High Degree Of Phosphorylation Research Articles

Specific cellular microenvironments for spatiotemporal regulation of StAR and steroid synthesis.

For many years, research in the field of steroid synthesis has aimed to understand the regulation of the rate-limiting step of steroid synthesis, i.e. the transport of cholesterol from the outer to the inner mitochondrial membrane, and identify the protein involved in the conversion of cholesterol into pregnenolone. The extraordinary work by B Clark, J Wells, S R King, and D M Stocco eventually identified this protein and named it steroidogenic acute regulatory protein (StAR). The group's finding was also one of the milestones in understanding the mechanism of nonvesicular lipid transport between organelles. A notable feature of StAR is its high degree of phosphorylation. In fact, StAR phosphorylation in the acute phase is required for full steroid biosynthesis. As a contribution to this subject, our work has led to the characterization of StAR as a substrate of kinases and phosphatases and as an integral part of a mitochondrion-associated multiprotein complex, essential for StAR function and cholesterol binding and mitochondrial transport to yield maximum steroid production. Results allow us to postulate the existence of a specific cellular microenvironment where StAR protein synthesis and activation, along with steroid synthesis and secretion, are performed in a compartmentalized manner, at the site of hormone receptor stimulation, and involving the compartmentalized formation of the steroid molecule-synthesizing complex.

Interaction research of resveratrol and phosvitin based on fluorescence spectroscopy and molecular docking analysis.

Phosvitin (PV) is the main phosphoprotein in egg yolk, with the highest degree of phosphorylation known in nature. The PV and resveratrol (Res) can form a complex, thus effectively improve the solubility of Res. In this work, the interaction between Res and PV was investigated by the fluorescence spectroscopy and molecular docking. The fluorescence emission intensity of PV became weak along with a red shift when it interacted with Res and the antioxidant activity was enhanced. The quenching constants of the interaction systems were 1.12×104 M-1 and 9.40×103 M-1 at 25°C and 35°C, respectively, which indicated the presence of static quenching phenomena between them. The binding constant was 1.80×104 M-1 , and the number of corresponding binding sites was approximately equal to one. The thermodynamic results revealed the combination was spontaneous, and the change of enthalpy and entropy was ∆H=53.50kJ/mol, ∆S=261.00 J/mol·K, respectively. It indicated that the interaction forces between Res and PV were mainly hydrophobic interaction and hydrogen bonding. Molecular docking showed the binding mode, which was consistent with the experiment results. The research on the interaction between Res and PV provided theoretical guidance for the application of Res in food. PRACTICAL APPLICATION: PV is the most highly phosphorylated protein in nature and has pro-calcium absorption effects. Res is a polyphenol with strong antioxidant and anti-inflammatory activity, but its poor solubility limits its application. In this study, the solubility of Res was considerably enhanced by compounding Res and PV, and the antioxidant activity of Res was well retained. It increases the value of Res in food and other applications and opens up new possibilities for processing and utilization of PV.

The Effects of Phosphorylation and Microwave Treatment on the Functional Characteristics of Freeze-Dried Egg White Powder.

The effects of phosphorylation pre-treatments at 1.5, 2.5, and 3.5% levels, as well as microwave application at 200, 400, and 700 watts levels for 2 min, on the functional parameters of egg white powder obtained by the freeze dryer procedure were investigated. P1.5-M200 had the highest oil-holding capacity, emulsion stability, and emulsion activity, while P2.5-M200 had the highest foam capacity. The P2.5-M400 had the largest particle size, and P3.5-M200 had the highest degree of phosphorylation and protein solubility. On the other hand, P3.5-M200 had the highest solution viscosity by 1% (w/v), water-holding capacity, and foam stability, in the treatments that used phosphorylation and microwave treatment simultaneously. FTIR spectroscopy of the unfolding structure of egg white protein revealed changes in the protein’s secondary structure, such as the development of β-sheets and β-turns, as well as the binding of negatively charged phosphate groups on the serine, threonine, and tyrosine side chains. The phosphorylation and microwave treatments reduced the particle size of the egg white protein powder while increasing the surface area of the protein molecules, according to SEM analyses.

Calcineurin-dependent dephosphorylation of the transcription factor CrzA at specific sites controls conidiation, stress tolerance, and virulence of Aspergillus fumigatus.

Calcium signaling through calcineurin and its major transcription factor (TF), CrzA, is integral to hyphal growth, stress response and virulence of the pathogenic fungus Aspergillus fumigatus, the leading etiology of invasive aspergillosis. Dephosphorylation of CrzA by calcineurin activates the TF, but the specific phosphorylation sites and their roles in the activation/inactivation mechanism are unknown. Mass spectroscopic analysis identified 20 phosphorylation sites, the majority of which were specific to filamentous fungi and distributed throughout the CrzA protein, with particular concentration in a serine-rich region N-terminal to the conserved DNA-binding domain (DBD). Site-directed mutagenesis of phosphorylated residues revealed that CrzA activity during calcium stimulation can only be suppressed by a high degree of phosphorylation in multiple regions of the protein. Our findings further suggest that this regulation is not solely accomplished through control of CrzA nuclear import. Additionally, we demonstrate the importance of the CrzA phosphorylation state in regulating growth, conidiation, calcium and cell wall stress tolerance, and virulence. Finally, we identify two previously undescribed nuclear localization sequences in the DBD. These findings provide novel insight into the phosphoregulation of CrzA which may be exploited to selectively target A. fumigatus.

An efficient method for straightforward phosphorylation of ethylene/vinyl alcohol copolymers using trialkyl phosphite/iodine

The phosphoric acid group was introduced into the poly(vinyl alcohol) (PVA) and ethylene/vinyl alcohol copolymers (EVA) structures via H3PO4/urea and POCl3/pyridine systems. The structure of phosphorylated polymers was characterized by 1H and 31P {1H} NMR and pH titration analyses. It was found that H3PO4/urea system leads to formation of soluble phosphorylated polymers with a low degree of phosphorylation (DP) or insoluble phosphorylated polymers with a high DP while POCl3/pyridine system results in the crosslinked structure. Then, an efficient and straightforward method for the introduction of pendant diemethyl phosphate groups into EVA via reaction with in situ synthesized dimethyl iodoophosphate was proposed. Resulting polymers were characterized by 1H and 31P {1H} NMR spectroscopies. The phosphorylated polymers with high DP and without any crosslinked structure were achieved. To remove one or both methyl groups, the phosphate groups were subsequently (mono)-dealkylated using NaI and ClSi(Me)3/NaI, respectively.

Genome-wide association study for αS1- and αS2-casein phosphorylation in Dutch Holstein Friesian

Phosphorylation of caseins (CN) is a crucial post-translational modification that allows caseins to form colloid particles known as casein micelles. Both αS1- and αS2-CN show varying degrees of phosphorylation (isoforms) in cow milk and were suggested to be more relevant for stabilizing internal micellar structure than β- and κ-CN. However, little is known about the genetic background of individual αS2-CN phosphorylation isoforms and the phosphorylation degrees of αS1- and αS2-CN (αS1-CN PD and αS2-CN PD), defined as the proportion of isoforms with higher degrees of phosphorylation in total αS1- and αS2-CN, respectively. We aimed to identify genomic regions associated with these traits using 50K single nucleotide polymorphisms for 1,857 Dutch Holstein Friesian cows. A total of 10 quantitative trait loci (QTL) regions were identified for all studied traits on 10 Bos taurus autosomes (BTA1, 2, 6, 9, 11, 14, 15, 18, 24, and 28). Regions associated with multiple traits were found on BTA1, 6, 11, and 14. We showed 2 QTL regions on BTA1, one affecting αS2-CN production and the other harboring the SLC37A1 gene, which encodes a phosphorus antiporter and affects αS1- and αS2-CN PD. The QTL on BTA6 harbors the casein gene cluster and affects individual αS2-CN phosphorylation isoforms. The QTL on BTA11 harbors the PAEP gene that encodes for β-lactoglobulin and affects relative concentrations of αS2-CN-10P and αS2-CN-11P as well as αS1-CN PD and αS2-CN PD. The QTL on BTA14 harbors the DGAT1 gene and affects relative concentrations of αS2-CN-10P and αS2-CN-11P as well as αS1-CN PD and αS2-CN PD. Our results suggest that effects of identified genomic regions on phosphorylation of αS1- and αS2-CN are related to changes in milk synthesis and phosphorus secretion in milk. The actual roles of SLC37A1, PAEP, and DGAT1 in αS1- and αS2-CN phosphorylation in Dutch Holstein Friesian require further investigation.

Phosphoesterification of soybean and peanut proteins with sodium trimetaphosphate (STMP): Changes in structure to improve functionality for food applications

Soybean and peanut protein isolates underwent phosphorylation using sodium trimetaphosphate (STMP). Changes in functional properties and the influence of STMP (1, 2 or 3% w/w), pH (11.5 or 12.5), temperature (35 or 55 °C) and time (3 or 5 h) were evaluated. The highest degree of phosphorylation was achieved at 2% of STMP and pH 12.5. The best specific conditions varied according to the raw material: in soybean, 25% phosphorylation was achieved at 55 °C and 5 h whereas in peanut, 30% was reached at 35 °C and 3 h. The modified proteins showed an improved emulsifying activity (27.3% for soybean and 6.6% for peanut), whereas NSI for soybean increased more than three times and for peanut decreased by half. In vitro digestibility improved in both isolates around 1.5%. These results showed that phosphorylation with STMP of peanut and soybean proteins yielded isolates with a wide array of potential applications in food systems.

Genetic parameters for αS1-casein and αS2-casein phosphorylation isoforms in Dutch Holstein Friesian

Relative concentrations of αS1-casein and αS2-casein (αS1-CN and αS2-CN) phosphorylation isoforms vary considerably among milk of individual cows. We estimated heritabilities for αS2-CN phosphorylation isoforms, determined by capillary zone electrophoresis from 1,857 morning milk samples, and genetic correlations among αS2-CN phosphorylation isoforms in Dutch Holstein Friesian. To investigate if phosphorylation of αS1-CN and αS2-CN are due to the same genetic mechanism, we also estimated genetic correlations between αS1-CN and αS2-CN phosphorylation isoforms as well as the genetic correlations between the phosphorylation degrees (PD) of αS1-CN and αS2-CN defined as the proportion of isoforms with higher degrees of phosphorylation in total αS1-CN and αS2-CN, respectively. The intra-herd heritabilities for the relative concentrations of αS2-CN phosphorylation isoforms were high and ranged from 0.54 for αS2-CN-10P to 0.89 for αS2-CN-12P. Furthermore, the high intra-herd heritabilities of αS1-CN PD and αS2-CN PD imply a strong genetic control of the phosphorylation process, which is independent of casein production. The genetic correlations between αS2-CN phosphorylation isoforms are positive and moderate to high (0.33-0.90). Furthermore, the strong positive genetic correlation (0.94) between αS1-CN PD and αS2-CN PD suggests that the phosphorylation processes of αS1-CN and αS2-CN are related. This study shows the possibility of breeding for specific αS1-CN and αS2-CN phosphorylation isoforms, and relations between the phosphorylation degrees of αS1-CN and αS2-CN and technological properties of milk need to be further investigated to identify potential benefits for the dairy industry.

Genetic and nongenetic factors contributing to differences in αS-casein phosphorylation isoforms and other major milk proteins

Relative concentrations of αS-casein (αS-CN) phosphorylation isoforms vary considerably among milk of individual cows. We aimed to explore to what extent genetic and other factors contribute to the variation in relative concentrations of αS-CN phosphorylation isoforms and the phosphorylation degree of αS-CN defined as the proportion of isoforms with higher degrees of phosphorylation. We also investigated the associations of genetic variants of milk proteins and casein haplotypes with relative concentrations of αS-CN phosphorylation isoforms and with the phosphorylation degree of αS-CN in French Montbéliarde cattle from the cheese production area of Franche-Comté. Detailed milk protein composition was determined by liquid chromatography coupled with electrospray ionization mass spectrometry from 531 test-day morning milk samples. Parity, lactation stage, and genetic variation of cows contributed to the phenotypic variation in relative concentrations of individual αS-CN phosphorylation isoforms and in the phosphorylation degree of αS-CN. As lactation progressed, we observed a significant increase for relative concentrations of αS-CN isoforms with higher degrees of phosphorylation (αS1-CN-9P, αS2-CN-13P, and αS2-CN-14P) as well as for the phosphorylation degree of both αS1-CN and αS2-CN. Furthermore, the β-CN I variant was associated with a greater proportion of isoforms with lower degrees of phosphorylation (αS1-CN-8P, αS2-CN-10P, and αS2-CN-11P); the β-CN B variant was associated with a greater proportion of isoforms with higher degrees of phosphorylation (αS1-CN-9P, αS2-CN-12P to αS2-CN-14P). The heritability estimates were low to moderate for relative concentrations of αS2-CN phosphorylation isoforms (0.07 to 0.32), high for relative concentrations of αS1-CN-8P (0.84) and αS1-CN-9P (0.56), and moderate for phosphorylation degrees of αS1-CN (0.37) and αS2-CN (0.23). Future studies investigating relations between the phosphorylation degree of αS-CN and technological properties of milk will be beneficial for the dairy industry.

Understanding the influence of phosphorylation and polysialylation of gelatin on mineralization and osteogenic differentiation

Post-translational modifications such as phosphorylation and sialylation impart crucial functions such as mineral deposition and osteogenic differentiation to non-collagenous bone matrix proteins. In this work, the influence of phosphorylation and polysialylation of gelatin on mineralization in simulated body fluid (SBF) and on osteogenic differentiation of mesenchymal stem cells (MSC) was studied. It was observed that increase in phosphorylation could be directly correlated with the mineralization ability of phosphorylated gelatin in SBF. The total calcium and phosphate deposited increased with increase in degree of phosphorylation and was >3 fold higher on the highest degree of phosphorylation. Whereas, polysialylation did not have any significant influence on mineral deposition in SBF. On the other hand, when MSCs were cultured on polysialylated surfaces they showed relatively higher cell elongation with 1.5 fold higher cell aspect ratio, higher alkaline phosphatase activity and 3 fold higher mineral deposition when compared to control and phosphorylated gelatin surfaces. In conclusion, phosphorylation and polysialylation of gelatin show a significant influence on mineralization and osteogenic differentiation respectively which can be advantageously used for bone tissue engineering.

Induction of Endotoxin Tolerance by Pathogenic Neisseria Is Correlated with the Inflammatory Potential of Lipooligosaccharides and Regulated by MicroRNA-146a

In this article, we report that retreatment of human monocytic THP-1 cells and primary monocytes with pathogenic Neisseria or with purified lipooligosaccharides (LOS) after previous exposure to LOS induced immune tolerance, as evidenced by reduced TNF-α and IL-1β cytokine expression. LOS that we have previously shown to vary in their potential to activate TLR4 signaling, which was correlated with differences in levels of lipid A phosphorylation, had similarly variable ability to induce tolerance. Efficacy for induction of tolerance was proportional to the level of lipid A phosphorylation, as LOS from meningococcal strain 89I with the highest degree of phosphorylation was the most tolerogenic following retreatment with LOS or whole bacteria, compared with LOS from gonococcal strains 1291 and GC56 with reduced levels of phosphorylation. Hydrogen fluoride treatment of 89I LOS to remove phosphates rendered the LOS nontolerogenic. Tolerance induced by the more highly inflammatory meningococcal LOS was correlated with significantly greater downregulation of p38 activation, greater induction of the expression of A20 and of microRNA-146a, and greater reductions in IL-1R-associated kinase 1 and TRAF6 levels following LOS retreatment of cells. The role of miR-146a in regulation of induction of TNF-α was confirmed by transfecting cells with an inhibitor and a mimic of miR-146a. Our results provide a mechanistic framework for understanding the variable pathophysiology of meningococcal and gonococcal infections given that after an initial exposure, greater upregulation of microRNA-146a by more highly inflammatory LOS conversely leads to the suppression of immune responses, which would be expected to facilitate bacterial survival and dissemination.

Upregulation of Inhibitory Molecules in T Cells is Associated with Altered Functions of Dendritic Cells by HIV-1 and Activation of the P38MAPK/STAT3 Signaling Pathways

Upregulation of Inhibitory Molecules in T Cells is Associated with Altered Functions of Dendritic Cells by HIV-1 and Activation of the P38MAPK/STAT3 Signaling Pathways

Rotavirus viroplasm proteins interact with the cellular SUMOylation system: implications for viroplasm-like structure formation.

Posttranslational modification by SUMO provides functional flexibility to target proteins. Viruses interact extensively with the cellular SUMO modification system in order to improve their replication, and there are numerous examples of viral proteins that are SUMOylated. However, thus far the relevance of SUMOylation for rotavirus replication remains unexplored. In this study, we report that SUMOylation positively regulates rotavirus replication and viral protein production. We show that SUMO can be covalently conjugated to the viroplasm proteins VP1, VP2, NSP2, VP6, and NSP5. In addition, VP1, VP2, and NSP2 can also interact with SUMO in a noncovalent manner. We observed that an NSP5 SUMOylation mutant protein retains most of its activities, such as its interaction with VP1 and NSP2, the formation of viroplasm-like structures after the coexpression with NSP2, and the ability to complement in trans the lack of NSP5 in infected cells. However, this mutant is characterized by a high degree of phosphorylation and is impaired in the formation of viroplasm-like structures when coexpressed with VP2. These results reveal for the first time a positive role for SUMO modification in rotavirus replication, describe the SUMOylation of several viroplasm resident rotavirus proteins, and demonstrate a requirement for NSP5 SUMOylation in the production of viroplasm-like structures.

C-terminal Modification of Osteopontin Inhibits Interaction with the αVβ3-Integrin

Osteopontin (OPN) is a multifunctional phosphorylated protein containing the integrin binding sequence Arg-Gly-Asp through which it interacts with several integrin receptors, such as the α(V)β(3)-integrin. OPN exists in many different isoforms differing in phosphorylation status that are likely to interact differently with integrins. The C-terminal region of OPN is particularly well conserved among mammalian species, which suggests an important functional role of this region. In this study, we show that modification of the extreme C terminus of OPN plays an important regulatory role for the interaction with the α(V)β(3)-integrin. It is demonstrated that highly phosphorylated OPN has a much reduced capability to promote cell adhesion via the α(V)β(3)-integrin compared with lesser phosphorylated forms. The cell attachment promoted by highly phosphorylated OPN could be greatly increased by both dephosphorylation and proteolytic removal of the C terminus. Using recombinantly expressed OPN containing a tag in the N or C terminus, it is shown that a modification in the C-terminal part significantly reduces the adhesion of cells to OPN via the α(V)β(3)-integrin, whereas modification of the N terminus does not influence the binding. The inhibited binding of the α(V)β(3)-integrin to OPN could be restored by proteolytic removal of the C terminus by thrombin and plasmin. These data illustrate a novel mechanism regulating the interaction of OPN and the α(V)β(3)-integrin by modification of the highly conserved C-terminal region of the protein.

Phosphoproteome analysis during larval development and metamorphosis in the spionid polychaete Pseudopolydora vexillosa

BackgroundThe metamorphosis of the spionid polychaete Pseudopolydora vexillosa includes spontaneous settlement onto soft-bottom habitats and morphogenesis that can be completed in a very short time. A previous study on the total changes to the proteome during the various developmental stages of P. vexillosa suggested that little or no de novo protein synthesis occurs during metamorphosis. In this study, we used multicolor fluorescence detection of proteins in 2-D gels for differential analysis of proteins and phosphoproteins to reveal the dynamics of post-translational modification proteins in this species. A combination of affinity chromatography, 2D-PAGE, and mass spectrometry was used to identify the phosphoproteins in pre-competent larvae, competent larvae, and newly metamorphosed juveniles.ResultsWe reproducibly detected 210, 492, and 172 phosphoproteins in pre-competent larvae, competent larvae, and newly metamorphosed juveniles, respectively. The highest percentage of phosphorylation was observed during the competent larval stage. About 64 stage-specific phosphoprotein spots were detected in the competent stage, and 32 phosphoproteins were found to be significantly differentially expressed in the three stages. We identified 38 phosphoproteins, 10 of which were differentially expressed during metamorphosis. These phosphoproteins belonged to six categories of biological processes: (1) development, (2) cell differentiation and integrity, (3) transcription and translation, (4) metabolism, (5) protein-protein interaction and proteolysis, and (6) receptors and enzymes.ConclusionThis is the first study to report changes in phosphoprotein expression patterns during the metamorphosis of the marine polychaete P. vexillosa. The higher degree of phosphorylation during the process of attaining competence to settle and metamorphose may be due to fast morphological transitions regulated by various mechanisms. Our data are consistent with previous studies showing a high percentage of phosphorylation during competency in the barnacle Balanus amphitrite and the bryozoan Bugula neritina. The identified phosphoproteins may play an important role during metamorphosis, and further studies on the location and functions of important proteins during metamorphosis are warranted.

Abstract 2171: The fusion protein PAX5-JAK2 constitutively activates JAK-STAT signaling

Abstract In B-cell precursor acute lymphoblastic leukemia the transcription factor PAX5, a master regulator of B-cell commitment and maintenance, is recurrently fused to several different partner proteins including other transcription factors, structural proteins, and the tyrosine kinase (TK) JAK2. Mutations and translocations affecting JAK2 are found in a variety of hematopoietic malignancies and lead to its constitutive activation and cytokine-independent JAK-STAT signaling. The chimeric PAX5-JAK2 protein contains the DNA binding domain of PAX5 and the JH1 kinase domain of JAK2. Reciprocal JAK2-PAX5 transcripts are also expressed, and the respective JAK2-PAX5 fusion protein consists of the JAK2 JH2-JH7 domains fused to the PAX5 partial homeodomain and the transactivation and inhibitory domains. In a first step towards the understanding of the function of these chimeric proteins, their subcellular localization was determined. Transfection of tagged fusion proteins into HEK293 and HeLa cells displayed nuclear localization of PAX5-JAK2. In contrast, JAK2-PAX5 fusion proteins were mainly localized in the cytoplasm. In order to determine whether PAX5-JAK2 is tyrosine phosphorylated and whether PAX5-JAK2 or JAK2-PAX5 is capable of activating STAT proteins, intracellular phosphoprotein analyses using flow cytometry were performed. Transiently transfected unstimulated JAK2-deficient human gamma2A cells exhibited a high degree of phosphorylation of PAX5-JAK2 compared to JAK2 wild-type protein, strongly suggesting cytokine stimulation-independent TK activity of the fusion protein. Additionally, expression of PAX5-JAK2 but not JAK2-PAX5 resulted in constitutive phosphorylation of STATs 1, 3, and 5. The capability of PAX5-JAK2 to induce STAT5 was confirmed by reporter gene assays. Mutation of the respective tyrosine residues in PAX5-JAK2 completely abolished the phosphorylation of STATs demonstrating that their activation is phospho-PAX5-JAK2-dependent. As it has been shown that PAX5 fusion proteins can act as competitive inhibitors of wild-type PAX5, we also performed a CD19 luciferase assay which showed that PAX5-JAK2 has a negative effect on the activation capacity of wild-type protein. Additional analysis including gene expression profiling of patient material will be performed to further evaluate the function of the chimeric proteins. Together, PAX5-JAK2 appears to affect JAK-STAT signaling and may at the same time interfere with normal B-cell development. In contrast to ETV6-JAK2 – the only other JAK2 chimeric protein analyzed in detail to date – which resides in the cytoplasm and is tyrosine phosphorylated upon dimerization, PAX5-JAK2 localizes to the nucleus and lacks any putative self-association motif. Hence, PAX5-JAK2 represents the first nuclear JAK2-fusion protein, which constitutively activates the JAK-STAT signaling pathway. This project is funded by the Austrian Science Fund FWF (P21554). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2171. doi:10.1158/1538-7445.AM2011-2171

Effect of phytic acid degradation by soaking and exogenous phytase on the bioavailability of magnesium and zinc from Pisum sativum, L.

The effect of dephytinization of Pisum sativum, L. flour on the bioavailability of Mg and Zn was evaluated in growing rats. Processing of legume flours under optimal conditions for phytase activity (pH 5.5, 37 °C, 60 min) and subsequent removal of the soaking solution led to a 42 and 61% reduction in the content of Mg and Zn, respectively. Treatment with phytase led to an additional reduction in the concentration of the above-mentioned seed flour components, compared to the raw pea flour (69% and 74% for Mg and Zn, respectively). The considerable reduction in the content of inositol phosphates with high degree of phosphorylation attained under both processing conditions did not affect the digestive utilization of Mg, whereas the metabolic utilization of this mineral increased significantly. The digestive and metabolic utilization of Zn increased significantly in response to both processes assayed, reaching the highest values in the experimental group that was fed the phytase-treated pea flour diet. The amount of Mg retained by the experimental animals was reflected in the content of this mineral in the different tissues studied (femur, sternum, kidney, and heart), whereas no correlation was found in the case of Zn.

Differential proteome analysis of tonsils from children with chronic tonsillitis or with hyperplasia reveals disease-associated protein expression differences

A proteomic approach has been used to establish a proteome map and differentiate between the protein composition of tonsils from patients with chronic tonsillitis (CT) and that of tonsils with hyperplasia (HPL). Two-dimensional gel analysis was performed with material from four patients with HPL and five patients with CT. An average of approximately 600 spots were detected in each gel. A total of 127 different proteins were identified in 158 spots analyzed by mass spectrometry. Our study revealed disease-associated differences between protein abundance for two protein spots, an HSP27 isoform and UMP-CMP kinase. Both protein spots were more abundant in the CT group. HSP27 ELISA was performed for 32 patients, 12 belonging to the HPL group and 20 to the CT group. ELISA could not be used to differentiate HSP27 isoforms nor to distinguish CT from HPL. HSP27 was found to migrate to two further protein spots in the 2D gels. The differently expressed HSP27 isoform migrated as the most acidic of all the HSP27 isoforms detected, indicating the highest degree of phosphorylation. The sum of all three HSP27 abundances in the gels from the CT group was not different from that of the HPL group, consistent with the ELISA results. Our results suggest that phosphorylation differences caused the observed migration differences of HSP27. Together with the UMP-CMP kinase abundance differences, we conclude that kinase and/or phosphatase activity are different in CT and HPL.

Phosphorylation of transitory starch is increased during degradation.

The starch excess phenotype of Arabidopsis mutants defective in the starch phosphorylating enzyme glucan, water dikinase (EC 2.7.9.4) indicates that phosphorylation of starch is required for its degradation. However, the underlying mechanism has not yet been elucidated. In this study, two in vivo systems have been established that allow the analysis of phosphorylation of transitory starch during both biosynthesis in the light and degradation in darkness. First, a photoautotrophic culture of the unicellular green alga Chlamydomonas reinhardtii was used to monitor the incorporation of exogenously supplied (32)P orthophosphate into starch. Illuminated cells incorporated (32)P into starch with a constant rate during 2 h. By contrast, starch phosphorylation in darkened cells exceeded that in illuminated cells within the first 30 min, but subsequently phosphate incorporation declined. Pulse-chase experiments performed with (32)P/(31)P orthophosphate revealed a high turnover of the starch-bound phosphate esters in darkened cells but no detectable turnover in illuminated cells. Secondly, leaf starch granules were isolated from potato (Solanum tuberosum) plants grown under controlled conditions and glucan chains from the outer granule layer were released by isoamylase. Phosphorylated chains were purified and analyzed using high performance anion-exchange chromatography and matrix-assisted laser desorption/ionization mass spectrometry. Glucans released from the surface of starch granules that had been isolated from darkened leaves possessed a considerably higher degree of phosphorylation than those prepared from leaves harvested during the light period. Thus, in the unicellular alga as well as in potato leaves, net starch degradation is accompanied with an increased phosphorylation of starch.

Deoxyhypusine synthase is phosphorylated by protein kinase C in vivo as well as in vitro.

Deoxyhypusine synthase catalyzes the first step in the posttranslational synthesis of an unusual amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein. We earlier observed that yeast recombinant deoxyhypusine synthase was phosphorylated by protein kinase C (PKC) in vitro (Kang and Chung, 1999) and the phosphorylation rate was synergistically increased to a 3.5-fold following treatment with phosphatidylserine (P.Ser)/diacylglycerol (DAG)/ Ca(2+), suggesting a possible involvement of PKC. We have extended study on the phosphorylation of deoxyhypusine synthase in vivo in different cell lines in order to define its role on the regulation of eIF5A in the cell. Deoxyhypusine synthase was found to be phosphorylated by endogenous kinases in CHO, NIH3T3, and chicken embryonic cells. The highest degree of phosphorylation was found in CHO cells. Moreover, phosphorylation of deoxyhypusine synthase in intact CHO cells was revealed and the expression of phosphorylated deoxyhypusine synthase was significantly diminished by diacyl ethylene glycol (DAEG), a PKC inhibitor, and enhanced by phorbol 12-myristate 13-acetate (PMA) or Ca(2+)/DAG. Endogenous PKC in CHO cell and cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced by PMA or Ca(2+) plus DAG. Close association of PKC with deoxyhypusine synthase in the CHO cells was evident in the immune coprecipitation and was PMA-, and Ca(2+)/phospholipid dependent. These results suggest that phosphorylation of deoxyhypusine synthase was PKC-dependent cellular event and open a path for possible regulation in the interaction with eIF5A precursor for hypusine synthesis.