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The Journal of Biological Chemistry Dec 2014O-GlcNAc signaling is regulated by an opposing pair of enzymes: O-GlcNAc transferase installs and O-GlcNAcase (OGA) removes the modification from proteins. The dynamics... (Review)
Review
O-GlcNAc signaling is regulated by an opposing pair of enzymes: O-GlcNAc transferase installs and O-GlcNAcase (OGA) removes the modification from proteins. The dynamics and regulation of this process are only beginning to be understood as the physiological functions of both enzymes are being probed using genetic and pharmacological approaches. This minireview charts the discovery and functional and structural analysis of OGA and summarizes the insights gained from recent studies using OGA inhibition, gene knock-out, and overexpression. We identify several areas of "known unknowns" that would benefit from future research, such as the enigmatic C-terminal domain of OGA.
Topics: Acetylglucosamine; Animals; Enzyme Inhibitors; Humans; Signal Transduction; Substrate Specificity; beta-N-Acetylhexosaminidases
PubMed: 25336650
DOI: 10.1074/jbc.R114.609198 -
Journal of the American Chemical Society Mar 2022Posttranslational modifications alter the biophysical properties of proteins and thereby influence cellular physiology. One emerging manner by which such modifications...
Posttranslational modifications alter the biophysical properties of proteins and thereby influence cellular physiology. One emerging manner by which such modifications regulate protein functions is through their ability to perturb protein stability. Despite the increasing interest in this phenomenon, there are few methods that enable global interrogation of the biophysical effects of posttranslational modifications on the proteome. Here, we describe an unbiased proteome-wide approach to explore the influence of protein modifications on the thermodynamic stability of thousands of proteins in parallel. We apply this profiling strategy to study the effects of O-linked -acetylglucosamine (O-GlcNAc), an abundant modification found on hundreds of proteins in mammals that has been shown in select cases to stabilize proteins. Using this thermal proteomic profiling strategy, we identify a set of 72 proteins displaying O-GlcNAc-dependent thermostability and validate this approach using orthogonal methods targeting specific proteins. These collective observations reveal that the majority of proteins influenced by O-GlcNAc are, surprisingly, destabilized by O-GlcNAc and cluster into distinct macromolecular complexes. These results establish O-GlcNAc as a bidirectional regulator of protein stability and provide a blueprint for exploring the impact of any protein modification on the meltome of, in principle, any organism.
Topics: Acetylglucosamine; Animals; Mammals; Protein Processing, Post-Translational; Proteome; Proteomics
PubMed: 35230102
DOI: 10.1021/jacs.1c10621 -
Molecular Medicine (Cambridge, Mass.) Sep 2022O-linked β-D-N-acetylglucosamine (O-GlcNAc) is an important post-translational modification of serine or threonine residues on thousands of proteins in the nucleus and... (Review)
Review
O-linked β-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.
Topics: Acetylglucosamine; Animals; Diabetes Mellitus; Humans; Neoplasms; Neovascularization, Pathologic; Protein Processing, Post-Translational; Proteins
PubMed: 36104770
DOI: 10.1186/s10020-022-00544-y -
Cell Metabolism Aug 2014The nutrient sensor, O-linked N-acetylglucosamine (O-GlcNAc), cycles on and off nuclear and cytosolic proteins to regulate many cellular processes, including... (Review)
Review
The nutrient sensor, O-linked N-acetylglucosamine (O-GlcNAc), cycles on and off nuclear and cytosolic proteins to regulate many cellular processes, including transcription and signaling. Dysregulated O-GlcNAcylation and its interplay with phosphorylation contribute to the etiology of diabetes, cancer, and neurodegeneration. Herein, we review recent findings about O-GlcNAc's regulation of cell physiology.
Topics: AMP-Activated Protein Kinases; Acetylglucosamine; Diabetes Mellitus; Epigenomics; Glycosylation; Humans; Neoplasms; Signal Transduction; Transcription, Genetic
PubMed: 25100062
DOI: 10.1016/j.cmet.2014.07.014 -
Acta Crystallographica. Section F,... Aug 2018The lacto-N-biose I (Galβ1-3GlcNAc; LNB) disaccharide is present as a core unit of type-1 blood group antigens of animal glycoconjugates and milk oligosaccharides.... (Review)
Review
The lacto-N-biose I (Galβ1-3GlcNAc; LNB) disaccharide is present as a core unit of type-1 blood group antigens of animal glycoconjugates and milk oligosaccharides. Type-1 antigens often serve as cell-surface receptors for infection by pathogens. LNB in human milk oligosaccharides functions as a prebiotic for bifidobacteria and plays a key role in the symbiotic relationship of commensal gut microbes in infants. Protein Data Bank (PDB) entries exhibiting the LNB unit were investigated using the GlycoMapsDB web tool. There are currently 159 β-LNB and nine α-LNB moieties represented in ligands in the database. β-LNB and α-LNB moieties occur in 74 and six PDB entries, respectively, as NCS copies. The protein and enzyme structures are from various organisms including humans (galectins), viruses (haemagglutinin and capsid proteins), a pathogenic fungus, a parasitic nematode and protist, pathogenic bacteria (adhesins) and a symbiotic bacterium (a solute-binding protein of an ABC transporter). The conformations of LNB-containing glycans in enzymes vary significantly according to their mechanism of substrate recognition and catalysis. Analysis of glycosidic bond conformations indicated that the binding modes are significantly different in proteins adapted for modified or unmodified glycans.
Topics: Acetylglucosamine; Animals; Blood Group Antigens; Crystallography, X-Ray; Databases, Protein; Humans; Protein Conformation
PubMed: 30084396
DOI: 10.1107/S2053230X18006568 -
Nature Communications May 2022All living organisms have the ability to sense nutrient levels to coordinate cellular metabolism. Despite the importance of nutrient-sensing pathways that detect the...
All living organisms have the ability to sense nutrient levels to coordinate cellular metabolism. Despite the importance of nutrient-sensing pathways that detect the levels of amino acids and glucose, how the availability of these two types of nutrients is integrated is unclear. Here, we show that glucose availability regulates the central nutrient effector mTORC1 through intracellular leucine sensor leucyl-tRNA synthetase 1 (LARS1). Glucose starvation results in O-GlcNAcylation of LARS1 on residue S1042. This modification inhibits the interaction of LARS1 with RagD GTPase and reduces the affinity of LARS1 for leucine by promoting phosphorylation of its leucine-binding site by the autophagy-activating kinase ULK1, decreasing mTORC1 activity. The lack of LARS1 O-GlcNAcylation constitutively activates mTORC1, supporting its ability to sense leucine, and deregulates protein synthesis and leucine catabolism under glucose starvation. This work demonstrates that LARS1 integrates leucine and glucose availability to regulate mTORC1 and the metabolic fate of leucine.
Topics: Acetylglucosamine; Autophagy; Glucose; Humans; Leucine; Leucine-tRNA Ligase; Mechanistic Target of Rapamycin Complex 1
PubMed: 35614056
DOI: 10.1038/s41467-022-30696-8 -
Frontiers in Immunology 2022The Apextrin C-terminal (ApeC) domain is a new protein domain largely specific to aquatic invertebrates. In amphioxus, a short-form ApeC-containing protein (ACP) family...
The Apextrin C-terminal (ApeC) domain is a new protein domain largely specific to aquatic invertebrates. In amphioxus, a short-form ApeC-containing protein (ACP) family is capable of binding peptidoglycan (PGN) and agglutinating bacteria its ApeC domain. However, the functions of ApeC in other phyla remain unknown. Here we examined 130 ACPs from gastropods and bivalves, the first and second biggest mollusk classes. They were classified into nine groups based on their phylogenetics and architectures, including three groups of short-form ACPs, one group of apextrins and two groups of ACPs of complex architectures. No groups have orthologs in other phyla and only four groups have members in both gastropods and bivalves, suggesting that mollusk ACPs are highly diversified. We selected one bivalve ACP (CgACP1; from the oyster ) and one gastropod ACP (BgACP1; from the snail ) for functional experiments. Both are highly-expressed, secreted short-form ACPs and hence comparable to the amphioxus ACPs previously reported. We found that recombinant CgACP1 and BgACP1 bound with yeasts and several bacteria with different affinities. They also agglutinated these microbes, but showed no inhibiting or killing effects. Further analyses show that both ACPs had high affinities to the Lys-type PGN from S. but weak or no affinities to the DAP-type PGN from Bacillus . Both recombinant ACPs displayed weak or no affinities to other microbial cell wall components, including lipopolysaccharide (LPS), lipoteichoic acid (LTA), zymosan A, chitin, chitosan and cellulose, as well as to several PGN moieties, including muramyl dipeptide (MDP), N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). Besides, CgACP1 had the highest expression in the gill and could be greatly up-regulated quickly after bacterial challenge. This is reminiscent of the amphioxus ACP1/2 which serve as essential mucus lectins in the gill. Taken together, the current findings from mollusk and amphioxus ACPs suggest several basic common traits for the ApeC domains, including the high affinity to Lys-type PGN, the bacterial binding and agglutinating capacity, and the role as mucus proteins to protect the mucosal surface.
Topics: Animals; Peptidoglycan; Lipopolysaccharides; Acetylmuramyl-Alanyl-Isoglutamine; Staphylococcus aureus; Acetylglucosamine; Zymosan; Chitosan; Lancelets; Bacteria; Cell Wall; Lectins; Mollusca; Cellulose
PubMed: 36275759
DOI: 10.3389/fimmu.2022.971883 -
Current Protocols Jul 2021This protocol enables identification of the interaction partners of O-GlcNAcylated proteins. The method involves the introduction of the diazirine photocrosslinker onto...
This protocol enables identification of the interaction partners of O-GlcNAcylated proteins. The method involves the introduction of the diazirine photocrosslinker onto the O-GlcNAc modification within living cells. The photocrosslinker is activated by UV light to yield covalent crosslinking between O-GlcNAcylated proteins and neighboring molecules. The binding partners can be further characterized by immunoblot or proteomics mass spectrometry methods. The benefits of using the photocrosslinker include the capacity to trap low-affinity binding interactions and the ability to selectively target the interaction partners of the O-GlcNAcylated form of the protein of interest. © 2021 Wiley Periodicals LLC. Basic Protocol 1: In-cell production and crosslinking of O-GlcNDAzylated proteins Basic Protocol 2: Immunoblot analysis to assess O-GlcNDAz crosslinking Support Protocol: Detection of UDP-GlcNDAz from cell lysates.
Topics: Acetylglucosamine; Diazomethane; Mass Spectrometry; Protein Processing, Post-Translational; Proteins
PubMed: 34288588
DOI: 10.1002/cpz1.201 -
Glycobiology Aug 2014O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation, the covalent attachment of N-acetylglucosamine to serine and threonine residues of proteins, is a...
O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation, the covalent attachment of N-acetylglucosamine to serine and threonine residues of proteins, is a post-translational modification that shares many features with protein phosphorylation. O-GlcNAc is essential for cell survival and plays important role in many biological processes (e.g. transcription, translation, cell division) and human diseases (e.g. diabetes, Alzheimer's disease, cancer). However, detection of O-GlcNAc is challenging. Here, a method for O-GlcNAc detection using in vitro sulfation with two N-acetylglucosamine (GlcNAc)-specific sulfotransferases, carbohydrate sulfotransferase 2 and carbohydrate sulfotransferase 4, and the radioisotope (35)S is described. Sulfation on free GlcNAc is first demonstrated, and then on O-GlcNAc residues of peptides as well as nuclear and cytoplasmic proteins. It is also demonstrated that the sulfation on O-GlcNAc is sensitive to OGT and O-β-N-acetylglucosaminidase treatment. The labeled samples are separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography. Overall, the method is sensitive, specific and convenient.
Topics: Acetylglucosamine; Acetylglucosaminidase; Glycosylation; HEK293 Cells; Humans; Sulfates; Sulfotransferases; Carbohydrate Sulfotransferases
PubMed: 24799377
DOI: 10.1093/glycob/cwu037 -
The Journal of Cell Biology Mar 2015Unlike the complex glycans decorating the cell surface, the O-linked β-N-acetyl glucosamine (O-GlcNAc) modification is a simple intracellular Ser/Thr-linked... (Review)
Review
Unlike the complex glycans decorating the cell surface, the O-linked β-N-acetyl glucosamine (O-GlcNAc) modification is a simple intracellular Ser/Thr-linked monosaccharide that is important for disease-relevant signaling and enzyme regulation. O-GlcNAcylation requires uridine diphosphate-GlcNAc, a precursor responsive to nutrient status and other environmental cues. Alternative splicing of the genes encoding the O-GlcNAc cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) yields isoforms targeted to discrete sites in the nucleus, cytoplasm, and mitochondria. OGT and OGA also partner with cellular effectors and act in tandem with other posttranslational modifications. The enzymes of O-GlcNAc cycling act preferentially on intrinsically disordered domains of target proteins impacting transcription, metabolism, apoptosis, organelle biogenesis, and transport.
Topics: Acetylglucosamine; Alternative Splicing; Animals; Cell Nucleus; Cytoplasm; Humans; Mitochondria; N-Acetylglucosaminyltransferases; Protein Isoforms; Protein Processing, Post-Translational; Stem Cells; Uridine Diphosphate
PubMed: 25825515
DOI: 10.1083/jcb.201501101