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Journal of the American Chemical Society May 2024While the function of protein phosphorylation in eukaryotic cell signaling is well established, the role of a closely related modification, protein pyrophosphorylation,...
While the function of protein phosphorylation in eukaryotic cell signaling is well established, the role of a closely related modification, protein pyrophosphorylation, is just starting to surface. A recent study has identified several targets of endogenous protein pyrophosphorylation in mammalian cell lines, including -acetylglucosamine kinase (NAGK). Here, a detailed functional analysis of NAGK phosphorylation and pyrophosphorylation on serine 76 (S76) has been conducted. This analysis was enabled by using amber codon suppression to obtain phosphorylated pS76-NAGK, which was subsequently converted to site-specifically pyrophosphorylated NAGK (ppS76-NAGK) with a phosphorimidazolide reagent. A significant reduction in GlcNAc kinase activity was observed upon phosphorylation and near-complete inactivation upon pyrophosphorylation. The formation of ppS76-NAGK proceeded via an ATP-dependent autocatalytic process, and once formed, ppS76-NAGK displayed notable stability toward dephosphorylation in mammalian cell lysates. Proteomic examination unveiled a distinct set of protein-protein interactions for ppS76-NAGK, suggesting an alternative function, independent of its kinase activity. Overall, a significant regulatory role of pyrophosphorylation on NAGK activity was uncovered, providing a strong incentive to investigate the influence of this unusual phosphorylation mode on other kinases.
Topics: Phosphorylation; Humans; Phosphotransferases (Alcohol Group Acceptor); HEK293 Cells
PubMed: 38733353
DOI: 10.1021/jacs.4c03069 -
International Journal of Molecular... Apr 2024Fatty acid synthesis has been extensively investigated as a therapeutic target in cancers, including colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of...
Overexpression of Fatty Acid Synthase Upregulates Glutamine-Fructose-6-Phosphate Transaminase 1 and O-Linked N-Acetylglucosamine Transferase to Increase O-GlcNAc Protein Glycosylation and Promote Colorectal Cancer Growth.
Fatty acid synthesis has been extensively investigated as a therapeutic target in cancers, including colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of de novo lipid synthesis, is significantly upregulated in CRC, and therapeutic approaches of targeting this enzyme are currently being tested in multiple clinical trials. However, the mechanisms behind the pro-oncogenic action of FASN are still not completely understood. Here, for the first time, we show that overexpression of FASN increases the expression of glutamine-fructose-6-phosphate transaminase 1 (GFPT1) and O-linked N-acetylglucosamine transferase (OGT), enzymes involved in hexosamine metabolism, and the level of O-GlcNAcylation in vitro and in vivo. Consistently, expression of FASN significantly correlates with expression of GFPT1 and OGT in human CRC tissues. shRNA-mediated downregulation of GFPT1 and OGT inhibits cellular proliferation and the level of protein O-GlcNAcylation in vitro, and knockdown of GFPT1 leads to a significant decrease in tumor growth and metastasis in vivo. Pharmacological inhibition of GFPT1 and OGT leads to significant inhibition of cellular proliferation and colony formation in CRC cells. In summary, our results show that overexpression of FASN increases the expression of GFPT1 and OGT as well as the level of protein O-GlcNAcylation to promote progression of CRC; targeting the hexosamine biosynthesis pathway could be a therapeutic approach for this disease.
Topics: Humans; Colorectal Neoplasms; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); N-Acetylglucosaminyltransferases; Cell Proliferation; Glycosylation; Animals; Mice; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Up-Regulation; Mice, Nude; Fatty Acid Synthase, Type I
PubMed: 38732103
DOI: 10.3390/ijms25094883 -
Scientific Reports May 2024Anaesthetics are used daily in human and veterinary medicine as well as in scientific research. Anaesthetics have an impact on cell homeostasis especially through...
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.
Topics: Animals; Male; Rats, Wistar; Rats; Isoflurane; Ketamine; Xylazine; Anesthesia; Acetylglucosamine; Protein Processing, Post-Translational; Brain; N-Acetylglucosaminyltransferases; Heart Rate; Lung; Anesthetics; Blood Pressure; Hemodynamics
PubMed: 38724577
DOI: 10.1038/s41598-024-61445-0 -
Journal of Applied Microbiology May 2024We aimed to investigate the function of an unidentified gene annotated as a PIG-L domain deacetylase (cspld) in Chitiniphilus shinanonensis SAY3. cspld was identified...
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.
Topics: Chitin; Amidohydrolases; Acetylglucosamine; Bacterial Proteins; Escherichia coli; Gammaproteobacteria
PubMed: 38724455
DOI: 10.1093/jambio/lxae117 -
The Journal of Sexual Medicine Jun 2024Intravesical instillation of hyaluronic acid (HA) has been associated with reduced sexual dysfunction in participants with recurrent urinary tract infections (rUTIs),... (Randomized Controlled Trial)
Randomized Controlled Trial
Oral preparation of hyaluronic acid, chondroitin sulfate, N-acetylglucosamine, and vitamin C improves sexual and urinary symptoms in participants with recurrent urinary tract infections: a randomized crossover trial.
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).
Topics: Humans; Ascorbic Acid; Chondroitin Sulfates; Hyaluronic Acid; Female; Cross-Over Studies; Male; Adult; Urinary Tract Infections; Acetylglucosamine; Administration, Oral; Pilot Projects; Sexual Dysfunction, Physiological; Middle Aged; Recurrence; Propolis; Mannose
PubMed: 38721680
DOI: 10.1093/jsxmed/qdae052 -
Cell Death & Disease May 2024RAD18, an important ubiquitin E3 ligase, plays a dual role in translesion DNA synthesis (TLS) and homologous recombination (HR) repair. However, whether and how the...
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.
Topics: Humans; DNA-Binding Proteins; Ubiquitin-Protein Ligases; Recombinational DNA Repair; Acetylglucosamine; Rad51 Recombinase; Proliferating Cell Nuclear Antigen; Phosphorylation; DNA Replication; Ubiquitination; DNA Breaks, Double-Stranded; DNA-Directed DNA Polymerase; Cell Cycle Proteins; DNA Damage; DNA; HEK293 Cells; Ultraviolet Rays; Protein Binding; Glycosylation; Translesion DNA Synthesis
PubMed: 38719812
DOI: 10.1038/s41419-024-06700-y -
The Journal of Biological Chemistry Jun 2024The dynamic and reversible modification of nuclear and cytoplasmic proteins by O-GlcNAcylation significantly impacts the function and dysfunction of the immune system.... (Review)
Review
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.
Topics: Humans; Signal Transduction; Animals; Acetylglucosamine; Immune System; Protein Processing, Post-Translational; Glycosylation
PubMed: 38718861
DOI: 10.1016/j.jbc.2024.107349 -
Nucleic Acids Research Jun 2024In recent years, several noncanonical RNA caps derived from cofactors and metabolites have been identified. Purine-containing RNA caps have been extensively studied,...
In recent years, several noncanonical RNA caps derived from cofactors and metabolites have been identified. Purine-containing RNA caps have been extensively studied, with multiple decapping enzymes identified and efficient capture and sequencing protocols developed for nicotinamide adenine dinucleotide (NAD)-RNA, which allowed for a stepwise elucidation of capping functions. Despite being identified as an abundant noncanonical RNA-cap, UDP-sugar-capped RNA remains poorly understood, which is partly due to its complex in vitro preparation. Here, we describe a scalable synthesis of sugar-capped uridine-guanosine dinucleotides from readily available protected building blocks and their enzymatic conversion into several cell wall precursor-capped dinucleotides. We employed these capped dinucleotides in T7 RNA polymerase-catalyzed in vitro transcription reactions to efficiently generate RNAs capped with uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), its N-azidoacetyl derivative UDP-GlcNAz, and various cell wall precursors. We furthermore identified four enzymes capable of processing UDP-GlcNAc-capped RNA in vitro: MurA, MurB and MurC from Escherichia coli can sequentially modify the sugar-cap structure and were used to introduce a bioorthogonal, clickable moiety, and the human Nudix hydrolase Nudt5 was shown to efficiently decap UDP-GlcNAc-RNA. Our findings underscore the importance of efficient synthetic methods for capped model RNAs. Additionally, we provide useful enzymatic tools that could be utilized in the development and application of UDP-GlcNAc capture and sequencing protocols. Such protocols are essential for deepening our understanding of the widespread yet enigmatic GlcNAc modification of RNA and its physiological significance.
Topics: Uridine Diphosphate N-Acetylglucosamine; RNA Caps; Endoribonucleases; DNA-Directed RNA Polymerases; Humans; Escherichia coli; Viral Proteins
PubMed: 38716860
DOI: 10.1093/nar/gkae353 -
Nature Communications May 2024c-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...
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.
Topics: Acetylglucosamine; Allosteric Regulation; Signal Transduction; Bacterial Proteins; Gene Expression Regulation, Bacterial; Dinucleoside Phosphates; Saccharopolyspora
PubMed: 38714645
DOI: 10.1038/s41467-024-48063-0 -
Bioorganic Chemistry Jun 2024Fluorination of carbohydrate ligands of lectins is a useful approach to examine their binding profile, improve their metabolic stability and lipophilicity, and convert...
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 F 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 β-glycosides of N,N'-diacetylchitobiose (GlcNAcβ(1-4)GlcNAcβ1-OMe) and LacdiNAc (GalNAcβ(1-4)GlcNAcβ1-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 F NMR-active probes.
Topics: Disaccharides; Wheat Germ Agglutinins; Halogenation; Molecular Structure; Acetylglucosamine; Lactose
PubMed: 38705105
DOI: 10.1016/j.bioorg.2024.107395