OGT (O-GlcNAc Transferase) Selectively Modifies Multiple Residues Unique to Lamin A.

The LMNA gene encodes lamins A and C with key roles in nuclear structure, signaling, gene regulation, and genome integrity. Mutations in LMNA cause over 12 diseases ('laminopathies'). Lamins A and C are identical for their first 566 residues. However, they form separate filaments in vivo, with apparently distinct roles. We report that lamin A is ß-O-linked N-acetylglucosamine-(O-GlcNAc)-modified in human hepatoma (Huh7) cells and in mouse liver. In vitro assays with purified O-GlcNAc transferase (OGT) enzyme showed robust O-GlcNAcylation of recombinant mature lamin A tails (residues 385⁻646), with no detectable modification of lamin B1, lamin C, or 'progerin' (Δ50) tails. Using mass spectrometry, we identified 11 O-GlcNAc sites in a 'sweet spot' unique to lamin A, with up to seven sugars per peptide. Most sites were unpredicted by current algorithms. Double-mutant (S612A/T643A) lamin A tails were still robustly O-GlcNAc-modified at seven sites. By contrast, O-GlcNAcylation was undetectable on tails bearing deletion Δ50, which causes Hutchinson⁻Gilford progeria syndrome, and greatly reduced by deletion Δ35. We conclude that residues deleted in progeria are required for substrate recognition and/or modification by OGT in vitro. Interestingly, deletion Δ35, which does not remove the majority of identified O-GlcNAc sites, does remove potential OGT-association motifs (lamin A residues 622⁻625 and 639⁻645) homologous to that in mouse Tet1. These biochemical results are significant because they identify a novel molecular pathway that may profoundly influence lamin A function. The hypothesis that lamin A is selectively regulated by OGT warrants future testing in vivo, along with two predictions: genetic variants may contribute to disease by perturbing OGT-dependent regulation, and nutrient or other stresses might cause OGT to misregulate wildtype lamin A.

O-GlcNAc site-mapping of liver X receptor-α and O-GlcNAc transferase.

The Liver X Receptor α (LXRα) belongs to the nuclear receptor superfamily and plays an essential role in regulating cholesterol, lipid and glucose metabolism and inflammatory responses. We have previously shown that LXRα is post-translationally modified by O-linked ß-N-acetyl-glucosamine (O-GlcNAc) with increased transcriptional activity. Moreover, we showed that LXRα associates with O-GlcNAc transferase (OGT) in vitro and in vivo in mouse liver. In this study, we report that human LXRα is O-GlcNAc modified in its N-terminal domain (NTD) by identifying a specific O-GlcNAc site S49 and a novel O-GlcNAc modified peptide 20LWKPGAQDASSQAQGGSSCILRE42. However, O-GlcNAc site-mutations did not modulate LXRα transactivation of selected target gene promoters in vitro. Peptide array and co-immunoprecipitation assays demonstrate that LXRα interacts with OGT in its NTD and ligand-binding domain (LBD) in a ligand-independent fashion. Moreover, we map two new O-GlcNAc sites in the longest OGT isoform (ncOGT): S437 in the tetratricopeptide repeat (TPR) 13 domain and T1043 in the far C-terminus, and a new O-GlcNAc modified peptide (amino acids 826-832) in the intervening region (Int-D) within the catalytic domain. We also map four new O-GlcNAc sites in the short isoform sOGT: S391, T393, S399 and S437 in the TPRs 11-13 domain. Future studies will reveal the biological role of identified O-GlcNAc sites in LXRα and OGT.

LXRα Regulates Hepatic ChREBPα Activity and Lipogenesis upon Glucose, but Not Fructose Feeding in Mice.

Liver X receptors (LXRα/ß) and carbohydrate response element-binding proteins (ChREBPα/ß) are key players in the transcriptional control of hepatic de novo lipogenesis. LXRα/ß double knockout (LXRα-/-/ß-/-) mice have reduced feeding-induced nuclear O-linked N-acetylglucosamine (O-GlcNAc) signaling, ChREBPα activity, and lipogenic gene expression in livers, suggesting important roles for LXRs in linking hepatic glucose utilization to lipid synthesis. However, the role of LXRs in fructose-induced ChREBP activation and lipogenesis is currently unknown. In this study, we studied the effects of high fructose or high glucose feeding on hepatic carbohydrate metabolism and lipogenic gene expression in livers from fasted (24 h) and fasted-refed (12 h) wild type and LXRα knockout (LXRα-/-) mice. Hepatic lipogenic gene expression was reduced in glucose fed, but not fructose fed LXRα-/- mice. This was associated with lower expression of liver pyruvate-kinase (L-pk) and Chrebpß, indicating reduced ChREBPα activity in glucose fed, but not fructose fed mice. Interestingly, ChREBP binding to the L-pk promoter was increased in fructose fed LXRα-/- mice, concomitant with increased glucose-6-phosphatase (G6pc) expression and O-GlcNAc modified LXRß, suggesting a role for LXRß in regulating ChREBPα activity upon fructose feeding. In conclusion, we propose that LXRα is an important regulator of hepatic lipogenesis and ChREBPα activity upon glucose, but not fructose feeding in mice.

Liver X receptor regulates hepatic nuclear O-GlcNAc signaling and carbohydrate responsive element-binding protein activity.

Liver X receptor (LXR)α and LXRß play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription in response to feeding, which is believed to be mediated by insulin. We have previously shown that LXRs are targets for glucose-hexosamine-derived O-linked ß-N-acetylglucosamine (O-GlcNAc) modification enhancing their ability to regulate SREBP-1c promoter activity in vitro. To elucidate insulin-independent effects of feeding on LXR-mediated lipogenic gene expression in vivo, we subjected control and streptozotocin-treated LXRα/ß(+/+) and LXRα/ß(-/-) mice to a fasting-refeeding regime. We show that under hyperglycemic and hypoinsulinemic conditions, LXRs maintain their ability to upregulate the expression of glycolytic and lipogenic enzymes, including glucokinase (GK), SREBP-1c, ChREBPα, and the newly identified shorter isoform ChREBPß. Furthermore, glucose-dependent increases in LXR/retinoid X receptor-regulated luciferase activity driven by the ChREBPα promoter was mediated, at least in part, by O-GlcNAc transferase (OGT) signaling in Huh7 cells. Moreover, we show that LXR and OGT interact and colocalize in the nucleus and that loss of LXRs profoundly reduced nuclear O-GlcNAc signaling and ChREBPα promoter binding activity in vivo. In summary, our study provides evidence that LXRs act as nutrient and glucose metabolic sensors upstream of ChREBP by modulating GK expression, nuclear O-GlcNAc signaling, and ChREBP expression and activity.

The molecular basis of emerin-emerin and emerin-BAF interactions.

Emerin is a conserved membrane component of nuclear lamina structure. Here, we report an advance in understanding the molecular basis of emerin function: intermolecular emerin-emerin association. There were two modes: one mediated by association of residues 170-220 in one emerin molecule to residues 170-220 in another, and the second involving residues 170-220 and 1-132. Deletion analysis showed residues 187-220 contain a positive element essential for intermolecular association in cells. By contrast, deletion of residues 168-186 inactivated a proposed negative element, required to limit or control association. Association of GFP-emerin with nuclear BAF in cells required the LEM domain (residues 1-47) and the positive element. Emerin peptide arrays revealed direct binding of residues 170-220 to residues 206-225 (the proposed positive element), residues 147-174 (particularly P(153)MYGRDSAYQSITHYRP(169)) and the LEM domain. Emerin residues 1-132 and 159-220 were each sufficient to bind lamin A or B1 tails in vitro, identifying two independent regions of molecular contact with lamins. These results, and predicted emerin intrinsic disorder, support the hypothesis that there are multiple 'backbone' and LEM-domain configurations in a proposed intermolecular emerin network at the nuclear envelope.

Cardiac O-GlcNAc signaling is increased in hypertrophy and heart failure.

Reversible protein O-GlcNAc modification has emerged as an essential intracellular signaling system in several tissues, including cardiovascular pathophysiology related to diabetes and acute ischemic stress. We tested the hypothesis that cardiac O-GlcNAc signaling is altered in chronic cardiac hypertrophy and failure of different etiologies. Global protein O-GlcNAcylation and the main enzymes regulating O-GlcNAc, O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), and glutamine-fructose-6-phosphate amidotransferase (GFAT) were measured by immunoblot and/or real-time RT-PCR analyses of left ventricular tissue from aortic stenosis (AS) patients and rat models of hypertension, myocardial infarction (MI), and aortic banding (AB), with and without failure. We show here that global O-GlcNAcylation was increased by 65% in AS patients, by 47% in hypertensive rats, by 81 and 58% post-AB, and 37 and 60% post-MI in hypertrophic and failing hearts, respectively (P < 0.05). Noticeably, protein O-GlcNAcylation patterns varied in hypertrophic vs. failing hearts, and the most extensive O-GlcNAcylation was observed on proteins of 20-100 kDa in size. OGT, OGA, and GFAT2 protein and/or mRNA levels were increased by pressure overload, while neither was regulated by myocardial infarction. Pharmacological inhibition of OGA decreased cardiac contractility in post-MI failing hearts, demonstrating a possible role of O-GlcNAcylation in development of chronic cardiac dysfunction. Our data support the novel concept that O-GlcNAc signaling is altered in various etiologies of cardiac hypertrophy and failure, including human aortic stenosis. This not only provides an exciting basis for discovery of new mechanisms underlying pathological cardiac remodeling but also implies protein O-GlcNAcylation as a possible new therapeutic target in heart failure.

Nuclear receptor liver X receptor is O-GlcNAc-modified in response to glucose.

Post-translational modification of nucleocytoplasmic proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) has for the last 25 years emerged as an essential glucose-sensing mechanism. The liver X receptors (LXRs) function as nutritional sensors for cholesterol-regulating lipid metabolism, glucose homeostasis, and inflammation. LXRs are shown to be post-translationally modified by phosphorylation, acetylation, and sumoylation, affecting their target gene specificity, stability, and transactivating and transrepressional activity, respectively. In the present study, we show for the first time that LXRalpha and LXRbeta are targets for glucose-hexosamine-derived O-GlcNAc modification in human Huh7 cells. Furthermore, we observed increased hepatic LXRalpha O-GlcNAcylation in vivo in refed mice and in streptozotocin-induced refed diabetic mice. Importantly, induction of LXRalpha O-GlcNAcylation in both mouse models was concomitant with increased expression of the lipogenic gene SREBP-1c (sterol regulatory element-binding protein 1c). Furthermore, glucose increased LXR/retinoic acid receptor-dependent activation of luciferase reporter activity driven by the mouse SREBP-1c promoter via the hexosamine biosynthetic pathway in Huh7 cells. Altogether, our results suggest that O-GlcNAcylation of LXR is a novel mechanism by which LXR acts as a glucose sensor affecting LXR-dependent gene expression, substantiating the crucial role of LXR as a nutritional sensor in lipid and glucose metabolism.

Adrenaline potentiates insulin-stimulated PKB activation in the rat fast-twitch epitrochlearis muscle without affecting IRS-1-associated PI 3-kinase activity.

We have previously shown in the rat slow-twitch soleus muscle that adrenaline greatly potentiates insulin-stimulated protein kinase B (PKB) phosphorylation without having an effect alone. However, insulin signalling capacity through the PKB pathway is higher in soleus than in fast-twitch muscles, whereas adrenaline activates phosphorylase more strongly in epitrochlearis. Therefore, the aim of the present study was to investigate the interaction between adrenaline and insulin signalling in the fast-twitch epitrochlearis muscle. Insulin increased insulin receptor substrate-1 (IRS-1)-associated phosphoinositide (PI) 3-kinase activity threefold, and adrenaline did not influence basal or insulin-stimulated PI 3-kinase activity. Insulin but not adrenaline increased PKB activity and phosphorylation of Ser(473) and Thr(308). It is interesting to note that adrenaline potentiated insulin-stimulated PKB activity and PKB Ser(473) and Thr(308) phosphorylation. These effects were mimicked by dibutyryl-cyclic adenosine monophosphate (db-cAMP). Adrenaline and db-cAMP increased glycogen synthase kinase (GSK)-3beta Ser(9) phosphorylation independently of PKB activation and enhanced insulin-stimulated GSK-3beta Ser(9) phosphorylation. Although adrenaline increased GSK-3 phosphorylation (inhibiting activity), phosphorylation of its target sites on glycogen synthase was increased, and adrenaline blocked insulin-stimulated glycogen synthase dephosphorylation of Ser(641) and Ser(645,649,653,657), glycogen synthase activation and glycogen synthesis. Insulin-stimulated glucose transport was not influenced by adrenaline despite the increased PKB activation. In conclusion, as in the slow-twitch soleus muscle, adrenaline potentiates insulin-stimulated PKB activation in the fast-twitch glycolytic epitrochlearis muscle without increasing IRS-1-associated PI 3-kinase activity. Furthermore, adrenaline induces phosphorylation of a pool of GSK-3 that is not involved in the regulation of glycogen metabolism. These results indicate that the combination of adrenaline and insulin may activate novel signalling molecules rather than just summing up their effects on linear pathways.