Overview of the Assays to Probe O-Linked β-N-Acetylglucosamine Transferase Binding and Activity

O-GlcNAcylation is a posttranslational modification that occurs at serine and threonine residues of protein substrates by the addition of O-linked β-d-N-acetylglucosamine (GlcNAc) moiety. Two enzymes are involved in this modification: O-GlcNac transferase (OGT), which attaches the GlcNAc residue to the protein substrate, and O-GlcNAcase (OGA), which removes it. This biological balance is important for many biological processes, such as protein expression, cell apoptosis, and regulation of enzyme activity. The extent of this modification has sparked interest in the medical community to explore OGA and OGT as therapeutic targets, particularly in degenerative diseases. While some OGA inhibitors are already in phase 1 clinical trials for the treatment of Alzheimer’s disease, OGT inhibitors still have a long way to go. Due to complex expression and instability, the discovery of potent OGT inhibitors is challenging. Over the years, the field has grappled with this problem, and scientists have developed a number of techniques and assays. In this review, we aim to highlight assays and techniques for OGT inhibitor discovery, evaluate their strength for the field, and give us direction for future bioassay methods.

Download Full-text

Molecular pathways of Interferon–Stimulated Gene 15: Implications in cancer

Current Protein and Peptide Science ◽

10.2174/1389203721999201208200747 ◽

2020 ◽

Vol 21 ◽

Author(s):

Angeles C. Tecalco–Cruz

Keyword(s):

Posttranslational Modification ◽

Human Interferon ◽

Molecular Pathways ◽

Biological Processes ◽

Small Protein ◽

Target Proteins ◽

Central Elements ◽

Cancer Types ◽

Interferon Stimulated Gene 15

Abstract:: Human interferon–stimulated gene 15 (ISG15) is a 15–kDa ubiquitin–like protein that can be detected as either free ISG15 or covalently associated with its target proteins through a process termed ISGylation. Interestingly, extracellular free ISG15 has been proposed as a cytokine–like protein, whereas ISGylation is a posttranslational modification. ISG15 is a small protein with implications in some biological processes and pathologies that include cancer. This review highlights the findings of both free ISG15 and protein ISGylation involved in several molecular pathways, emerging as central elements in some cancer types.

Download Full-text

Myeloid-derived cullin 3 promotes STAT3 phosphorylation by inhibiting OGT expression and protects against intestinal inflammation

Journal of Experimental Medicine ◽

10.1084/jem.20161105 ◽

2017 ◽

Vol 214 (4) ◽

pp. 1093-1109 ◽

Cited By ~ 29

Author(s):

Xinghui Li ◽

Zhibin Zhang ◽

Lupeng Li ◽

Wei Gong ◽

Audrey J. Lazenby ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Posttranslational Modification ◽

Intestinal Inflammation ◽

Inhibitory Effect ◽

Related Factor ◽

Colonic Inflammation ◽

Stat3 Phosphorylation ◽

Cullin 3 ◽

Glcnac Transferase ◽

Transcription 3

Signal transducer and activator of transcription 3 (STAT3) is a key mediator of intestinal inflammation and tumorigenesis. However, the molecular mechanism that modulates STAT3 phosphorylation and activation is not fully understood. Here, we demonstrate that modification of STAT3 with O-linked β-N-acetylglucosamine (O-GlcNAc) on threonine 717 (T717) negatively regulates its phosphorylation and targets gene expression in macrophages. We further found that cullin 3 (CUL3), a cullin family E3 ubiquitin ligase, down-regulates the expression of the O-GlcNAc transferase (OGT) and inhibits STAT3 O-GlcNAcylation. The inhibitory effect of CUL3 on OGT expression is dependent on nuclear factor E2–related factor-2 (Nrf2), which binds to the Ogt promoter region and increases gene transcription. Myeloid deletion of Cul3 led to defective STAT3 phosphorylation in colon macrophages, which was accompanied by exacerbated colonic inflammation and inflammation-driven tumorigenesis. Thus, this study identifies a new form of posttranslational modification of STAT3, modulating its phosphorylation, and suggests the importance of immunometabolism on colonic inflammation and tumorigenesis.

Download Full-text

Inhibition of O-GlcNAc Transferase Alters the Differentiation and Maturation Process of Human Monocyte Derived Dendritic Cells

Cells ◽

10.3390/cells10123312 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3312

Author(s):

Matjaž Weiss ◽

Marko Anderluh ◽

Martina Gobec

Keyword(s):

Dendritic Cells ◽

Posttranslational Modification ◽

Human Monocyte ◽

T Cell Differentiation ◽

Maturation Process ◽

Hla Dr ◽

Glcnac Transferase ◽

And Function

The O-GlcNAcylation is a posttranslational modification of proteins regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase. These enzymes regulate the development, proliferation and function of cells, including the immune cells. Herein, we focused on the role of O-GlcNAcylation in human monocyte derived dendritic cells (moDCs). Our study suggests that inhibition of OGT modulates AKT and MEK/ERK pathways in moDCs. Changes were also observed in the expression levels of relevant surface markers, where reduced expression of CD80 and DC-SIGN, and increased expression of CD14, CD86 and HLA-DR occurred. We also noticed decreased IL-10 and increased IL-6 production, along with diminished endocytotic capacity of the cells, indicating that inhibition of O-GlcNAcylation hampers the transition of monocytes into immature DCs. Furthermore, the inhibition of OGT altered the maturation process of immature moDCs, since a CD14medDC-SIGNlowHLA-DRmedCD80lowCD86high profile was noticed when OGT inhibitor, OSMI-1, was present. To evaluate DCs ability to influence T cell differentiation and polarization, we co-cultured these cells. Surprisingly, the observed phenotypic changes of mature moDCs generated in the presence of OSMI-1 led to an increased proliferation of allogeneic T cells, while their polarization was not affected. Taken together, we confirm that shifting the O-GlcNAcylation status due to OGT inhibition alters the differentiation and function of moDCs in in vitro conditions.

Download Full-text

Methylation of Histone 4's Lysine 20: A critical analysis of the state of the field

Physiological Genomics ◽

10.1152/physiolgenomics.00128.2020 ◽

2020 ◽

Author(s):

Adriana Z. Corvalan ◽

Hilary A. Coller

Keyword(s):

Posttranslational Modification ◽

Current Knowledge ◽

Dynamic Structure ◽

Future Research ◽

Biological Processes ◽

Histone H4 ◽

Histone Tails ◽

Nonhistone Proteins ◽

Multiple Processes ◽

Cycle Regulation

Chromatin is a highly dynamic structure whose plasticity is achieved through multiple processes including the posttranslational modification of histone tails. Histone modifications function through the recruitment of nonhistone proteins to chromatin and thus have the potential to influence many fundamental biological processes. Here, we focus on the function and regulation of lysine 20 of histone H4 (H4K20) methylation in multiple biological processes including DNA repair, cell cycle regulation and DNA replication. The purpose of this review is to highlight recent studies that elucidate the functions associated with each of the methylation states of H4K20, their modifying enzymes, and their protein readers. Based on our current knowledge of H4K20 methylation, we critically analyze the data supporting these functions and outline questions for future research.

Download Full-text

Recognition of a glycosylation substrate by the O-GlcNAc transferase TPR repeats

Open Biology ◽

10.1098/rsob.170078 ◽

2017 ◽

Vol 7 (6) ◽

pp. 170078 ◽

Cited By ~ 27

Author(s):

Karim Rafie ◽

Olawale Raimi ◽

Andrew T. Ferenbach ◽

Vladimir S. Borodkin ◽

Vaibhav Kapuria ◽

...

Keyword(s):

Innate Immunity ◽

Substrate Specificity ◽

Active Site ◽

Target Site ◽

Post Translational Modification ◽

Tetratricopeptide Repeats ◽

Protein Substrates ◽

Nucleocytoplasmic Proteins ◽

Glcnac Transferase ◽

Single Enzyme

O-linked N -acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification found on hundreds of nucleocytoplasmic proteins in metazoa. Although a single enzyme, O-GlcNAc transferase (OGT), generates the entire cytosolic O-GlcNAc proteome, it is not understood how it recognizes its protein substrates, targeting only a fraction of serines/threonines in the metazoan proteome for glycosylation. We describe a trapped complex of human OGT with the C-terminal domain of TAB1, a key innate immunity-signalling O-GlcNAc protein, revealing extensive interactions with the tetratricopeptide repeats of OGT. Confirmed by mutagenesis, this interaction suggests that glycosylation substrate specificity is achieved by recognition of a degenerate sequon in the active site combined with an extended conformation C-terminal of the O-GlcNAc target site.

Download Full-text

Quantitative proteomics reveals extensive lysine ubiquitination in the Arabidopsis root proteome and uncovers novel transcription factor stability states

10.1101/2021.01.07.425780 ◽

2021 ◽

Author(s):

Gaoyuan Song ◽

Damilola Olatunji ◽

Christian Montes ◽

Natalie M Clark ◽

Yunting Pu ◽

...

Keyword(s):

Posttranslational Modification ◽

Site Directed Mutagenesis ◽

Biological Processes ◽

Protein Activity ◽

Helix Loop Helix ◽

Primary Roots ◽

Lysine Residues ◽

Ubiquitin Proteasome ◽

The Stability

Protein activity, abundance, and stability can be regulated by posttranslational modification including ubiquitination. Ubiquitination is conserved among eukaryotes and plays a central role in modulating cellular function and yet we lack comprehensive catalogs of proteins that are modified by ubiquitin in plants. In this study, we describe an antibody-based approach to enrich peptides containing the di-glycine (diGly) remnant of ubiquitin and coupled that with isobaric labeling to enable quantification, from up to 16-multiplexed samples, for plant tissues. Collectively, we identified 7,130 diGly-modified lysine residues sites arising from 3,178 proteins in Arabidopsis primary roots. These data include ubiquitin proteasome dependent ubiquitination events as well as ubiquitination events associated with auxin treatment. Gene Ontology analysis indicated that ubiquitinated proteins are associated with numerous biological processes including hormone signaling, plant defense, protein homeostasis, and root morphogenesis. We determined the ubiquitinated lysine residues that directly regulate the stability of the transcription factors CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1 (CIB1), CIB1 LIKE PROTEIN 2 (CIL2), and SENSITIVE TO PROTON RHIZOTOXICITY (STOP1) using site directed mutagenesis and in vivo degradation assays. These comprehensive site-level ubiquitinome profiles provide a wealth of data for future studies related to modulation of biological processes mediated by this posttranslational modification in plants.

Download Full-text

‘O-GlcNAc Code’ Mediated Biological Functions of Downstream Proteins

Molecules ◽

10.3390/molecules23081967 ◽

2018 ◽

Vol 23 (8) ◽

pp. 1967 ◽

Cited By ~ 7

Author(s):

Linhong Zhao ◽

Junaid Ali Shah ◽

Yong Cai ◽

Jingji Jin

Keyword(s):

Neurodegenerative Disease ◽

Glycoside Hydrolase ◽

Research Data ◽

Cellular Proteins ◽

Specific Substrate ◽

Biological Processes ◽

Biological Functions ◽

Post Translational Modifications ◽

Glcnac Transferase ◽

Protein Recruitment

As one of the post-translational modifications, O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) often occurs on serine (Ser) and threonine (Thr) residues of specific substrate cellular proteins via the addition of O-GlcNAc group by O-GlcNAc transferase (OGT). Maintenance of normal intracellular levels of O-GlcNAcylation is controlled by OGT and glycoside hydrolase O-GlcNAcase (OGA). Unbalanced O-GlcNAcylation levels have been involved in many diseases, including diabetes, cancer, and neurodegenerative disease. Recent research data reveal that O-GlcNAcylation at histones or non-histone proteins may provide recognition platforms for subsequent protein recruitment and further initiate intracellular biological processes. Here, we review the current understanding of the ‘O-GlcNAc code’ mediated intracellular biological functions of downstream proteins.

Download Full-text

O-GlcNAc Transferase Recognizes Protein Substrates Using an Asparagine Ladder in the Tetratricopeptide Repeat (TPR) Superhelix

Journal of the American Chemical Society ◽

10.1021/jacs.7b13546 ◽

2018 ◽

Vol 140 (10) ◽

pp. 3510-3513 ◽

Cited By ~ 33

Author(s):

Zebulon G. Levine ◽

Chenguang Fan ◽

Michael S. Melicher ◽

Marina Orman ◽

Tania Benjamin ◽

...

Keyword(s):

Tetratricopeptide Repeat ◽

Protein Substrates ◽

Glcnac Transferase

Download Full-text

Cross-talk between GlcNAcylation and phosphorylation: roles in insulin resistance and glucose toxicity

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.90281.2008 ◽

2008 ◽

Vol 295 (1) ◽

pp. E17-E28 ◽

Cited By ~ 167

Author(s):

Ronald J. Copeland ◽

John W. Bullen ◽

Gerald W. Hart

Keyword(s):

Insulin Resistance ◽

Cross Talk ◽

Hydroxyl Groups ◽

Spinal Cord Tissue ◽

Strong Positive Correlation ◽

Biological Processes ◽

Glucose Toxicity ◽

Site Specific ◽

Chronic Hyperglycemia ◽

Glcnac Transferase

O-linked β- N-acetylglucosamine ( O-GlcNAc) is a dynamic posttranslational modification that, analogous to phosphorylation, cycles on and off serine and/or threonine hydroxyl groups. Cycling of O-GlcNAc is regulated by the concerted actions of O-GlcNAc transferase and O-GlcNAcase. GlcNAcylation is a nutrient/stress-sensitive modification that regulates proteins involved in a wide array of biological processes, including transcription, signaling, and metabolism. GlcNAcylation is involved in the etiology of glucose toxicity and chronic hyperglycemia-induced insulin resistance, a major hallmark of type 2 diabetes. Several reports demonstrate a strong positive correlation between GlcNAcylation and the development of insulin resistance. However, recent studies suggest that inhibiting GlcNAcylation does not prevent hyperglycemia-induced insulin resistance, suggesting that other mechanisms must also be involved. To date, proteomic analyses have identified more than 600 GlcNAcylated proteins in diverse functional classes. However, O-GlcNAc sites have been mapped on only a small percentage (<15%) of these proteins, most of which were isolated from brain or spinal cord tissue and not from other metabolically relevant tissues. Mapping the sites of GlcNAcylation is not only necessary to elucidate the complex cross-talk between GlcNAcylation and phosphorylation but is also key to the design of site-specific mutational studies and necessary for the generation of site-specific antibodies, both of which will help further decipher O-GlcNAc's functional roles. Recent technical advances in O-GlcNAc site-mapping methods should now finally allow for a much-needed increase in site-specific analyses to address the functional significance of O-GlcNAc in insulin resistance and glucose toxicity as well as other major biological processes.

Download Full-text

Protein S-palmitoylation in cellular differentiation

Biochemical Society Transactions ◽

10.1042/bst20160236 ◽

2017 ◽

Vol 45 (1) ◽

pp. 275-285 ◽

Cited By ~ 13

Author(s):

Mingzi M. Zhang ◽

Howard C. Hang

Keyword(s):

Posttranslational Modification ◽

Protein Function ◽

Cellular Differentiation ◽

Protein S ◽

Biological Processes ◽

Global Studies ◽

Technological Advances ◽

Protein Palmitoylation ◽

Spatiotemporal Control

Reversible protein S-palmitoylation confers spatiotemporal control of protein function by modulating protein stability, trafficking and activity, as well as protein–protein and membrane–protein associations. Enabled by technological advances, global studies revealed S-palmitoylation to be an important and pervasive posttranslational modification in eukaryotes with the potential to coordinate diverse biological processes as cells transition from one state to another. Here, we review the strategies and tools to analyze in vivo protein palmitoylation and interrogate the functions of the enzymes that put on and take off palmitate from proteins. We also highlight palmitoyl proteins and palmitoylation-related enzymes that are associated with cellular differentiation and/or tissue development in yeasts, protozoa, mammals, plants and other model eukaryotes.

Download Full-text