Hyung Wook Nam

Modification of p53 with O -linked N -acetylglucosamine regulates p53 activity and stability

Post-translational addition of O-linked N-acetylglucosamine (O-GlcNAc) to p53 is known to occur, but the site of O-GlcNAcylation and its effects on p53 are not understood. Here, we show that Ser 149 of p53 is O-GlcNAcylated and that this modification is associated with decreased phosphorylation of p53 at Thr 155, which is a site that is targeted by the COP9 signalosome, resulting in decreased p53 ubiquitination. Accordingly, O-GlcNAcylation at Ser 149 stabilizes p53 by blocking ubiquitin-dependent proteolysis. Our results indicate that the dynamic interplay between O-GlcNAc and O-phosphate modifications coordinately regulate p53 stability and activity.

NFκB activation is associated with its O-GlcNAcylation state under hyperglycemic conditions

The transcription factor NFκB is activated by phosphorylation and acetylation and plays important roles in inflammatory and immune responses in the cell. Additionally, posttranslational modification of the NFκB p65 subunit by O-linked N-acetylglucosamine (O-GlcNAc) has been reported, but the modification site of O-GlcNAc on NFκB p65 and its exact function have not been elucidated. In this work, we show that O-GlcNAcylation of NFκB p65 decreases binding to IκBα and increases transcriptional activity under hyperglycemic conditions. Also, we demonstrate that both Thr-322 and Thr-352 of NFκB p65 can be modified with O-GlcNAc, but modification on Thr-352, not Thr-322, is important for transcriptional activation. Our findings suggest that site-specific O-GlcNAcylation may be a reason why NFκB activity increases continuously under hyperglycemic conditions.

Irreversible Oxidation of the Active-site Cysteine of Peroxiredoxin to Cysteine Sulfonic Acid for Enhanced Molecular Chaperone Activity

The thiol (–SH) of the active cysteine residue in peroxiredoxin (Prx) is known to be reversibly hyperoxidized to cysteine sulfinic acid (–SO2H), which can be reduced back to thiol by sulfiredoxin/sestrin. However, hyperoxidized Prx of an irreversible nature has not been reported yet. Using an antibody developed against the sulfonylated (–SO3H) yeast Prx (Tsa1p) active-site peptide (AFTFVCPTEI), we observed an increase in the immunoblot intensity in proportion to the H2O2 concentrations administered to the yeast cells. We identified two species of hyperoxidized Tsa1p: one can be reduced back (reversible) with sulfiredoxin, and the other cannot (irreversible). Irreversibly hyperoxidized Tsa1p was identified as containing the active-site cysteine sulfonic acid (Tsa1p-SO3H) by mass spectrometry. Tsa1p-SO3H was not an autoxidation product of Tsa1p-SO2H and was maintained in yeast cells even after two doubling cycles. Tsa1p-SO3H self-assembled into a ring-shaped multimeric form was shown by electron microscopy. Although the Tsa1p-SO3H multimer lost its peroxidase activity, it gained ∼4-fold higher chaperone activity compared with Tsa1p-SH. In this study, we identify an irreversibly hyperoxidized Prx, Tsa1p-SO3H, with enhanced molecular chaperone activity and suggest that Tsa1p-SO3H is a marker of cumulative oxidative stress in cells.

Snail1 is stabilized by O-GlcNAc modification in hyperglycaemic condition.

Protein O‐phosphorylation often occurs reciprocally with O‐GlcNAc modification and represents a regulatory principle for proteins. O‐phosphorylation of serine by glycogen synthase kinase‐3β on Snail1, a transcriptional repressor of E‐cadherin and a key regulator of the epithelial–mesenchymal transition (EMT) programme, results in its proteasomal degradation. We show that by suppressing O‐phosphorylation‐mediated degradation, O‐GlcNAc at serine112 stabilizes Snail1 and thus increases its repressor function, which in turn attenuates E‐cadherin mRNA expression. Hyperglycaemic condition enhances O‐GlcNAc modification and initiates EMT by transcriptional suppression of E‐cadherin through Snail1. Thus, dynamic reciprocal O‐phosphorylation and O‐GlcNAc modification of Snail1 constitute a molecular link between cellular glucose metabolism and the control of EMT.

Adenosine Transporter ENT1 Regulates the Acquisition of Goal-Directed Behavior and Ethanol Drinking through A2A Receptor in the Dorsomedial Striatum

Adenosine signaling has been implicated in the pathophysiology of many psychiatric disorders including alcoholism. Striatal adenosine A2A receptors (A2AR) play an essential role in both ethanol drinking and the shift from goal-directed action to habitual behavior. However, direct evidence for a role of striatal A2AR signaling in ethanol drinking and habit development has not been established. In the present study, we found that decreased A2AR-mediated CREB activity in the dorsomedial striatum (DMS) enhanced initial behavioral acquisition of goal-directed behaviors and the vulnerability to progress to excessive ethanol drinking during operant conditioning in mice lacking ethanol-sensitive adenosine transporter ENT1 (ENT1−/−). Using mice expressing β-galactosidase (lacZ) under the control of seven repeated CRE sites in both genotypes (CRE-lacZ/ENT1+/+ mice and CRE-lacZ/ENT1−/− mice) and the dominant-negative form of CREB, we found that reduced CREB activity in the DMS was causally associated with decreased A2AR signaling and increased goal-directed ethanol drinking. Finally, we have demonstrated that the A2AR antagonist ZM241385 dampened protein kinase A activity–mediated signaling in the DMS and promoted excessive ethanol drinking in ENT1+/+ mice, but not in ENT1−/− mice. Our results indicate that A2AR-mediated CREB signaling in the DMS is a key determinant in enhancing the development of goal-directed ethanol drinking in mice.

Altered glutamatergic neurotransmission in the striatum regulates ethanol sensitivity and intake in mice lacking ENT1

Alcohol-sensitive type 1 equilibrative nucleoside transporter (ENT1) regulates adenosine-mediated glutamate neurotransmission in the brain. Our behavioral studies suggest that the diminished aversive effects of ethanol and the increased resistance to acute ethanol intoxication in mice lacking ENT1, could be related to increased voluntary ethanol self-seeking behavior. In addition, we found that ENT1 null mice were resistant to the ataxic effects of glutamate antagonists when tested on a rotarod. Using microdialysis experiments, we examined glutamate levels in the dorsal and ventral striatum in response to ethanol. In the dorsal striatum of ENT1 null mice, a low intoxicating dose of ethanol (1.5 g/kg) induced a greater increase of glutamate levels, while a higher hypnotic dose of ethanol (3.0 g/kg) decreased to a lesser degree the glutamate levels, compared with that of wild-type mice. In the ventral striatum, however, the low (1.5 g/kg) and the high (3.0 g/kg) ethanol doses altered glutamate levels similarly in both genotypes. Our results suggest that adenosine-regulated glutamatergic signaling contributes to a reduced level of alcohol response, which might be associated with a higher susceptibility for alcoholism in humans.

Comprehensive Royal Jelly (RJ) Proteomics Using One- and Two-Dimensional Proteomics Platforms Reveals Novel RJ Proteins and Potential Phospho/Glycoproteins

Royal jelly (RJ) is an exclusive food for queen honey bee (Apis mellifera L.) that is synthesized and secreted by young worker bees. RJ is also widely used in medical products, cosmetics, and as health foods. However, little is known about RJ functionality and the total protein components, although recent research is attempting to unravel the RJ proteome. We have embarked on a detailed investigation of the RJ proteome, using a modified protein extraction protocol and two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with tandem mass spectrometry. Simultaneously, we examined total soluble protein from RJ collected at 24, 48, and 72 h after honey bee larvae deposition twice (in two flower blooming seasons), to check differences, if any, in RJ proteome therein. Both 1- and 2-D gels stained with silver nitrate revealed similar protein profiles among these three time points. However, we observed a clear difference in two bands (ca. MW of 55 and 75 kDa) on 1-D gel between the first and the second collection of RJ. A similar difference was also observed in the 2-D gel. Except for this difference, the protein profiles were similar at the 3 time points. As the RJ from 48 (or sometimes 72) is commercially used, we selected the RJ sample at 48 h for detailed analysis with the first collection. 1-DGE identified 90 and 15 proteins from the first and second selection, respectively; in total, 47 nonredundant proteins were identified. 2-DGE identified 105 proteins comprising 14 nonredundant proteins. In total, 52 nonredundant proteins were identified in this study, and other than the major royal jelly protein family and some other previously identified proteins, 42 novel proteins were identified. Furthermore, we also report potentially post-translationally modified (phosphorylation and glycosylation) RJ proteins based on the Pro-Q diamond/emerald phosphoprotein/glycoprotein gel stains; MRJP 2p and 7p were suggested as potential phosphoproteins. The 2-DGE data were integrated to develop a 2-D gel reference map, and all data are accessible through RJ proteomics portal (http://foodfunc.agr.ibaraki.ac.jp/RJP.html).

Novel Protective Mechanism against Irreversible Hyperoxidation of Peroxiredoxin Nα-TERMINAL ACETYLATION OF HUMAN PEROXIREDOXIN II

Peroxiredoxins (Prxs) are a group of peroxidases containing a cysteine thiol at their catalytic site. During peroxidase catalysis, the catalytic cysteine, referred to as the peroxidatic cysteine (CP), cycles between thiol (CP-SH) and disulfide (–S–S–) states via a sulfenic (CP-SOH) intermediate. Hyperoxidation of the CP thiol to its sulfinic (CP-SO2H) derivative has been shown to be reversible, but its sulfonic (CP-SO3H) derivative is irreversible. Our comparative study of hyperoxidation and regeneration of Prx I and Prx II in HeLa cells revealed that Prx II is more susceptible than Prx I to hyperoxidation and that the majority of the hyperoxidized Prx II formation is reversible. However, the hyperoxidized Prx I showed much less reversibility because of the formation of its irreversible sulfonic derivative, as verified with CP-SO3H-specific antiserum. In an attempt to identify the multiple hyperoxidized spots of the Prx I on two-dimensional PAGE analysis, an N-acetylated Prx I was identified as part of the total Prx I using anti-acetylated Lys antibody. Using peptidyl-Asp metalloendopeptidase (EC 3.4.24.33) peptide fingerprints, we found that Nα-terminal acetylation (Nα-Ac) occurred exclusively on Prx II after demethionylation. Nα-Ac of Prx II blocks Prx II from irreversible hyperoxidation without altering its affinity for hydrogen peroxide. A comparative study of non-Nα-acetylated and Nα-terminal acetylated Prx II revealed that Nα-Ac of Prx II induces a significant shift in the circular dichroism spectrum and elevation of Tm from 59.6 to 70.9 °C. These findings suggest that the structural maintenance of Prx II by Nα-Ac may be responsible for preventing its hyperoxidation to form CP-SO3H.

Type 1 Equilibrative Nucleoside Transporter Regulates Ethanol Drinking Through Accumbal N-Methyl-D-Aspartate Receptor Signaling

BACKGROUND Mice lacking type 1 equilibrative nucleoside transporter (ENT1(-/-)) exhibit increased ethanol-preferring behavior compared with wild-type littermates. This phenotype of ENT1(-/-) mice appears to be correlated with increased glutamate levels in the nucleus accumbens (NAc). However, little is known about the downstream consequences of increased glutamate signaling in the NAc. METHODS To investigate the significance of the deletion of ENT1 and its effect on glutamate signaling in the NAc, we employed microdialysis and iTRAQ proteomics. We validated altered proteins using Western blot analysis. We then examined the pharmacological effects of the inhibition of the N-methyl-D-aspartate (NMDA) glutamate receptor and protein kinase Cγ (PKCγ) on alcohol drinking in wild-type mice. In addition, we investigated in vivo cyclic adenosine monophosphate response element binding activity using cyclic adenosine monophosphate response element-β-galactosidase mice in an ENT1(-/-) background. RESULTS We identified that NMDA glutamate receptor-mediated downregulation of intracellular PKCγ-neurogranin-calcium-calmodulin dependent protein kinase type II signaling is correlated with reduced cyclic adenosine monophosphate response element binding activity in ENT1(-/-) mice. Inhibition of PKCγ promotes ethanol drinking in wild-type mice to levels similar to those of ENT1(-/-) mice. In contrast, an NMDA glutamate receptor antagonist reduces ethanol drinking of ENT1(-/-) mice. CONCLUSIONS These findings demonstrate that the genetic deletion or pharmacological inhibition of ENT1 regulates NMDA glutamate receptor-mediated signaling in the NAc, which provides a molecular basis that underlies the ethanol-preferring behavior of ENT1(-/-) mice.

Regulation of Telomeric Repeat Binding Factor 1 Binding to Telomeres by Casein Kinase 2-mediated Phosphorylation

Telomere maintenance is essential for continued cell proliferation and chromosome stability. Telomeres are maintained by telomerase and a collection of associated proteins. The telomeric protein telomeric repeat binding factor 1 (TRF1) negatively regulates telomere length by inhibiting access of telomerase at telomere termini. Here we report that TRF1 interacts with the β subunit of casein kinase 2 (CK2) and serves as a substrate for CK2. CK2-mediated phosphorylation is required for the efficient telomere binding of TRF1 in vitro and in vivo. Inhibition of CK2 by the CK2 inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole decreased the ability of TRF1 to bind telomeric DNA. The resulting telomere-unbound form of TRF1 was then ubiquitinated and degraded by the proteasome. Partial knockdown of CK2 by small interfering RNA resulted in removal of TRF1 from telomeres and subsequent degradation of TRF1. Mapping of the CK2 target site identified threonine 122 as a substrate in TRF1. A threonine to alanine change at this position led to a diminished DNA binding due to reduced dimerization of TRF1. In addition, phosphorylation of threonine 122 seemed critical for TRF1-mediated telomere length control. Our findings suggest that CK2-mediated phosphorylation of TRF1 plays an important role in modulating telomere length homeostasis by determining the levels of TRF1 at telomeres.