Targeted Protein O-GlcNAcylation Using Bifunctional Small Molecules.

Protein O-linked ß-N-acetylglucosamine modification (O-GlcNAcylation) plays a crucial role in regulating essential cellular processes. The disruption of the homeostasis of O-GlcNAcylation has been linked to various human diseases, including cancer, diabetes, and neurodegeneration. However, there are limited chemical tools for protein- and site-specific O-GlcNAc modification, rendering the precise study of the O-GlcNAcylation challenging. To address this, we have developed heterobifunctional small molecules, named O-GlcNAcylation TArgeting Chimeras (OGTACs), which enable protein-specific O-GlcNAcylation in living cells. OGTACs promote O-GlcNAcylation of proteins such as BRD4, CK2α, and EZH2 in cellulo by recruiting FKBP12F36V-fused O-GlcNAc transferase (OGT), with temporal, magnitude, and reversible control. Overall, the OGTACs represent a promising approach for inducing protein-specific O-GlcNAcylation, thus enabling functional dissection and offering new directions for O-GlcNAc-targeting therapeutic development.

The Immunometabolic Gene N-Acetylglucosamine Kinase Is Uniquely Involved in the Heritability of Multiple Sclerosis Severity.

The clinical severity of multiple sclerosis (MS), an autoimmune disorder of the central nervous system, is thought to be determined by environmental and genetic factors that have not yet been identified. In a recent genome-wide association study (GWAS), a single nucleotide polymorphism (SNP), rs10191329, has been associated with MS severity in two large independent cohorts of patients. Different approaches were followed by the authors to prioritize the genes that are transcriptionally regulated by such an SNP. It was concluded that the identified SNP regulates a group of proximal genes involved in brain resilience and cognitive abilities rather than immunity. Here, by conducting an alternative strategy for gene prioritization, we reached the opposite conclusion. According to our re-analysis, the main target of rs10191329 is N-Acetylglucosamine Kinase (NAGK), a metabolic gene recently shown to exert major immune functions via the regulation of the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) pathway. To gain more insights into the immunometabolic functions of NAGK, we analyzed the currently known list of NAGK protein partners. We observed that NAGK integrates a dense network of human proteins that are involved in glucose metabolism and are highly expressed by classical monocytes. Our findings hold potentially major implications for the understanding of MS pathophysiology.

Malnutrition enteropathy in Zambian and Zimbabwean children with severe acute malnutrition: A multi-arm randomized phase II trial.

Malnutrition underlies almost half of all child deaths globally. Severe Acute Malnutrition (SAM) carries unacceptable mortality, particularly if accompanied by infection or medical complications, including enteropathy. We evaluated four interventions for malnutrition enteropathy in a multi-centre phase II multi-arm trial in Zambia and Zimbabwe and completed in 2021. The purpose of this trial was to identify therapies which could be taken forward into phase III trials. Children of either sex were eligible for inclusion if aged 6-59 months and hospitalised with SAM (using WHO definitions: WLZ <-3, and/or MUAC <11.5 cm, and/or bilateral pedal oedema), with written, informed consent from the primary caregiver. We randomised 125 children hospitalised with complicated SAM to 14 days treatment with (i) bovine colostrum (n = 25), (ii) N-acetyl glucosamine (n = 24), (iii) subcutaneous teduglutide (n = 26), (iv) budesonide (n = 25) or (v) standard care only (n = 25). The primary endpoint was a composite of faecal biomarkers (myeloperoxidase, neopterin, α1-antitrypsin). Laboratory assessments, but not treatments, were blinded. Per-protocol analysis used ANCOVA, adjusted for baseline biomarker value, sex, oedema, HIV status, diarrhoea, weight-for-length Z-score, and study site, with pre-specified significance of P < 0.10. Of 143 children screened, 125 were randomised. Teduglutide reduced the primary endpoint of biomarkers of mucosal damage (effect size -0.89 (90% CI: -1.69,-0.10) P = 0.07), while colostrum (-0.58 (-1.4, 0.23) P = 0.24), N-acetyl glucosamine (-0.20 (-1.01, 0.60) P = 0.67), and budesonide (-0.50 (-1.33, 0.33) P = 0.32) had no significant effect. All interventions proved safe. This work suggests that treatment of enteropathy may be beneficial in children with complicated malnutrition. The trial was registered at ClinicalTrials.gov with the identifier NCT03716115.

Lysinibacillus piscis sp. nov. isolated from the gut of mottled spinefoot Siganus fuscescens.

A novel Lysinibacillus strain, designated KH24T, was isolated from the gut of Siganus fuscescens, a herbivorous fish, which was captured off the coast of Okinawa, Japan. Strain KH24T is a rod-shaped, Gram-stain-positive, spore-forming, and motile bacterium that forms off-white colonies. The 16S rRNA gene sequence of strain KH24T showed the highest similarity (97.4%) with Lysinibacillus pakistanensis JCM 18776T and L. irui IRB4-01T. Genomic similarities between strain KH24T and Lysinibacillus type strains, based on average nucleotide identity, digital DNA-DNA hybridization (genome-to-genome distance calculation), and average amino acid identity were 70.4-77.7%, 17.1-24.4%, and 69.2-81.2%, respectively, which were lower than species delineation thresholds. Strain KH24T growth occurred at pH values of 5.5-8.5, temperatures of 20-40 °C, and NaCl concentrations of 0-4.0%, and optimally at pH 7.0, 30 °C, and 0%, respectively. Unlike related Lysinibacillus type strains, strain KH24T could assimilate D-glucose, D-fructose, N-acetyl-glucosamine, amygdalin, arbutin, esculin, ferric citrate, salicin, D-cellobiose, D-maltose, D-sucrose, and gentiobiose. Major fatty acids included iso-C15:0 (45.8%), anteiso-C15:0 (15.1%), iso-C17:0 (12.6%), and anteiso-C17:0 (10.9%). Menaquinone-7 was the predominant quinone, and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and lysophosphatidylethanolamine. Based on its genetic and phenotypic properties, strain KH24T represents a novel species of the genus Lysinibacillus, for which the name Lysinibacillus piscis sp. nov. is proposed. The type strain is KH24T (= JCM 36611 T = KCTC 43676 T).

Efficient Escorting Strategy for Aggregation-Prone Notch EGF Repeats with Sparcl1.

Epidermal growth factor (EGF) repeats are present in various proteins and form well-defined structures with three disulfide bonds. One representative protein is the Notch receptor. Each EGF repeat contains unique atypical O-linked glycans, such as O-linked N-acetylglucosamine (O-GlcNAc). To generate a monoclonal antibody against the O-GlcNAc moiety in mouse Notch1, we expressed the recombinant C-terminal His6-tagged Notch1 EGF14-15 protein in HEK293T cells to prepare the immunogen. Most of the proteins were not secreted and showed higher molecular weight ladders in the cell lysate, suggesting protein aggregation. To overcome this issue, we fused Sparcl1 as an extracellular escorting tag to the N-terminus of Notch1 EGF14-15. The fusion protein was efficiently secreted extracellularly without protein aggregates in the lysates. Following PreScission protease treatment, Notch1 EGF14-15 was efficiently released from the escorting tag. Notch1 EGF14-15 prepared using this method was indeed O-GlcNAcylated. The optimal length of the escorting tag was determined by generating deletion mutants to improve the extracellular secretion of EGF14-15. Hence, a large amount of EGF14-15 was successfully prepared from the culture supernatant of HEK293T cells, which were otherwise prone to aggregation.

Influence of N-Acetylglucosamine and Melatonin Interaction in Modeling the Photosynthetic Component and Metabolomics of Cucumber under Salinity Stress.

The application of N-acetylglucosamine (GlcNAc) and melatonin (Mel) in agriculture could be a promising avenue for improving crop resilience and productivity, especially under challenging environmental conditions. In the current study, we treated the cucumber plant with GlcNAc and Mel solely and combinedly under salt stress (150 mM) then studied photosynthetic attributes using the transient OJIP fluorescence method. The results showed that the combination of GlcNAc × Mel significantly improved the plant morphological attributes, such as root and shoot biomass, and also improved chlorophyll and photosynthetic components. The mineral elements such as K, Mg, Ca, and P were significantly elevated, whereas a lower influx of Na was observed in GlcNAc × Mel treated cucumber shoots. A significant reduction in abscisic acid was observed, which was validated by the reduction in proline content and the increase in stomatal conductance (Gs), transpiration rate (E), and substomatal CO2 concentration (Ci). Furthermore, the activities of antioxidants such as polyphenol and flavonoid were considerably improved, resulting in a decrease in SOD and CAT with GlcNAc × Mel treatment. In addition, GlcNAc × Mel treatment dropped levels of the toxic radical Malondialdehyde (MDA) and elevated amino acids in cucumber shoots. These findings suggest that the combination of GlcNAc × Mel could be an effective elicitor for modeling plant metabolism to confer stress tolerance in crops.

Efficient Biosynthesis of Lacto-N-Biose I, a Building Block of Type I Human Milk Oligosaccharides, by a Metabolically Engineered Escherichia coli.

Lacto-N-biose I (LNB), termed a Type 1 disaccharide, is an important building block of human milk oligosaccharides. It shows promising prebiotic activity by stimulating the proliferation of many gut-associated bifidobacteria and thus displays good potential in infant foods or supplements. Enzymatic and microbial approaches to LNB synthesis have been studied, almost all of which involve glycosylation of LNB phosphorylase as the final step. Herein, we report a new and easier microbial LNB synthesis strategy through the route "lactose → lacto-N-triose II (LNTri II) → lacto-N-tetraose (LNT) → LNB". A previously constructed LNT-producing Escherichia coli BL21(DE3) strain was engineered for LNB biosynthesis by introducing Bifidobacterium bifidum LnbB. LNB was efficiently produced, accompanied by lactose regeneration. Genomic integration of key pathway genes related to LNTri II and LNT synthesis was performed to enhance LNB titers. The final engineered strain produced 3.54 and 26.88 g/L LNB by shake-flask and fed-batch cultivation, respectively.

O-GlcNAc regulates anti-fibrotic genes in lung fibroblasts through EZH2.

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.

The O-GlcNAc Modification of Recombinant Tau Protein and Characterization of the O-GlcNAc Pattern for Functional Study.

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.

The interaction between intratumoral bacteria and metabolic distortion in hepatocellular carcinoma.

BACKGROUND: Intratumoral bacteria might play essential roles in tumorigenesis in different cancer types. However, its features and potential roles in hepatocellular carcinoma (HCC) are largely unknown. METHODS: In this study, we assessed bacterial RNA by 16S rRNA fluorescence in situ hybridization and detected bacterial lipopolysaccharide (LPS) via immunohistochemistry. Hepa1-6 cells were used to establish orthotopic HCC models in mice. 2bRAD sequencing for microbiome was performed to determine the intratumoral bacterial characteristics, and liquid chromatography-mass spectrometry was conducted to explore the metabolic profile. The potential association between different intratumoral microbiota and metabolites were evaluated. RESULTS: We detected bacterial 16S rRNA and LPS in HCC tissues from the patients with HCC. In HCC mouse model, we found that the intratumor bacteria in HCC tissues were significantly different to adjacent nontumor tissues. Furthermore, we observed different metabolites in HCC tissues and adjacent nontumor tissues, such as N-acetyl-D-glucosamine and a-lactose. Our results showed that several bacteria were significantly associated with metabolites, such as Pseudomonas koreensis, which was positively correlated with N-acetyl-D-glucosamine and negatively correlated with citrulline. CONCLUSIONS: This study confirmed the close association between different bacteria and metabolites, which might provide novel opportunities for developing new biomarkers and therapeutic targets for HCC.

Bacterial Enzyme Assay for Mucin Glycan Degradation.

Bacterial sialidase and sulfoglycosidase may act on the acidic modifications of mucin O-glycans, producing sialic acid and 6-sulfated N-acetylglucosamine, respectively. Assays for these enzymes, using mucin as a substrate, are enabled by the detection and/or quantification of the free monosaccharides that are released by these enzymes. This chapter describes enzyme reactions with mucin, detection by thin-layer chromatography of sialic acid, and quantification of 6-sulfated N-acetylglucosamine by liquid chromatography-tandem mass spectrometry.

The role of O-GlcNAcylation in RNA polymerase II transcription.

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.

Nitrogen assimilation by E. coli in the mammalian intestine.

Nitrogen is an essential element for all living organisms, including Escherichia coli. Potential nitrogen sources are abundant in the intestine, but knowledge of those used specifically by E. coli to colonize remains limited. Here, we sought to determine the specific nitrogen sources used by E. coli to colonize the streptomycin-treated mouse intestine. We began by investigating whether nitrogen is limiting in the intestine. The NtrBC two-component system upregulates approximately 100 genes in response to nitrogen limitation. We showed that NtrBC is crucial for E. coli colonization, although most genes of the NtrBC regulon are not induced, which indicates that nitrogen is not limiting in the intestine. RNA-seq identified upregulated genes in colonized E. coli involved in transport and catabolism of seven amino acids, dipeptides and tripeptides, purines, pyrimidines, urea, and ethanolamine. Competitive colonization experiments revealed that L-serine, N-acetylneuraminic acid, N-acetylglucosamine, and di- and tripeptides serve as nitrogen sources for E. coli in the intestine. Furthermore, the colonization defect of a L-serine deaminase mutant was rescued by excess nitrogen in the drinking water but not by an excess of carbon and energy, demonstrating that L-serine serves primarily as a nitrogen source. Similar rescue experiments showed that N-acetylneuraminic acid serves as both a carbon and nitrogen source. To a minor extent, aspartate and ammonia also serve as nitrogen sources. Overall, these findings demonstrate that E. coli utilizes multiple nitrogen sources for successful colonization of the mouse intestine, the most important of which is L-serine. IMPORTANCE: While much is known about the carbon and energy sources that are used by E. coli to colonize the mammalian intestine, very little is known about the sources of nitrogen. Interrogation of colonized E. coli by RNA-seq revealed that nitrogen is not limiting, indicating an abundance of nitrogen sources in the intestine. Pathways for assimilation of nitrogen from several amino acids, dipeptides and tripeptides, purines, pyrimidines, urea, and ethanolamine were induced in mice. Competitive colonization assays confirmed that mutants lacking catabolic pathways for L-serine, N-acetylneuraminic acid, N-acetylglucosamine, and di- and tripeptides had colonization defects. Rescue experiments in mice showed that L-serine serves primarily as a nitrogen source, whereas N-acetylneuraminic acid provides both carbon and nitrogen. Of the many nitrogen assimilation mutants tested, the largest colonization defect was for an L-serine deaminase mutant, which demonstrates L-serine is the most important nitrogen source for colonized E. coli.

Functional significance of O-linked N-acetylglucosamine protein modification in regulating autophagy.

Autophagy is a core molecular pathway that preserves cellular and organismal homeostasis. Being susceptible to nutrient availability and stress, eukaryotic cells recycle or degrade internal components via membrane transport pathways to provide sustainable biological molecules and energy sources. The dysregulation of this highly conserved physiological process has been strongly linked to human disease. Post-translational modification, a mechanism that regulates protein function, plays a crucial role in autophagy regulation. O-linked N-acetylglucosamine protein modification (O-GlcNAcylation), a monosaccharide post-translational modification of intracellular proteins, is essential in nutritional and stress regulatory mechanisms. O-GlcNAcylation has emerged as an essential regulatory mechanism of autophagy. It regulates autophagy throughout its lifetime by targeting the core components of the autophagy regulatory network. This review provides an overview of the O-GlcNAcylation of autophagy-associated proteins and their regulation and function in the autophagy pathway. Therefore, this article may contribute to further understanding of the role of O-GlcNAc-regulated autophagy and provide new perspectives for the treatment of human diseases.

Characterization of galactosyltransferase and sialyltransferase genes mediating the elongation of the extracellular O-GlcNAc glycans.

O-GlcNAc is a unique post-translational modification found in cytoplasmic, nuclear, and mitochondrial proteins. In a limited number of extracellular proteins, O-GlcNAc modifications occur through the action of EOGT, which specifically modifies subsets of epidermal growth factor-like (EGF) domain-containing proteins such as Notch receptors. The abnormalities due to EOGT mutations in mice and humans and the increased EOGT expression in several cancers signify the importance of EOGT pathophysiology and extracellular O-GlcNAc. Unlike intracellular O-GlcNAc monosaccharides, extracellular O-GlcNAc extends to form elongated glycan structures. However, the enzymes involved in the O-GlcNAc glycan extension have not yet been reported. In our study, we comprehensively screened potential galactosyltransferase and sialyltransferase genes related to the canonical O-GlcNAc glycan pathway and revealed the essential roles of B4GALT1 and ST3GAL4 in O-GlcNAc glycan elongation in human HEK293 cells. These findings were confirmed by sequential glycosylation of Drosophila EGF20 in vitro by EOGT, ß4GalT-1, and ST3Gal-IV. Thus, the findings from our study throw light on the specific glycosyltransferases that mediate O-GlcNAc glycan elongation in human HEK293 cells.

Caffeine-induced protein kinase A activation restores cognitive deficits induced by sleep deprivation by regulating O-GlcNAc cycling in adult zebrafish.

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.

Targeting Breast Cancer with N-Acetyl-D-Glucosamine: Integrating Machine Learning and Cellular Assays for Promising Results.

BACKGROUND: Breast cancer is a common cancer with high mortality rates. Early diagnosis is crucial for reducing the prognosis and mortality rates. Therefore, the development of alternative treatment options is necessary. OBJECTIVE: This study aimed to investigate the inhibitory effect of N-acetyl-D-glucosamine (D-GlcNAc) on breast cancer using a machine learning method. The findings were further confirmed through assays on breast cancer cell lines. METHODS: MCF-7 and 4T1 cell lines (ATCC) were cultured in the presence and absence of varying concentrations of D-GlcNAc (0.5 mM, 1 mM, 2 mM, and 4 mM) for 72 hours. A xenograft mouse model for breast cancer was established by injecting 4T1 cells into mammary glands. D-GlcNAc (2 mM) was administered intraperitoneally to mice daily for 28 days, and histopathological effects were evaluated at pre-tumoral and post-tumoral stages. RESULTS: Treatment with 2 mM and 4 mM D-GlcNAc significantly decreased cell proliferation rates in MCF-7 and 4T1 cell lines and increased Fas expression. The number of apoptotic cells was significantly higher than untreated cell cultures (p < 0.01 - p < 0.0001). D-GlcNAc administration also considerably reduced tumour size, mitosis, and angiogenesis in the post-treatment group compared to the control breast cancer group (p < 0.01 - p < 0.0001). Additionally, molecular docking/dynamic analysis revealed a high binding affinity of D-GlcNAc to the marker protein HER2, which is involved in tumour progression and cell signalling. CONCLUSION: Our study demonstrated the positive effect of D-GlcNAc administration on breast cancer cells, leading to increased apoptosis and Fas expression in the malignant phenotype. The binding affinity of D-GlcNAc to HER2 suggests a potential mechanism of action. These findings contribute to understanding D-GlcNAc as a potential anti-tumour agent for breast cancer treatment.

O-GlcNAc signaling increases neuron regeneration through one-carbon metabolism in Caenorhabditis elegans.

Cellular metabolism plays an essential role in the regrowth and regeneration of a neuron following physical injury. Yet, our knowledge of the specific metabolic pathways that are beneficial to neuron regeneration remains sparse. Previously, we have shown that modulation of O-linked ß-N-acetylglucosamine (O-GlcNAc) signaling, a ubiquitous post-translational modification that acts as a cellular nutrient sensor, can significantly enhance in vivo neuron regeneration. Here, we define the specific metabolic pathway by which O-GlcNAc transferase (ogt-1) loss of function mediates increased regenerative outgrowth. Performing in vivo laser axotomy and measuring subsequent regeneration of individual neurons in C. elegans, we find that glycolysis, serine synthesis pathway (SSP), one-carbon metabolism (OCM), and the downstream transsulfuration metabolic pathway (TSP) are all essential in this process. The regenerative effects of ogt-1 mutation are abrogated by genetic and/or pharmacological disruption of OCM and the SSP linking OCM to glycolysis. Testing downstream branches of this pathway, we find that enhanced regeneration is dependent only on the vitamin B12 independent shunt pathway. These results are further supported by RNA sequencing that reveals dramatic transcriptional changes by the ogt-1 mutation, in the genes involved in glycolysis, OCM, TSP, and ATP metabolism. Strikingly, the beneficial effects of the ogt-1 mutation can be recapitulated by simple metabolic supplementation of the OCM metabolite methionine in wild-type animals. Taken together, these data unearth the metabolic pathways involved in the increased regenerative capacity of a damaged neuron in ogt-1 animals and highlight the therapeutic possibilities of OCM and its related pathways in the treatment of neuronal injury.

Chemo-enzymatic synthesis of tetrasaccharide linker peptides to study the divergent step in glycosaminoglycan biosynthesis.

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.

Targeting O-GlcNAcylation in cancer therapeutic resistance: The sugar Saga continues.

O-linked-N-acetylglucosaminylation (O-GlcNAcylation), a dynamic post-translational modification (PTM), holds profound implications in controlling various cellular processes such as cell signaling, metabolism, and epigenetic regulation that influence cancer progression and therapeutic resistance. From the therapeutic perspective, O-GlcNAc modulates drug efflux, targeting and metabolism. By integrating signals from glucose, lipid, amino acid, and nucleotide metabolic pathways, O-GlcNAc acts as a nutrient sensor and transmits signals to exerts its function on genome stability, epithelial-mesenchymal transition (EMT), cell stemness, cell apoptosis, autophagy, cell cycle. O-GlcNAc also attends to tumor microenvironment (TME) and the immune response. At present, several strategies aiming at targeting O-GlcNAcylation are under mostly preclinical evaluation, where the newly developed O-GlcNAcylation inhibitors markedly enhance therapeutic efficacy. Here we systematically outline the mechanisms through which O-GlcNAcylation influences therapy resistance and deliberate on the prospects and challenges associated with targeting O-GlcNAcylation in future cancer treatments.