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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 -
In Vivo (Athens, Greece) 2024Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation or environment, recognized by a site-specific monoclonal mouse IgM...
BACKGROUND/AIM
Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation or environment, recognized by a site-specific monoclonal mouse IgM antibody H. O-GlcNAcH occurs in several normal and pathological cells and in several polypeptides, including keratin-8 and vimentin, on the latter in cells under stress.
MATERIALS AND METHODS
In this work, we studied the distribution of O-GlcNAcH on cells of endocervical mucosa in 60 specimens of endocervical curettings, 10 of which contained 15 inflamed polyps.
RESULTS
In our results, expression of O-GlcNAcH was weak in the mucosa with <5% mucin-secreting cells and up to 30% of the polyps staining positively. All non-ciliated, non-mucin-secreting cells, normal and hyperplastic 'reserve' cells, as well as the cells of immature squamous metaplasia, showed strong diffuse cytoplasmic staining for O-GlcNAcH. In mature squamous epithelium, fewer than 5% of basal cells and all the intermediate and superficial cells showed cytoplasmic staining for O-GlcNAcH, whereas parabasal cells were negative. All ciliated cells showed patchy or diffuse cytoplasmic staining. Nuclear staining for O-GlcNAcH was weak with fewer than 5% of hyperplastic 'reserve' and ciliated cells staining positively. Moreover, mucosal fibroblasts were negative, whereas all stromal cells of the polyps showed strong cytoplasmic staining for O-GlcNAcH.
CONCLUSION
O-GlcNAcH is: a) differentially expressed among the cellular elements of mucosa and polyps, b) upregulated in mucin-secreting cells of polyps, c) induced in stromal cells of inflamed polyps, and d) can be used as a marker to differentiate between 'reserve' (positive) and parabasal (negative) cells, which have similar morphology using conventional cytological stains.
Topics: Humans; Female; Acetylglucosamine; Cervix Uteri; Epitopes; Mucous Membrane; Adult; Middle Aged; Immunohistochemistry
PubMed: 38688609
DOI: 10.21873/invivo.13545 -
Cell Reports May 2024Dysregulation of O-GlcNAcylation has emerged as a potential biomarker for several diseases, particularly cancer. The role of OGT (O-GlcNAc transferase) in maintaining...
Dysregulation of O-GlcNAcylation has emerged as a potential biomarker for several diseases, particularly cancer. The role of OGT (O-GlcNAc transferase) in maintaining O-GlcNAc homeostasis has been extensively studied; nevertheless, the regulation of OGA (O-GlcNAcase) in cancer remains elusive. Here, we demonstrated that the multifunctional protein RBM14 is a regulator of cellular O-GlcNAcylation. By investigating the correlation between elevated O-GlcNAcylation and increased RBM14 expression in lung cancer cells, we discovered that RBM14 promotes ubiquitin-dependent proteasomal degradation of OGA, ultimately mediating cellular O-GlcNAcylation levels. In addition, RBM14 itself is O-GlcNAcylated at serine 521, regulating its interaction with the E3 ligase TRIM33, consequently affecting OGA protein stability. Moreover, we demonstrated that mutation of serine 521 to alanine abrogated the oncogenic properties of RBM14. Collectively, our findings reveal a previously unknown mechanism for the regulation of OGA and suggest a potential therapeutic target for the treatment of cancers with dysregulated O-GlcNAcylation.
Topics: Humans; Acetylglucosamine; Antigens, Neoplasm; beta-N-Acetylhexosaminidases; Cell Line, Tumor; Glycosylation; HEK293 Cells; Histone Acetyltransferases; Hyaluronoglucosaminidase; Lung Neoplasms; N-Acetylglucosaminyltransferases; Proteasome Endopeptidase Complex; Protein Stability; RNA-Binding Proteins; Tripartite Motif Proteins; Ubiquitin-Protein Ligases
PubMed: 38678556
DOI: 10.1016/j.celrep.2024.114163 -
Analytical and Bioanalytical Chemistry Jun 2024Nucleotide sugars (NS) fulfil important roles in all living organisms and in humans, related defects result in severe clinical syndromes. NS can be seen as the...
Nucleotide sugars (NS) fulfil important roles in all living organisms and in humans, related defects result in severe clinical syndromes. NS can be seen as the "activated" sugars used for biosynthesis of a wide range of glycoconjugates and serve as substrates themselves for the synthesis of other nucleotide sugars. NS analysis is complicated by the presence of multiple stereoisomers without diagnostic transition ions, therefore requiring separation by liquid chromatography. In this paper, we explored weak anion-exchange/reversed-phase chromatography on a hybrid column for the separation of 17 nucleotide sugars that can occur in humans. A robust and reproducible method was established with intra- and inter-day coefficients of variation below 10% and a linear range spanning three orders of magnitude. Application to patient fibroblasts with genetic defects in mannose-1-phosphate guanylyltransferase beta, CDP-L-ribitol pyrophosphorylase A, and UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase showed abnormal levels of guanosine-5'-diphosphate-α-D-mannose (GDP-Man), cytidine-5'-diphosphate-L-ribitol (CDP-ribitol), and cytidine-5'-monophosphate-N-acetyl-β-D-neuraminic acid (CMP-Neu5Ac), respectively, in consonance with expectations based on the diagnosis. In conclusion, a novel, semi-quantitative method was established for the analysis of nucleotide sugars that can be applied to diagnose several genetic glycosylation disorders in fibroblasts and beyond.
Topics: Humans; Fibroblasts; Tandem Mass Spectrometry; Chromatography, Ion Exchange; Chromatography, Reverse-Phase; Nucleotides; Anions; Liquid Chromatography-Mass Spectrometry
PubMed: 38676823
DOI: 10.1007/s00216-024-05313-w -
Vaccines Apr 2024Vaccine development against group A (GAS) has gained traction in the last decade, fuelled by recognition of the significant worldwide burden of the disease. Several...
Vaccine development against group A (GAS) has gained traction in the last decade, fuelled by recognition of the significant worldwide burden of the disease. Several vaccine candidates are currently being evaluated in preclinical and early clinical studies. Here, we investigate two conjugate vaccine candidates that have shown promise in mouse models of infection. Two antigens, the J8 peptide from the conserved C-terminal end of the M protein, and the group A carbohydrate lacking -acetylglucosamine side chain (ΔGAC) were each conjugated to arginine deiminase (ADI), an anchorless surface protein from GAS. Both conjugate vaccine candidates combined with alum adjuvant were tested in a non-human primate (NHP) model of pharyngeal infection. High antibody titres were detected against J8 and ADI antigens, while high background antibody titres in NHP sera hindered accurate quantification of ΔGAC-specific antibodies. The severity of pharyngitis and tonsillitis signs, as well as the level of GAS colonisation, showed no significant differences in NHPs immunised with either conjugate vaccine candidate compared to NHPs in the negative control group.
PubMed: 38675764
DOI: 10.3390/vaccines12040382 -
Microorganisms Apr 2024Chitin, a polymer of β-1,4-linked -acetylglucosamine (GlcNAc), can be degraded into valuable oligosaccharides by various chitinases. In this study, the genome of JW44,...
Chitin, a polymer of β-1,4-linked -acetylglucosamine (GlcNAc), can be degraded into valuable oligosaccharides by various chitinases. In this study, the genome of JW44, displaying remarkable chitinolytic activity, was investigated to understand its chitin-degradation potential. A chitinase gene from this strain was then cloned, expressed, and purified to characterize its enzymatic properties and substrate hydrolysis. Genome analysis showed that, of the 14 genes related to chitin utilization in JW44, six belonged to glycoside hydrolase (GH) families because of their functional domains for chitin binding and catalysis. The recombinant chitinase SkChi65, consisting of 1129 amino acids, was identified as a member of the GH18 family and possessed two chitin-binding domains with a typical motif of [A/N]KWWT[N/S/Q] and one catalytic domain with motifs of DxxDxDxE, SxGG, YxR, and [E/D]xx[V/I]. SkChi65 was heterologously expressed as an active protein of 139.95 kDa best at 37 °C with 1.0 mM isopropyl-β-d-thiogalactopyranoside induction for 6 h. Purified SkChi65 displayed high stability over the ranges of 30-50 °C and pH 5.5-8.0 with optima at 40 °C and pH 7.0. The kinetic parameters , , and of SkChi65 towards colloidal chitin were 27.2 μM, 299.2 μMs, and 10,203 s, respectively. In addition to colloidal chitin, SkChi65 showed high activity towards glycol chitosan and crystalline chitin. After analysis by thin-layer chromatography, the main products were ,'-diacetylchitobiose, and GlcNAc with (GlcNAc) used as substrates. Collectively, SkChi65 could exhibit both exo- and endochitinase activities towards diverse substrates, and strain JW44 has a high potential for industrial application with an excellent capacity for chitin bioconversion.
PubMed: 38674717
DOI: 10.3390/microorganisms12040774 -
Recessive Mutations in Korean Nonaka Distal Myopathy Patients with or without Peripheral Neuropathy.Genes Apr 2024Autosomal recessive Nonaka distal myopathy is a rare autosomal recessive genetic disease characterized by progressive degeneration of the distal muscles, causing muscle...
Autosomal recessive Nonaka distal myopathy is a rare autosomal recessive genetic disease characterized by progressive degeneration of the distal muscles, causing muscle weakness and decreased grip strength. It is primarily associated with mutations in the gene, which encodes a key enzyme of sialic acid biosynthesis (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase). This study was performed to find mutations in six independent distal myopathy patients with or without peripheral neuropathy using whole-exome sequencing (WES). In silico pathogenic prediction and simulation of 3D structural changes were performed for the mutant GNE proteins. As a result, we identified five pathogenic or likely pathogenic missense variants: c.86T>C (p.Met29Thr), c.527A>T (p.Asp176Val), c.782T>C (p.Met261Thr), c.1714G>C (p.Val572Leu), and c.1771G>A (p.Ala591Thr). Five affected individuals showed compound heterozygous mutations, while only one patient revealed a homozygous mutation. Two patients revealed unreported combinations of combined heterozygous mutations. We observed some specific clinical features, such as complex phenotypes of distal myopathy with distal hereditary peripheral neuropathy, an earlier onset of weakness in legs than that of hands, and clinical heterogeneity between two patients with the same set of compound heterozygous mutations. Our findings on these genetic causes expand the clinical spectrum associated with the mutations and can help prepare therapeutic strategies.
Topics: Humans; Distal Myopathies; Male; Female; Adult; Republic of Korea; Exome Sequencing; Peripheral Nervous System Diseases; Mutation, Missense; Middle Aged; Multienzyme Complexes; Pedigree; Mutation; Genes, Recessive
PubMed: 38674419
DOI: 10.3390/genes15040485 -
International Journal of Molecular... Apr 2024During choriogenesis in insects, chorion (eggshell) is formed by surrounding follicular epithelial cells in ovarioles. However, the regulatory endocrine factor(s)...
During choriogenesis in insects, chorion (eggshell) is formed by surrounding follicular epithelial cells in ovarioles. However, the regulatory endocrine factor(s) activating choriogenesis and the effect of chemical components on eggshell deserve further exploration. In two representative coleopterans, a coccinellid and a chrysomelid , genes encoding the 20-hydroxyecdysone (20E) receptor heterodimer, ecdysone receptor (EcR) and ultraspiracle (USP), and two chitin biosynthesis enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP) and chitin synthase (ChS1), were highly expressed in ovaries of the young females. RNA interference (RNAi)-aided knockdown of either or in inhibited oviposition, suppressed the expression of , and lessened the positive signal of Calcofluor staining on the chorions, which suggests the reduction of a chitin-like substance (CLS) deposited on eggshells. Similarly, RNAi of or in constrained oviposition, decreased the expression of and , and reduced CLS contents in the resultant ovaries. Knockdown of or caused similar defective phenotypes, i.e., reduced oviposition and CLS contents in the ovaries. These results, for the first time, indicate that 20E signaling activates choriogenesis in two coleopteran species. Moreover, our findings suggest the deposition of a CLS on the chorions.
Topics: Animals; Coleoptera; RNA Interference; Signal Transduction; Female; Ecdysone; Insect Proteins; Oviposition; Egg Shell; Ovary; Receptors, Steroid
PubMed: 38674140
DOI: 10.3390/ijms25084555 -
Cells Apr 2024Chitinase 3-like 1 (also known as CHI3L1 or YKL-40) is a mammalian chitinase that has no enzymatic activity, but has the ability to bind to chitin, the polymer of... (Review)
Review
Chitinase 3-like 1 (also known as CHI3L1 or YKL-40) is a mammalian chitinase that has no enzymatic activity, but has the ability to bind to chitin, the polymer of N-acetylglucosamine (GlcNAc). Chitin is a component of fungi, crustaceans, arthropods including insects and mites, and parasites, but it is completely absent from mammals, including humans and mice. In general, chitin-containing organisms produce mammalian chitinases, such as CHI3L1, to protect the body from exogenous pathogens as well as hostile environments, and it was thought that it had a similar effect in mammals. However, recent studies have revealed that CHI3L1 plays a pathophysiological role by inducing anti-apoptotic activity in epithelial cells and macrophages. Under chronic inflammatory conditions such as inflammatory bowel disease and chronic obstructive pulmonary disease, many groups already confirmed that the expression of CHI3L1 is significantly induced on the apical side of epithelial cells, and activates many downstream pathways involved in inflammation and carcinogenesis. In this review article, we summarize the expression of CHI3L1 under chronic inflammatory conditions in various disorders and discuss the potential roles of CHI3L1 in those disorders on various cell types.
Topics: Humans; Chitinase-3-Like Protein 1; Animals; Inflammation; Chronic Disease
PubMed: 38667293
DOI: 10.3390/cells13080678