Mechanical stress alters protein O‐GlcNAc in human periodontal ligament cells

Protein O‐linked N‐acetylglucosamine (O‐GlcNAc) is a post‐translational modification of intracellular proteins that regulates several physiological and pathophysiological process, including response to various stressors. However, O‐GlcNAc's response to mechanical stress has not been investigated yet. As human periodontal ligament (PDL) cells are stimulated by compression force during orthodontic tooth movement that results in structural remodelling, in this study we investigated whether mechanical stress induces any alteration in protein O‐GlcNAc in PDL cells. In this study, PDL cells isolated from premolars extracted for orthodontic indications were exposed to 0, 1.5, 3, 7 and 14 g/cm² compression forces for 12 hours. Cell viability was measured by flow cytometry, and protein O‐GlcNAc was analysed by Western blot. Cellular structure and intracellular distribution of O‐GlcNAc was studied by immunofluorescence microscopy. We found that between 1.5 and 3 g/cm² mechanical compression, O‐GlcNAc significantly elevated; however, at higher forces O‐GlcNAc level was not increased. We also found that intracellular localization of O‐GlcNAc proteins became more centralized under 2 g/cm² compression force. Our results suggest that structural changes stimulated by compression forces have a significant effect on the regulation of O‐GlcNAc; thus, it might play a role in the mechanical stress adaptation of PDL cells.     

TET‐catalyzed oxidation of intragenic 5‐methylcytosine regulates CTCF‐dependent alternative splicing

Intragenic 5‐methylcytosine and CTCF mediate opposing effects on pre‐mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5‐methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF‐binding sites were unclear. Here, we reveal the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF‐mediated alternative splicing through conversion of 5‐methylcytosine to its oxidation derivatives. 5‐hydroxymethylcytosine and 5‐carboxylcytosine are enriched at an intragenic CTCF‐binding sites in the CD45 model gene and are associated with alternative exon inclusion. Reduced TET levels culminate in increased 5‐methylcytosine, resulting in CTCF eviction and exon exclusion. In vitro analyses establish the oxidation derivatives are not sufficient to stimulate splicing, but efficiently promote CTCF association. We further show genomewide that reciprocal exchange of 5‐hydroxymethylcytosine and 5‐methylcytosine at downstream CTCF‐binding sites is a general feature of alternative splicing in naïve and activated CD4⁺ T cells. These findings significantly expand our current concept of the pre‐mRNA “splicing code” to include dynamic intragenic DNA methylation catalyzed by the TET proteins.     

A flavonoid 3‐O‐glucoside:2″‐O‐glucosyltransferase responsible for terminal modification of pollen‐specific flavonols in Arabidopsis thaliana

Flavonol 3‐O‐diglucosides with a 1→2 inter‐glycosidic linkage are representative pollen‐specific flavonols that are widely distributed in plants, but their biosynthetic genes and physiological roles are not well understood. Flavonoid analysis of four Arabidopsis floral organs (pistils, stamens, petals and calyxes) and flowers of wild‐type and male sterility 1 (ms1) mutants, which are defective in normal development of pollen and tapetum, showed that kaempferol/quercetin 3‐O‐β‐d ‐glucopyranosyl‐(1→2)‐β‐d ‐glucopyranosides accumulated in Arabidopsis pollen. Microarray data using wild‐type and ms1 mutants, gene expression patterns in various organs, and phylogenetic analysis of UDP‐glycosyltransferases (UGTs) suggest that UGT79B6 (At5g54010) is a key modification enzyme for determining pollen‐specific flavonol structure. Kaempferol and quercetin 3‐O‐glucosyl‐(1→2)‐glucosides were absent from two independent ugt79b6 knockout mutants. Transgenic ugt79b6 mutant lines transformed with the genomic UGT79B6 gene had the same flavonoid profile as wild‐type plants. Recombinant UGT79B6 protein converted kaempferol 3‐O‐glucoside to kaempferol 3‐O‐glucosyl‐(1→2)‐glucoside. UGT79B6 recognized 3‐O‐glucosylated/galactosylated anthocyanins/flavonols but not 3,5‐ or 3,7‐diglycosylated flavonoids, and prefers UDP‐glucose, indicating that UGT79B6 encodes flavonoid 3‐O‐glucoside:2″‐O‐glucosyltransferase. A UGT79B6‐GUS fusion showed that UGT79B6 was localized in tapetum cells and microspores of developing anthers.     

Oxidative stress induces transient O‐GlcNAc elevation and tau dephosphorylation in SH‐SY5Y cells

O‐linked β‐N‐acetlyglucosamine or O‐GlcNAc modification is a dynamic post‐translational modification occurring on the Ser/Thr residues of many intracellular proteins. The chronic imbalance between phosphorylation and O‐GlcNAc on tau protein is considered as one of the main hallmarks of Alzheimer's disease. In recent years, many studies also showed that O‐GlcNAc levels can elevate upon acute stress and suggested that this might facilitate cell survival. However, many consider chronic stress, including oxidative damage as a major risk factor in the development of the disease. In this study, using the neuronal cell line SH‐SY5Y we investigated the dynamic nature of O‐GlcNAc after treatment with 0.5 mM H₂O₂ for 30 min. to induce oxidative stress. We found that overall O‐GlcNAc quickly increased and reached peak level at around 2 hrs post‐stress, then returned to baseline levels after about 24 hrs. Interestingly, we also found that tau protein phosphorylation at site S262 showed parallel, whereas at S199 and PHF1 sites showed inverse dynamic to O‐Glycosylation. In conclusion, our results show that temporary elevation in O‐GlcNAc modification after H₂O₂‐induced oxidative stress is detectable in cells of neuronal origin. Furthermore, oxidative stress changes the dynamic balance between O‐GlcNAc and phosphorylation on tau proteins.     

Glutathione S‐transferase π complexes with and stimulates Na+,K+‐ATPase

Glutathione S‐transferase (GST) was found to complex with the Na⁺,K⁺‐ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α‐subunit of the Na⁺,K⁺‐ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺‐ATPase. Co‐immunoprecipitation experiments, using the anti‐α‐subunit antiserum to precipitate the GST‐Na⁺,K⁺‐ATPase complex and then using antibodies specific to an isoform of GST to identify the co‐precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺‐ATPase. GST stimulated the Na⁺,K⁺‐ATPase activity up to 1.4‐fold. The level of stimulation exhibited a saturable dose–response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺‐ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺‐ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺‐ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Mapping of O‐linked β‐N‐acetylglucosamine modification sites in key contractile proteins of rat skeletal muscle

O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is a widespread modification of serine/threonine residues of nucleocytoplasmic proteins. Recently, several key contractile proteins in rat skeletal muscle (i.e., myosin heavy and light chains and actin) were identified as O‐GlcNAc modified. Moreover, it was demonstrated that O‐GlcNAc moieties involved in contractile protein interactions could modulate Ca²⁺ activation parameters of contraction. In order to better understand how O‐GlcNAc can modulate the contractile activity of muscle fibers, we decided to identify the sites of O‐GlcNAc modification in purified contractile protein homogenates. Using an MS‐based method that relies on mild β‐elimination followed by Michael addition of DTT (BEMAD), we determined the localization of one O‐GlcNAc site in the subdomain four of actin and four O‐GlcNAc sites in the light meromyosin region of myosin heavy chains (MHC). According to previous reports concerning the role of these regions, our data suggest that O‐GlcNAc sites might modulate the actin–tropomyosin interaction, and be involved in MHC polymerization or interactions between MHC and other contractile proteins. Thus, the results suggest that this PTM might be involved in protein–protein interactions but could also modulate the contractile properties of skeletal muscle.     

Conformational evaluation of labeled C3′‐O‐P‐13CH2‐O‐C4″ phosphonate internucleotide linkage,a phosphodiester isostere

Modified internucleotide linkage featuring the C3′‐O‐P‐CH₂‐O‐C4″ phosphonate grouping as an isosteric alternative to the phosphodiester C3′‐O‐P‐O‐CH₂‐C4″ bond was studied in order to learn more on its stereochemical arrangement, which we showed earlier to be of prime importance for the properties of the respective oligonucleotide analogues. Two approaches were pursued: First, the attempt to prepare the model dinucleoside phosphonate with ¹³C‐labeled CH₂ group present in the modified internucleotide linkage that would allow for a more detailed evaluation of the linkage conformation by NMR spectroscopy. Second, the use of ab initio calculations along with molecular dynamics (MD) simulations in order to observe the most populated conformations and specify main structural elements governing the conformational preferences. To deal with the former aim, a novel synthesis of key labeled reagent (CH₃O)₂P(O)¹³CH₂OH for dimer preparation had to be elaborated using aqueous ¹³C‐formaldehyde. The results from both approaches were compared and found consistent. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 514–529, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Characterization of population structure from the mitochondrial DNA vis‐à‐vis language and geography in Papua New Guinea

Situated along a corridor linking the Asian continent with the outer islands of the Pacific, Papua New Guinea has long played a key role in understanding the initial peopling of Oceania. The vast diversity in languages and unique geographical environments in the region have been central to the debates on human migration and the degree of interaction between the Pleistocene settlers and newer migrants. To better understand the role of Papua New Guinea in shaping the region's prehistory, we sequenced the mitochondrial DNA (mtDNA) control region of three populations, a total of 94 individuals, located in the East Sepik Province of Papua New Guinea. We analyzed these samples with a large data set of Oceania populations to examine the role of geography and language in shaping population structure within New Guinea and between the region and Island Melanesia. Our results from median‐joining networks, star‐cluster age estimates, and population genetic analyses show that while highland New Guinea populations seem to be the oldest settlers, there has been significant gene flow within New Guinea with little influence from geography or language. The highest genetic division is between Papuan speakers of New Guinea versus East Papuan speakers located outside of mainland New Guinea. Our study supports the weak language barriers to genetic structuring among populations in close contact and highlights the complexity of understanding the genetic histories of Papua New Guinea in association with language and geography. Am J Phys Anthropol 142:613–624, 2010. © 2010 Wiley‐Liss, Inc.     

Substituent effects on chiral resolutions of derivatized 1‐phenylalkylamines by heptakis(2,3‐di‐O‐methyl‐6‐O‐tert‐butyldimethylsilyl)‐β‐cyclodextrin GC stationary phase

Chiral resolutions of trifluoroacetyl‐derivatized 1‐phenylalkylamines with different type and position of substituent were investigated by capillary gas chromatography by using heptakis(2,3‐di‐O‐methyl‐6‐O‐tert‐butyldimethylsilyl)‐β‐cyclodextrin diluted in OV‐1701 as a chiral stationary phase. The influence of column temperature on retention and enantioselectivity was examined. All enantiomers of meta‐substituted analytes as well as fluoro‐substituted analytes could be resolved. Temperature had a favorable influence on enantioselectivity for small amines with substituents at the ortho‐position. The type of substituent at the stereogenic center of amines also had a crucial effect as the ethyl group led to poor enantioseparation. Among all analytes studied, trifluoroacetyl‐derivatized 1‐(2′‐fluorophenyl)ethylamine exhibited baseline resolution with the shortest analysis time.     

6‐O‐(3″, 4″‐di‐O‐trans‐cinnamoyl)‐α‐l‐rhamnopyranosylcatalpol and verbascoside: Cytotoxicity,cell cycle kinetics,apoptosis, and ROS production evaluation in tumor cells

The aim of this study was to evaluate the impact that 6‐O‐(3″, 4″‐di‐O‐trans‐cinnamoyl)‐α‐ l ‐rhamnopyranosylcatalpol (Dicinn) and verbascoside (Verb), two compounds simultaneously reported in Verbascum ovalifolium, have on tumor cell viability, apoptosis, cell cycle kinetics, and intracellular reactive oxygen species (ROS) level. At 100 µg/mL and 48 hours incubation time, Dicinn and Verb produced good cytotoxic effects in A549, HT‐29, and MCF‐7 cells. Dicinn induced cell‐cycle arrest at the G₀/G₁ phase and apoptosis, whereas Verb increased the population of subG₁ cells and cell apoptosis rates. Furthermore, the two compounds exhibited time‐dependent ROS generating effects in tumor cells (1‐24 hours). Importantly, no cytotoxic effects were induced in nontumor MCF‐10A cells by the two compounds up to 100 µg/mL. Overall, the effects exhibited by Verb in tumor cells were more potent, which can be correlated with its structural features, such as the presence of phenolic hydroxyl groups.     

Crystal structures of the archaeal UDP‐GlcNAc 2‐epimerase from Methanocaldococcus jannaschii reveal a conformational change induced by UDP‐GlcNAc

Uridine diphosphate N ‐ acetylglucosamine (UDP‐GlcNAc) 2‐epimerase catalyzes the interconversion of UDP‐GlcNAc to UDP‐N‐acetylmannosamine (UDP‐ManNAc), which is used in the biosynthesis of cell surface polysaccharides in bacteria. Biochemical experiments have demonstrated that mutation of this enzyme causes changes in cell morphology and the thermoresistance of the cell wall. Here, we present the crystal structures of Methanocaldococcus jannaschii UDP‐GlcNAc 2‐epimerase in open and closed conformations. A comparison of these crystal structures shows that upon UDP and UDP‐GlcNAc binding, the enzyme undergoes conformational changes involving a rigid‐body movement of the C‐terminal domain. We also present the crystal structure of Bacillus subtilis UDP‐GlcNAc 2‐epimerase in the closed conformation in the presence of UDP and UDP‐GlcNAc. Although a structural overlay of these two closed‐form structures reveals that the substrate‐binding site is evolutionarily conserved, some areas of the allosteric site are distinct between the archaeal and bacterial UDP‐GlcNAc 2‐epimerases. This is the first report on the crystal structure of archaeal UDP‐GlcNAc 2‐epimerase, and our results clearly demonstrate the changes between the open and closed conformations of this enzyme. Proteins 2014; 82:1519–1526. © 2014 Wiley Periodicals, Inc.     

Preventing N‐ and O‐formylation of proteins when incubated in concentrated formic acid

Concentrated formic acid is among the most effective solvents for protein solubilization. Unfortunately, this acid also presents a risk of inducing chemical modifications thereby limiting its use in proteomics. Previous reports have supported the esterification of serine and threonine residues (O‐formylation) for peptides incubated in formic acid. However as shown here, exposure of histone H4 to 80% formic (1 h, 20^oC) induces N‐formylation of two independent lysine residues. Furthermore, incubating a mixture of Escherichia coli proteins in formic acid demonstrates a clear preference toward lysine modification over reactions at serine/threonine. N‐formylation accounts for 84% of the 225 uniquely identified formylation sites. To prevent formylation, we provide a detailed investigation of reaction conditions (temperature, time, acid concentration) that define the parameters permitting the use of concentrated formic acid in a proteomics workflow for MS characterization. Proteins can be maintained in 80% formic acid for extended periods (24 h) without inducing modification, so long as the temperature is maintained at or below –20^oC.     

Mechanism of dihydrofolate reductase downregulation in melanoma by 3‐O‐(3,4,5‐trimethoxybenzoyl)‐(−)‐epicatechin

In our search to improve the stability and cellular absorption of tea polyphenols, we synthesized 3‐O‐(3,4,5‐trimethoxybenzoyl)‐(?)‐epicatechin (TMECG), which showed high antiproliferative activity against melanoma. TMECG downregulates dihydrofolate reductase (DHFR) expression in melanoma cells and we detail the sequential mechanisms that result from this even. TMECG is specifically activated in melanoma cells to form a stable quinone methide (TMECG‐QM). TMECG‐QM has a dual action on these cells. First, it acts as a potent antifolate compound, disrupting folate metabolism and increasing intracellular oxidized folate coenzymes, such as dihydrofolate, which is a non‐competitive inhibitor of dihydropterine reductase, an enzyme essential for tetrahydrobiopterin (H₄B) recycling. Such inhibition results in H₄B deficiency, endothelial nitric oxide synthase (eNOS) uncoupling and superoxide production. Second, TMECG‐QM acts as an efficient superoxide scavenger and promotes intra‐cellular H₂O₂ accumulation. Here, we present evidence that TMECG markedly reduces melanoma H₄B and NO bioavailability and that TMECG action is abolished by the eNOS inhibitor N_ω‐nitro‐L ‐arginine methyl ester or the H₂O₂ scavenger catalase, which strongly suggests H₂O₂‐dependent DHFR downregulation. In addition, the data presented here indicate that the simultaneous targeting of important pathways for melanoma survival, such as the folate cycle, H₄B recycling, and the eNOS reaction, could represent an attractive strategy for fighting this malignant skin pathology. J. Cell. Biochem. 110: 1399–1409, 2010. © 2010 Wiley‐Liss, Inc.