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1.
Biochim Biophys Acta Gen Subj ; 1867(1): 130250, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36228878

RESUMO

Metabolite sensing, a fundamental biological process, plays a key role in metabolic signaling circuit rewiring. Hexosamine biosynthetic pathway (HBP) is a glucose metabolic pathway essential for the synthesis of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), which senses key nutrients and integrally maintains cellular homeostasis. UDP-GlcNAc dynamically regulates protein N-glycosylation and O-linked-N-acetylglucosamine modification (O-GlcNAcylation). Dysregulated HBP flux leads to abnormal protein glycosylation, and contributes to cancer development and progression by affecting protein function and cellular signaling. Furthermore, O-GlcNAcylation regulates cellular signaling pathways, and its alteration is linked to various cancer characteristics. Additionally, recent findings have suggested a close association between HBP stimulation and cancer stemness; an elevated HBP flux promotes cancer cell conversion to cancer stem cells and enhances chemotherapy resistance via downstream signal activation. In this review, we highlight the prominent roles of HBP in metabolic signaling and summarize the recent advances in HBP and its downstream signaling, relevant to cancer.


Assuntos
Fenômenos Biológicos , Neoplasias , Humanos , Hexosaminas/metabolismo , Vias Biossintéticas , Acetilglucosamina/metabolismo , Neoplasias/metabolismo , Difosfato de Uridina
2.
Adv Neurobiol ; 29: 95-116, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255673

RESUMO

Glycoproteins carrying O-linked N-acetylgalactosamine, N-acetylglucosamine, mannose, fucose, glucose, and xylose are found in the nervous system. Lipids are glycosylated by distinct glycosylation enzymes as well. Membrane lipid, ceramide, is modified by the addition of either glucose or galactose to form glycosphingolipid, galactosylceramide, or glucosylceramide. Recent careful analyses by MS have identified glucosylated lipids of cholesterol and phosphatidic acid. These O-linked carbohydrate residues are found primarily on the outer surface of the plasma membrane or in the extracellular space. Their expression is cell or tissue specific and developmentally regulated. Due to their structural diversity, they play important roles in a variety of biological processes such as membrane transport, metabolic stress responses, cell-cell interactions and so on. Discoveries of human diseases associated with glycosylation enzyme deficits have proved modification of lipids and proteins with carbohydrates play critical roles in human health and disease in the nervous systems.


Assuntos
Acetilgalactosamina , Fucose , Humanos , Fucose/metabolismo , Acetilgalactosamina/metabolismo , Acetilglucosamina/metabolismo , Galactose/metabolismo , Manose , Glucosilceramidas , Xilose , Galactosilceramidas , Glicoconjugados/metabolismo , Carboidratos/análise , Glicoproteínas/metabolismo , Sistema Nervoso , Glucose , Ácidos Fosfatídicos
3.
Adv Neurobiol ; 29: 255-280, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255678

RESUMO

O-GlcNAc is the attachment of ß-N-acetylglucosamine to the hydroxyl group of serine and threonine in nuclear and cytoplasmic proteins. It is generally not further elongated but exists as a monosaccharide that can be rapidly added or removed. Thousands of proteins involved in gene transcription, protein translation and degradation as well as the regulation of signal transduction contain O-GlcNAc. Brain is one of the tissues where O-GlcNAc is the most highly expressed and deletion of neuronal O-GlcNAc leads to death early in development. O-GlcNAc is also important for normal adult brain function, where dynamic processes like learning and memory at least in part depend on the modification of specific proteins by O-GlcNAc. Conversely, too much or too little O-GlcNAc in the brain contributes to several disorders including obesity, intellectual disability and Alzheimer's disease. In this chapter, we describe the expression and regulation of O-GlcNAc in the nervous system.


Assuntos
Acetilglucosamina , Encéfalo , Acetilglucosamina/metabolismo , Encéfalo/metabolismo , Fenótipo , Serina , Monossacarídeos , Treonina
4.
Sci Rep ; 12(1): 19251, 2022 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-36357422

RESUMO

Posttranslational modifications (PTMs) on histone tails regulate eukaryotic gene expression by impacting the chromatin structure and by modulating interactions with other cellular proteins. One such PTM has been identified as serine and threonine glycosylation, the introduction of the ß-N-acetylglucosamine (GlcNAc) moiety on histone H3 tail at position Ser10 and Thr32. The addition of the ß-O-GlcNAc moiety on serine or threonine residues is facilitated by the O-GlcNAc transferase (OGT), and can be removed by the action of O-GlcNAcase (OGA). Conflicting reports on histone tail GlcNAc modification in vivo prompted us to investigate whether synthetic histone H3 tail peptides in conjunction with other PTMs are substrates for OGT and OGA in vitro. Our enzymatic assays with recombinantly expressed human OGT revealed that the unmodified and PTM-modified histone H3 tails are not substrates for OGT at both sites, Ser10 and Thr32. In addition, full length histone H3 was not a substrate for OGT. Conversely, our work demonstrates that synthetic peptides containing the GlcNAc functionality at Ser10 are substrates for recombinantly expressed human OGA, yielding deglycosylated histone H3 peptides. We also show that the catalytic domains of human histone lysine methyltransferases G9a, GLP and SETD7 and histone lysine acetyltransferases PCAF and GCN5 do somewhat tolerate glycosylated H3Ser10 close to lysine residues that undergo methylation and acetylation reactions, respectively. Overall, this work indicates that GlcNAcylation of histone H3 tail peptide in the presence of OGT does not occur in vitro.


Assuntos
Histonas , Lisina , Humanos , Histonas/metabolismo , Glicosilação , Lisina/metabolismo , N-Acetilglucosaminiltransferases/genética , Acetilglucosamina/metabolismo , Processamento de Proteína Pós-Traducional , Treonina/metabolismo , Peptídeos/metabolismo , Serina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo
5.
Cells ; 11(21)2022 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-36359905

RESUMO

The modification of nuclear, mitochondrial, and cytosolic proteins by O-linked ßN-acetylglucosamine (O-GlcNAc) has emerged as a dynamic and essential post-translational modification of mammalian proteins. O-GlcNAc is cycled on and off over 5000 proteins in response to diverse stimuli impacting protein function and, in turn, epigenetics and transcription, translation and proteostasis, metabolism, cell structure, and signal transduction. Environmental and physiological injury lead to complex changes in O-GlcNAcylation that impact cell and tissue survival in models of heat shock, osmotic stress, oxidative stress, and hypoxia/reoxygenation injury, as well as ischemic reperfusion injury. Numerous mechanisms that appear to underpin O-GlcNAc-mediated survival include changes in chaperone levels, impacts on the unfolded protein response and integrated stress response, improvements in mitochondrial function, and reduced protein aggregation. Here, we discuss the points at which O-GlcNAc is integrated into the cellular stress response, focusing on the roles it plays in the cardiovascular system and in neurodegeneration.


Assuntos
Acetilglucosamina , Processamento de Proteína Pós-Traducional , Animais , Acetilglucosamina/metabolismo , Glicosilação , Estresse Oxidativo , Transdução de Sinais/fisiologia , Proteínas/metabolismo , Mamíferos/metabolismo
6.
PLoS One ; 17(10): e0276285, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36288343

RESUMO

Protein posttranslational modifications (PTMs) by O-GlcNAc globally rise during pressure-overload hypertrophy (POH). However, a major knowledge gap exists on the specific proteins undergoing changes in O-GlcNAc levels during POH primarily because this PTM is low abundance and easily lost during standard mass spectrometry (MS) conditions used for protein identification. Methodologies have emerged to enrich samples for O-GlcNAcylated proteins prior to MS analysis. Accordingly, our goal was to identify the specific proteins undergoing changes in O-GlcNAc levels during POH. We used C57/Bl6 mice subjected to Sham or transverse aortic constriction (TAC) to create POH. From the hearts, we labelled the O-GlcNAc moiety with tetramethylrhodamine azide (TAMRA) before sample enrichment by TAMRA immunoprecipitation (IP). We used LC-MS/MS to identify and quantify the captured putative O-GlcNAcylated proteins. We identified a total of 700 putative O-GlcNAcylated proteins in Sham and POH. Two hundred thirty-three of these proteins had significantly increased enrichment in POH over Sham suggesting higher O-GlcNAc levels whereas no proteins were significantly decreased by POH. We examined two MS identified metabolic enzymes, CPT1B and the PDH complex, to validate by immunoprecipitation. We corroborated increased O-GlcNAc levels during POH for CPT1B and the PDH complex. Enzyme activity assays suggests higher O-GlcNAcylation increases CPT1 activity and decreases PDH activity during POH. In summary, we generated the first comprehensive list of proteins with putative changes in O-GlcNAc levels during POH. Our results demonstrate the large number of potential proteins and cellular processes affected by O-GlcNAc and serve as a guide for testing specific O-GlcNAc-regulated mechanisms during POH.


Assuntos
Acetilglucosamina , Azidas , Animais , Camundongos , Acetilglucosamina/metabolismo , Cromatografia Líquida , Hipertrofia , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Espectrometria de Massas em Tandem/métodos
7.
Int J Mol Sci ; 23(20)2022 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-36293310

RESUMO

ß-N-Acetylhexosaminidase from Talaromyces flavus (TfHex; EC 3.2.1.52) is an exo-glycosidase with dual activity for cleaving N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from carbohydrates. By targeting a mutation hotspot of the active site residue Glu332, we prepared a library of ten mutant variants with their substrate specificity significantly shifted towards GlcNAcase activity. Suitable mutations were identified by in silico methods. We optimized a microtiter plate screening method in the yeast Pichia pastoris expression system, which is required for the correct folding of tetrameric fungal ß-N-acetylhexosaminidases. While the wild-type TfHex is promiscuous with its GalNAcase/GlcNAcase activity ratio of 1.2, the best single mutant variant Glu332His featured an 8-fold increase in selectivity toward GlcNAc compared with the wild-type. Several prepared variants, in particular Glu332Thr TfHex, had significantly stronger transglycosylation capabilities than the wild-type, affording longer chitooligomers - they behaved like transglycosidases. This study demonstrates the potential of mutagenesis to alter the substrate specificity of glycosidases.


Assuntos
Acetilglucosamina , beta-N-Acetil-Hexosaminidases , beta-N-Acetil-Hexosaminidases/metabolismo , Especificidade por Substrato , Acetilglucosamina/metabolismo , Acetilgalactosamina/metabolismo , Cinética , Acetilglucosaminidase , Mutação
8.
Microbiol Spectr ; 10(5): e0129022, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36200915

RESUMO

Secretion of high-molecular-weight polysaccharides across the bacterial envelope is ubiquitous, as it enhances prokaryotic survival in (a)biotic settings. Such polymers are often assembled by Wzx/Wzy- or ABC transporter-dependent schemes implicating outer membrane (OM) polysaccharide export (OPX) proteins in cell-surface polymer translocation. In the social predatory bacterium Myxococcus xanthus, the exopolysaccharide (EPS) pathway WzaX, major spore coat (MASC) pathway WzaS, and biosurfactant polysaccharide (BPS) pathway WzaB were herein found to be truncated OPX homologues of Escherichia coli Wza lacking OM-spanning α-helices. Comparative genomics across all bacteria (>91,000 OPX proteins identified and analyzed), complemented with cryo-electron tomography cell-envelope analyses, revealed such "truncated" WzaX/S/B architecture to be the most common among three defined OPX-protein structural classes independent of periplasm thickness. Fold recognition and deep learning revealed the conserved M. xanthus proteins MXAN_7418/3226/1916 (encoded beside wzaX/S/B, respectively) to be integral OM ß-barrels, with structural homology to the poly-N-acetyl-d-glucosamine synthase-dependent pathway porin PgaA. Such bacterial porins were identified near numerous genes for all three OPX protein classes. Interior MXAN_7418/3226/1916 ß-barrel electrostatics were found to match properties of their associated polymers. With MXAN_3226 essential for MASC export, and MXAN_7418 herein shown to mediate EPS translocation, we have designated this new secretion machinery component "Wzp" (i.e., Wz porin), with the final step of M. xanthus EPS/MASC/BPS secretion across the OM now proposed to be mediated by WzpX/S/B (i.e., MXAN_7418/3226/1916). Importantly, these data support a novel and widespread secretion paradigm for polysaccharide biosynthesis pathways in which those containing OPX components that cannot span the OM instead utilize ß-barrel porins to mediate polysaccharide transport across the OM. IMPORTANCE Diverse bacteria assemble and secrete polysaccharides that alter their physiologies through modulation of motility, biofilm formation, and host immune system evasion. Most such pathways require outer membrane (OM) polysaccharide export (OPX) proteins for sugar-polymer transport to the cell surface. In the prototypic Escherichia coli Group-1-capsule biosynthesis system, eight copies of this canonical OPX protein cross the OM with an α-helix, forming a polysaccharide-export pore. Herein, we instead reveal that most OPX proteins across all bacteria lack this α-helix, raising questions as to the manner by which most secreted polysaccharides actually exit cells. In the model developmental bacterium Myxococcus xanthus, we show this process to depend on OPX-coupled OM-spanning ß-barrel porins, with similar porins encoded near numerous OPX genes in diverse bacteria. Knowledge of the terminal polysaccharide secretion step will enable development of antimicrobial compounds targeted to blocking polymer export from outside the cell, thus bypassing any requirements for antimicrobial compound uptake by the cell.


Assuntos
Proteínas de Escherichia coli , Porinas , Porinas/genética , Porinas/metabolismo , Membrana Externa Bacteriana , Polímeros/química , Polímeros/metabolismo , Acetilglucosamina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Polissacarídeos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Açúcares/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo
9.
ACS Appl Mater Interfaces ; 14(42): 47482-47490, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36240223

RESUMO

As an essential modification, O-linked ß-N-acetylglucosamine (O-GlcNAc) modulates the functions of many proteins. However, site-specific characterization of O-GlcNAcylated proteins remains challenging. Herein, an innovative material grafted with nitro-oxide (N→O) groups was designed for high affinity enrichment for O-GlcNAc peptides from native proteins. By testing with synthetic O-GlcNAc peptides and standard proteins, the synthesized material exhibited high affinity and selectivity. Based on the material prepared, we developed a workflow for site-specific analysis of O-GlcNAcylated proteins in complex samples. We performed O-GlcNAc proteomics with the PANC-1 cell line, a representative model for pancreatic ductal adenocarcinoma. In total 364 O-GlcNAc peptides from 267 proteins were identified from PANC-1 cells. Among them, 183 proteins were newly found to be O-GlcNAcylated in humans (with 197 O-GlcNAc sites newly reported). The materials and methods can be facilely applied for site-specific O-GlcNAc proteomics in other complex samples.


Assuntos
Acetilglucosamina , Nanosferas , Humanos , Acetilglucosamina/análise , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Ligação de Hidrogênio , Óxidos , Proteínas , Peptídeos
10.
Sci Rep ; 12(1): 16991, 2022 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-36216916

RESUMO

N-acetylglucosamine (GlcNAc) is a key component of glycans such as glycoprotein and the cell wall. GlcNAc kinase is an enzyme that transfers a phosphate onto GlcNAc to generate GlcNAc-6-phosphate, which can be a precursor for glycan synthesis. GlcNAc kinases have been found in a broad range of organisms, including pathogenic yeast, human and bacteria. However, this enzyme has never been discovered in Saccharomyces cerevisiae, a eukaryotic model. In this study, the first GlcNAc kinase from S. cerevisiae was identified and named Ngk1. The Km values of Ngk1 for GlcNAc and glucose were 0.11 mM and 71 mM, respectively, suggesting that Ngk1 possesses a high affinity for GlcNAc, unlike hexokinases. Ngk1 showed the GlcNAc phosphorylation activity with various nucleoside triphosphates, namely ATP, CTP, GTP, ITP, and UTP, as phosphoryl donors. Ngk1 is phylogenetically distant from known enzymes, as the amino acid sequence identity with others is only about 20% or less. The physiological role of Ngk1 in S. cerevisiae is also discussed.


Assuntos
Acetilglucosamina , Fosfotransferases (Aceptor do Grupo Álcool) , Saccharomyces cerevisiae , Acetilglucosamina/metabolismo , Trifosfato de Adenosina/metabolismo , Citidina Trifosfato/metabolismo , Glucose/metabolismo , Glicoproteínas/metabolismo , Guanosina Trifosfato/metabolismo , Nucleosídeos/metabolismo , Fosfatos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Polissacarídeos/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Uridina Trifosfato/metabolismo
11.
Int J Mol Sci ; 23(19)2022 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-36232558

RESUMO

Kidneys maintain internal milieu homeostasis through a well-regulated manipulation of body fluid composition. This task is performed by the correlation between structure and function in the nephron. Kidney diseases are chronic conditions impacting healthcare programs globally, and despite efforts, therapeutic options for its treatment are limited. The development of chronic degenerative diseases is associated with changes in protein O-GlcNAcylation, a post-translation modification involved in the regulation of diverse cell function. O-GlcNAcylation is regulated by the enzymatic balance between O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) which add and remove GlcNAc residues on target proteins, respectively. Furthermore, the hexosamine biosynthetic pathway provides the substrate for protein O-GlcNAcylation. Beyond its physiological role, several reports indicate the participation of protein O-GlcNAcylation in cardiovascular, neurodegenerative, and metabolic diseases. In this review, we discuss the impact of protein O-GlcNAcylation on physiological renal function, disease conditions, and possible future directions in the field.


Assuntos
Acetilglucosamina , N-Acetilglucosaminiltransferases , Acetilglucosamina/metabolismo , Hexosaminas/metabolismo , Homeostase , Rim/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional
12.
Glycobiology ; 32(12): 1153-1163, 2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36106687

RESUMO

N-glycans are modified by glycosyltransferases in the endoplasmic reticulum and Golgi apparatus. N-acetylglucosaminyltransferase IV (GnT-IV) is a Golgi-localized glycosyltransferase that synthesizes complex-type N-glycans in vertebrates. This enzyme attaches N-acetylglucosamine (GlcNAc) to the α-1,3-linked mannose branch of the N-glycan core structure via a ß-1,4 linkage. Deficiency of this enzyme is known to cause abnormal cellular functions, making it a vital enzyme for living organisms. However, there has been no report on its 3-dimensional structure to date. Here, we demonstrated that the C-terminal regions (named CBML) of human GnT-IVa and Bombyx mori ortholog have the ability to bind ß-N-acetylglucosamine. In addition, we determined the crystal structures of human CBML, B. mori CBML, and its complex with ß-GlcNAc at 1.97, 1.47, and 1.15 Å resolutions, respectively, and showed that they adopt a ß-sandwich fold, similar to carbohydrate-binding module family 32 (CBM32) proteins. The regions homologous to CBML (≥24% identity) were found in GnT-IV isozymes, GnT-IVb, and GnT-IVc (known as GnT-VI), and the structure of B. mori CBML in complex with ß-GlcNAc indicated that the GlcNAc-binding residues were highly conserved among these isozymes. These residues are also conserved with the GlcNAc-binding CBM32 domain of ß-N-acetylhexosaminidase NagH from Clostridium perfringens despite the low sequence identity (<20%). Taken together with the phylogenetic analysis, these findings indicate that these CBMLs may be novel CBM family proteins with GlcNAc-binding ability.


Assuntos
Acetilglucosamina , Isoenzimas , Animais , Humanos , Acetilglucosamina/metabolismo , Isoenzimas/metabolismo , Filogenia , N-Acetilglucosaminiltransferases/genética , Glicosiltransferases/metabolismo , Polissacarídeos/química , Manose/química
13.
Open Biol ; 12(9): 220215, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36099933

RESUMO

O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a nutrient-sensitive protein modification that alters the structure and function of a wide range of proteins involved in diverse cellular processes. Similar to phosphorylation, another protein modification that targets serine and threonine residues, O-GlcNAcylation occupancy on cellular proteins exhibits daily rhythmicity and has been shown to play critical roles in regulating daily rhythms in biology by modifying circadian clock proteins and downstream effectors. We recently reported that daily rhythm in global O-GlcNAcylation observed in Drosophila tissues is regulated via the integration of circadian and metabolic signals. Significantly, mistimed feeding, which disrupts coordination of these signals, is sufficient to dampen daily O-GlcNAcylation rhythm and is predicted to negatively impact animal biological rhythms and health span. In this review, we provide an overview of published and potential mechanisms by which metabolic and circadian signals regulate hexosamine biosynthetic pathway metabolites and enzymes, as well as O-GlcNAc processing enzymes to shape daily O-GlcNAcylation rhythms. We also discuss the significance of functional interactions between O-GlcNAcylation and other post-translational modifications in regulating biological rhythms. Finally, we highlight organ/tissue-specific cellular processes and molecular pathways that could be modulated by rhythmic O-GlcNAcylation to regulate time-of-day-specific biology.


Assuntos
Acetilglucosamina , Relógios Circadianos , Acetilglucosamina/metabolismo , Animais , Proteínas CLOCK/genética , Relógios Circadianos/fisiologia , Drosophila/metabolismo , Nutrientes , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo
14.
Biochim Biophys Acta Rev Cancer ; 1877(6): 188806, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36152903

RESUMO

O-GlcNAcylation is a posttranslational modification that attaches O-linked ß-N-acetylglucosamine (O-GlcNAc) to the serine and threonine residues of proteins. Such a glycosylation would alter the activities, stabilities, and interactions of target proteins that are functional in a wide range of biological processes and diseases. Accumulating evidence indicates that O-GlcNAcylation is tightly associated with hepatocellular carcinoma (HCC) in its onset, growth, invasion and metastasis, drug resistance, and stemness. Here we summarize the discoveries of the role of O-GlcNAcylation in HCC and its function mechanism, aiming to deepen our understanding of HCC pathology, generate more biomarkers for its diagnosis and prognosis, and offer novel molecular targets for its treatment.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Acetilglucosamina/metabolismo , Processamento de Proteína Pós-Traducional , Glicosilação
15.
DNA Repair (Amst) ; 119: 103394, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36095925

RESUMO

O-Linked ß-N-acetylglucosamine glycosylation (O-GlcNAcylation) to serine or threonine residues is a reversible and dynamic post-translational modification. O-GlcNAc transferase (OGT) is the only enzyme for O-GlcNAcylation, and is a potential cancer therapeutic target in combination with clastogenic (i.e., chromosomal breaking) therapeutics. Thus, we sought to examine the influence of O-GlcNAcylation on chromosomal break repair. Using a set of DNA double strand break (DSB) reporter assays, we found that the depletion of OGT, and its inhibition with a small molecule each caused a reduction in repair pathways that involve use of homology: RAD51-dependent homology-directed repair (HDR), and single strand annealing. In contrast, such OGT disruption did not obviously affect chromosomal break end joining, and furthermore caused an increase in homology-directed gene targeting. Such disruption in OGT also caused a reduction in clonogenic survival, as well as modifications to cell cycle profiles, particularly an increase in G1-phase cells. We also examined intermediate steps of HDR, finding no obvious effects on an assay for DSB end resection, nor for RAD51 recruitment into ionizing radiation induced foci (IRIF) in proliferating cells. However, we also found that the influence of OGT on HDR and homology-directed gene targeting were dependent on RAD52, and that OGT is important for RAD52 IRIF in proliferating cells. Thus, we suggest that OGT is important for regulation of HDR that is partially linked to RAD52 function.


Assuntos
Acetilglucosamina , Quebra Cromossômica , Acetilglucosamina/metabolismo , DNA , Humanos , N-Acetilglucosaminiltransferases , Serina/metabolismo , Treonina/metabolismo
16.
J Biomed Sci ; 29(1): 64, 2022 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-36058931

RESUMO

O-GlcNAcylation corresponds to the addition of N-Acetylglucosamine (GlcNAc) on serine or threonine residues of cytosolic, nuclear and mitochondrial proteins. This reversible modification is catalysed by a unique couple of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). OGT uses UDP-GlcNAc produced in the hexosamine biosynthesis pathway, to modify proteins. UDP-GlcNAc is at the cross-roads of several cellular metabolisms, including glucose, amino acids and fatty acids. Therefore, OGT is considered as a metabolic sensor that post-translationally modifies proteins according to nutrient availability. O-GlcNAcylation can modulate protein-protein interactions and regulate protein enzymatic activities, stability or subcellular localization. In addition, it can compete with phosphorylation on the same serine or threonine residues, or regulate positively or negatively the phosphorylation of adjacent residues. As such, O-GlcNAcylation is a major actor in the regulation of cell signaling and has been implicated in numerous physiological and pathological processes. A large body of evidence have indicated that increased O-GlcNAcylation participates in the deleterious effects of glucose (glucotoxicity) in metabolic diseases. However, recent studies using mice models with OGT or OGA knock-out in different tissues have shown that O-GlcNAcylation protects against various cellular stresses, and indicate that both increase and decrease in O-GlcNAcylation have deleterious effects on the regulation of energy homeostasis.


Assuntos
Acetilglucosamina , N-Acetilglucosaminiltransferases , Acetilglucosamina/metabolismo , Animais , Glucose , Homeostase , Camundongos , Camundongos Knockout , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Proteínas , Serina , Treonina , Difosfato de Uridina
17.
Front Endocrinol (Lausanne) ; 13: 943576, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36111295

RESUMO

Although traditionally considered a glucose metabolism-associated modification, the O-linked ß-N-Acetylglucosamine (O-GlcNAc) regulatory system interacts extensively with lipids and is required to maintain lipid homeostasis. The enzymes of O-GlcNAc cycling have molecular properties consistent with those expected of broad-spectrum environmental sensors. By direct protein-protein interactions and catalytic modification, O-GlcNAc cycling enzymes may provide both acute and long-term adaptation to stress and other environmental stimuli such as nutrient availability. Depending on the cell type, hyperlipidemia potentiates or depresses O-GlcNAc levels, sometimes biphasically, through a diversity of unique mechanisms that target UDP-GlcNAc synthesis and the availability, activity and substrate selectivity of the glycosylation enzymes, O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA). At the same time, OGT activity in multiple tissues has been implicated in the homeostatic regulation of systemic lipid uptake, storage and release. Hyperlipidemic patterns of O-GlcNAcylation in these cells are consistent with both transient physiological adaptation and feedback uninhibited obesogenic and metabolic dysregulation. In this review, we summarize the numerous interconnections between lipid and O-GlcNAc metabolism. These links provide insights into how the O-GlcNAc regulatory system may contribute to lipid-associated diseases including obesity and metabolic syndrome.


Assuntos
Acetilglucosamina , Glucose , Acetilglucosamina/metabolismo , Glicosilação , Lipídeos , Difosfato de Uridina/metabolismo
18.
Mol Med ; 28(1): 115, 2022 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-36104770

RESUMO

O-linked ß-D-N-acetylglucosamine (O-GlcNAc) is an important post-translational modification of serine or threonine residues on thousands of proteins in the nucleus and cytoplasm of all animals and plants. In eukaryotes, only two conserved enzymes are involved in this process. O-GlcNAc transferase is responsible for adding O-GlcNAc to proteins, while O-GlcNAcase is responsible for removing it. Aberrant O-GlcNAcylation is associated with a variety of human diseases, such as diabetes, cancer, neurodegenerative diseases, and cardiovascular diseases. Numerous studies have confirmed that O-GlcNAcylation is involved in the occurrence and progression of cancers in multiple systems throughout the body. It is also involved in regulating multiple cancer hallmarks, such as metabolic reprogramming, proliferation, invasion, metastasis, and angiogenesis. In this review, we first describe the process of O-GlcNAcylation and the structure and function of O-GlcNAc cycling enzymes. In addition, we detail the occurrence of O-GlcNAc in various cancers and the role it plays. Finally, we discuss the potential of O-GlcNAc as a promising biomarker and novel therapeutic target for cancer diagnosis, treatment, and prognosis.


Assuntos
Diabetes Mellitus , Neoplasias , Acetilglucosamina/metabolismo , Animais , Humanos , Neoplasias/metabolismo , Neoplasias/terapia , Neovascularização Patológica , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo
19.
Aging Cell ; 21(10): e13711, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36124412

RESUMO

Glucosamine feeding and genetic activation of the hexosamine biosynthetic pathway (HBP) have been linked to improved protein quality control and lifespan extension. However, as an energy sensor, the HBP has been implicated in tumor progression and diabetes. Given these opposing outcomes, it is imperative to explore the long-term effects of chronic HBP activation in mammals. Thus, we asked if HBP activation affects metabolism, coordination, memory, and survival in mice. N-acetyl-D-glucosamine (GlcNAc) supplementation in the drinking water had no adverse effect on weight in males but increased weight in young females. Glucose or insulin tolerance was not affected up to 20 months of age. Of note, we observed improved memory in young male mice supplemented with GlcNAc. Survival was not changed by GlcNAc treatment. To assess the effects of genetic HBP activation, we overexpressed the pathway's key enzyme GFAT1 and a constitutively activated mutant form in all mouse tissues. We detected elevated levels of the HBP product UDP-GlcNAc in mouse brains, but did not find any effects on behavior, memory, or survival. Together, while dietary GlcNAc supplementation did not extend survival in mice, it positively affected memory and is generally well tolerated.


Assuntos
Água Potável , Insulinas , Acetilglucosamina/metabolismo , Animais , Feminino , Glucosamina , Glucose/metabolismo , Glicosilação , Hexosaminas/metabolismo , Insulinas/metabolismo , Longevidade , Masculino , Mamíferos , Camundongos , Difosfato de Uridina/metabolismo
20.
Nature ; 610(7931): 402-408, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36131020

RESUMO

Chitin, the most abundant aminopolysaccharide in nature, is an extracellular polymer consisting of N-acetylglucosamine (GlcNAc) units1. The key reactions of chitin biosynthesis are catalysed by chitin synthase2-4, a membrane-integrated glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain. However, the precise mechanism of this process has yet to be elucidated. Here we report five cryo-electron microscopy structures of a chitin synthase from the devastating soybean root rot pathogenic oomycete Phytophthora sojae (PsChs1). They represent the apo, GlcNAc-bound, nascent chitin oligomer-bound, UDP-bound (post-synthesis) and chitin synthase inhibitor nikkomycin Z-bound states of the enzyme, providing detailed views into the multiple steps of chitin biosynthesis and its competitive inhibition. The structures reveal the chitin synthesis reaction chamber that has the substrate-binding site, the catalytic centre and the entrance to the polymer-translocating channel that allows the product polymer to be discharged. This arrangement reflects consecutive key events in chitin biosynthesis from UDP-GlcNAc binding and polymer elongation to the release of the product. We identified a swinging loop within the chitin-translocating channel, which acts as a 'gate lock' that prevents the substrate from leaving while directing the product polymer into the translocating channel for discharge to the extracellular side of the cell membrane. This work reveals the directional multistep mechanism of chitin biosynthesis and provides a structural basis for inhibition of chitin synthesis.


Assuntos
Quitina , Microscopia Crioeletrônica , Acetilglucosamina/metabolismo , Aminoglicosídeos/farmacologia , Sítios de Ligação , Membrana Celular/metabolismo , Quitina/biossíntese , Quitina/química , Quitina/metabolismo , Quitina/ultraestrutura , Quitina Sintase/metabolismo , Phytophthora/enzimologia , Difosfato de Uridina/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismo
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