scholarly journals The Metabolic Chemical Reporter 6-Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of O-GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by O-Glucose

2018 ◽  
Vol 140 (23) ◽  
pp. 7092-7100 ◽  
Author(s):  
Narek Darabedian ◽  
Jinxu Gao ◽  
Kelly N. Chuh ◽  
Christina M. Woo ◽  
Matthew R. Pratt
Keyword(s):  
Protein Modification ◽  
Intracellular Protein ◽  
Chemical Reporter ◽  
Substrate Promiscuity ◽  
Glcnac Transferase

scholarly journals The Metabolic Chemical Reporter Ac46AzGal Could Incorporate Intracellular Protein Modification in the Form of UDP-6AzGlc Mediated by OGT and Enzymes in the Leloir Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Jiajia Wang ◽  
Biao Dou ◽  
Lu Zheng ◽  
Wei Cao ◽  
Peiyu Dong ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Protein Modification ◽  
Time Dependent ◽  
Dependent Manner ◽  
Intracellular Protein ◽  
Galactose Metabolism ◽  
Naturally Occurring ◽  
Leloir Pathway ◽  
Chemical Reporter ◽  
Complex Glycans

Galactose is a naturally occurring monosaccharide used to build complex glycans that has not been targeted for labeling as a metabolic reporter. Here, we characterize the cellular modification of proteins by using Ac46AzGal in a dose- and time-dependent manner. It is noted that a vast majority of this labeling of Ac46AzGal occurs intracellularly in a range of mammalian cells. We also provided evidence that this labeling is dependent on not only the enzymes of OGT responsible for O-GlcNAcylation but also the enzymes of GALT and GALE in the Leloir pathway. Notably, we discover that Ac46AzGal is not the direct substrate of OGT, and the labeling results may attribute to UDP-6AzGlc after epimerization of UDP-6AzGal via GALE. Together, these discoveries support the conclusion that Ac46AzGal as an analogue of galactose could metabolically label intracellular O-glycosylation modification, raising the possibility of characterization with impaired functions of the galactose metabolism in the Leloir pathway under certain conditions, such as galactosemias.

scholarly journals Intracellular protein modification associated with altered T cell functions in autoimmunity

2006 ◽  
Vol 177 (12) ◽  
pp. 8878.1-8878
Author(s):  
M.-L. Yang ◽  
H. A. Doyle ◽  
R. J. Gee ◽  
J. D. Lowenson ◽  
S. Clarke ◽  
...  
Keyword(s):  
T Cell ◽  
Protein Modification ◽  
Intracellular Protein ◽  
Cell Functions

Role of O-Linked N-acetylglucosamine (O-GlcNAc) Protein Modification in Cellular (Patho)Physiology

2020 ◽  
Author(s):  
John C. Chatham ◽  
Jianhua Zhang ◽  
Adam Raymond Wende
Keyword(s):  
Chromatin Remodeling ◽  
Protein Modification ◽  
Protein Localization ◽  
Cellular Functions ◽  
Secretory Pathways ◽  
Cellular Physiology ◽  
Cytoplasmic Proteins ◽  
Wide Range ◽  
Glcnac Transferase

In the mid 1980s, the identification of serine and threonine residues on nuclear and cytoplasmic proteins modified by an O-linkage by a N-acetylglucosamine moiety (O-GlcNAc) overturned the widely held assumption that glycosylation only occurred in the endoplasmic reticulum, Golgi apparatus, and secretory pathways. In contrast to traditional glycosylation, the O-GlcNAc modification does not lead to complex branched glycan structures and is rapidly cycled on and off proteins by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Since its discovery O-GlcNAcylation has been shown to contribute to numerous cellular functions including signaling, protein localization and stability, transcription, chromatin remodeling, mitochondrial function, and cell survival. Dysregulation in O-GlcNAc cycling has been implicated in the progression of a wide range of diseases such as diabetes, diabetic complications, cancer, cardiovascular, and neurodegenerative diseases. This review will outline our current understanding of the processes involved in regulating O-GlcNAc turnover, the role of O-GlcNAcylation in regulating cellular physiology, and how dysregulation in O-GlcNAc cycling contributes to pathophysiological processes.

scholarly journals Correction to “An Alkyne–Aspirin Chemical Reporter for the Detection of Aspirin-Dependent Protein Modification in Living Cells”

2018 ◽  
Vol 140 (14) ◽  
pp. 4954-4954
Author(s):  
Leslie A. Bateman ◽  
Balyn W. Zaro ◽  
Stephanie M. Miller ◽  
Matthew R. Pratt
Keyword(s):  
Protein Modification ◽  
Living Cells ◽  
Chemical Reporter ◽  
Dependent Protein

scholarly journals Intracellular Protein Modification Associated with Altered T Cell Functions in Autoimmunity

2006 ◽  
Vol 177 (7) ◽  
pp. 4541-4549 ◽  
Author(s):  
Mei-Ling Yang ◽  
Hester A. Doyle ◽  
Renelle J. Gee ◽  
Jonathan D. Lowenson ◽  
Steven Clarke ◽  
...  
Keyword(s):  
T Cell ◽  
Protein Modification ◽  
Intracellular Protein ◽  
Cell Functions

scholarly journals Protein O-GlcNAcylation Promotes Trophoblast Differentiation at Implantation

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2246
Author(s):  
Peter T. Ruane ◽  
Cheryl M. J. Tan ◽  
Daman J. Adlam ◽  
Susan J. Kimber ◽  
Daniel R. Brison ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Protein Modification ◽  
Fusion Gene ◽  
Embryo Implantation ◽  
Adult Health ◽  
Trophoblast Differentiation ◽  
Human Trophoblast ◽  
Endometrial Epithelium ◽  
Glcnac Transferase

Embryo implantation begins with blastocyst trophectoderm (TE) attachment to the endometrial epithelium, followed by the breaching of this barrier by TE-derived trophoblast. Dynamic protein modification with O-linked β-N-acetylglucosamine (O-GlcNAcylation) is mediated by O-GlcNAc transferase and O-GlcNAcase (OGA), and couples cellular metabolism to stress adaptation. O-GlcNAcylation is essential for blastocyst formation, but whether there is a role for this system at implantation remains unexplored. Here, we used OGA inhibitor thiamet g (TMG) to induce raised levels of O-GlcNAcylation in mouse blastocysts and human trophoblast cells. In an in vitro embryo implantation model, TMG promoted mouse blastocyst breaching of the endometrial epithelium. TMG reduced expression of TE transcription factors Cdx2, Gata2 and Gata3, suggesting that O-GlcNAcylation stimulated TE differentiation to invasive trophoblast. TMG upregulated transcription factors OVOL1 and GCM1, and cell fusion gene ERVFRD1, in a cell line model of syncytiotrophoblast differentiation from human TE at implantation. Therefore O-GlcNAcylation is a conserved pathway capable of driving trophoblast differentiation. TE and trophoblast are sensitive to physical, chemical and nutritive stress, which can occur as a consequence of maternal pathophysiology or during assisted reproduction, and may lead to adverse neonatal outcomes and associated adult health risks. Further investigation of how O-GlcNAcylation regulates trophoblast populations arising at implantation is required to understand how peri-implantation stress affects reproductive outcomes.

scholarly journals Genetically encoded green fluorescent biosensors for monitoring UDP-GlcNAc in live cells

2021 ◽  
Author(s):  
Zefan Li ◽  
Jing Zhang ◽  
Huiwang Ai
Keyword(s):  
Mammalian Cells ◽  
Protein Modification ◽  
Fluorescent Protein ◽  
Culture Conditions ◽  
Live Cells ◽  
Uridine Diphosphate ◽  
Fluorescent Biosensors ◽  
Nucleotide Sugars ◽  
Green Fluorescent ◽  
Glcnac Transferase

Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is a nucleotide sugar used by glycosyltransferases to synthesize glycoproteins, glycosaminoglycans, glycolipids, and glycoRNA. UDP-GlcNAc also serves as the donor substrate for the formation of O-GlcNAc, a dynamic intracellular protein modification involved in diverse signaling and disease processes. UDP-GlcNAc is thus a central metabolite connecting nutrition, metabolism, signaling, and disease. There is a great interest in monitoring UDP-GlcNAc in biological systems. Here, we present the first genetically encoded, green fluorescent UDP-GlcNAc sensor (UGAcS), an optimized insertion of a circularly permuted green fluorescent protein (cpGFP) into an inactive mutant of an E. coli UDP-GlcNAc transferase, for ratiometric monitoring of UDP-GlcNAc dynamics in live mammalian cells. Although UGAcS responds to UDP-GlcNAc quite selectively among various nucleotide sugars, UDP and UTP interfere with the response. We thus developed another biosensor named UXPS, which is responsive to UDP and UTP but not UDP-GlcNAc. We demonstrated the use of the biosensors to follow UDP-GlcNAc levels in cultured mammalian cells perturbed with nutritional changes, pharmacological inhibition, and knockdown or overexpression of key enzymes in the UDP-GlcNAc synthesis pathway. We further utilized the biosensors to monitor UDP-GlcNAc concentrations in pancreatic MIN6 β-cells under various culture conditions.

scholarly journals Comparative O-GlcNAc Proteomic Analysis Reveals a Role of O-GlcNAcylated SAM68 in Lung Cancer Aggressiveness

Cancers ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 243
Author(s):  
Chia-Hung Lin ◽  
Chen-Chung Liao ◽  
Shu-Ying Wang ◽  
Chia-Yi Peng ◽  
Yi-Chen Yeh ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Protein Modification ◽  
Cancer Metastasis ◽  
Rna Binding ◽  
Migration And Invasion ◽  
Cancer Aggressiveness ◽  
Invasion Analysis ◽  
Proteome Profile ◽  
Invasive Lung Adenocarcinoma ◽  
Glcnac Transferase

O-GlcNAcylation is a reversible and dynamic post-translational protein modification catalyzed by O-GlcNAc transferase (OGT). Despite the reported association of O-GlcNAcylation with cancer metastasis, the O-GlcNAc proteome profile for cancer aggressiveness remains largely uncharacterized. Here, we report our comparative O-GlcNAc proteome profiling of two differentially invasive lung adenocarcinoma cell lines, which identified 158 down-regulated and 106 up-regulated candidates in highly invasive cells. Among these differential proteins, a nuclear RNA-binding protein, SAM68 (SRC associated in mitosis of 68 kDa), was further investigated. Results showed that SAM68 is O-GlcNAcylated and may interact with OGT in the nucleus. Eleven O-GlcNAcylation sites were identified, and data from mutant analysis suggested that multiple serine residues in the N-terminal region are important for O-GlcNAcylation and the function of SAM68 in modulating cancer cell migration and invasion. Analysis of clinical specimens found that high SAM68 expression was associated with late cancer stages, and patients with high-OGT/high-SAM68 expression in their tumors had poorer overall survival compared to those with low-OGT/low-SAM68 expression. Our study revealed an invasiveness-associated O-GlcNAc proteome profile and connected O-GlcNAcylated SAM68 to lung cancer aggressiveness.

scholarly journals Exploring the Potential of β-Catenin O-GlcNAcylation by Using Fluorescence-Based Engineering and Imaging

Molecules ◽  
2020 ◽  
Vol 25 (19) ◽  
pp. 4501
Author(s):  
Angelina Kasprowicz ◽  
Corentin Spriet ◽  
Christine Terryn ◽  
Vincent Rigolot ◽  
Stephan Hardiville ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Sequence ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Large Family ◽  
Post Translational Modifications ◽  
Chemical Reporter ◽  
Green Fluorescent ◽  
Glcnac Transferase

Monitoring glycosylation changes within cells upon response to stimuli remains challenging because of the complexity of this large family of post-translational modifications (PTMs). We developed an original tool, enabling labeling and visualization of the cell cycle key-regulator β-catenin in its O-GlcNAcylated form, based on intramolecular Förster resonance energy transfer (FRET) technology in cells. We opted for a bioorthogonal chemical reporter strategy based on the dual-labeling of β-catenin with a green fluorescent protein (GFP) for protein sequence combined with a chemically-clicked imaging probe for PTM, resulting in a fast and easy to monitor qualitative FRET assay. We validated this technology by imaging the O-GlcNAcylation status of β-catenin in HeLa cells. The changes in O-GlcNAcylation of β-catenin were varied by perturbing global cellular O-GlcNAc levels with the inhibitors of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Finally, we provided a flowchart demonstrating how this technology is transposable to any kind of glycosylation.

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