Overexpression Of O-GlcNAc Transferase Research Articles

Non-canonical glycosyltransferase modulates post-hypoxic cardiac myocyte death and mitochondrial permeability transition

O-linked β- N-acetylglucosamine ( O-GlcNAc) is a dynamic, inducible, and reversible post-translational modification of nuclear and cytoplasmic proteins on Ser/Thr amino acid residues. In addition to its putative role as a nutrient sensor, we have recently shown pharmacologic elevation of O-GlcNAc levels positively affected myocyte survival during oxidant stress. However, no rigorous assessment of the contribution of O-GlcNAc transferase has been performed, particularly in the post-hypoxic setting. Therefore, we hypothesized that pharmacological or genetic manipulation of O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc to proteins, would affect cardiac myocyte survival following hypoxia/reoxygenation (H/R). Adenoviral overexpression of OGT (AdOGT) in cardiac myocytes augmented O-GlcNAc levels and reduced post-hypoxic damage. Conversely, pharmacologic inhibition of OGT significantly attenuated O-GlcNAc levels, exacerbated post-hypoxic cardiac myocyte death, and sensitized myocytes to mitochondrial membrane potential collapse. Both genetic deletion of OGT using a cre-lox approach and translational silencing via RNAi also resulted in significant reductions in OGT protein and O-GlcNAc levels, and, exacerbated post-hypoxic cardiac myocyte death. Inhibition of OGT reduced O-GlcNAc levels on voltage dependent anion channel (VDAC) in isolated mitochondria and sensitized to calcium-induced mitochondrial permeability transition pore (mPTP) formation, indicating that mPTP may be an important target of O-GlcNAc signaling and confirming the aforementioned mitochondrial membrane potential results. These data demonstrate that OGT exerts pro-survival actions during hypoxia-reoxygenation in cardiac myocytes, particularly at the level of mitochondria.

Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein O-GlcNAc and increased mitochondrial Bcl-2.

We have previously reported that glucosamine protected neonatal rat ventricular myocytes against ischemia-reperfusion (I/R) injury, and this was associated with an increase in protein O-linked-N-acetylglucosamine (O-GlcNAc) levels. However, the protective effect of glucosamine could be mediated via pathways other that O-GlcNAc formation; thus the initial goal of the present study was to determine whether increasing O-GlcNAc transferase (OGT) expression, which catalyzes the formation of O-GlcNAc, had a protective effect similar to that of glucosamine. To better understand the potential mechanism underlying O-GlcNAc-mediated cytoprotection, we examined whether increased O-GlcNAc levels altered the expression and translocation of members of the Bcl-2 protein family. Both glucosamine (5 mM) and OGT overexpression increased basal and I/R-induced O-GlcNAc levels, significantly decreased cellular injury, and attenuated loss of cytochrome c. Both interventions also attenuated the loss of mitochondrial membrane potential induced by H2O2 and were also associated with an increase in mitochondrial Bcl-2 levels but had no effect on Bad or Bax levels. Compared with glucosamine and OGT overexpression, NButGT (100 microM), an inhibitor of O-GlcNAcase, was less protective against I/R and H2O2 and did not affect Bcl-2 expression, despite a 5- to 10-fold greater increase in overall O-GlcNAc levels. Decreased OGT expression resulted in lower basal O-GlcNAc levels, prevented the I/R-induced increase in O-GlcNAc and mitochondrial Bcl-2, and increased cellular injury. These results demonstrate that the protective effects of glucosamine are mediated via increased formation of O-GlcNAc and suggest that this is due, in part, to enhanced mitochondrial Bcl-2 translocation.

Coactivator Associated Arginine Methyltransferase 1 (CARM1) is reciprocally regulated by phosphorylation and O‐GlcNAcylation

Enzymatic modification of serine and threonine residues on nucleocytoplasmic proteins with single N‐acetylglucosamine moiety through a β‐glycosidic linkage (O‐GlcNAc) is a dynamic post‐translational modification. Much like phosphorylation, protein O‐GlcNAcylation is responsive to cell stimuli and serves to regulate protein function. However unlike phosphorylation, which is mediated by more than 500 kinases, GlcNAcylation is regulated by transient targeting of a single catalytic subunit, O‐GlcNAc Transferase (OGT), to a multitude of substrates by interactor proteins. Recently, through a yeast two‐hybrid approach, we identified Coactivator Associated Arginine Methyltransferase (CARM1) as a putative binding partner with OGT. CARM1 methylates Arg residues of proteins, such as p300 and histone H3. Here, we demonstrate that not only does CARM1 associate with OGT, it also is O‐GlcNAcylated. Additionally, we show that it is reciprocally regulated by phosphorylation and O‐GlcNAcylation. When CARM1 becomes hyper‐phosphorylated during M phase, O‐GlcNAc on CARM1 decreases. This reciprocal relationship regulates substrate specificity, since methylation on Arg 17 of histone H3, a CARM1 specific substrate, increases dramatically during M phase. Overexpression of OGT alters not only the modification status of CARM1, but also the methylation of histone H3 by decreasing mitotic phosphorylation of CARM1 and concomitantly decreasing histone H3 methylation. Aberrant methylation of histone H3 caused by OGT overexpression could explain the gross defects associated with altering O‐GlcNAc levels during cell cycle. Supported by NIH HD13563. Dr. Hart receives royalty received by the university on the sales of the 110.6 antibody. Terms of the arrangement are managed by JHU.

In isolated cardiomyocytes NF‐κB activation is modulated by alterations in protein O‐GlcNAc levels

O‐Linked N‐acetylglucosamine (O‐GlcNAc) modification of proteins plays an important role in transcription, translation, nuclear transport and cytoskeletal assembly. We have shown that glucosamine (GlcN) increased O‐GlcNAc levels and attenuated activation of NF‐κB signaling following trauma hemorrhage and resuscitation in vivo and in response to lipopolysaccharide (LPS) treatment of in cardiomyocytes in vitro. Therefore the goal of this study was to determine whether the effect of GlcN on NF‐κB activation was mediated via O‐GlcNAc transferase (OGT), which catalyzes O‐GlcNAc synthesis. Neonatal rat ventricular myocytes(NRVMs) were untreated, treated with 5mM GlcN, transfected with OGT adenovirus or OGT siRNA and then stimulated with LPS (2μg/ml) for 6h. In untreated cells LPS significantly increased IκB‐α phosphorylation, TNF‐α and ICAM1 expression and increased nuclear NF‐κB levels. Both GlcN and OGT overexpression significantly increased O‐GlcNAc levels, attenuated LPS‐induced TNF‐α and ICAM1 expression and decreased IκB‐α phosphorylation and nuclear NF‐κB levels. In contrast, OGT siRNA treatment decreased OGT and O‐GlcNAc levels and enhanced LPS‐induced increase in IκB‐α phosphorylation. These results demonstrate that modulation of cellular O‐GlcNAc levels alters the response to activation of NF‐κB signaling pathway, and may contribute the protective effect of GlcN in vivo.

Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance

Glucose flux through the hexosamine biosynthetic pathway leads to the post-translational modification of cytoplasmic and nuclear proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc). This tandem system serves as a nutrient sensor to couple systemic metabolic status to cellular regulation of signal transduction, transcription, and protein degradation. Here we show that O-GlcNAc transferase (OGT) harbours a previously unrecognized type of phosphoinositide-binding domain. After induction with insulin, phosphatidylinositol 3,4,5-trisphosphate recruits OGT from the nucleus to the plasma membrane, where the enzyme catalyses dynamic modification of the insulin signalling pathway by O-GlcNAc. This results in the alteration in phosphorylation of key signalling molecules and the attenuation of insulin signal transduction. Hepatic overexpression of OGT impairs the expression of insulin-responsive genes and causes insulin resistance and dyslipidaemia. These findings identify a molecular mechanism by which nutritional cues regulate insulin signalling through O-GlcNAc, and underscore the contribution of this modification to the aetiology of insulin resistance and type 2 diabetes.