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1.
Sci Rep ; 10(1): 15938, 2020 09 29.
Article in English | MEDLINE | ID: mdl-32994436

ABSTRACT

In bacteria, glucosamine-6-phosphate (GlcN6P) synthase, GlmS, is an enzyme required for the synthesis of Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a precursor of peptidoglycan. In Bacillus subtilis, an UDP-GlcNAc binding protein, GlmR (formerly YvcK), essential for growth on non-glycolytic carbon sources, has been proposed to stimulate GlmS activity; this activation could be antagonized by UDP-GlcNAc. Using purified proteins, we demonstrate that GlmR directly stimulates GlmS activity and the presence of UDP-GlcNAc (at concentrations above 0.1 mM) prevents this regulation. We also showed that YvcJ, whose gene is associated with yvcK (glmR), interacts with GlmR in an UDP-GlcNAc dependent manner. Strains producing GlmR variants unable to interact with YvcJ show decreased transformation efficiency similar to that of a yvcJ null mutant. We therefore propose that, depending on the intracellular concentration of UDP-GlcNAc, GlmR interacts with either YvcJ or GlmS. When UDP-GlcNAc concentration is high, this UDP-sugar binds to YvcJ and to GlmR, blocking the stimulation of GlmS activity and driving the interaction between GlmR and YvcJ to probably regulate the cellular role of the latter. When the UDP-GlcNAc level is low, GlmR does not interact with YvcJ and thus does not regulate its cellular role but interacts with GlmS to stimulate its activity.


Subject(s)
Bacillus subtilis/metabolism , Uridine Diphosphate N-Acetylglucosamine/genetics , Uridine Diphosphate N-Acetylglucosamine/metabolism , Amino Acid Sequence , Bacterial Proteins/metabolism , Cell Wall/metabolism , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/metabolism , Peptidoglycan/metabolism , Uridine Diphosphate/metabolism , Uridine Diphosphate N-Acetylglucosamine/physiology
3.
Sci STKE ; 2005(312): re13, 2005 Nov 29.
Article in English | MEDLINE | ID: mdl-16317114

ABSTRACT

A dynamic cycle of addition and removal of O-linked N-acetylglucosamine (O-GlcNAc) at serine and threonine residues is emerging as a key regulator of nuclear and cytoplasmic protein activity. Like phosphorylation, protein O-GlcNAcylation dramatically alters the posttranslational fate and function of target proteins. Indeed, O-GlcNAcylation may compete with phosphorylation for certain Ser/Thr target sites. Like kinases and phosphatases, the enzymes of O-GlcNAc metabolism are highly compartmentalized and regulated. Yet, O-GlcNAc addition is subject to an additional and unique level of metabolic control. O-GlcNAc transfer is the terminal step in a "hexosamine signaling pathway" (HSP). In the HSP, levels of uridine 5'-diphosphate (UDP)-GlcNAc respond to nutrient excess to activate O-GlcNAcylation. Removal of O-GlcNAc may also be under similar metabolic regulation. Differentially targeted isoforms of the enzymes of O-GlcNAc metabolism allow the participation of O-GlcNAc in diverse intracellular functions. O-GlcNAc addition and removal are key to histone remodeling, transcription, proliferation, apoptosis, and proteasomal degradation. This nutrient-responsive signaling pathway also modulates important cellular pathways, including the insulin signaling cascade in animals and the gibberellin signaling pathway in plants. Alterations in O-GlcNAc metabolism are associated with various human diseases including diabetes mellitus and neurodegeneration. This review will focus on current approaches to deciphering the "O-GlcNAc code" in order to elucidate how O-GlcNAc participates in its diverse functions. This ongoing effort requires analysis of the enzymes of O-GlcNAc metabolism, their many targets, and how the O-GlcNAc modification may be regulated.


Subject(s)
Acetylglucosamine/physiology , Hexosamines/physiology , Protein Processing, Post-Translational/physiology , Signal Transduction/physiology , Uridine Diphosphate N-Acetylglucosamine/physiology , Acetylglucosamine/analysis , Acetylglucosaminidase/deficiency , Acetylglucosaminidase/genetics , Acetylglucosaminidase/physiology , Animals , Antigens, Neoplasm , Caenorhabditis elegans Proteins/physiology , Computational Biology , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/physiopathology , Genetic Predisposition to Disease , Histone Acetyltransferases/physiology , Humans , Hyaluronoglucosaminidase , Insulin Resistance , Intracellular Signaling Peptides and Proteins/physiology , Mammals , Mice , Mice, Transgenic , Multienzyme Complexes/physiology , N-Acetylglucosaminyltransferases/physiology , Neoplasm Proteins/deficiency , Neoplasm Proteins/genetics , Neoplasm Proteins/physiology , Neurodegenerative Diseases/physiopathology , Plant Proteins/physiology , Stress, Physiological/metabolism , beta-N-Acetylhexosaminidases
4.
Biochem J ; 360(Pt 2): 401-12, 2001 Dec 01.
Article in English | MEDLINE | ID: mdl-11716769

ABSTRACT

Glutamine:fructose-6-phosphate aminotransferase (GFAT; EC 2.6.1.16) expression is tightly regulated in the context of amino sugar synthesis in many organisms from yeast to humans by transcriptional and post-translational processes. We have cloned the cDNA of the GFAT1 of Drosophila melanogaster (Dmel/Gfat1). One of the two putative protein kinase A (PKA) phosphorylation sites proposed for the regulation of human GFAT1 [Zhou, Huynh, Hoffmann, Crook, Daniels, Gulve and McClain (1998) Diabetes 47, 1836-1840] is conserved in Dmel/GFAT1. In the other one the reactive serine has been converted to a cysteine, making further access by PKA unlikely. The Dmel/Gfat1 gene is localized at position 81F on the right arm of chromosome 3. By whole-mount in situ hybridization specific expression of Dmel/GFAT1 was detected in embryonic chitin-synthesizing tissues and in the corpus cells of salivary glands from late third larval instar. Expressing Dmel/GFAT1 in yeast we showed that Dmel/GFAT1 activity is controlled by UDP-N-acetylglucosamine and PKA in the yeast total protein extract system. We propose a model for the independent regulation of the Dmel/GFAT1 enzyme by feedback inhibition and PKA.


Subject(s)
Cyclic AMP/physiology , Drosophila melanogaster/enzymology , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/metabolism , Uridine Diphosphate N-Acetylglucosamine/physiology , Amino Acid Sequence , Animals , Blotting, Northern , Cloning, Molecular , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA, Complementary/analysis , DNA, Complementary/isolation & purification , Drosophila melanogaster/genetics , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic , Genes, Insect , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/antagonists & inhibitors , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/biosynthesis , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/genetics , In Situ Hybridization , Molecular Sequence Data , Phosphorylation , Promoter Regions, Genetic , Sequence Analysis, DNA , Sequence Analysis, Protein , Transcription Factors/genetics , Transcription Factors/metabolism
5.
Vet Pathol ; 33(1): 1-13, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8826001

ABSTRACT

A 7-month-old female cat was seen for abnormal facial features and abnormality of gait. Facial dysmorphism, large paws in relation to body size, dysostosis multiplex, and poor growth were noted, and mucopolysaccharidosis was suspected. A negative urine test for sulfated glycosaminoglycans and extreme stiffness of skin indicated a mucolipidosis hitherto unknown in animals. Deficiency of UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase, EC 2.7.8.17) activity was demonstrated in leukocytes and cultured fibroblasts, which had the appearance of inclusion cells (I-cells). Activities of a set of lysosomal hydrolases were abnormally low in fibroblasts and excessive in blood plasma. Postmortem morphology revealed lysosomal inclusions predominantly in fibroblasts but also in endothelial cells and chondrocytes, i.e., in cells of mesenchymal origin. Storage lysosomes contained oligosaccharides, mucopolysaccharides, and lipids. Tissues most affected were bones, cartilage, skin, and other connective tissues such as those in heart valves, aortic wall, and vocal cords. Parenchymal cells of liver and kidney were unaffected, as was skeletal muscle. Only a few of the cerebral cortical neurons had lipid inclusions; in sciatic nerve some axons were affected, but other peripheral nerves were normal. There were striking clinical, biochemical, and morphologic similarities between the disorder in this cat and the human I-cell disease.


Subject(s)
Cat Diseases/diagnosis , Cat Diseases/metabolism , Cats/metabolism , Mucolipidoses/veterinary , Animals , Aorta/pathology , Body Constitution/physiology , Bone and Bones/diagnostic imaging , Bone and Bones/pathology , Cat Diseases/pathology , Cats/growth & development , Cats/physiology , Disease Models, Animal , Female , Gait/physiology , Glycosaminoglycans/urine , Kidney/pathology , Kidney/ultrastructure , Leukocytes/chemistry , Leukocytes/pathology , Liver/pathology , Liver/ultrastructure , Mucolipidoses/diagnosis , Mucolipidoses/metabolism , Radiography , Retinal Degeneration/pathology , Retinal Degeneration/physiopathology , Sciatic Nerve/pathology , Sciatic Nerve/ultrastructure , Skin/pathology , Skin/ultrastructure , Transferases (Other Substituted Phosphate Groups)/analysis , Transferases (Other Substituted Phosphate Groups)/deficiency , Transferases (Other Substituted Phosphate Groups)/physiology , Uridine Diphosphate N-Acetylglucosamine/analysis , Uridine Diphosphate N-Acetylglucosamine/deficiency , Uridine Diphosphate N-Acetylglucosamine/physiology
6.
Anticancer Res ; 14(3A): 793-8, 1994.
Article in English | MEDLINE | ID: mdl-8074480

ABSTRACT

NMR spectroscopy was used to analyse perchloric acid extracts of normal human brain, murine brain cell cultures, glioblastoma tissue and the glioblastoma cell line U-87. 1H NMR spectra revealed the presence of elevated levels of UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine in glioblastoma extracts and the glioblastoma cell line U-87, in comparison with normal brain tissue and primary cell cultures of neurons and astrocytes. UDP-N-acetylhexosamines appear to accumulate in cells that are unable to differentiate. Furthermore, it was found that the culture medium had an effect on the concentration of UDP-N-acetygalactosamine in the glioblastoma cell line. Hypotaurine, previously only associated with oligodendrocytes, has been identified in astrocyte cultures and in cerebellar granule cells. In normal brain it was not observed by NMR spectroscopy, but was easily detectable in glioblastoma tissue extracts. UDP-N-acetylhexoseamines and hypotaurine might be useful markers for brain pathology and play a role in cell differentiation and cell division.


Subject(s)
Brain Chemistry , Glioblastoma/chemistry , Taurine/analogs & derivatives , Uridine Diphosphate N-Acetylgalactosamine/analysis , Uridine Diphosphate N-Acetylglucosamine/analysis , Animals , Cells, Cultured , Humans , Magnetic Resonance Spectroscopy , Mice , Taurine/analysis , Taurine/physiology , Uridine Diphosphate N-Acetylgalactosamine/physiology , Uridine Diphosphate N-Acetylglucosamine/physiology
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