O-Linked β-N-Acetylglucosaminyltransferase Substrate Specificity Is Regulated by Myosin Phosphatase Targeting and Other Interacting Proteins

Cheung, Win D.; Sakabe, Kaoru; Housley, Michael P.; Dias, Wagner B.; Hart, Gerald W.

doi:10.1074/jbc.m806199200

Cited by 152 publications

(167 citation statements)

References 37 publications

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“…The overall in vitro activity of mitochondrial OGT from purified mitochondrial lysate was observed to be similar for the normal and diabetic samples ( Fig. 1E) (23). Thus, more OGT protein is required to maintain activity levels similar to normal, indicating a potential reduction in specific activity of mito-specific OGT in diabetic samples.…”

Section: Mitochondrial Ogt Is Increased and Mislocalized In Diabetic Ratmentioning

confidence: 82%

Diabetes-associated dysregulation of O -GlcNAcylation in rat cardiac mitochondria

Banerjee¹,

Ma²,

Hart³

2015

Proc. Natl. Acad. Sci. U.S.A.

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178

181

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Elevated mitochondrial O-GlcNAcylation caused by hyperglycemia, as occurs in diabetes, significantly contributes to mitochondrial dysfunction and to diabetic cardiomyopathy. However, little is known about the enzymology of mitochondrial O-GlcNAcylation. Herein, we investigated the enzymes responsible for cycling O-GlcNAc on mitochondrial proteins and studied the mitochondrial transport of UDP-GlcNAc. Analyses of purified rat heart mitochondria from normal and streptozocin-treated diabetic rats show increased mitochondrial O-GlcNAc transferase (OGT) and a concomitant decrease in the mito-specific O-GlcNAcase (OGA). Strikingly, OGT is mislocalized in cardiac mitochondria from diabetic rats. Interaction of OGT and complex IV observed in normal rat heart mitochondria is visibly reduced in diabetic samples, where OGT is mislocalized to the matrix. Live cell OGA activity assays establish the presence of O-GlcNAcase within the mitochondria. Furthermore, we establish that the inner mitochondrial membrane transporter, pyrimidine nucleotide carrier, transports UDP-GlcNAc from the cytosol to the inside of the mitochondria. Knockdown of this transporter substantially lowers mitochondrial O-GlcNAcylation. Inhibition of OGT or OGA activity within neonatal rat cardiomyocytes significantly affects energy production, mitochondrial membrane potential, and mitochondrial oxygen consumption. These data suggest that cardiac mitochondria not only have robust O-GlcNAc cycling, but also that dysregulation of O-GlcNAcylation likely plays a key role in mitochondrial dysfunction associated with diabetes.

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Section: Mitochondrial Ogt Is Increased and Mislocalized In Diabetic Ratmentioning

confidence: 82%

Diabetes-associated dysregulation of O -GlcNAcylation in rat cardiac mitochondria

Banerjee¹,

Ma²,

Hart³

2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

178

181

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“…Interestingly, in this assay, the major targets for OGT appeared to be histone H2A and H4. It is possible that the permeabilization technique we used prevented an accessory factor from correctly targeting OGT to recognize H3 and H2B, and, in fact, several studies have demonstrated that OGT uses interacting partners to recognize specific substrates (47,48). We also determined OGT-specific activity by immunoprecipitating OGT from control cells, heat-shocked cells, and heat-shocked cells that had been allowed to recover for 1 h at 37°C and performing in vitro OGT assays using a casein kinase II (CK2) peptide as substrate.…”

Section: Resultsmentioning

confidence: 99%

β- N -acetylglucosamine (O-GlcNAc) is part of the histone code

Sakabe

Wang

Hart

2010

Proc. Natl. Acad. Sci. U.S.A.

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328

282

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Dynamic posttranslational modification of serine and threonine residues of nucleocytoplasmic proteins by β-N-acetylglucosamine (O-GlcNAc) is a regulator of cellular processes such as transcription, signaling, and protein-protein interactions. Like phosphorylation, O-GlcNAc cycles in response to a wide variety of stimuli. Although cycling of O-GlcNAc is catalyzed by only two highly conserved enzymes, O-GlcNAc transferase (OGT), which adds the sugar, and β-N-acetylglucosaminidase (O-GlcNAcase), which hydrolyzes it, the targeting of these enzymes is highly specific and is controlled by myriad interacting subunits. Here, we demonstrate by multiple specific immunological and enzymatic approaches that histones, the proteins that package DNA within the nucleus, are O-GlcNAcylated in vivo. Histones also are substrates for OGT in vitro. We identify O-GlcNAc sites on histones H2A, H2B, and H4 using mass spectrometry. Finally, we show that histone O-GlcNAcylation changes during mitosis and with heat shock. Taken together, these data show that O-GlcNAc cycles dynamically on histones and can be considered part of the histone code.epigenetics | histones

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“…For OGA, the difficulty is compounded by the need to obtain such proteins in a form having significant levels of O-GlcNAc modification. To address these issues, we looked to previous reports that revealed a number of hOGT target proteins (23,38) as well as studies showing that co-expression of substrate proteins with hOGT in E. coli results in their high level O-GlcNAc modification (33,39). Based on these reports, we selected the genes encoding TAB1, CaMKIV, CARM1, microtubule-associated protein Tau, and Nup62 for cloning and co-expression with hOGT in E. coli ( Fig.…”

Section: Generation Of Diverse Substrate Proteins As Substrates Formentioning

confidence: 99%

Insights into O-Linked N-Acetylglucosamine ([0-9]O-GlcNAc) Processing and Dynamics through Kinetic Analysis of O-GlcNAc Transferase and O-GlcNAcase Activity on Protein Substrates

Shen

Gloster

Yuzwa

et al. 2012

Journal of Biological Chemistry

107

116

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O-Linked β-N-Acetylglucosaminyltransferase Substrate Specificity Is Regulated by Myosin Phosphatase Targeting and Other Interacting Proteins

Cited by 152 publications

References 37 publications

Diabetes-associated dysregulation of O -GlcNAcylation in rat cardiac mitochondria

Diabetes-associated dysregulation of O -GlcNAcylation in rat cardiac mitochondria

β- N -acetylglucosamine (O-GlcNAc) is part of the histone code

Insights into O-Linked N-Acetylglucosamine ([0-9]O-GlcNAc) Processing and Dynamics through Kinetic Analysis of O-GlcNAc Transferase and O-GlcNAcase Activity on Protein Substrates

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