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

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.

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Arginase induction by sodium phenylbutyrate in mouse tissues and human cell lines

Molecular Genetics and Metabolism ◽

10.1016/j.ymgme.2006.07.002 ◽

2007 ◽

Vol 90 (1) ◽

pp. 37-41 ◽

Cited By ~ 7

Author(s):

R.M. Kern ◽

Z. Yang ◽

P.S. Kim ◽

W.W. Grody ◽

R.K. Iyer ◽

...

Keyword(s):

Cell Lines ◽

Human Cell ◽

Human Cell Lines ◽

Mouse Tissues ◽

Sodium Phenylbutyrate

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HBO1 (KAT7) Does Not Have an Essential Role in Cell Proliferation, DNA Replication, or Histone 4 Acetylation in Human Cells

Molecular and Cellular Biology ◽

10.1128/mcb.00506-19 ◽

2019 ◽

Vol 40 (4) ◽

Author(s):

Andrew J. Kueh ◽

Samantha Eccles ◽

Leonie Tang ◽

Alexandra L. Garnham ◽

Rose E. May ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Adhesion ◽

Dna Replication ◽

Cell Lines ◽

Human Cell ◽

Human Cells ◽

Human Cell Lines ◽

Hela Cell Lines ◽

Mouse Tissues ◽

Sirna Knockdown

ABSTRACT HBO1 (MYST2/KAT7) is essential for histone 3 lysine 14 acetylation (H3K14ac) but is dispensable for H4 acetylation and DNA replication in mouse tissues. In contrast, previous studies using small interfering RNA (siRNA) knockdown in human cell lines have suggested that HBO1 is essential for DNA replication. To determine if HBO1 has distinctly different roles in immortalized human cell lines and normal mouse cells, we performed siRNA knockdown of HBO1. In addition, we used CRISPR/Cas9 to generate 293T, MCF7, and HeLa cell lines lacking HBO1. Using both techniques, we show that HBO1 is essential for all H3K14ac in human cells and is unlikely to have a direct effect on H4 acetylation and only has minor effects on cell proliferation. Surprisingly, the loss of HBO1 and H3K14ac in HeLa cells led to the secondary loss of almost all H4 acetylation after 4 weeks. Thus, HBO1 is dispensable for DNA replication and cell proliferation in immortalized human cells. However, while cell proliferation proceeded without HBO1 and H3K14ac, HBO1 gene deletion led to profound changes in cell adhesion, particularly in 293T cells. Consistent with this phenotype, the loss of HBO1 in both 293T and HeLa principally affected genes mediating cell adhesion, with comparatively minor effects on other cellular processes.

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

Molecules ◽

10.3390/molecules25153381 ◽

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.

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Revealing dynamic protein acetylation across subcellular compartments

10.1101/472530 ◽

2018 ◽

Cited By ~ 1

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.

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Mirror expression of adrenoleukodystrophy and adrenoleukodystrophy related genes in mouse tissues and human cell lines

European Journal of Cell Biology ◽

10.1016/s0171-9335(98)80121-0 ◽

1998 ◽

Vol 75 (3) ◽

pp. 254-264 ◽

Cited By ~ 64

Author(s):

Nathalie Troffer-Charlier ◽

Nathalie Doerflinger ◽

Elisabeth Metzger ◽

Françoise Fouquet ◽

Jean-Louis Mandel ◽

...

Keyword(s):

Cell Lines ◽

Human Cell ◽

Human Cell Lines ◽

Mouse Tissues

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A cross-organism framework for supervised enhancer prediction with epigenetic pattern recognition and targeted validation

10.1101/385237 ◽

2018 ◽

Cited By ~ 5

Author(s):

Anurag Sethi ◽

Mengting Gu ◽

Emrah Gumusgoz ◽

Landon Chan ◽

Koon-Kiu Yan ◽

...

Keyword(s):

Cell Lines ◽

Human Cell ◽

Mammalian Species ◽

Machine Learning Algorithms ◽

Supervised Machine Learning ◽

Support Vector ◽

Human Cell Lines ◽

Mouse Tissues

AbstractEnhancers are important noncoding elements, but they have been traditionally hard to characterize experimentally. Only a few mammalian enhancers have been validated, making it difficult to train statistical models for their identification properly. Instead, postulated patterns of genomic features have been used heuristically for identification. The development of massively parallel assays allows for the characterization of large numbers of enhancers for the first time. Here, we developed a framework that uses Drosophila STARR-seq data to create shape-matching filters based on enhancer-associated meta-profiles of epigenetic features. We combined these features with supervised machine learning algorithms (e.g., support vector machines) to predict enhancers. We demonstrated that our model could be applied to predict enhancers in mammalian species (i.e., mouse and human). We comprehensively validated the predictions using a combination of in vivo and in vitro approaches, involving transgenic assays in mouse and transduction-based reporter assays in human cell lines. Overall, the validations involved 153 enhancers in 6 mouse tissues and 4 human cell lines. The results confirmed that our model can accurately predict enhancers in different species without re-parameterization. Finally, we examined the transcription-factor binding patterns at predicted enhancers and promoters in human cell lines. We demonstrated that these patterns enable the construction of a secondary model effectively discriminating between enhancers and promoters.

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Complex expression pattern of the Barth syndrome gene product tafazzin in human cell lines and murine tissues

Biochemistry and Cell Biology ◽

10.1139/o04-055 ◽

2004 ◽

Vol 82 (5) ◽

pp. 569-576 ◽

Cited By ~ 23

Author(s):

Biao Lu ◽

Marguerite R Kelher ◽

Douglas P Lee ◽

Tal M Lewin ◽

Rosalind A Coleman ◽

...

Keyword(s):

Cell Lines ◽

Human Cell ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Barth Syndrome ◽

In Vitro Translation ◽

Human Cell Lines ◽

Mouse Tissues ◽

Species Specific

Tafazzins, a group of proteins that are defective in patients with Barth syndrome, are produced by alternate splicing of the gene G4.5 or TAZ. RT-PCR and transcription-coupled in vitro translation analysis were undertaken to determine the expression of alternatively spliced TAZ mRNA in mouse tissues and human cell lines. Only two tafazzin transcripts, both lacking exon 5, were expressed in murine tissues, whereas four tafazzin transcripts, all lacking exon 5, were observed in human umbilical vein vascular endothelial cells and U937 human monoblasts indicating a species-specific difference in the expression of TAZ mRNAs in mouse and humans. Only TAZ lacking exon 5 was expressed in murine heart. Differentiation of U937 human monoblasts into macrophages did not alter expression of the tafazzin transcripts indicating that TAZ expression is independent of monocyte differentiation. Cloning and in vitro expression of both murine and human tafazzin cDNA revealed two prominent protein bands that corresponded to the expected sizes of alternative translation. A novel fifth motif, identified as critical for the glycerolphosphate acyltransferase family, was observed in human tafazzin. The presence of a mutation in this region in Barth syndrome patients indicates that this motif is essential for tafazzin function.Key words: cardiolipin, murine, heart, Barth Syndrome, phospholipid, acyltransferase, tafazzin.

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

10.1101/2020.08.19.258079 ◽

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.

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