Active sites of human MEPE-ASARM regulating bone matrix mineralization.

The proteolytic fragment ASARM (acidic serine- and aspartate-rich motif) of MEPE (matrix extracellular phosphoglycoprotein) (MEPE-ASARM) may act as an endogenous anti-mineralization factor involved in X-linked hypophosphatemic rickets/osteomalacia (XLH). We synthesized MEPE-ASARM peptides and relevant peptide fragments with or without phosphorylated Ser residues (pSer) to determine the active site(s) of MEPE-ASARM in a rat calvaria cell culture model. None of the synthetic peptides elicited changes in cell death, proliferation or differentiation, but the peptide (pASARM) with three pSer residues inhibited mineralization without causing changes in gene expression of osteoblast markers tested. The anti-mineralization effect was maintained in peptides in which any one of three pSer residues was deleted. Polyclonal antibodies recognizing pASARM but not ASARM abolished the pASARM effect. Deletion of six N-terminal residues but leaving the recognition sites for PHEX (phosphate regulating endopeptidase homolog, X-linked), a membrane endopeptidase responsible for XLH, intact and two C-terminal amino acid residues did not alter the anti-mineralization activity of pASARM. Our results strengthen understanding of the active sites of MEPE-pASARM and allowed us to identify a shorter more stable sequence with fewer pSer residues still exhibiting hypomineralization activity, reducing peptide synthesis cost and increasing reliability for exploring biological and potential therapeutic effects.

Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists.

Agonists of the nociceptin/orphanin FQ opioid peptide (NOP) receptor, a member of the opioid receptor family, are under active investigation as novel analgesics, but their modes of signaling are less well characterized than those of other members of the opioid receptor family. Therefore, we investigated whether different NOP receptor ligands showed differential signaling or functional selectivity at the NOP receptor. Using newly developed phosphosite-specific antibodies to the NOP receptor, we found that agonist-induced NOP receptor phosphorylation occurred primarily at four carboxyl-terminal serine (Ser) and threonine (Thr) residues, namely, Ser346, Ser351, Thr362, and Ser363, and proceeded with a temporal hierarchy, with Ser346 as the first site of phosphorylation. G protein-coupled receptor kinases 2 and 3 (GRK2/3) cooperated during agonist-induced phosphorylation, which, in turn, facilitated NOP receptor desensitization and internalization. A comparison of structurally distinct NOP receptor agonists revealed dissociation in functional efficacies between G protein-dependent signaling and receptor phosphorylation. Furthermore, in NOP-eGFP and NOP-eYFP mice, NOP receptor agonists induced multisite phosphorylation and internalization in a dose-dependent and agonist-selective manner that could be blocked by specific antagonists. Our study provides new tools to study ligand-activated NOP receptor signaling in vitro and in vivo. Differential agonist-selective NOP receptor phosphorylation by chemically diverse NOP receptor agonists suggests that differential signaling by NOP receptor agonists may play a role in NOP receptor ligand pharmacology.

Free Radical-Initiated Peptide Sequencing Mass Spectrometry for Phosphopeptide Post-translational Modification Analysis.

Free radical-initiated peptide sequencing mass spectrometry (FRIPS MS) was employed to analyze a number of representative singly or doubly protonated phosphopeptides (phosphoserine and phosphotyrosine peptides) in positive ion mode. In contrast to collision-activated dissociation (CAD) results, a loss of a phosphate group occurred to a limited degree for both phosphoserine and phosphotyrosine peptides, and thus, localization of a phosphorylated site was readily achieved. Considering that FRIPS MS supplies a substantial amount of collisional energy to peptides, this result was quite unexpected because a labile phosphate group was conserved. Analysis of the resulting peptide fragments revealed the extensive production of a-, c-, x-, and z-type fragments (with some minor b- and y-type fragments), suggesting that radical-driven peptide fragmentation was the primary mechanism involved in the FRIPS MS of phosphopeptides. Results of this study clearly indicate that FRIPS MS is a promising tool for the characterization of post-translational modifications such as phosphorylation. Graphical Abstract.

Genetically Encoded Protein Phosphorylation in Mammalian Cells.

Protein phosphorylation regulates diverse processes in eukaryotic cells. Strategies for installing site-specific phosphorylation in target proteins in eukaryotic cells, through routes that are orthogonal to enzymatic post-translational modification, would provide a powerful route for defining the consequences of particular phosphorylations. Here we show that the SepRSv1.0/tRNAv1.0CUA pair (created from the Methanococcus maripaludis phosphoseryl-transfer RNA synthetase [MmSepRS]/Methanococcus janaschii [Mj]tRNAGCACys pair) is orthogonal in mammalian cells. We create a eukaryotic elongation factor 1 alpha (EF-1α) variant, EF-1α-Sep, that enhances phosphoserine incorporation, and combine this with a mutant of eRF1, and manipulations of the cell's phosphoserine biosynthetic pathway, to enable the genetically encoded incorporation of phosphoserine and its non-hydrolyzable phosphonate analog. Using this approach we demonstrate synthetic activation of a protein kinase in mammalian cells.

A novel phosphoserine motif in the LCMV matrix protein Z regulates the release of infectious virus and defective interfering particles.

We report that the lymphocytic choriomeningitis virus (LCMV) matrix protein, which drives viral budding, is phosphorylated at serine 41 (S41). A recombinant (r)LCMV bearing a phosphomimetic mutation (S41D) was impaired in infectious and defective interfering (DI) particle release, while a non-phosphorylatable mutant (S41A) was not. The S41D mutant was disproportionately impaired in its ability to release DI particles relative to infectious particles. Thus, DI particle production by LCMV may be dynamically regulated via phosphorylation of S41.

Gas chromatography with mass spectrometry analysis of phosphoserine, phosphoethanolamine, phosphoglycerol, and phosphate.

A new, rapid, sensitive, robust, and reliable method has been developed for the qualitative analysis of phosphoserine, phosphoethanolamine, phosphoglycerol, and phosphate using gas chromatography with mass spectrometry and two-step trimethylsilylation. The method employs hexamethyldisilazane for silylation of the phosphate and hydroxyl groups in the first phase and bis(trimethylsilyl)trifluoroacetamide for silylation of the less-reactive amino groups in the second phase. This order is of key importance for the method because of the different reactivities of the two reagents and the mechanism of derivatization of the active groups of the analytes. Trimethylsilylated derivatives of the analytes were identified on the basis of their retention times and mass spectra. The probable structures of the major fragments were identified in the spectra of the trimethylsilylated derivatives and characteristic m/z fragments were selected for each analyte. Fragments with m/z 73 and 299 occurred in the spectra of all the analytes. The characteristic retention data were employed to calculate the retention indices of the individual silylated phosphorylated substances in the hydrocarbon range C12-C19 for the DB-5ms column. The method was employed to measure the polar fraction of the hydrolysate of the cytoplasmic membrane of Bacillus subtilis. The detection limits vary between 5 µg/mL (trimethylsilylated phosphate) and 72 µg/mL (trimethylsilylated phosphoethanolamine).

Cyclosporine A enhances gingival ß-catenin stability via Wnt signaling.

BACKGROUND: Cyclosporine A (CsA) increases ß-catenin messenger RNA (mRNA) and protein expression. The present study demonstrates that Wnt/ß-catenin signaling inhibits ß-catenin degradation in the gingiva. METHODS: Forty 5-week-old male Sprague-Dawley rats were assigned to two study groups after healing from right maxillary molar extractions. The rats in the experimental group were fed 30 mg/kg CsA daily for 4 weeks, whereas the control rats were fed mineral oil. At the end of the study, all rats were sacrificed, and the gingivae were obtained. The gingival morphology after CsA treatment was evaluated by histology, and the genes related to Wnt/ß-catenin signaling were initially screened by microarray. Polymerase chain reaction, Western blotting, and immunohistochemistry were used to examine the mRNA and protein expression of proliferating cell nuclear antigen, cyclin D1, E-cadherin, ß-catenin, Dvl-1, glycogen synthase kinase-3ß, axin-1, and adenomatous polyposis coli (APC). Phosphoserine and ubiquitinylated ß-catenin were detected after immunoprecipitation. RESULTS: In rats treated with CsA, overgrowth of gingivae was observed, and altered expression of genes related to Wnt/ß-catenin signaling was detected by the microarray. The gingival mRNA and protein expression profiles for genes associated with Wnt/ß-catenin signaling further confirmed the effect of CsA: ß-catenin and Dvl-1 expression increased, but APC and axin-1 expression decreased. Western blotting and immunohistochemistry showed decreases in ß-catenin serine phosphorylation (33/37) and ubiquitinylation in the gingivae of CsA-treated rats. CONCLUSION: CsA-enhanced gingival ß-catenin stability may be involved in gene upregulation or ß-catenin degradation via the Wnt/ß-catenin pathway.

Beryllium is an inhibitor of cellular GSK-3ß that is 1,000-fold more potent than lithium.

Glycogen synthase kinase 3ß (GSK-3ß) is a key regulator in signaling networks that control cell proliferation, metabolism, development, and other processes. Lithium chloride is a GSK-3 family inhibitor that has been a mainstay of in vitro and in vivo studies for many years. Beryllium salt has the potential to act as a lithium-like inhibitor of GSK-3, but it is not known whether this agent is effective under physiologically relevant conditions. Here we show that BeSO4 inhibits endogenous GSK-3ß in cultured human cells. Exposure to 10 µM Be(2+) produced a decrease in GSK-3ß kinase activity that was comparable to that produced by 10 mM Li(+), indicating that beryllium is about 1,000-fold more potent than the classical inhibitor when treating intact cells. There was a statistically significant dose-dependent reduction in specific activity of GSK-3ß immunoprecipitated from cells that had been treated with either agent. Lithium inhibited GSK-3ß kinase activity directly, and it also caused GSK-3ß in cells to become phosphorylated at serine-9 (Ser-9), a post-translational modification that occurs as part of a well-known positive feedback loop that suppresses the kinase activity. Beryllium also inhibited the kinase directly, but unlike lithium it had little effect on Ser-9 phosphorylation in the cell types tested, suggesting that alternative modes of feedback inhibition may be elicited by this agent. These results indicate that beryllium, like lithium, can induce perturbations in the GSK-3ß signaling network of treated cells.

A selective fluorescent chemosensor for phosphoserine.

A fluorescent chemosensor for the detection of phosphoserine is reported. The ditopic sensor features a phosphate-coordinating zinc(II)-dipicolylamine (Zn(2+)-DPA) unit tethered to an amine-binding coumarin aldehyde fluorophore. With phosphoserine, the sensor demonstrates a 30-fold fluorescence enhancement under buffered aqueous conditions.

Determination of phosphoserine/threonine by nano ultra-performance liquid chromatography-tandem mass spectrometry coupled with microscale labeling.

Protein phosphorylation is an important regulatory post-translational modification in many biochemical processes. The phosphopeptide analysis strategies developed in this study were all at microscale. After using a standard microwave oven to assist protein digestion, phosphoserine and phosphothreonine were tagged with chemical analogues, such as 2-mercaptoethanol and 3-mercapto-1-propanol, to enable simultaneously relative quantitation and identification. This method enabled the use of thio alcohols for direct labeling of phosphorylated sites (not labeled at the mercapto, amino, hydroxyl, or carboxyl groups) of phosphopeptides. Various digestion parameters (e.g., microwave power, reaction time, NH4HCO3 concentration) and derivatization efficiency parameters (e.g., reaction time, labeling tag concentration) were studied and optimized. In both control and experimental samples, microwave-assisted digestion coupled with relative quantitation using analogue tags enabled calculation of phosphopeptide ratios in the same sequence. A non-labeling method was also established for quantifying phosphopeptides in human plasma by using the abundant protein albumin as an internal control for normalizing relative quantities of phosphopeptides. Nano ultra-performance liquid chromatography (nanoUPLC) was combined with LTQ Orbitrap to enable simultaneous protein relative quantitation and identification. These strategies proved to be effective for quantifying phosphopeptides in biological samples.

An high-performance liquid chromatographic method for the simultaneous analysis of acetylcarnitine taurinate, carnosine, asparagine and potassium aspartate and for the analysis of phosphoserine in alimentary supplements.

A RP-HPLC method with pre-column derivatization was developed and validated for the simultaneous quantification of carnosine (Carn), acetylcarnitine taurinate (AC-Tau), asparagine (Asn), potassium aspartate (Asp) and for the determination of phosphoserine (p-Ser) in new and commercial alimentary supplements. The effect of complex matrices was evaluated by the study of the amino acid derivatization reaction with 2,4-dinitrofluorobenzene (DNFB) both in standard and placebo solutions. The reaction was carried out for 20 min at 70 °C in alkaline medium (pH10) for p-Ser analysis, whereas for 60 min in the case of Carn, AC-Tau, Asn and Asp analysis. The adducts have been separated on a Discovery RP Amide C16 (250 mm×4.6mm, i.d.) column using a mobile phase consisting of acetonitrile (ACN) and triethylammonium (TEA) phosphate buffer (pH 3, 0.05 M) under gradient elution conditions at a flow-rate of 0.8 mL/min. Detection was set at λ=360 nm. The validation parameters such as linearity, sensitivity, accuracy, precision and specificity were found to be highly satisfactory. Linear responses were observed by placebo solutions (determination coefficient ≤0.9996). Intra-day precision (relative standard deviation, RSD) was ≤1.06% for corrected peak area and ≤0.99% for retention times (tR) without significant differences between intra- and inter-day data. Recovery studies showed good results for all examined compounds (from 97.7% to 101.5%) with RSD ranging from 0.5% to 1.3%). The high stability of derivatized compound solutions at room temperature means an undoubted advantage of the method allowing the simultaneous preparation of a large number of samples and consecutive chromatographic analyses by the use of an autosampler. The developed method can be considered suitable for the quality control of new and commercial products.

Role of individual MARK isoforms in phosphorylation of tau at Ser²6² in Alzheimer's disease.

The microtubule-affinity regulating kinase (MARK) family consists of four highly conserved members that have been implicated in phosphorylation of tau protein, causing formation of neurofibrillary tangles in Alzheimer's disease (AD). Understanding of roles by individual MARK isoform in phosphorylating tau has been limited due to lack of antibodies selective for each MARK isoform. In this study, we first applied the proximity ligation assay on cells to select antibodies specific for each MARK isoform. In cells, a CagA peptide specifically and significantly inhibited tau phosphorylation at Ser²6² mediated by MARK4 but not other MARK isoforms. We then used these antibodies to study expression levels of MARK isoforms and interactions between tau and individual MARK isoforms in postmortem human brains. We found a strong and significant elevation of MARK4 expression and MARK4-tau interactions in AD brains, correlating with the Braak stages of the disease. These results suggest the MARK4-tau interactions are of functional importance in the progression of AD and the results also identify MARK4 as a promising target for AD therapy.

Ganglioside GM1 induces phosphorylation of mutant huntingtin and restores normal motor behavior in Huntington disease mice.

Huntington disease (HD) is a progressive neurodegenerative monogenic disorder caused by expansion of a polyglutamine stretch in the huntingtin (Htt) protein. Mutant huntingtin triggers neural dysfunction and death, mainly in the corpus striatum and cerebral cortex, resulting in pathognomonic motor symptoms, as well as cognitive and psychiatric decline. Currently, there is no effective treatment for HD. We report that intraventricular infusion of ganglioside GM1 induces phosphorylation of mutant huntingtin at specific serine amino acid residues that attenuate huntingtin toxicity, and restores normal motor function in already symptomatic HD mice. Thus, our studies have identified a potential therapy for HD that targets a posttranslational modification of mutant huntingtin with critical effects on disease pathogenesis.

'In vitro' capacitation and further 'in vitro' progesterone-induced acrosome exocytosis are linked to specific changes in the expression and acrosome location of protein phosphorylation in serine residues of boar spermatozoa.

The aim of this study is to determine changes in the expression and location of protein serine phosphorylation (pSer) during 'in vitro' capacitation (IVC) and 'in vitro' acrosome exocytosis (IVAE) in boar spermatozoa. This was performed in both mono- and bi-dimensional analyses of protein expression through Western blot, as well as through immunocytochemistry. Furthermore, IVC was induced through incubation in an IVC medium, and afterwards, progesterone-induced IVAE was performed. The mono-dimensional Western blot analysis showed the presence of a predominant pSer band of approximately 70-75 kDa, which was accompanied by fainter bands, especially three with molecular weights of approximately 50, 35 and 32 kDa. Neither IVC nor IVAE significantly modified this pattern. Bi-dimensional analyses showed a more complex pattern, with at least five protein clusters. The attainment of IVC caused the disappearance of the proteins with the highest molecular weight concomitantly with the appearance of pSer proteins of 75-kDa/pI 9.5 and 80-kDa/pI 10. The induction of IVAE caused the appearance of new pSer proteins of a 75-kDa/pI 6.5-7.5 and 75-kDa/pI 10. Immunocytochemistry showed that the main pSer expression in boar expression before the attainment of IVC was located at the midpiece. The IVC induced the appearance of acrosomal pSer, which was greatly increased during IVAE. Our results indicate that the changes in serine protein phosphorylation associated with IVC and IVAE comprise not only the appearance of specific phosphorylated proteins, such as the pSer-75 kDa, but also changes in pI and displacements in the sperm location of phosphorylated proteins, like the specific acrosomal pSer signal induced during IVC.

Functionalized graphene oxide as a nanocarrier in a multienzyme labeling amplification strategy for ultrasensitive electrochemical immunoassay of phosphorylated p53 (S392).

P53 phosphorylation plays an important role in many biological processes and might be used as a potential biomarker in clinical diagnoses. We report a new electrochemical immunosensor for ultrasensitive detection of phosphorylated p53 at Ser392 (phospho-p53(392)) based on graphene oxide (GO) as a nanocarrier in a multienzyme amplification strategy. Greatly enhanced sensitivity was achieved by using the bioconjugates featuring horseradish peroxidase (HRP) and p53(392) signal antibody (p53(392)Ab(2)) linked to functionalized GO (HRP-p53(392)Ab(2)-GO) at a high ratio of HRP/p53(392)Ab(2). After a sandwich immunoreaction, the HRP-p53(392)Ab(2)-GO captured onto the electrode surface produced an amplified electrocatalytic response by the reduction of enzymatically oxidized thionine in the presence of hydrogen peroxide. The increase of response current was proportional to the phospho-p53(392) concentration in the range of 0.02-2 nM with the detection limit of 0.01 nM, which was 10-fold lower than that of the traditional sandwich electrochemical measurement for p53(392). The amplified immunoassay developed in this work shows acceptable stability and reproducibility, and the assay results for phospho-p53(392) spiked in human plasma also show good recovery (92-103.8%). This simple and low-cost immunosensor shows great promise for detection of other phosphorylated proteins and clinical applications.

mTOR partly mediates insulin resistance by phosphorylation of insulin receptor substrate-1 on serine(307) residues after burn.

Mammalian target of rapamycin (mTOR) is an important mediator for cross talk between nutritional signals and metabolic signals of insulin by downregulating insulin receptor substrate proteins. Therefore, mTOR inhibition could become a therapeutic strategy in insulin-resistant states, including insulin resistance induced by burn. We tested this hypothesis in the rat model of 30% TBSA full thickness burn, using the mTOR inhibitor rapamycin. Rapamycin (0.4 mg/kg, i.p.) was injected 2 h before euglycemic-hyperinsulinemic glucose clamps at 4 days after burn. IRS-1, phospho-serine³°7, phospho-tyrosine of IRS-1 and phospho-mTOR in muscle tissue were determined by immunoprecipitation and Western blot analysis or immunohistochemistry. Plasma TNF-α, insulin and C-peptide were determined before and after euglycemic-hyperinsulinemic glucose clamps. Our data showed that TNF-α, insulin and C-peptide significantly increased in the early stage after burn (P < 0.01). The infused rates of total 10% glucose (GIR, mg/kg min) significantly decreased at 4 days after burn. The level of IRS-1 serine³°7 phosphorylation in muscle in vivo significantly increased after burn (P < 0.01), while insulin-induced tyrosine phosphorylation of IRS-1 significantly decreased (P < 0.01). Inhibition of mTOR by rapamycin inhibited the phosphorylation of mTOR, reduced serine³°7 phosphorylation, elevated tyrosine phosphorylation and partly prevented the decrease of GIR after burn. However, TNF-α, insulin and C-peptide were not decreased by rapamycin treatment postburn. Taken together, these results indicate that the mTOR pathway is an important modulator of the signals involved in the acute regulation of insulin-stimulated glucose metabolism, and at least, partly contributes to burn-induced insulin resistance. mTOR inhibition may become a therapeutic strategy in insulin-resistant states after burn.

Determination of L-serine-O-phosphate in rat and mouse brain tissue using high-performance liquid chromatography and fluorimetric detection.

L-Serine-O-phosphate (L-SOP), the precursor of l-serine, is an agonist at group III metabotropic glutamate receptors. Despite the interest in L-SOP, very few articles have reported its brain levels. Here we report a convenient and reproducible method for simultaneous analysis of L-SOP and several other important amino acids in brain tissue using high-performance liquid chromatography (HPLC) with fluorimetric detection after derivatization with o-phthaldialdehyde and N-isobutyl-L-cysteine. Analyses were carried out in rat whole brain and cerebellum and in mouse whole brain, forebrain, amygdala, and prefrontal cortex. The method should be useful for future comprehensive neurochemical and pharmacological studies on neuropsychiatric disorders.

Reciprocal epigenetic modification of histone H2B occurs in chromatin during apoptosis in vitro and in vivo.

Histone H2B phosphorylation at Serine 14 (phosS14) has been proposed as an epigenetic marker of apoptotic cells, whereas acetylation at the adjacent Lysine 15 (acK15) is a property of non-dying cells. We investigated the relationship and the potential regulatory mechanisms between these two epigenetic histone modifications and internucleosomal DNA degradation during apoptosis. Using rat primary thymocytes induced to undergo apoptosis with glucocorticoids we found that H2B phosphorylated at Ser14 was associated with soluble, cleaved DNA in apoptotic nuclei. In contrast acK15 was prevalent in non-apoptotic nuclei and scarce in apoptotic nuclei. This switch between K15 acetylation and S14 phosphorylation on H2B was also observed in apoptotic thymocytes from animals treated in vivo with glucocorticoids and in a rat hepatoma cell line (HTC) induced to die by UV-C or Fas ligand. It is interesting to note that the combined use of a histone deacetylase inhibitor and glucocorticoid suppressed both S14 phosphorylation and internucleosomal DNA degradation without inhibiting apoptosis in thymocytes. Using synthetic peptides and a PKC phosphorylation assay system, we show that the deacetylation of K15 was necessary to allow the S14 phosphorylation. These findings suggest that selective chromatin post-translational modifications are associated with DNA degradation during apoptosis.

Short communication: ischemia/reperfusion tolerance is time-of-day-dependent: mediation by the cardiomyocyte circadian clock.

RATIONALE: Cardiovascular physiology and pathophysiology vary dramatically over the course of the day. For example, myocardial infarction onset occurs with greater incidence during the early morning hours in humans. However, whether myocardial infarction tolerance exhibits a time-of-day dependence is unknown. OBJECTIVE: To investigate whether time of day of an ischemic insult influences clinically relevant outcomes in mice. METHODS AND RESULTS: Wild-type mice were subjected to ischemia/reperfusion (I/R) (45 minutes of ischemia followed by 1 day or 1 month of reperfusion) at distinct times of the day, using the closed-chest left anterior descending coronary artery occlusion model. Following 1 day of reperfusion, hearts subjected to ischemia at the sleep-to-wake transition (zeitgeber time [ZT]12) resulted in 3.5-fold increases in infarct size compared to hearts subjected to ischemia at the wake-to-sleep transition (ZT0). Following 1 month of reperfusion, prior ischemic event at ZT12 versus ZT0 resulted in significantly greater infarct volume, fibrosis, and adverse remodeling, as well as greater depression of contractile function. Genetic ablation of the cardiomyocyte circadian clock (termed cardiomyocyte-specific circadian clock mutant [CCM] mice) attenuated/abolished time-of-day variations in I/R outcomes observed in wild-type hearts. Investigation of Akt and glycogen synthase kinase-3beta in wild-type and CCM hearts identified these kinases as potential mechanistic ties between the cardiomyocyte circadian clock and I/R tolerance. CONCLUSIONS: We expose a profound time-of-day dependence for I/R tolerance, which is mediated by the cardiomyocyte circadian clock. Further understanding of I/R tolerance rhythms will potentially provide novel insight regarding the etiology and treatment of ischemia-induced cardiac dysfunction.