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 AcetiltransferasesRESUMO
Intellectual Disability (ID) is the major cause of handicap, affecting nearly 3% of the general population, and is highly genetically heterogenous with more than a thousand genes involved. Exome sequencing performed in two independent families identified the same missense variant, p.(Gly611Ser), in the NDST1 (N-deacetylase/N-sulfotransferase member 1) gene. This variant had been previously found in ID patients of two other families but has never been functionally characterized. The NDST1 gene encodes a bifunctional enzyme that catalyzes both N-deacetylation and N-sulfation of N-acetyl-glucosamine residues during heparan sulfate (HS) biosynthesis. This step is essential because it influences the downstream enzymatic modifications and thereby determines the overall structure and sulfation degree of the HS polysaccharide chain. To discriminate between a rare polymorphism and a pathogenic variant, we compared the enzymatic properties of wild-type and mutant NDST1 proteins. We found that the p.(Gly611Ser) variant results in a complete loss of N-sulfotransferase activity while the N-deacetylase activity is retained. NDST1 shows the highest and the most homogeneous expression in the human cerebral structures compared to the other members of the NDST gene family. These results indicate that a loss of NDST1 N-sulfation activity is associated with impaired cognitive functions.
Assuntos
Deficiência Intelectual , Humanos , Deficiência Intelectual/genética , Acetilglucosamina , Cognição , Padrões de Herança , Proteínas Mutantes , Sulfotransferases/genéticaRESUMO
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/genéticaRESUMO
Amyloid-forming proteins such α-synuclein and tau, which are implicated in Alzheimer's and Parkinson's disease, can form different fibril structures or strains with distinct toxic properties, seeding activities and pathology. Understanding the determinants contributing to the formation of different amyloid features could open new avenues for developing disease-specific diagnostics and therapies. Here we report that O-GlcNAc modification of α-synuclein monomers results in the formation of amyloid fibril with distinct core structure, as revealed by cryogenic electron microscopy, and diminished seeding activity in seeding-based neuronal and rodent models of Parkinson's disease. Although the mechanisms underpinning the seeding neutralization activity of the O-GlcNAc-modified fibrils remain unclear, our in vitro mechanistic studies indicate that heat shock proteins interactions with O-GlcNAc fibril inhibit their seeding activity, suggesting that the O-GlcNAc modification may alter the interactome of the α-synuclein fibrils in ways that lead to reduce seeding activity in vivo. Our results show that posttranslational modifications, such as O-GlcNAc modification, of α-synuclein are key determinants of α-synuclein amyloid strains and pathogenicity.
Assuntos
Amiloide , alfa-Sinucleína , alfa-Sinucleína/metabolismo , alfa-Sinucleína/química , Amiloide/metabolismo , Humanos , Animais , Camundongos , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Acetilglucosamina/metabolismo , Acetilglucosamina/química , Processamento de Proteína Pós-Traducional , Microscopia Crioeletrônica , Neurônios/metabolismo , Neurônios/patologiaRESUMO
Using an Ig H chain conferring specificity for N-acetyl-d-glucosamine (GlcNAc), we developed transgenic (VHHGAC39 TG) mice to study the role of self-antigens in GlcNAc-reactive B-1 B cell development. In VHHGAC39 TG mice, GlcNAc-reactive B-1 B cell development during ontogeny and in adult bone marrow was normal. However, adult TG mice exhibited a block at transitional-2 immature B cell stages, resulting in impaired allelic exclusion and accumulation of a B cell subset coexpressing endogenous Ig gene rearrangements. Similarly, VHHGAC39 B cell fitness was impeded compared with non-self-reactive VHJ558 B TG cells in competitive mixed bone marrow chimeras. Nonetheless, adult VHHGAC39 mice immunized with Streptococcus pyogenes produce anti-GlcNAc Abs. Peritoneal cavity B cells transferred from VHHGAC39 TG mice into RAG-/- mice also exhibited robust expansion and anti-GlcNAc Ab production. However, chronic treatment of young VHHGAC39 mice with GlcNAc-specific mAbs leads to lower GlcNAc-binding B cell frequencies while increasing the proportion of GlcNAc-binding B1-a cells, suggesting that Ag masking or clearance of GlcNAc Ags impedes maturation of newly formed GlcNAc-reactive B cells. Finally, BCR H chain editing promotes expression of endogenous nontransgenic BCR alleles, allowing potentially self-reactive TG B cells to escape anergy or deletion at the transitional stage of precursor B cell development. Collectively, these observations indicate that GlcNAc-reactive B cell development is sensitive to the access of autologous Ags.
Assuntos
Acetilglucosamina , Camundongos Transgênicos , Animais , Camundongos , Acetilglucosamina/imunologia , Diferenciação Celular/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Imunidade Inata/imunologia , Subpopulações de Linfócitos B/imunologia , Camundongos Endogâmicos C57BL , Autoantígenos/imunologia , Streptococcus pyogenes/imunologia , Linfócitos B/imunologiaRESUMO
O-GlcNAcylation is a critical post-translational modification of proteins observed in both plants and animals and plays a key role in growth and development. While considerable knowledge exists about over 3000 substrates in animals, our understanding of this modification in plants remains limited. Unlike animals, plants possess two putative homologs: SECRET AGENT (SEC) and SPINDLY, with SPINDLY also exhibiting O-fucosylation activity. To investigate the role of SEC as a major O-GlcNAc transferase in plants, we utilized lectin-weak affinity chromatography enrichment and stable isotope labeling in Arabidopsis labeling, quantifying at both MS1 and MS2 levels. Our findings reveal a significant reduction in O-GlcNAc levels in the sec mutant, indicating the critical role of SEC in mediating O-GlcNAcylation. Through a comprehensive approach, combining higher-energy collision dissociation and electron-transfer high-energy collision dissociation fragmentation with substantial fractionations, we expanded our GlcNAc profiling, identifying 436 O-GlcNAc targets, including 227 new targets. The targets span diverse cellular processes, suggesting broad regulatory functions of O-GlcNAcylation. The expanded targets also enabled exploration of crosstalk between O-GlcNAcylation and O-fucosylation. We also examined electron-transfer high-energy collision dissociation fragmentation for site assignment. This report advances our understanding of O-GlcNAcylation in plants, facilitating further research in this field.
Assuntos
Proteínas de Arabidopsis , N-Acetilglucosaminiltransferases , Acetilglucosamina/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Glicosilação , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional/genéticaRESUMO
Cells continuously fine-tune signaling pathway proteins to match nutrient and stress levels in their local environment by modifying intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc) sugars, an essential process for cell survival and growth. The small size of these monosaccharide modifications poses a challenge for functional determination, but the chemistry and biology communities have together created a collection of precision tools to study these dynamic sugars. This review presents the major themes by which O-GlcNAc influences signaling pathway proteins, including G-protein coupled receptors, growth factor signaling, mitogen-activated protein kinase (MAPK) pathways, lipid sensing, and cytokine signaling pathways. Along the way, we describe in detail key chemical biology tools that have been developed and applied to determine specific O-GlcNAc roles in these pathways. These tools include metabolic labeling, O-GlcNAc-enhancing RNA aptamers, fluorescent biosensors, proximity labeling tools, nanobody targeting tools, O-GlcNAc cycling inhibitors, light-activated systems, chemoenzymatic labeling, and nutrient reporter assays. An emergent feature of this signaling pathway meta-analysis is the intricate interplay between O-GlcNAc modifications across different signaling systems, underscoring the importance of O-GlcNAc in regulating cellular processes. We highlight the significance of O-GlcNAc in signaling and the role of chemical and biochemical tools in unraveling distinct glycobiological regulatory mechanisms. Collectively, our field has determined effective strategies to probe O-GlcNAc roles in biology. At the same time, this survey of what we do not yet know presents a clear roadmap for the field to use these powerful chemical tools to explore cross-pathway O-GlcNAc interactions in signaling and other major biological pathways.
Assuntos
Acetilglucosamina , Técnicas de Química Analítica , Transdução de Sinais , Acetilglucosamina/análise , Acetilglucosamina/metabolismo , Técnicas de Química Analítica/métodos , Receptores Acoplados a Proteínas G/metabolismo , Bioquímica/métodos , Biotecnologia/métodosRESUMO
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 , GlicosilaçãoRESUMO
Eukaryotic RNA polymerase II (RNAPII) is responsible for the transcription of the protein-coding genes in the cell. Enormous progress has been made in discovering the protein activities that are required for transcription to occur, but the effects of post-translational modifications (PTMs) on RNAPII transcriptional regulation are much less understood. Most of our understanding relates to the cyclin-dependent kinases (CDKs), which appear to act relatively early in transcription. However, it is becoming apparent that other PTMs play a crucial role in the transcriptional cycle, and it is doubtful that any sort of complete understanding of this regulation is attainable without understanding the spectra of PTMs that occur on the transcriptional machinery. Among these is O-GlcNAcylation. Recent experiments have shown that the O-GlcNAc PTM likely has a prominent role in transcription. This review will cover the role of the O-GlcNAcylation in RNAPII transcription during initiation, pausing, and elongation, which will hopefully be of interest to both O-GlcNAc and RNAPII transcription researchers.
Assuntos
Regulação da Expressão Gênica , RNA Polimerase II , Transcrição Gênica , Acetilglucosamina/genética , Acetilglucosamina/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , RNA Polimerase II/genética , RNA Polimerase II/metabolismoRESUMO
O-linked ß-N-acetylglucosamine (O-GlcNAcylation) is a dynamic post-translational modification that regulates thousands of proteins and almost all cellular processes. Aberrant O-GlcNAcylation has been associated with numerous diseases, including cancer, neurodegenerative diseases, cardiovascular diseases, and type 2 diabetes. O-GlcNAcylation is highly nutrient-sensitive since it is dependent on UDP-GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP). We previously observed daily rhythmicity of protein O-GlcNAcylation in a Drosophila model that is sensitive to the timing of food consumption. We showed that the circadian clock is pivotal in regulating daily O-GlcNAcylation rhythms given its control of the feeding-fasting cycle and hence nutrient availability. Interestingly, we reported that the circadian clock also modulates daily O-GlcNAcylation rhythm by regulating molecular mechanisms beyond the regulation of food consumption time. A large body of work now indicates that O-GlcNAcylation is likely a generalized cellular status effector as it responds to various cellular signals and conditions, such as ER stress, apoptosis, and infection. In this review, we summarize the metabolic regulation of protein O-GlcNAcylation through nutrient availability, HBP enzymes, and O-GlcNAc processing enzymes. We discuss the emerging roles of circadian clocks in regulating daily O-GlcNAcylation rhythm. Finally, we provide an overview of other cellular signals or conditions that impact O-GlcNAcylation. Many of these cellular pathways are themselves regulated by the clock and/or metabolism. Our review highlights the importance of maintaining optimal O-GlcNAc rhythm by restricting eating activity to the active period under physiological conditions and provides insights into potential therapeutic targets of O-GlcNAc homeostasis under pathological conditions.
Assuntos
Relógios Circadianos , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Animais , Acetilglucosamina/metabolismo , Relógios Circadianos/fisiologia , Açúcares de Uridina Difosfato/metabolismo , HumanosRESUMO
The modification of nuclear, cytoplasmic, and mitochondrial proteins by O-linked ß-N-actylglucosamine (O-GlcNAc) is an essential posttranslational modification that is common in metozoans. O-GlcNAc is cycled on and off proteins in response to environmental and physiological stimuli impacting protein function, which, in turn, tunes pathways that include transcription, translation, proteostasis, signal transduction, and metabolism. One class of stimulus that induces rapid and dynamic changes to O-GlcNAc is cellular injury, resulting from environmental stress (for instance, heat shock), hypoxia/reoxygenation injury, ischemia reperfusion injury (heart attack, stroke, trauma hemorrhage), and sepsis. Acute elevation of O-GlcNAc before or after injury reduces apoptosis and necrosis, suggesting that injury-induced changes in O-GlcNAcylation regulate cell fate decisions. However, prolonged elevation or reduction in O-GlcNAc leads to a maladaptive response and is associated with pathologies such as hypertrophy and heart failure. In this review, we discuss the impact of O-GlcNAc in both acute and prolonged models of injury with a focus on the heart and biological mechanisms that underpin cell survival.
Assuntos
Cardiopatias , Humanos , Glicosilação , Animais , Cardiopatias/metabolismo , Cardiopatias/patologia , Citoproteção , Processamento de Proteína Pós-Traducional , Acetilglucosamina/metabolismoRESUMO
Histone 2A monoubiquitination (uH2A) underscores a key epigenetic regulation of gene expression. In this report, we show that the deubiquitinase for uH2A, ubiquitin-specific peptidase 16 (USP16), is modified by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation involves the installation of the O-GlcNAc moiety to Ser/Thr residues. It crosstalks with Ser/Thr phosphorylation, affects protein-protein interaction, alters enzyme activity or protein folding, and changes protein subcellular localization. In our study, we first confirmed that USP16 is glycosylated on Thr203 and Ser214, as reported in a previous chemoenzymatic screen. We then discovered that mutation of the O-GlcNAcylation site Thr203, which is adjacent to deubiquitination-required Cys204, reduces the deubiquitination activity toward H2AK119ub in vitro and in cells, while mutation on Ser214 had the opposite effects. Using USP16 Ser552 phosphorylation-specific antibodies, we demonstrated that O-GlcNAcylation antagonizes cyclin-dependent kinase 1-mediated phosphorylation and promotes USP16 nuclear export. O-GlcNAcylation of USP16 is also required for deubiquitination of Polo-like kinase 1, a mitotic master kinase, and the subsequent chromosome segregation and cytokinesis. In summary, our study revealed that O-GlcNAcylation of USP16 at Thr203 and Ser214 coordinates deubiquitination of uH2A and Polo-like kinase 1, thus ensuring proper cell cycle progression.
Assuntos
Acetilglucosamina , Ubiquitina Tiolesterase , Ubiquitinação , Humanos , Acetilglucosamina/metabolismo , Transporte Ativo do Núcleo Celular , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Glicosilação , Células HEK293 , Células HeLa , Histonas/metabolismo , Fosforilação , Quinase 1 Polo-Like , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genéticaRESUMO
The emerging roles of O-GlcNAcylation, a distinctive post-translational modification, are increasingly recognized for their involvement in the intricate processes of protein trafficking and secretion. This modification exerts its influence on both conventional and unconventional secretory pathways. Under healthy and stress conditions, such as during diseases, it orchestrates the transport of proteins within cells, ensuring timely delivery to their intended destinations. O-GlcNAcylation occurs on key factors, like coat protein complexes (COPI and COPII), clathrin, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), and GRASP55 (Golgi reassembly stacking protein of 55 kDa) that control vesicle budding and fusion in anterograde and retrograde trafficking and unconventional secretion. The understanding of O-GlcNAcylation offers valuable insights into its critical functions in cellular physiology and the progression of diseases, including neurodegeneration, cancer, and metabolic disorders. In this review, we summarize and discuss the latest findings elucidating the involvement of O-GlcNAc in protein trafficking and its significance in various human disorders.
Assuntos
Clatrina , Proteínas SNARE , Humanos , Acetilglucosamina/metabolismo , Clatrina/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico/fisiologia , Proteínas SNARE/metabolismo , Animais , Acetilação , Glucose/metabolismoRESUMO
Sleep deprivation (SD) is widely acknowledged as a significant risk factor for cognitive impairment. In this study, intraperitoneal caffeine administration significantly ameliorated the learning and memory (L/M) deficits induced by SD and reduced aggressive behaviors in adult zebrafish. SD led to a reduction in protein kinase A (PKA) phosphorylation, phosphorylated-cAMP response element-binding protein (p-CREB), and c-Fos expression in zebrafish brain. Notably, these alterations were effectively reversed by caffeine. In addition, caffeine mitigated neuroinflammation induced by SD, as evident from suppression of the SD-mediated increase in glial fibrillary acidic protein (GFAP) and nuclear factor-κB (NF-κB) activation. Caffeine restored normal O-GlcNAcylation and O-GlcNAc transferase (OGT) levels while reversing the increased expression of O-GlcNAcase (OGA) in zebrafish brain after SD. Intriguingly, rolipram, a selective phosphodiesterase 4 (PDE4) inhibitor, effectively mitigated cognitive deficits, restored p-CREB and c-Fos levels, and attenuated the increase in GFAP in brain induced by SD. In addition, rolipram reversed the decrease in O-GlcNAcylation and OGT expression as well as elevation of OGA expression following SD. Treatment with H89, a PKA inhibitor, significantly impaired the L/M functions of zebrafish compared with the control group, inducing a decrease in O-GlcNAcylation and OGT expression and, conversely, an increase in OGA expression. The H89-induced changes in O-GlcNAc cycling and L/M dysfunction were effectively reversed by glucosamine treatment. H89 suppressed, whereas caffeine and rolipram promoted O-GlcNAc cycling in Neuro2a cells. Our collective findings underscore the interplay between PKA signaling and O-GlcNAc cycling in the regulation of cognitive function in the brain, offering potential therapeutic targets for cognitive deficits associated with SD.NEW & NOTEWORTHY Our observation highlights the intricate interplay between cAMP/PKA signaling and O-GlcNAc cycling, unveiling a novel mechanism that potentially governs the regulation of learning and memory functions. The dynamic interplay between these two pathways provides a novel and nuanced perspective on the molecular foundation of learning and memory regulation. These insights open avenues for the development of targeted interventions to treat conditions that impact cognitive function, including SD.
Assuntos
Disfunção Cognitiva , Isoquinolinas , Privação do Sono , Sulfonamidas , Animais , Privação do Sono/tratamento farmacológico , Peixe-Zebra/metabolismo , Cafeína/farmacologia , Rolipram , Acetilglucosamina/metabolismo , Processamento de Proteína Pós-Traducional , Cognição , Disfunção Cognitiva/tratamento farmacológico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismoRESUMO
Phenotypic changes to endometrial epithelial cells underpin receptivity to embryo implantation at the onset of pregnancy but the effect of hyperglycemia on these processes remains poorly understood. Here, we show that physiological levels of glucose (5 mM) abolished receptivity in the endometrial epithelial cell line, Ishikawa. However, embryo attachment was supported by 17 mM glucose as a result of glucose flux through the hexosamine biosynthetic pathway (HBP) and modulation of cell function via protein O-GlcNAcylation. Pharmacological inhibition of HBP or protein O-GlcNAcylation reduced embryo attachment in cocultures at 17 mM glucose. Mass spectrometry analysis of the O-GlcNAcylated proteome in Ishikawa cells revealed that myosin phosphatase target subunit 1 (MYPT1) is more highly O-GlcNAcylated in 17 mM glucose, correlating with loss of its target protein, phospho-myosin light chain 2, from apical cell junctions of polarized epithelium. Two-dimensional (2-D) and three-dimensional (3-D) morphologic analysis demonstrated that the higher glucose level attenuates epithelial polarity through O-GlcNAcylation. Inhibition of Rho (ras homologous)A-associated kinase (ROCK) or myosin II led to reduced polarity and enhanced receptivity in cells cultured in 5 mM glucose, consistent with data showing that MYPT1 acts downstream of ROCK signaling. These data implicate regulation of endometrial epithelial polarity through RhoA signaling upstream of actomyosin contractility in the acquisition of endometrial receptivity. Glucose levels impinge on this pathway through O-GlcNAcylation of MYPT1, which may impact endometrial receptivity to an implanting embryo in women with diabetes.NEW & NOTEWORTHY Understanding how glucose regulates endometrial function will support preconception guidance and/or the development of targeted interventions for individuals living with diabetes wishing to embark on pregnancy. We found that glucose can influence endometrial epithelial cell receptivity to embryo implantation by regulating posttranslational modification of proteins involved in the maintenance of cell polarity. Impaired or inappropriate endometrial receptivity could contribute to fertility and/or early pregnancy complications caused by poor glucose control.
Assuntos
Citoesqueleto , Implantação do Embrião , Endométrio , Glucose , Fosfatase de Miosina-de-Cadeia-Leve , Feminino , Implantação do Embrião/fisiologia , Humanos , Endométrio/metabolismo , Glucose/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Citoesqueleto/metabolismo , Quinases Associadas a rho/metabolismo , Células Epiteliais/metabolismo , Cadeias Leves de Miosina/metabolismo , Animais , Gravidez , Acetilglucosamina/metabolismo , Glicosilação , Polaridade Celular/fisiologia , Hexosaminas/metabolismo , Hexosaminas/biossíntese , Miosinas CardíacasRESUMO
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 ProteicaRESUMO
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 , AnimaisRESUMO
Epigenetic modifications are involved in fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), and contribute to the silencing of anti-fibrotic genes. H3K27me3, a key repressive histone mark, is catalysed by the methyltransferase enhancer of Zeste homologue 2 (EZH2), which is regulated by the post-translational modification, O-linked N-Acetylglucosamine (O-GlcNAc). In this study, we explored the effects of O-GlcNAc and EZH2 on the expression of antifibrotic genes, cyclooxygenase-2 (Cox2) and Heme Oxygenase (Homx1). The expression of Cox2 and Hmox1 was examined in primary IPF or non-IPF lung fibroblasts with or without EZH2 inhibitor EZP6438, O-GlcNAc transferase (OGT) inhibitor (OSMI-1) or O-GlcNAcase (OGA) inhibitor (thiamet G). Non-IPF cells were also subjected to TGF-ß1 with or without OGT inhibition. The reduced expression of Cox2 and Hmox1 in IPF lung fibroblasts is restored by OGT inhibition. In non-IPF fibroblasts, TGF-ß1 treatment reduces Cox2 and Hmox1 expression, which was restored by OGT inhibition. ChIP assays demonstrated that the association of H3K27me3 is reduced at the Cox2 and Hmox1 promoter regions following OGT or EZH2 inhibition. EZH2 levels and stability were decreased by reducing O-GlcNAc. Our study provided a novel mechanism of O-GlcNAc modification in regulating anti-fibrotic genes in lung fibroblasts and in the pathogenesis of IPF.
Assuntos
Histonas , Fibrose Pulmonar Idiopática , Humanos , Histonas/metabolismo , Acetilglucosamina/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Pulmão/metabolismo , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismoRESUMO
O-GlcNAcylation is a dynamic modulator of signaling pathways, equal in magnitude to the widely studied phosphorylation. With the rapid development of tools for its detection at the single protein level, the O-GlcNAc modification rapidly emerged as a novel diagnostic and therapeutic target in human diseases. Yet, mapping the human O-GlcNAcome in various tissues is essential for generating relevant biomarkers. In this study, we used human banked tissue as a sample source to identify O-GlcNAcylated protein targets relevant to human diseases. Using human term placentas, we propose (1) a method to clean frozen banked tissue of blood proteins; (2) an optimized protocol for the enrichment of O-GlcNAcylated proteins using immunoaffinity purification; and (3) a bioinformatic workflow to identify the most promising O-GlcNAc targets. As a proof-of-concept, we used 45 mg of banked placental samples from two pregnancies to generate intracellular protein extracts depleted of blood protein. Then, antibody-based O-GlcNAc enrichment on denatured samples yielded over 2000 unique HexNAc PSMs and 900 unique sites using 300 µg of protein lysate. Due to efficient sample cleanup, we also captured 82 HexNAc proteins with high placental expression. Finally, we provide a bioinformatic tool (CytOVS) to sort the HexNAc proteins based on their cellular localization and extract the most promising O-GlcNAc targets to explore further. To conclude, we provide a simple 3-step workflow to generate a manageable list of O-GlcNAc proteins from human tissue and improve our understanding of O-GlcNAcylation's role in health and diseases.
Assuntos
Placenta , Proteínas , Humanos , Feminino , Gravidez , Placenta/metabolismo , Proteínas/metabolismo , Fosforilação , Acetilglucosamina/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Glycosaminoglycans are extended linear polysaccharides present on cell surfaces and within the extracellular matrix that play crucial roles in various biological processes. Two prominent glycosaminoglycans, heparan sulfate and chondroitin sulfate, are covalently linked to proteoglycan core proteins through a common tetrasaccharide linker comprising glucuronic acid, galactose, galactose, and xylose moities. This tetrasaccharide linker is meticulously assembled step by step by four Golgi-localized glycosyltransferases. The addition of the fifth sugar moiety, either N-acetylglucosamine or N-acetylgalactosamine, initiates further chain elongation, resulting in the formation of heparan sulfate or chondroitin sulfate, respectively. Despite the fundamental significance of this step in glycosaminoglycan biosynthesis, its regulatory mechanisms have remained elusive. In this study, we detail the expression and purification of the four linker-synthesizing glycosyltransferases and their utilization in the production of fluorescent peptides carrying the native tetrasaccharide linker. We generated five tetrasaccharide peptides, mimicking the core proteins of either heparan sulfate or chondroitin sulfate proteoglycans. These peptides were readily accepted as substrates by the EXTL3 enzyme, which adds an N-acetylglucosamine moiety, thereby initiating heparan sulfate biosynthesis. Importantly, EXTL3 showed a preference towards peptides mimicking the core proteins of heparan sulfate proteoglycans over the ones from chondroitin sulfate proteoglycans. This suggests that EXTL3 could play a role in the decision-making step during glycosaminoglycan biosynthesis. The innovative strategy for chemo-enzymatic synthesis of fluorescent-labeled linker-peptides promises to be instrumental in advancing future investigations into the initial steps and the divergent step of glycosaminoglycan biosynthesis.