scholarly journals CRISPR-Cas9-mediated depletion of O-GlcNAc hydrolase and transferase for functional dissection of O-GlcNAcylation in human cells

2020 ◽  
Author(s):  
Andrii Gorelik ◽  
Andrew T. Ferenbach
Keyword(s):  
Rna Interference ◽  
Human Cell ◽  
Compensatory Mechanism ◽  
Human Cell Lines ◽  
Post Translational Modification ◽  
Short Term ◽  
Protein Substrates ◽  
Cytoplasmic Proteins ◽  
Pharmacological Studies ◽  
Glcnac Transferase

AbstractO-GlcNAcylation is an abundant post-translational modification (PTM) on serine and threonine residues of nuclear and cytoplasmic proteins. Although this PTM has been reported on thousands of proteins, O-GlcNAc transferase (OGT) and hydrolase (OGA) are the only two enzymes that perform the respective addition and removal of O-GlcNAc on protein substrates. To examine the consequences of deregulated O-GlcNAcylation, the O-GlcNAc field has mostly relied on the use of RNA interference to knockdown OGT/OGA and inhibitors to block their activities in cells. Here, we describe the first complete CRISPR-Cas9 knockouts of OGA and a knockdown of OGT (with a maximal decrease in expression of over 80%) in two human cell lines. Notably, constitutive depletion of one O-GlcNAc cycling enzyme not only led to a respective increase or decrease in total O-GlcNAcylation levels but also resulted in diminished expression of the opposing enzyme, as a compensatory mechanism, observed in previous short-term pharmacological studies. The OGA knockout system presents a convenient platform to dissect OGA mutations and was used to further characterise the single Ser405 O-GlcNAc site of human OGA using the S-GlcNAc genetic recoding approach, helping to identify an S-GlcNAc-specific antibody which was previously thought to primarily detect O-GlcNAc.

scholarly journals Intracellular Hydrolysis of Small-Molecule O-Linked N-Acetylglucosamine Transferase Inhibitors Differs among Cells and Is Not Required for Its Inhibition

Molecules ◽  
2020 ◽  
Vol 25 (15) ◽  
pp. 3381
Author(s):  
Elena Maria Loi ◽  
Matjaž Weiss ◽  
Stane Pajk ◽  
Martina Gobec ◽  
Tihomir Tomašič ◽  
...  
Keyword(s):  
Cell Lines ◽  
Active Metabolite ◽  
Research Field ◽  
Human Cell Lines ◽  
Post Translational Modification ◽  
Potential Therapeutic Target ◽  
Cytoplasmic Proteins ◽  
Ester Moiety ◽  
Glcnac Transferase ◽  
Hydrolysis Of

O-GlcNAcylation is an essential post-translational modification that occurs on nuclear and cytoplasmic proteins, regulating their function in response to cellular stress and altered nutrient availability. O-GlcNAc transferase (OGT) is the enzyme that catalyzes this reaction and represents a potential therapeutic target, whose biological role is still not fully understood. To support this research field, a series of cell-permeable, low-nanomolar OGT inhibitors were recently reported. In this study, we resynthesized the most potent OGT inhibitor of the library, OSMI-4, and we used it to investigate OGT inhibition in different human cell lines. The compound features an ethyl ester moiety that is supposed to be cleaved by carboxylesterases to generate its active metabolite. Our LC-HRMS analysis of the cell lysates shows that this is not always the case and that, even in the cell lines where hydrolysis does not occur, OGT activity is inhibited.

scholarly journals Protein substrates engage the lumen of O-GlcNac transferase’s tetratricopeptide repeat domain in different ways

2020 ◽  
Author(s):  
Cassandra M. Joiner ◽  
Forrest A. Hammel ◽  
John Janetzko ◽  
Suzanne Walker
Keyword(s):  
Tetratricopeptide Repeat ◽  
Substrate Selection ◽  
Post Translational Modification ◽  
Protein Substrates ◽  
Cytoplasmic Proteins ◽  
C Terminus ◽  
Glycosylation Sites ◽  
Repeat Domain ◽  
Tetratricopeptide Repeat Domain ◽  
Glcnac Transferase

ABSTRACTGlycosylation of nuclear and cytoplasmic proteins is an essential post-translational modification in mammals. O-GlcNAc transferase (OGT), the sole enzyme responsible for this modification, glycosylates over a thousand unique nuclear and cytoplasmic substrates. How OGT selects its substrates is a fundamental question that must be answered to understand OGT’s unusual biology. OGT contains a long tetratricopeptide repeat (TPR) domain that has been implicated in substrate selection, but there is almost no information about how changes to this domain affect glycosylation of individual substrates. Here, we used proteome-wide glycosylation profiling and probed glycosylation of selected purified substrates to show that asparagine and aspartate ladders that extend the full length of OGT’s TPR lumen control substrate glycosylation. We also found that substrates with glycosylation sites close to the C-terminus bypass lumenal binding. Our findings demonstrate that substrates can engage OGT in a variety of different ways for glycosylation.

scholarly journals Nucleocytoplasmic human O-GlcNAc transferase is sufficient for O-GlcNAcylation of mitochondrial proteins

2016 ◽  
Vol 473 (12) ◽  
pp. 1693-1702 ◽  
Author(s):  
Riccardo Trapannone ◽  
Daniel Mariappa ◽  
Andrew T. Ferenbach ◽  
Daan M.F. van Aalten
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Neuronal Development ◽  
Mitochondrial Proteins ◽  
Human Cell Lines ◽  
Post Translational Modification ◽  
Mitochondrial Proteome ◽  
Mouse Tissues ◽  
Dependent Protein ◽  
Glcnac Transferase

O-linked N-acetylglucosamine modification (O-GlcNAcylation) is a nutrient-dependent protein post-translational modification (PTM), dynamically and reversibly driven by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that catalyse the addition and the removal of the O-GlcNAc moieties to/from serine and threonine residues of target proteins respectively. Increasing evidence suggests involvement of O-GlcNAcylation in many biological processes, including transcription, signalling, neuronal development and mitochondrial function. The presence of a mitochondrial O-GlcNAc proteome and a mitochondrial OGT (mOGT) isoform has been reported. We explored the presence of mOGT in human cell lines and mouse tissues. Surprisingly, analysis of genomic sequences indicates that this isoform cannot be expressed in most of the species analysed, except some primates. In addition, we were not able to detect endogenous mOGT in a range of human cell lines. Knockdown experiments and Western blot analysis of all the predicted OGT isoforms suggested the expression of only a single OGT isoform. In agreement with this, we demonstrate that overexpression of the nucleocytoplasmic OGT (ncOGT) isoform leads to increased O-GlcNAcylation of mitochondrial proteins, suggesting that ncOGT is necessary and sufficient for the generation of the O-GlcNAc mitochondrial proteome.

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

Open Biology ◽  
2017 ◽  
Vol 7 (6) ◽  
pp. 170078 ◽  
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.

scholarly journals Revealing dynamic protein acetylation across subcellular compartments

2018 ◽  
Author(s):  
Josue Baeza ◽  
Alexis J. Lawton ◽  
Jing Fan ◽  
Michael J. Smallegan ◽  
Ian Lienert ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Growth Conditions ◽  
Regulatory Control ◽  
Human Cell Lines ◽  
Protein Acetylation ◽  
Post Translational Modification ◽  
Site Specific ◽  
Daunting Task ◽  
Cellular Compartments

ABSTRACTProtein acetylation is a widespread post-translational modification implicated in many cellular processes. Recent advances in mass spectrometry have enabled the cataloging of thousands of sites throughout the cell, however identifying regulatory acetylation marks have proven to be a daunting task. Knowledge of the kinetics and stoichiometry of site-specific acetylation are important factors to uncover function. Here, an improved method of quantifying acetylation stoichiometry was developed and validated, providing a detailed landscape of dynamic acetylation stoichiometry within cellular compartments. The dynamic nature of site-specific acetylation in response to serum stimulation was revealed. In two distinct human cell lines, growth factor stimulation led to site-specific, temporal acetylation changes, revealing diverse kinetic profiles that clustered into several groups. Overlap of dynamic acetylation sites among two different human cell lines suggested similar regulatory control points across major cellular pathways that include splicing, translation, and protein homeostasis. Rapid increases in acetylation on protein translational machinery suggest a positive regulatory role under pro-growth conditions. Lastly, higher median stoichiometry was observed in cellular compartments where active acetyltransferases are well-described.

Abstract 336: Acute Increase In O-GlcNAcylation Reduces The Release Of Cytokines And Attenuates Hypotension In Mice With LPS-induced Sepsis.

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Vania C Olivon ◽  
Raphael G Ferreira ◽  
Fabíola L Mestriner ◽  
José C Alves-Filho ◽  
Fernando Q Cunha ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Inflammatory Mediators ◽  
Vascular Reactivity ◽  
Inflammatory Cells ◽  
Serum Levels ◽  
Post Translational Modification ◽  
Cytoplasmic Proteins ◽  
Tnf Α ◽  
Acute Increase ◽  
Glcnac Transferase

The attachment of β-O-linked N-Acetylglucosamine (O-GlcNAc) in nuclear and cytoplasmic proteins, known as O-GlcNAcylation, is a common post-translational modification controlled by two enzymes: O-GlcNAc transferase (OGT) and β-N-acetylglucosaminidase (OGA). Acute increases in O-GlcNAc reduce migration of inflammatory cells and the release of pro-inflammatory mediators, important events in sepsis caused by bacterial infection or multiple non-infectious causes. We postulated that acute increases of O-GlcNAc reduce sepsis-associated mortality, release of inflammatory mediators and changes in blood pressure and vascular reactivity. C57BL/6 mice received lipopolysaccharide (LPS) injections to produce mild (LPS M, 10mg/Kg, i.p.) or severe (LPS S, 20mg/Kg, i.p.) sepsis. LPS-treated and naive (N) mice received glucosamine (GlcN), 300mg/Kg, i.v.) or vehicle (30 min before the LPS administration) and were euthanized 6 h later. GlcN treatment augmented O-GlcNAc levels and increased survival in mice with LPS-induced sepsis (LPS M=20%*; LPS S=50%*). GlcN treatment reduced serum levels (pg/mL) of IL-1β [N and N+GlcN = not detected (nd); LPS M = 286.3±14.5*, LPS M+GlcN = 212.9±3.1*#; LPS S= 343.9±29.1*, LPS S+GlcN = 128.4±11*#], IL-6 (N and N+GlcN = nd; LPS M = 448.0±11.5*, LPS M+GlcN = 241.2±11.6*#; LPS S = 508.6±21.7*, LPS S+GlcN = 451.6±8.9*) and TNF-α ((N and N+GlcN = nd; LPS M = 311.3±15.7*, LPS M+GlcN = 76.5±5.5*#; LPS S = 354.2±32.1*, LPS S+GlcN = 136.2±10.2*#) and vascular mRNA expression (2-ΔΔCT) of these cytokines: IL-1β (N and N+GlcN = nd; LPS M = 20.6±1.2*, LPS M+GlcN = 14.0±1.4*#; LPS S = 21.46±1.5*, LPS S+GlcN = 15.87±0.9*#), TNF-α [N and N+GlcN = nd; LPS M = 0.07±0.004*, LPS M+GlcN = 0.02±0.004*#; LPS S = 0.13±0.01*, LPS S+GlcN = 0.02±0.006*#. GlcN treatment attenuated LPS-induced decrease in blood pressure [(mmHg), N = 108±9.0, N+GlcN = 112±10.0; LPS M = 72±6.0*, LPS M+GlcN = 92±8.5*#; LPS S = 59±4.5*, LPS S+GlcN = 88±7.5*# mmHg). No changes in vascular reactivity (thoracic aorta) to phenylephrine or acetylcholine were detected 6 h after LPS administration; (* p<0.05 vs. N; # p<0.05 vs. LPS). In conclusion, O-GlcNAc reduces sepsis-associated inflammatory and cardiovascular events, making this pathway a potential target for therapeutic intervention.

scholarly journals O-Linked N-Acetylglucosamine Cycling Regulates Mitotic Spindle Organization

2013 ◽  
Vol 288 (38) ◽  
pp. 27085-27099 ◽  
Author(s):  
Ee Phie Tan ◽  
Sarah Caro ◽  
Anish Potnis ◽  
Christopher Lanza ◽  
Chad Slawson
Keyword(s):  
Mitotic Spindle ◽  
Aurora Kinase ◽  
Post Translational Modification ◽  
Gain Of Function ◽  
Aurora Kinase B ◽  
H3 Phosphorylation ◽  
Cytoplasmic Proteins ◽  
New Perspective ◽  
Glcnac Transferase ◽  
Histone H3 Phosphorylation

Any defects in the correct formation of the mitotic spindle will lead to chromosomal segregation errors, mitotic arrest, or aneuploidy. We demonstrate that O-linked N-acetylglucosamine (O-GlcNAc), a post-translational modification of serine and threonine residues in nuclear and cytoplasmic proteins, regulates spindle function. In O-GlcNAc transferase or O-GlcNAcase gain of function cells, the mitotic spindle is incorrectly assembled. Chromosome condensation and centrosome assembly is impaired in these cells. The disruption in spindle architecture is due to a reduction in histone H3 phosphorylation by Aurora kinase B. However, gain of function cells treated with the O-GlcNAcase inhibitor Thiamet-G restored the assembly of the spindle and partially rescued histone phosphorylation. Together, these data suggest that the coordinated addition and removal of O-GlcNAc, termed O-GlcNAc cycling, regulates mitotic spindle organization and provides a potential new perspective on how O-GlcNAc regulates cellular events.

The PML gene is not involved in the regulation of MHC class I expression in human cell lines

Blood ◽  
2003 ◽  
Vol 101 (9) ◽  
pp. 3514-3519 ◽  
Author(s):  
Silvia Bruno ◽  
Fabio Ghiotto ◽  
Franco Fais ◽  
Marta Fagioli ◽  
Lucilla Luzi ◽  
...  
Keyword(s):  
Rna Interference ◽  
Mhc Class I ◽  
Cell Lines ◽  
Human Cell ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Class I ◽  
Dominant Negative Effect ◽  
Human Cell Lines ◽  
Down Regulation

The promyelocytic leukemia gene, PML, is a growth and transformation suppressor. An additional role forPML as a regulator of major histocompatibility complex (MHC) class I antigen presentation has been proposed in a murine model, which would account for evasion from host immunity of tumors bearing malfunctioning PML, such as acute promyelocytic leukemia. Here we investigated a possible role ofPML for the control MHC class I expression in human cells. PML function was perturbed in human cell lines either byPML/RARα transfection or by PML- specific RNA interference. Impairment of wild-type PML function was proved by a microspeckled disassembly of nuclear bodies (NBs), where the protein is normally localized, or by their complete disappearance. However, no MHC class I down-regulation was observed in both instances. We next constructed a PML mutant, PML mut ex3,that is a human homolog of the murine PML mutant, truncated in exon 3, that was shown to down-regulate murine MHC class I. PML mut ex3 transfected in human cell lines exerted a dominant-negative effect since no PML molecules were detected in NBs but, instead, in perinuclear and cytoplasmic larger dotlike structures. Nevertheless, no down-regulation of MHC class I expression was evident. Moreover, neither transfection with PML mut ex3 nor PML-specific RNA interference affected the ability of γ-interferon to up-regulate MHC class I expression. We conclude that, in human cell lines, PML is not involved directly in the regulation of MHC class I expression.

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