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Frontiers in Genetics 2024This report outlines the case of a child affected by a type of congenital disorder of glycosylation (CDG) known as ALG2-CDG (OMIM 607906), presenting as a congenital...
This report outlines the case of a child affected by a type of congenital disorder of glycosylation (CDG) known as ALG2-CDG (OMIM 607906), presenting as a congenital myasthenic syndrome (CMS) caused by variants identified in , which encodes an α1,3-mannosyltransferase (EC 2.4.1.132) involved in the early steps of N-glycosylation. To date, fourteen cases of ALG2-CDG have been documented worldwide. From birth, the child experienced perinatal asphyxia, muscular weakness, feeding difficulties linked to an absence of the sucking reflex, congenital hip dislocation, and hypotonia. Over time, additional complications emerged, such as inspiratory stridor, gastroesophageal reflux, low intake, recurrent seizures, respiratory infections, an inability to maintain the head upright, and a global developmental delay. Whole genome sequencing (WGS) revealed the presence of two variants in compound heterozygosity: a novel variant c.1055_1056delinsTGA p.(Ser352Leufs*3) and a variant of uncertain significance (VUS) c.964C>A p.(Pro322Thr). Additional studies, including determination of carbohydrate-deficient transferrin (CDT) revealed a mild type I CDG pattern and the presence of an abnormal transferrin glycoform containing a linear heptasaccharide consisting of one sialic acid, one galactose, one N-acetyl-glucosamine, two mannoses and two N-acetylglucosamines (NeuAc-Gal-GlcNAc-Man2-GlcNAc2), ALG2-CDG diagnostic biomarker, confirming the pathogenicity of these variants.
PubMed: 38770420
DOI: 10.3389/fgene.2024.1363558 -
Heliyon May 2024Acute kidney injury (AKI), a condition associated with reactive oxygen species (ROS), causes high mortality in clinics annually. Active targeted antioxidative therapy is...
Acute kidney injury (AKI), a condition associated with reactive oxygen species (ROS), causes high mortality in clinics annually. Active targeted antioxidative therapy is emerging as a novel strategy for AKI treatment. In this study, we developed a polymeric prodrug that targets the highly expressed Megalin receptor on proximal tubule cells, enabling direct delivery of N-Acetylcysteine (NAC) for the treatment of ischemia reperfusion injury (IRI)-induced AKI. We conjugated NAC with low molecular weight chitosan (LMWC), a biocompatible and biodegradable polymer consisting of glucosamine and N-acetylglucosamine, to enhance its internalization by tubular epithelial cells. Moreover, we further conjugated triphenylphosphonium (TPP), a lipophilic cation with a delocalized positive charge, to low molecular weight chitosan-NAC in order to enhance the distribution of NAC in mitochondria. Our study confirmed that triphenylphosphonium-low molecular weight chitosan-NAC (TLN) exhibits remarkable therapeutic effects on IRI-AKI mice. This was evidenced by improvements in renal function, reduction in oxidative stress, mitigation of pathological progress, and decreased levels of kidney injury molecule-1. These findings suggested that the polymeric prodrug TLN holds promising potential for IRI-AKI treatment.
PubMed: 38770316
DOI: 10.1016/j.heliyon.2024.e30947 -
Scientific Reports May 2024N-acetyl glucosamine (NAG) is a natural amino sugar found in various human tissues with previously described anti-inflammatory effects. Various chemical modifications of...
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.
Topics: Animals; Acetylglucosamine; Mice; Anti-Inflammatory Agents; Lipopolysaccharides; Macrophages, Peritoneal; Tumor Necrosis Factor-alpha; Interleukin-6; Inflammation; Male; Disease Models, Animal
PubMed: 38745047
DOI: 10.1038/s41598-024-61780-2 -
JIMD Reports May 2024ALG1-CDG is a rare, clinically variable metabolic disease, caused by the defect of adding the first mannose (Man) to N-acetylglucosamine (GlcNAc)-pyrophosphate...
ALG1-CDG is a rare, clinically variable metabolic disease, caused by the defect of adding the first mannose (Man) to N-acetylglucosamine (GlcNAc)-pyrophosphate (PP)-dolichol to the growing oligosaccharide chain, resulting in impaired N-glycosylation of proteins. N-glycosylation has a key role in functionality, stability, and half-life of most proteins. Therefore, congenital defects of glycosylation typically are multisystem disorders. Here we report a 3-year-old patient with severe neurological, cardiovascular, respiratory, musculoskeletal and gastrointestinal symptoms. ALG1-CDG was suggested based on exome sequencing and Western blot analysis. Despite her severe clinical manifestations and genetic diagnosis, serum transferrin glycoform analysis was normal. Western blot analysis of highly glycosylated proteins in fibroblasts revealed decreased intercellular adhesion molecule 1 (ICAM1), but normal lysosomal associated membrane protein 1 and 2 (LAMP1 and LAMP2) expression levels. Glycoproteomics in fibroblasts showed the presence of the abnormal tetrasacharide. Reviewing the literature, we found 86 reported ALG1-CDG patients, but only one with normal transferrin analysis. Based on our results we would like to highlight the importance of multiple approaches in diagnosing ALG1-CDG, as normal serum transferrin glycosylation or other biomarkers with normal expression levels can occur.
PubMed: 38736633
DOI: 10.1002/jmd2.12415 -
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 -
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 -
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