Yu Sam Kim

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.

Effects of pH and Dissolved Oxygen on Cellulose Production by Acetobacter xylinum BRC5 in Agitated Culture

Acetobacter xylinum BRC5 was cultivated in a jar fermentor using glucose as the sole carbon source. Strain BRC5 oxidized almost all of the glucose to gluconic acid; thereafter, it biosynthesized cellulose by utilizing gluconic acid accumulated in the broth. The optimal pH for metabolizing glucose to gluconic acid was 4.0, while a pH of 5.5 was preferred for cell growth and cellulose production from the accumulated gluconic acid in the medium. Shifting the pH from 4.0 to 5.5 during the cellulose production phase in batch cultures improved cellulose production and reduced the total fermentation time, compared to batch cultures at constant pH. In constant fed-batch culture, 10 g/l of cellulose was obtained from 40 g/l of glucose, a yield which was approximately 2-fold higher than in batch culture with the same initial glucose concentration, even without control of the level of dissolved oxygen. The highest cellulose yield was obtained in fed-batch cultures in which the dissolved oxygen concentration was controlled at 10% saturation. Control of pH and dissolved oxygen to optimal levels was effective for improving the production rate and yield of cellulose, to achieve a high cellulose productivity of 0.3 g cellulose/l x h. Approximately 15 g/l of cellulose was considered to be the highest yield obtainable using conventional fermentors because the culture broth then became too viscous to allow satisfactory aeration.

Peroxisomal-proliferator-activated receptor alpha activates transcription of the rat hepatic malonyl-CoA decarboxylase gene: a key regulation of malonyl-CoA level.

MCD (malonyl-CoA decarboxylase), which catalyses decarboxylation of malonyl-CoA, is known to play an important role in the regulation of malonyl-CoA concentration. Recently, it has been observed that the expression of MCD is significantly decreased in the hearts of the PPARalpha (peroxisome-proliferator-activated receptor alpha) (-/-) mice, where the rate of fatty-acid oxidation is decreased by the increased malonyl-CoA level [Campbell, Kozak, Wagner, Altarejos, Dyck, Belke, Severson, Kelly and Lopaschuk (2002) J. Biol. Chem. 277, 4098-4103]. This suggests that MCD may be transcriptionally regulated by PPARalpha. To investigate whether PPARalpha is truly responsible for transcriptional regulation of the rat MCD gene, transient reporter assay was performed in CV-1 cells. The promoter activity was increased by 17-fold in CV-1 cells co-transfected with PPARalpha/retinoid X receptor alpha expression plasmid. In sequence analysis of the promoter region, three putative PPREs (PPAR response elements) were identified, and promoter deletion analysis showed that PPRE2 and PPRE3 were functional. Electrophoretic mobility-shift assays revealed that PPARalpha/retinoid X receptor alpha heterodimer indeed bound to the two PPREs, and the binding specificity of PPARalpha on PPRE was also confirmed by experiments with mutated oligonucleotides. These results indicate that the elements behaved as a responsive site to PPARalpha activation. MCD mRNA levels in WY14643-treated rat hepatoma cells as well as in the liver of fenofibrate-fed Otsuka Long-Evans Tokushima fatty rats were also found to be increased, suggesting that PPARalpha can activate the rat hepatic MCD transcription by binding to the PPREs in the promoter. We propose that MCD performs an important role in understanding the regulatory mechanism between activated PPARalpha and fatty-acid oxidation by altering the malonyl-CoA concentration.

MDGAs interact selectively with neuroligin-2 but not other neuroligins to regulate inhibitory synapse development

The MAM domain-containing GPI anchor proteins MDGA1 and MDGA2 are Ig superfamily adhesion molecules composed of six IG domains, a fibronectin III domain, a MAM domain, and a GPI anchor. MDGAs contribute to the radial migration and positioning of a subset of cortical neurons during early neural development. However, MDGAs continue to be expressed in postnatal brain, and their functions during postnatal neural development remain unknown. Here, we demonstrate that MDGAs specifically and with a nanomolar affinity bind to neuroligin-2, a cell-adhesion molecule of inhibitory synapses, but do not bind detectably to neuroligin-1 or neuroligin-3. We observed no cell adhesion between cells expressing neuroligin-2 and MDGA1, suggesting a cis interaction. Importantly, RNAi-mediated knockdown of MDGAs increased the abundance of inhibitory but not excitatory synapses in a neuroligin-2–dependent manner. Conversely, overexpression of MDGA1 decreased the numbers of functional inhibitory synapses. Likewise, coexpression of both MDGA1 and neuroligin-2 reduced the synaptogenic capacity of neuroligin-2 in an artificial synapse-formation assay by abolishing the ability of neuroligin-2 to form an adhesion complex with neurexins. Taken together, our data suggest that MDGAs inhibit the activity of neuroligin-2 in controlling the function of inhibitory synapses and that MDGAs do so by binding to neuroligin-2.

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.

Cellulose production by Acetobacter xylinum BRC5 under agitated condition

Abstract A potent cellulose producer, Acetobacter xylinum BRC5, was cultivated in shaking flasks and jar fermentors with glucose, fructose, sucrose and mixtures of these as carbon sources. It was confirmed that corn steep liquor (CSL) was cheap and a suitable organic nitrogen source for cellulose production by strain BRC5. When glucose was used as a sole carbon source, strain BRC5 oxidized almost all the glucose to gluconic acid, thereafter it biosynthesized cellulose by utilizing the gluconic acid accumulated in the broth under limited glucose conditions. Since strain BRC5 did not metabolize fructose to acid, the fermentation pattern of fructose was found to be typically growth associated with cellulose production. However, when glucose and fructose coexisted in the medium, strain BRC5 preferentially metabolized all the glucose to gluconic acid. Thereafter, it produced cellulose mainly by utilizing fructose. Overall cellulose productivity in a jar fermentor ranged from 0.071 to 0.086 g/ l /h.

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.