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1.
Proc Natl Acad Sci U S A ; 121(24): e2320867121, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38838015

RESUMO

O-GlcNAcase (OGA) is the only human enzyme that catalyzes the hydrolysis (deglycosylation) of O-linked beta-N-acetylglucosaminylation (O-GlcNAcylation) from numerous protein substrates. OGA has broad implications in many challenging diseases including cancer. However, its role in cell malignancy remains mostly unclear. Here, we report that a cancer-derived point mutation on the OGA's noncatalytic stalk domain aberrantly modulates OGA interactome and substrate deglycosylation toward a specific set of proteins. Interestingly, our quantitative proteomic studies uncovered that the OGA stalk domain mutant preferentially deglycosylated protein substrates with +2 proline in the sequence relative to the O-GlcNAcylation site. One of the most dysregulated substrates is PDZ and LIM domain protein 7 (PDLIM7), which is associated with the tumor suppressor p53. We found that the aberrantly deglycosylated PDLIM7 suppressed p53 gene expression and accelerated p53 protein degradation by promoting the complex formation with E3 ubiquitin ligase MDM2. Moreover, deglycosylated PDLIM7 significantly up-regulated the actin-rich membrane protrusions on the cell surface, augmenting the cancer cell motility and aggressiveness. These findings revealed an important but previously unappreciated role of OGA's stalk domain in protein substrate recognition and functional modulation during malignant cell progression.


Assuntos
Citoesqueleto , Proteínas com Domínio LIM , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Proteínas com Domínio LIM/metabolismo , Proteínas com Domínio LIM/genética , Citoesqueleto/metabolismo , Acetilglucosamina/metabolismo , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Linhagem Celular Tumoral , Glicosilação , Hidrólise , Mutação , Movimento Celular , Antígenos de Neoplasias , Hialuronoglucosaminidase , Histona Acetiltransferases
2.
Appl Microbiol Biotechnol ; 108(1): 373, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38878095

RESUMO

The lincoamide antibiotic lincomycin, derived from Streptomyces lincolnensis, is widely used for the treatment of infections caused by gram-positive bacteria. As a common global regulatory factor of GntR family, DasR usually exists as a regulatory factor that negatively regulates antibiotic synthesis in Streptomyces. However, the regulatory effect of DasR on lincomycin biosynthesis in S. lincolnensis has not been thoroughly investigated. The present study demonstrates that DasR functions as a positive regulator of lincomycin biosynthesis in S. lincolnensis, and its overexpression strain OdasR exhibits a remarkable 7.97-fold increase in lincomycin production compared to the wild-type strain. The effects of DasR overexpression could be attenuated by the addition of GlcNAc in the medium in S. lincolnensis. Combined with transcriptome sequencing and RT-qPCR results, it was found that most structural genes in GlcNAc metabolism and central carbon metabolism were up-regulated, but the lincomycin biosynthetic gene cluster (lmb) were down-regulated after dasR knock-out. However, DasR binding were detected with the DasR responsive elements (dre) of genes involved in GlcNAc metabolism pathway through electrophoretic mobility shift assay, while they were not observed in the lmb. These findings will provide novel insights for the genetic manipulation of S. lincolnensis to enhance lincomycin production. KEY POINTS: • DasR is a positive regulator that promotes lincomycin synthesis and does not affect spore production • DasR promotes lincomycin production through indirect regulation • DasR correlates with nutrient perception in S. lincolnensis.


Assuntos
Antibacterianos , Regulação Bacteriana da Expressão Gênica , Lincomicina , Streptomyces , Lincomicina/farmacologia , Lincomicina/biossíntese , Streptomyces/genética , Streptomyces/metabolismo , Streptomyces/efeitos dos fármacos , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Família Multigênica , Acetilglucosamina/metabolismo , Vias Biossintéticas/genética , Perfilação da Expressão Gênica
3.
Elife ; 122024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38884443

RESUMO

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.


Assuntos
Quitina , Quitinases , Simulação de Dinâmica Molecular , Quitinases/metabolismo , Quitinases/química , Animais , Concentração de Íons de Hidrogênio , Camundongos , Quitina/metabolismo , Quitina/química , Conformação Proteica , Cristalografia por Raios X , Ligação Proteica , Ligantes , Cinética , Acetilglucosamina/metabolismo , Acetilglucosamina/química , Modelos Moleculares
4.
J Agric Food Chem ; 72(25): 14255-14263, 2024 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-38867497

RESUMO

The addition of the O-linked N-acetylglucosamine (O-GlcNAc) is a significant modification for active molecules, such as proteins, carbohydrates, and natural products. However, the synthesis of terpenoid glycoside derivatives decorated with GlcNAc remains a challenging task due to the absence of glycosyltransferases, key enzymes for catalyzing the transfer of GlcNAc to terpenoids. In this study, we demonstrated that the enzyme mutant UGT74AC1T79Y/L48M/R28H/L109I/S15A/M76L/H47R efficiently transferred GlcNAc from uridine diphosphate (UDP)-GlcNAc to a variety of terpenoids. This powerful enzyme was employed to synthesize GlcNAc-decorated derivatives of terpenoids, including mogrol, steviol, andrographolide, protopanaxadiol, glycyrrhetinic acid, ursolic acid, and betulinic acid for the first time. To unravel the mechanism of UDP-GlcNAc recognition, we determined the X-ray crystal structure of the inactivated mutant UGT74AC1His18A/Asp111A in complex with UDP-GlcNAc at a resolution of 1.66 Å. Through molecular dynamic simulation and activity analysis, we revealed the molecular mechanism and catalytically important amino acids directly involved in the recognition of UDP-GlcNAc. Overall, this study not only provided a potent biocatalyst capable of glycodiversifying natural products but also elucidated the structural basis for UDP-GlcNAc recognition by glycosyltransferases.


Assuntos
Acetilglucosamina , Glicosídeos , Glicosiltransferases , Terpenos , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Glicosídeos/química , Glicosídeos/metabolismo , Glicosiltransferases/metabolismo , Glicosiltransferases/química , Glicosiltransferases/genética , Terpenos/química , Terpenos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Biocatálise
5.
Cell Death Dis ; 15(6): 391, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38830870

RESUMO

Tissue injury causes activation of mesenchymal lineage cells into wound-repairing myofibroblasts (MFs), whose uncontrolled activity ultimately leads to fibrosis. Although this process is triggered by deep metabolic and transcriptional reprogramming, functional links between these two key events are not yet understood. Here, we report that the metabolic sensor post-translational modification O-linked ß-D-N-acetylglucosaminylation (O-GlcNAcylation) is increased and required for myofibroblastic activation. Inhibition of protein O-GlcNAcylation impairs archetypal myofibloblast cellular activities including extracellular matrix gene expression and collagen secretion/deposition as defined in vitro and using ex vivo and in vivo murine liver injury models. Mechanistically, a multi-omics approach combining proteomic, epigenomic, and transcriptomic data mining revealed that O-GlcNAcylation controls the MF transcriptional program by targeting the transcription factors Basonuclin 2 (BNC2) and TEA domain transcription factor 4 (TEAD4) together with the Yes-associated protein 1 (YAP1) co-activator. Indeed, inhibition of protein O-GlcNAcylation impedes their stability leading to decreased functionality of the BNC2/TEAD4/YAP1 complex towards promoting activation of the MF transcriptional regulatory landscape. We found that this involves O-GlcNAcylation of BNC2 at Thr455 and Ser490 and of TEAD4 at Ser69 and Ser99. Altogether, this study unravels protein O-GlcNAcylation as a key determinant of myofibroblastic activation and identifies its inhibition as an avenue to intervene with fibrogenic processes.


Assuntos
Miofibroblastos , Transdução de Sinais , Miofibroblastos/metabolismo , Animais , Camundongos , Humanos , Fibrose/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP/metabolismo , Camundongos Endogâmicos C57BL , Fatores de Transcrição de Domínio TEA/metabolismo , Masculino , Processamento de Proteína Pós-Traducional , Acetilglucosamina/metabolismo , Transcrição Gênica , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética
6.
Int J Mol Sci ; 25(11)2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38892474

RESUMO

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 Celular
7.
PLoS Pathog ; 20(6): e1011979, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38900808

RESUMO

The cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and vital functions in the lytic cycle of this obligate intracellular parasite. Additionally, the cyst wall of bradyzoites, that shields the persistent form responsible for chronic infection from the immune system, is heavily glycosylated. Formation of glycoconjugates relies on activated sugar nucleotides, such as uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). The glucosamine-phosphate-N-acetyltransferase (GNA1) generates N-acetylglucosamine-6-phosphate critical to produce UDP-GlcNAc. Here, we demonstrate that downregulation of T. gondii GNA1 results in a severe reduction of UDP-GlcNAc and a concomitant drop in glycosylphosphatidylinositols (GPIs), leading to impairment of the parasite's ability to invade and replicate in the host cell. Surprisingly, attempts to rescue this defect through exogenous GlcNAc supplementation fail to completely restore these vital functions. In depth metabolomic analyses elucidate diverse causes underlying the failed rescue: utilization of GlcNAc is inefficient under glucose-replete conditions and fails to restore UDP-GlcNAc levels in GNA1-depleted parasites. In contrast, GlcNAc-supplementation under glucose-deplete conditions fully restores UDP-GlcNAc levels but fails to rescue the defects associated with GNA1 depletion. Our results underscore the importance of glucosamine-6-phosphate acetylation in governing T. gondii replication and invasion and highlight the potential of the evolutionary divergent GNA1 in Apicomplexa as a target for the development of much-needed new therapeutic strategies.


Assuntos
Acetilglucosamina , Glucose-6-Fosfato , Toxoplasma , Toxoplasma/metabolismo , Glucose-6-Fosfato/metabolismo , Glucose-6-Fosfato/análogos & derivados , Acetilglucosamina/metabolismo , Acetilação , Animais , Glucosamina 6-Fosfato N-Acetiltransferase/metabolismo , Humanos , Glucosamina/metabolismo , Glucosamina/análogos & derivados , Camundongos , Toxoplasmose/metabolismo , Toxoplasmose/parasitologia , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética
8.
Nat Commun ; 15(1): 3825, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38714645

RESUMO

c-di-AMP is an essential and widespread nucleotide second messenger in bacterial signaling. For most c-di-AMP synthesizing organisms, c-di-AMP homeostasis and the molecular mechanisms pertaining to its signal transduction are of great concern. Here we show that c-di-AMP binds the N-acetylglucosamine (GlcNAc)-sensing regulator DasR, indicating a direct link between c-di-AMP and GlcNAc signaling. Beyond its foundational role in cell-surface structure, GlcNAc is attractive as a major nutrient and messenger molecule regulating multiple cellular processes from bacteria to humans. We show that increased c-di-AMP levels allosterically activate DasR as a master repressor of GlcNAc utilization, causing the shutdown of the DasR-mediated GlcNAc signaling cascade and leading to a consistent enhancement in the developmental transition and antibiotic production in Saccharopolyspora erythraea. The expression of disA, encoding diadenylate cyclase, is directly repressed by the regulator DasR in response to GlcNAc signaling, thus forming a self-sustaining transcriptional feedback loop for c-di-AMP synthesis. These findings shed light on the allosteric regulation by c-di-AMP, which appears to play a prominent role in global signal integration and c-di-AMP homeostasis in bacteria and is likely widespread in streptomycetes that produce c-di-AMP.


Assuntos
Acetilglucosamina , Proteínas de Bactérias , Fosfatos de Dinucleosídeos , Regulação Bacteriana da Expressão Gênica , Saccharopolyspora , Transdução de Sinais , Acetilglucosamina/metabolismo , Regulação Alostérica , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Fosfatos de Dinucleosídeos/metabolismo , Saccharopolyspora/metabolismo , Saccharopolyspora/genética
9.
Cell Death Dis ; 15(5): 321, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38719812

RESUMO

RAD18, an important ubiquitin E3 ligase, plays a dual role in translesion DNA synthesis (TLS) and homologous recombination (HR) repair. However, whether and how the regulatory mechanism of O-linked N-acetylglucosamine (O-GlcNAc) modification governing RAD18 and its function during these processes remains unknown. Here, we report that human RAD18, can undergo O-GlcNAcylation at Ser130/Ser164/Thr468, which is important for optimal RAD18 accumulation at DNA damage sites. Mechanistically, abrogation of RAD18 O-GlcNAcylation limits CDC7-dependent RAD18 Ser434 phosphorylation, which in turn significantly reduces damage-induced PCNA monoubiquitination, impairs Polη focus formation and enhances UV sensitivity. Moreover, the ubiquitin and RAD51C binding ability of RAD18 at DNA double-strand breaks (DSBs) is O-GlcNAcylation-dependent. O-GlcNAcylated RAD18 promotes the binding of RAD51 to damaged DNA during HR and decreases CPT hypersensitivity. Our findings demonstrate a novel role of RAD18 O-GlcNAcylation in TLS and HR regulation, establishing a new rationale to improve chemotherapeutic treatment.


Assuntos
Acetilglucosamina , Proteínas de Ligação a DNA , Antígeno Nuclear de Célula em Proliferação , Rad51 Recombinase , Reparo de DNA por Recombinação , Ubiquitina-Proteína Ligases , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ubiquitina-Proteína Ligases/metabolismo , Acetilglucosamina/metabolismo , Rad51 Recombinase/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fosforilação , Replicação do DNA , Ubiquitinação , Quebras de DNA de Cadeia Dupla , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Dano ao DNA , DNA/metabolismo , Células HEK293 , Raios Ultravioleta , Ligação Proteica , Glicosilação , Síntese de DNA Translesão
10.
Sci Rep ; 14(1): 10669, 2024 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-38724577

RESUMO

Anaesthetics are used daily in human and veterinary medicine as well as in scientific research. Anaesthetics have an impact on cell homeostasis especially through modulation of protein post-translational modifications. O-GlcNAcylation, a ubiquitous post-translational modification, plays a role in many biological processes. The aims of this study were to evaluate whether (1) anaesthesia influences O-GlcNAcylation and (2) its stimulation affects physiological parameters. Male Wistar rats (n = 38) were anaesthetized with ketamine-xylazine or isoflurane. They randomly received either an intravenous injection of Ringer's lactate or NButGT (10mg/kg) in order to increase O-GlcNAcylation levels. One hour after induction of anaesthesia, haemodynamic parameters and plasmatic markers were evaluated. Heart, brain and lungs were harvested and O-GlcNAcylation levels and O-GlcNAc-related enzymes were evaluated by western blot. Cardiac and pulmonary O-GlcNAcylation levels and cardiac, cerebral and pulmonary O-GlcNAc associated enzyme expression were not impacted with anaesthesia. Compared with ketamine-xylazine, isoflurane had a lower impact on blood pressure, heart rate and glycaemia. Pharmacological stimulation of O-GlcNAcylation by NButGT did not affect the physiological parameters. This study offers unprecedented insights into the regulation of O-GlcNAcylation and O-GlcNAc related enzymes during anaesthesia. Pharmacological stimulation of O-GlcNAcylation over a 1-h period did not disrupt the physiological balance in healthy anaesthetized rats.


Assuntos
Isoflurano , Ketamina , Ratos Wistar , Xilazina , Animais , Masculino , Ratos , Isoflurano/farmacologia , Ketamina/farmacologia , Xilazina/farmacologia , Anestesia , Acetilglucosamina/metabolismo , Processamento de Proteína Pós-Traducional , Encéfalo/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Pulmão/metabolismo , Anestésicos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Hemodinâmica
11.
Biochemistry ; 63(12): 1513-1533, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38788673

RESUMO

Glycogen synthase kinase 3 (GSK3) plays a pivotal role in signaling pathways involved in insulin metabolism and the pathogenesis of neurodegenerative disorders. In particular, the GSK3ß isoform is implicated in Alzheimer's disease (AD) as one of the key kinases involved in the hyperphosphorylation of tau protein, one of the neuropathological hallmarks of AD. As a constitutively active serine/threonine kinase, GSK3 is inactivated by Akt/PKB-mediated phosphorylation of Ser9 in the N-terminal disordered domain, and for most of its substrates, requires priming (prephosphorylation) by another kinase that targets the substrate to a phosphate-specific pocket near the active site. GSK3 has also been shown to be post-translationally modified by O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation), with still unknown functions. Here, we have found that binding of Akt inhibits GSK3ß kinase activity on both primed and unprimed tau substrates. Akt-mediated Ser9 phosphorylation restores the GSK3ß kinase activity only on primed tau, thereby selectively inactivating GSK3ß toward unprimed tau protein. Additionally, we have shown that GSK3ß is highly O-GlcNAcylated at multiple sites within the kinase domain and the disordered N- and C-terminal domains, including Ser9. In contrast to Akt-mediated regulation, neither the O-GlcNAc transferase nor O-GlcNAcylation significantly alters GSK3ß kinase activity, but high O-GlcNAc levels reduce Ser9 phosphorylation by Akt. Reciprocally, Akt phosphorylation downregulates the overall O-GlcNAcylation of GSK3ß, indicating a crosstalk between both post-translational modifications. Our results indicate that specific O-GlcNAc profiles may be involved in the phosphorylation-dependent Akt-mediated regulation of GSK3ß kinase activity.


Assuntos
Glicogênio Sintase Quinase 3 beta , Proteínas Proto-Oncogênicas c-akt , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/química , Fosforilação , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Processamento de Proteína Pós-Traducional , Quinase 3 da Glicogênio Sintase/metabolismo , Acetilglucosamina/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/química , Glicosilação , Animais
12.
J Biol Chem ; 300(6): 107349, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38718861

RESUMO

The dynamic and reversible modification of nuclear and cytoplasmic proteins by O-GlcNAcylation significantly impacts the function and dysfunction of the immune system. O-GlcNAcylation plays crucial roles under both physiological and pathological conditions in the biochemical regulation of all immune cell functions. Three and a half decades of knowledge acquired in this field is merely sufficient to perceive that what we know is just the prelude. This review attempts to mark out the known regulatory roles of O-GlcNAcylation in key signal transduction pathways and specific protein functions in the immune system and adumbrate ensuing questions toward the unknown functions.


Assuntos
Acetilglucosamina , Transdução de Sinais , Humanos , Animais , Acetilglucosamina/metabolismo , Sistema Imunitário/metabolismo , Processamento de Proteína Pós-Traducional , Glicosilação
13.
J Sex Med ; 21(7): 627-634, 2024 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-38721680

RESUMO

BACKGROUND: Intravesical instillation of hyaluronic acid (HA) has been associated with reduced sexual dysfunction in participants with recurrent urinary tract infections (rUTIs), but the efficacy of an oral treatment has never been investigated. AIM: To investigate the efficacy of an oral preparation of HA, chondroitin sulfate, N-acetylglucosamine, and vitamin C in improving sexual and urinary symptoms in a cohort of reproductive-age participants with rUTI. METHODS: In a monocentric randomized crossover pilot trial, participants with rUTI who were referred to our institute between March 2022 and April 2023 were randomized 1:1 in 2 groups: intervention vs control. All participants had an oral preparation of cranberry, D-mannose, propolis extract, turmeric, and Boswellia twice a day for 3 months. The intervention group also included an oral preparation of HA, chondroitin sulfate, N-acetylglucosamine, and vitamin C once a day for 3 months. Crossover of treatment occurred at 3 months for an additional 3 months. At baseline and 3 and 6 months, participants were evaluated clinically and with the International Prostate Symptom Score (IPSS) and Female Sexual Function Index (FSFI). Descriptive statistics and logistic regression models tested the impact of the intervention on urinary and sexual symptoms at each follow-up assessment. OUTCOMES: Improvement in sexual and urinary symptoms as measured by the FSFI and IPSS. RESULTS: Overall, 27 (54%) participants had an FSFI score <26.5 at enrollment. At 3 months, FSFI scores were higher in the intervention group vs control (P < .001), but IPSS scores were lower (P = .03). After crossover of treatment, FSFI and IPSS scores remained stable in the intervention group. However, after crossover, the control group showed a significant improvement in IPSS and FSFI scores (all P < .01) vs the 3-month assessment. At last follow-up, urinary and sexual symptoms were comparable between groups. In logistic regression analyses, the intervention group was associated with early improvement in sexual symptoms (odds ratio, 3.9; P = .04) and urinary symptoms (odds ratio, 5.1; P = .01) after accounting for clinical confounders. CLINICAL IMPLICATIONS: Combination treatment with HA, chondroitin sulfate, N-acetylglucosamine, and vitamin C is effective if started immediately or even after a few months from symptoms in participants with rUTI. STRENGTHS AND LIMITATIONS: The main limitation is the lack of long-term follow-up. CONCLUSION: The oral formulation of HA, chondroitin sulfate, N-acetylglucosamine, and vitamin C could be an effective therapy against urinary and sexual distress in participants with rUTI (NCT06268483; ClinicalTrials.gov).


Assuntos
Acetilglucosamina , Ácido Ascórbico , Sulfatos de Condroitina , Estudos Cross-Over , Ácido Hialurônico , Infecções Urinárias , Humanos , Ácido Ascórbico/administração & dosagem , Ácido Ascórbico/uso terapêutico , Sulfatos de Condroitina/administração & dosagem , Sulfatos de Condroitina/uso terapêutico , Ácido Hialurônico/administração & dosagem , Ácido Hialurônico/uso terapêutico , Feminino , Masculino , Adulto , Infecções Urinárias/tratamento farmacológico , Acetilglucosamina/administração & dosagem , Acetilglucosamina/uso terapêutico , Administração Oral , Projetos Piloto , Disfunções Sexuais Fisiológicas/tratamento farmacológico , Pessoa de Meia-Idade , Recidiva , Própole/administração & dosagem , Própole/uso terapêutico , Manose/administração & dosagem , Manose/uso terapêutico
14.
Carbohydr Res ; 541: 109146, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38788561

RESUMO

A series of the first conjugates of N-acetyl-d-glucosamine with α-aminophosphonates was synthesized using the Kabachnik-Fields reaction, the Pudovik reaction, a copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC) and evaluated for the in vitro cytotoxicity against human cancer cell lines M - HeLa, HuTu-80, A549, PANC-1, MCF-7, T98G and normal lung fibroblast cells WI-38. The tested conjugates, with exception of compound 21b, considered as a lead compound, were either inactive against the used cancer cells or showed moderate cytotoxicity in the range of IC50 values 33-80 µM. The lead compound 21b, being non cytotoxic against normal human cells WI-38 (IC50 = 90 µM), demonstrated good activity (IC50 = 17 µM) against breast adenocarcinoma cells (MCF-7) which to be 1.5 times higher than the activity of the used reference anticancer drug tamoxifen (IC50 = 25.0 µM). A flexible receptor molecular docking simulation showed that the cytotoxicity of the synthesized conjugates of N-acetyl-d-glucosamine with α-aminophosphonates against breast adenocarcinoma MCF-7 cell line is due to their ability to inhibit EGFR kinase domain. In addition, it was found that conjugates 22a and 22b demonstrated antioxidant activity that was not typical for α-aminophosphonates.


Assuntos
Acetilglucosamina , Antineoplásicos , Antioxidantes , Simulação de Acoplamento Molecular , Organofosfonatos , Humanos , Organofosfonatos/química , Organofosfonatos/farmacologia , Organofosfonatos/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Acetilglucosamina/química , Acetilglucosamina/farmacologia , Antioxidantes/farmacologia , Antioxidantes/síntese química , Antioxidantes/química , Ensaios de Seleção de Medicamentos Antitumorais , Relação Estrutura-Atividade , Linhagem Celular Tumoral , Estrutura Molecular , Proliferação de Células/efeitos dos fármacos
15.
Curr Protoc ; 4(5): e1052, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38752278

RESUMO

Cells continuously remodel their intracellular proteins with the monosaccharide O-linked N-acetylglucosamine (O-GlcNAc) to regulate metabolism, signaling, and stress. This protocol describes the use of GlycoID tools to capture O-GlcNAc dynamics in live cells. GlycoID constructs contain an O-GlcNAc binding domain linked to a proximity labeling domain and a subcellular localization sequence. When expressed in mammalian cells, GlycoID tracks changes in O-GlcNAc-modified proteins and their interactomes in response to chemical induction with biotin over time. Pairing the subcellular localization of GlycoID with the chemical induction of activity enables spatiotemporal studies of O-GlcNAc biology during cellular events such as insulin signaling. However, optimizing intracellular labeling experiments requires attention to several variables. Here, we describe two protocols to adapt GlycoID methods to a cell line and biological process of interest. Next, we describe how to conduct a semiquantitative proteomic analysis of O-GlcNAcylated proteins and their interactomes using insulin versus glucagon signaling as a sample application. This articles aims to establish baseline GlycoID protocols for new users and set the stage for widespread use over diverse cellular applications for the functional study of O-GlcNAc glycobiology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Expression of targeted GlycoID constructs to verify subcellular location and labeling activity in mammalian cells Basic Protocol 2: GlycoID labeling in live HeLa cells for O-GlcNAc proteomic comparisons.


Assuntos
Acetilglucosamina , Humanos , Acetilglucosamina/metabolismo , Proteômica/métodos , Insulina/metabolismo , Animais , Coloração e Rotulagem/métodos , Transdução de Sinais , Proteínas/metabolismo , Células HeLa
16.
Sci Rep ; 14(1): 11079, 2024 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-38745047

RESUMO

N-acetyl glucosamine (NAG) is a natural amino sugar found in various human tissues with previously described anti-inflammatory effects. Various chemical modifications of NAG have been made to promote its biomedical applications. In this study, we synthesized two bi-deoxygenated NAG, BNAG1 and BNAG2 and investigated their anti-inflammatory properties, using an in vivo and in vitro inflammation mouse model induced by lipopolysaccharide (LPS). Among the parent molecule NAG, BNAG1 and BNAG2, BNAG1 showed the highest inhibition against serum levels of IL-6 and TNF α and the leukocyte migration to lungs and peritoneal cavity in LPS challenged mice, as well as IL-6 and TNF α production in LPS-stimulated primary peritoneal macrophages. BNAG2 displayed an anti-inflammatory effect which was comparable to NAG. These findings implied potential application of these novel NAG derivatives, especially BNAG1, in treatment of certain inflammation-related diseases.


Assuntos
Acetilglucosamina , Anti-Inflamatórios , Lipopolissacarídeos , Macrófagos Peritoneais , Fator de Necrose Tumoral alfa , Animais , Acetilglucosamina/farmacologia , Camundongos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/síntese química , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Interleucina-6/sangue , Inflamação/tratamento farmacológico , Masculino , Modelos Animais de Doenças
17.
Biochemistry ; 63(10): 1270-1277, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38770609

RESUMO

Cyanovirin-N (CV-N) binds high-mannose oligosaccharides on enveloped viruses with two carbohydrate-binding sites, one bearing high affinity and one low affinity to Manα(1-2)Man moieties. A tandem repeat of two CV-N molecules (CVN2) was tested for antiviral activity against human immunodeficiency virus type I (HIV-1) by using a domain-swapped dimer. CV-N was shown to bind N-acetylmannosamine (ManNAc) and N-acetyl-d-glucosamine (GlcNAc) when the carbohydrate-binding sites in CV-N were free to interact with these monosaccharides independently. CVN2 recognized ManNAc at a Kd of 1.4 µM and bound this sugar in solution, regardless of the lectin making amino acid side chain contacts on the targeted viral glycoproteins. An interdomain cross-contacting residue Glu41, which has been shown to be hydrogen bonding with dimannose, was substituted in the monomeric CV-N. The amide derivative of glucose, GlcNAc, achieved similar high affinity to the new variant CVN-E41T as high-mannose N-glycans, but binding to CVN2 in the nanomolar range with four binding sites involved or binding to the monomeric CVN-E41A. A stable dimer was engineered and expressed from the alanine-to-threonine-substituted monomer to confirm binding to GlcNAc. In summary, low-affinity binding was achieved by CVN2 to dimannosylated peptide or GlcNAc with two carbohydrate-binding sites of differing affinities, mimicking biological interactions with the respective N-linked glycans of interest and cross-linking of carbohydrates on human T cells for lymphocyte activation.


Assuntos
Acetilglucosamina , Proteínas de Bactérias , Proteínas de Transporte , Acetilglucosamina/metabolismo , Acetilglucosamina/química , Sítios de Ligação , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/química , Humanos , HIV-1/metabolismo , Ligação Proteica , Hexosaminas/metabolismo , Hexosaminas/química , Modelos Moleculares , Multimerização Proteica
18.
Cell Commun Signal ; 22(1): 279, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38773637

RESUMO

O-linked N-acetylglucosamine (O-GlcNAc) protein modification (O-GlcNAcylation) is a critical post-translational modification (PTM) of cytoplasmic and nuclear proteins. O-GlcNAcylation levels are regulated by the activity of two enzymes, O-GlcNAc transferase (OGT) and O­GlcNAcase (OGA). While OGT attaches O-GlcNAc to proteins, OGA removes O-GlcNAc from proteins. Since its discovery, researchers have demonstrated O-GlcNAcylation on thousands of proteins implicated in numerous different biological processes. Moreover, dysregulation of O-GlcNAcylation has been associated with several pathologies, including cancers, ischemia-reperfusion injury, and neurodegenerative diseases. In this review, we focus on progress in our understanding of the role of O-GlcNAcylation in bone pathophysiology, and we discuss the potential molecular mechanisms of O-GlcNAcylation modulation of bone-related diseases. In addition, we explore significant advances in the identification of O-GlcNAcylation-related regulators as potential therapeutic targets, providing novel therapeutic strategies for the treatment of bone-related disorders.


Assuntos
Acetilglucosamina , N-Acetilglucosaminiltransferases , Humanos , Animais , N-Acetilglucosaminiltransferases/metabolismo , Acetilglucosamina/metabolismo , Osso e Ossos/metabolismo , Processamento de Proteína Pós-Traducional , Doenças Ósseas/metabolismo
19.
Bioorg Chem ; 147: 107395, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38705105

RESUMO

Fluorination of carbohydrate ligands of lectins is a useful approach to examine their binding profile, improve their metabolic stability and lipophilicity, and convert them into 19F NMR-active probes. However, monofluorination of monovalent carbohydrate ligands often leads to a decreased or completely lost affinity. By chemical glycosylation, we synthesized the full series of methyl ß-glycosides of N,N'-diacetylchitobiose (GlcNAcß(1-4)GlcNAcß1-OMe) and LacdiNAc (GalNAcß(1-4)GlcNAcß1-OMe) systematically monofluorinated at all hydroxyl positions. A competitive enzyme-linked lectin assay revealed that the fluorination at the 6'-position of chitobioside resulted in an unprecedented increase in affinity to wheat germ agglutinin (WGA) by one order of magnitude. For the first time, we have characterized the binding profile of a previously underexplored WGA ligand LacdiNAc. Surprisingly, 4'-fluoro-LacdiNAc bound WGA even stronger than unmodified LacdiNAc. These observations were interpreted using molecular dynamic calculations along with STD and transferred NOESY NMR techniques, which gave evidence for the strengthening of CH/π interactions after deoxyfluorination of the side chain of the non-reducing GlcNAc. These results highlight the potential of fluorinated glycomimetics as high-affinity ligands of lectins and 19F NMR-active probes.


Assuntos
Dissacarídeos , Aglutininas do Germe de Trigo , Dissacarídeos/química , Dissacarídeos/síntese química , Aglutininas do Germe de Trigo/química , Aglutininas do Germe de Trigo/metabolismo , Halogenação , Estrutura Molecular , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Lactose/análogos & derivados
20.
J Appl Microbiol ; 135(5)2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38724455

RESUMO

AIMS: We aimed to investigate the function of an unidentified gene annotated as a PIG-L domain deacetylase (cspld) in Chitiniphilus shinanonensis SAY3. cspld was identified using transposon mutagenesis, followed by negatively selecting a mutant incapable of growing on chitin, a polysaccharide consisting of N-acetyl-d-glucosamine (GlcNAc). We focused on the physiological role of CsPLD protein in chitin utilization. METHODS AND RESULTS: Recombinant CsPLD expressed in Escherichia coli exhibited GlcNAc-6-phosphate deacetylase (GPD) activity, which is involved in the metabolism of amino sugars. However, SAY3 possesses two genes (csnagA1 and csnagA2) in its genome that code for proteins whose primary sequences are homologous to those of typical GPDs. Recombinant CsNagA1 and CsNagA2 also exhibited GPD activity with 23 and 1.6% of catalytic efficiency (kcat/Km), respectively, compared to CsPLD. The gene-disrupted mutant, Δcspld was unable to grow on chitin or GlcNAc, whereas the three mutants, ΔcsnagA1, ΔcsnagA2, and ΔcsnagA1ΔcsnagA2 grew similarly to SAY3. The determination of GPD activity in the crude extracts of each mutant revealed that CsPLD is a major enzyme that accounts for almost all cellular activities. CONCLUSIONS: Deacetylation of GlcNAc-6P catalyzed by CsPLD (but not by typical GPDs) is essential for the assimilation of chitin and its constituent monosaccharide, GlcNAc, as a carbon and energy source in C. shinanonensis.


Assuntos
Quitina , Quitina/metabolismo , Amidoidrolases/metabolismo , Amidoidrolases/genética , Acetilglucosamina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Gammaproteobacteria/genética , Gammaproteobacteria/enzimologia , Gammaproteobacteria/metabolismo
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