin-1ß (IL-1ß), Interleukin -6, and Tumor Necrosis Factor-α mRNA and anti-inflammatory cytokines such as IL-4, Arginase 1(Arg1), CD206, and Ym1 were detected by RT-qPCR. TUG1 was silenced using TUG1 siRNA and knocked out using CRISPR/Cas9. The mRNA and protein expression levels of key enzymes involved in glucose metabolism, such as Hexokinase2, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Lactate dehydrogenase, Glucose 6 phosphate dehydrogenase, and Pyruvate dehydrogenase (PDH), were determined by RT-qPCR and Western blotting. The glycolytic rate of microglial cells was measured using Seahorse. Differential metabolites were determined by metabolomics, and pathway enrichment was performed using these differential metabolites. Our findings revealed that the expression of TUG1 was elevated in proinflammatory-activated microglia and positively correlated with the levels of inflammatory factors. The expression of anti-inflammatory cytokines such as IL-4, Arg1, CD206, and Ym1 were decreased when induced with LPS/IFN-γ. However, this decrease was reversed by the treatment with 2-DG. Silencing of GAPDH led to an increase in the expression of TUG1 and inflammatory factors. TUG1 knockout (TUG1KO) inhibited the expression of glycolytic key enzymes and promoted the expression of oxidative phosphorylation key enzymes, shifting the metabolic profile of activated microglia from glycolysis to oxidative phosphorylation. Additionally, TUG1KO reduced the accumulation of metabolites, facilitating the restoration of the tricarboxylic acid cycle and enhancing oxidative phosphorylation in microglia. Furthermore, the downregulation of TUG1 was found to reduce the expression of both proinflammatory and anti-inflammatory cytokines under normal conditions. Interestingly, when induced with LPS/IFN-γ, TUG1 downregulation showed a potentially beneficial effect on microglia in terms of inflammation. Downregulation of TUG1 expression inhibits glycolysis and facilitates the shift of microglial glucose metabolism from glycolysis to oxidative phosphorylation, promoting their transformation towards an anti-inflammatory phenotype and exerting anti-inflammatory effects in BV2.
Assuntos
Glucose , Glicólise , Lipopolissacarídeos , Microglia , RNA Longo não Codificante , Microglia/metabolismo , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Glucose/metabolismo , Camundongos , Lipopolissacarídeos/farmacologia , Citocinas/metabolismo , Inflamação/metabolismo , Inflamação/genética , Interferon gama/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , beta-N-Acetil-Hexosaminidases/genética , Linhagem Celular , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Lectinas de Ligação a Manose/genética , Desoxiglucose/farmacologia , Interleucina-4/metabolismo , Interleucina-1beta/metabolismo , Reprogramação Metabólica , Arginase , Hexoquinase , LectinasRESUMO
Selenium-rich tea polysaccharides (Se-TPS) were extracted via high hydrostatic pressure technology with a pressure of 400 MPa (200-500 MPa) for 10 min (3-20 min) at a material-to-solvent ratio of 1:40 (1:20-1:50). Subsequently, Se-TPS1-4 were isolated and purified, with Se-TPS3-4 as the main components. A spectral analysis proved that Se, which has antioxidant activity, existed. An in vitro study found that among Se-TPS, Se-TPS3-4 attenuated the release of ß-hexosaminidase, histamine, and interleukin (IL)-4. Furthermore, in vivo experiments revealed that treatment with Se-TPS downregulated IL-4 levels and upregulated TGF-ß and interferon-γ levels to improve imbalanced Th1/Th2 immunity in tropomyosin-sensitized mice. Moreover, Se-TPS promoted Lactobacillus and norank_f_Muribaculaceaek growth and upregulated metabolites such as genipin and coniferyl alcohol. Overall, these results showed the strong anti-allergy potential of Se-TPS by regulating mast cell-mediated allergic inflammatory responses and microbiota regulation, highlighting the potential of Se-TPS as a novel therapeutic agent to regulate allergy-associated metabolic disorders.
Assuntos
Microbioma Gastrointestinal , Pressão Hidrostática , Polissacarídeos , Chá , Animais , Microbioma Gastrointestinal/efeitos dos fármacos , Polissacarídeos/farmacologia , Polissacarídeos/química , Camundongos , Chá/química , Mastócitos/metabolismo , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Antialérgicos/farmacologia , Antialérgicos/química , Antialérgicos/isolamento & purificação , beta-N-Acetil-Hexosaminidases/metabolismo , Citocinas/metabolismo , Masculino , Tropomiosina/metabolismo , Tropomiosina/imunologiaRESUMO
A new class of compounds, namely highly substituted diaminocyclopentane-l-lysine adducts, have been discovered as potent inhibitors of O-GlcNAcase, an enzyme crucial for protein de-O-glycosylation. These inhibitors exhibit exceptional selectivity and reversibility and are the first example of human O-GlcNAcase inhibitors that are structurally related to the transition state of the rate-limiting step with the "aglycon" still in bond-length proximity. The ease of their preparation, remarkable biological activities, stability, and non-toxicity make them promising candidates for the development of anti-tau-phosphorylation agents holding significant potential for the treatment of Alzheimer's disease.
Assuntos
Inibidores Enzimáticos , Lisina , Humanos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Relação Estrutura-Atividade , Lisina/química , Lisina/farmacologia , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/metabolismo , Ciclopentanos/química , Ciclopentanos/farmacologia , Ciclopentanos/síntese química , Estrutura Molecular , Relação Dose-Resposta a DrogaRESUMO
BACKGROUND: ß-N-acetylhexosaminidases (HEXs) are widely distributed in fungi and involved in cell wall chitin metabolism and utilization of chitin-containing substrates. However, details of the fungal pathogens-derived HEXs in the interaction with their hosts remain limited. RESULTS: An insect nutrients-induced ß-N-acetylhexosaminidase, BbHex1, was identified from the entomopathogenic fungus Beauveria bassiana, which was involved in cell wall modification and degradation of insect cuticle. BbHex1 was localized to cell wall and secreted, and displayed enzyme activity to degrade the chitinase-hydrolyzed product (GlcNAc)2. Disruption of BbHex1 resulted in a significant decrease in the level of cell wall chitin in the presence of insect nutrients and during infection of insects, with impaired ability to penetrate insect cuticle, accompanying downregulated cell wall metabolism-involved and cuticle-degrading chitinase genes. However, the opposite phenotypes were examined in the gene overexpression strain. Distinctly altered cell wall structures caused by BbHex1 mutation and overexpression led to the easy activation and evasion (respectively) of insect immune response during fungal infection. As a result, BbHex1 contributed to fungal virulence. Bioinformatics analysis revealed that promoters of some co-expressed chitinase genes with the BbHex1 promoter shared conserved transcription factors Skn7, Msn2 and Ste12, and CreA-binding motifs, implying co-regulation of those genes with BbHex1. CONCLUSION: These data support a mechanism that the fungal pathogen specifically expresses BbHex1, which is co-expressed with chitinases to modify cell wall for evasion of insect immune recognition and to degrade insect cuticle, and contributes to the fungal virulence against insects. © 2024 Society of Chemical Industry.
Assuntos
Beauveria , Parede Celular , Quitinases , beta-N-Acetil-Hexosaminidases , Animais , Parede Celular/metabolismo , Quitinases/genética , Quitinases/metabolismo , Beauveria/fisiologia , Beauveria/genética , Beauveria/enzimologia , beta-N-Acetil-Hexosaminidases/metabolismo , beta-N-Acetil-Hexosaminidases/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Virulência , Mariposas/microbiologia , Mariposas/imunologia , Mariposas/genéticaRESUMO
ß-N-Acetylgalactosamine-containing glycans play essential roles in several biological processes, including cell adhesion, signal transduction, and immune responses. ß-N-Acetylgalactosaminidases hydrolyze ß-N-acetylgalactosamine linkages of various glycoconjugates. However, their biological significance remains ambiguous, primarily because only one type of enzyme, exo-ß-N-acetylgalactosaminidases that specifically act on ß-N-acetylgalactosamine residues, has been documented to date. In this study, we identify four groups distributed among all three domains of life and characterize eight ß-N-acetylgalactosaminidases and ß-N-acetylhexosaminidase through sequence-based screening of deep-sea metagenomes and subsequent searching of public protein databases. Despite low sequence similarity, the crystal structures of these enzymes demonstrate that all enzymes share a prototype structure and have diversified their substrate specificities (oligosaccharide-releasing, oligosaccharide/monosaccharide-releasing, and monosaccharide-releasing) through the accumulation of mutations and insertional amino acid sequences. The diverse ß-N-acetylgalactosaminidases reported in this study could facilitate the comprehension of their structures and functions and present evolutionary pathways for expanding their substrate specificity.
Assuntos
Acetilgalactosamina , Glicosídeo Hidrolases , Metagenoma , Metagenoma/genética , Especificidade por Substrato , Acetilgalactosamina/metabolismo , Acetilgalactosamina/química , Glicosídeo Hidrolases/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/química , beta-N-Acetil-Hexosaminidases/metabolismo , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/química , Filogenia , Cristalografia por Raios X , Sequência de Aminoácidos , AnimaisRESUMO
This study aimed to elucidate the role of O-GlcNAc cycling in 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (PD)-like neurodegeneration and the underlying mechanisms. We observed dose-dependent downregulation of O-GlcNAcylation, accompanied by an increase in O-GlcNAcase following 6-OHDA treatment in both mouse brain and Neuro2a cells. Interestingly, elevating O-GlcNAcylation through glucosamine (GlcN) injection provided protection against PD pathogenesis induced by 6-OHDA. At the behavioral level, GlcN mitigated motor deficits induced by 6-OHDA, as determined using the pole, cylinder, and apomorphine rotation tests. Furthermore, GlcN attenuated 6-OHDA-induced neuroinflammation and mitochondrial dysfunction. Notably, augmented O-GlcNAcylation, achieved through O-GlcNAc transferase (OGT) overexpression in mouse brain, conferred protection against 6-OHDA-induced PD pathology, encompassing neuronal cell death, motor deficits, neuroinflammation, and mitochondrial dysfunction. These collective findings suggest that O-GlcNAcylation plays a crucial role in the normal functioning of dopamine neurons. Moreover, enhancing O-GlcNAcylation through genetic and pharmacological means could effectively ameliorate neurodegeneration and motor impairment in an animal model of PD. These results propose a potential strategy for safeguarding against the deterioration of dopamine neurons implicated in PD pathogenesis.
Assuntos
Camundongos Endogâmicos C57BL , N-Acetilglucosaminiltransferases , Oxidopamina , Doença de Parkinson , Animais , Oxidopamina/farmacologia , Camundongos , N-Acetilglucosaminiltransferases/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Masculino , Glucosamina/farmacologia , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Acetilglucosamina/metabolismo , Acetilglucosamina/farmacologia , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/efeitos dos fármacos , beta-N-Acetil-Hexosaminidases/metabolismo , Modelos Animais de DoençasRESUMO
The addition of O-linked ß-N-acetylglucosamine (O-GlcNAc) to proteins (referred to as O-GlcNAcylation) is a modification that is crucial for vertebrate development. O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). Missense variants of OGT have recently been shown to segregate with an X-linked syndromic form of intellectual disability, OGT-linked congenital disorder of glycosylation (OGT-CDG). Although the existence of OGT-CDG suggests that O-GlcNAcylation is crucial for neurodevelopment and/or cognitive function, the underlying pathophysiologic mechanisms remain unknown. Here we report a mouse line that carries a catalytically impaired OGT-CDG variant. These mice show altered O-GlcNAc homeostasis with decreased global O-GlcNAcylation and reduced levels of OGT and OGA in the brain. Phenotypic characterization of the mice revealed lower body weight associated with reduced body fat mass, short stature and microcephaly. This mouse model will serve as an important tool to study genotype-phenotype correlations in OGT-CDG in vivo and for the development of possible treatment avenues for this disorder.
Assuntos
Modelos Animais de Doenças , Deficiência Intelectual , N-Acetilglucosaminiltransferases , Animais , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/deficiência , Deficiência Intelectual/genética , Encéfalo/patologia , Encéfalo/metabolismo , Fenótipo , Camundongos , Transtornos do Neurodesenvolvimento/patologia , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/enzimologia , beta-N-Acetil-Hexosaminidases/metabolismo , Glicosilação , Peso CorporalRESUMO
Benastatin K (1), a new chlorinated benastatin derivative, was isolated from the culture broth of the actinomycete Streptomyces sp. HGTA384. The structure of 1 was determined on the basis of spectroscopic analysis, including 1D and 2D NMR, as well as HRESI-MS, UV and IR, and comparison with data reported in the literature. Compound 1 and benastatins A and B exhibited inhibitory activity against Micrococcus luteus (MIC 7.8, 31.3, and 3.9 µM, respectively), and IgE-mediated ß-hexosaminidase release in RBL-2H3 cells with IC50 values of 42, 79, and 19 µM, respectively.
Assuntos
Antibacterianos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Micrococcus luteus , Streptomyces , Streptomyces/química , Streptomyces/metabolismo , Micrococcus luteus/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/isolamento & purificação , Antibacterianos/química , Animais , Ratos , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/metabolismo , Concentração Inibidora 50 , Linhagem Celular Tumoral , Estrutura MolecularRESUMO
Dysregulation of O-GlcNAcylation has emerged as a potential biomarker for several diseases, particularly cancer. The role of OGT (O-GlcNAc transferase) in maintaining O-GlcNAc homeostasis has been extensively studied; nevertheless, the regulation of OGA (O-GlcNAcase) in cancer remains elusive. Here, we demonstrated that the multifunctional protein RBM14 is a regulator of cellular O-GlcNAcylation. By investigating the correlation between elevated O-GlcNAcylation and increased RBM14 expression in lung cancer cells, we discovered that RBM14 promotes ubiquitin-dependent proteasomal degradation of OGA, ultimately mediating cellular O-GlcNAcylation levels. In addition, RBM14 itself is O-GlcNAcylated at serine 521, regulating its interaction with the E3 ligase TRIM33, consequently affecting OGA protein stability. Moreover, we demonstrated that mutation of serine 521 to alanine abrogated the oncogenic properties of RBM14. Collectively, our findings reveal a previously unknown mechanism for the regulation of OGA and suggest a potential therapeutic target for the treatment of cancers with dysregulated O-GlcNAcylation.
Assuntos
Estabilidade Proteica , Proteínas de Ligação a RNA , Humanos , Acetilglucosamina/metabolismo , Antígenos de Neoplasias , beta-N-Acetil-Hexosaminidases/metabolismo , Linhagem Celular Tumoral , Glicosilação , Células HEK293 , Histona Acetiltransferases , Hialuronoglucosaminidase , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , N-Acetilglucosaminiltransferases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The past 4 decades have witnessed tremendous efforts in deciphering the role of O-GlcNAcylation in a plethora of biological processes. Chemists and biologists have joined hand in hand in the sweet adventure to unravel this unique and universal yet uncharted post-translational modification, and the recent advent of cutting-edge chemical biology and mass spectrometry tools has greatly facilitated the process. Compared with O-GlcNAc, DNA damage response (DDR) is a relatively intensively studied area that could be traced to before the elucidation of the structure of DNA. Unexpectedly, yet somewhat expectedly, O-GlcNAc has been found to regulate various DDR pathways: homologous recombination, nonhomologous end joining, base excision repair, and translesion DNA synthesis. In this review, we first cover the recent structural studies of the O-GlcNAc transferase and O-GlcNAcase, the elegant duo that "writes" and "erases" O-GlcNAc modification. Then we delineate the intricate roles of O-GlcNAc transferase and O-GlcNAcase in DDR. We envision that this is only the beginning of our full appreciation of how O-GlcNAc regulates the blueprint of life-DNA.
Assuntos
N-Acetilglucosaminiltransferases , Animais , Humanos , beta-N-Acetil-Hexosaminidases/metabolismo , beta-N-Acetil-Hexosaminidases/genética , DNA/metabolismo , DNA/química , Dano ao DNA , Reparo do DNA , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , Processamento de Proteína Pós-Traducional , GenomaRESUMO
The neuronal microtubule-associated tau protein is characterized in vivo by a large number of post-translational modifications along the entire primary sequence that modulates its function. The primary modification of tau is phosphorylation of serine/threonine or tyrosine residues that is involved in the regulation of microtubule binding and polymerization. In neurodegenerative disorders referred to as tauopathies including Alzheimer's disease, tau is abnormally hyperphosphorylated and forms fibrillar inclusions in neurons progressing throughout different brain area during the course of the disease. The O-ß-linked N-acetylglucosamine (O-GlcNAc) is another reversible post-translational modification of serine/threonine residues that is installed and removed by the unique O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA), respectively. This modification was described as a potential modulator of tau phosphorylation and functions in the physiopathology. Moreover, reducing protein O-GlcNAc levels in the brain upon treatment of tauopathy mouse models with an OGA inhibitor reveals a beneficial effect on tau pathology and neurodegeneration. However, whether the role of tau O-GlcNAcylation is responsible of the protective effect against tau toxicity remains to be determined. The production of O-GlcNAc modified recombinant tau protein is a valuable tool for the investigations of the impact of O-GlcNAcylation on tau functions, modulation of interactions with partners and crosstalk with other post-translational modifications, including but not restricted to phosphorylation. We describe here the in vitro O-GlcNAcylation of tau with recombinant OGT for which we provide an expression and purification protocol. The use of the O-GlcNAc tau protein in functional studies requires the analytical characterization of the O-GlcNAc pattern. Here, we describe a method for the O-GlcNAc modification of tau protein with recombinant OGT and the analytical characterization of the resulting O-GlcNAc pattern by a combination of methods for the overall characterization of tau O-GlcNAcylation by chemoenzymatic labeling and mass spectrometry, as well as the quantitative, site-specific pattern by NMR spectroscopy.
Assuntos
Tauopatias , Proteínas tau , Camundongos , Animais , Proteínas tau/metabolismo , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/metabolismo , Processamento de Proteína Pós-Traducional , Tauopatias/genética , Tauopatias/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Acetilglucosamina/metabolismo , Serina/metabolismo , Treonina/metabolismoRESUMO
BACKGROUND: Food allergy has become a global public health problem. This study aimed to explore the possible anti-allergic effect of vitamin C (VC). A rat basophilic leukemia (RBL)-2H3 cell degranulation model was used to assess the effect of VC on degranulation in vitro, and an ovalbumin (OVA)-induced BALB/c mouse allergy model was used to assess the anti-allergy effect of VC in vivo. RESULTS: In vitro, VC significantly attenuated the release of ß-hexosaminidase, tryptase and histamine, and also reduced cytokine production (interleukins 4 and 6, tumor necrosis factor α) significantly (P < 0.05), with the inhibitory effect demonstrating a positive correlation with VC dose. In vivo, compared with the OVA group, the levels of serum immunoglobulins E and G1 of the VC low-dose (VCL) group (50 mg kg-1) and high-dose (VCH) group (200 mg·kg-1) were significantly reduced (P < 0.05). Furthermore, the plasma histamine level was also significantly decreased (P < 0.05). Moreover, TH2 cell polarization in mice of the VCL and VCH groups was significantly inhibited (P < 0.05), promoting the TH1/TH2 cell polarization balance. Additionally, VC treatment enhanced the expression of CD80 (P < 0.05) in spleen and small intestine tissues, while significantly inhibiting the expression of CD86 (P < 0.05); notably, high-dose VC treatment was more effective. CONCLUSION: VC exerted an anti-allergic effect through inhibiting degranulation and regulating TH1/TH2 cell polarization balance. © 2024 Society of Chemical Industry.