Occurrence Of Phosphorylation Research Articles

Phosphorylating Tannin in Urea System: A Simple Approach for Enhanced Methylene Blue Removal from Aqueous Media.

Tannin, after lignin, is one of the most abundant sources of natural aromatic biomolecules. It has been used and chemically modified during the past few decades to create novel biobased materials. This work intended to functionalize for the first time quebracho Tannin (T) through a simple phosphorylation process in a urea system. The phosphorylation of tannin was studied by Fourier transform infrared spectroscopy (FTIR), NMR, inductively coupled plasma optical emission spectroscopy (ICP-OES), and X-ray fluorescence spectrometry (XRF), while further characterization was performed by scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDX) and thermogravimetric analysis (TGA) to investigate the morphology, composition, structure, and thermal degradation of the phosphorylated material. Results indicated the occurrence of phosphorylation, suggesting the insertion of phosphate-containing groups into the tannin structure, revealing a high content of phosphate for modified tannin (PT). This elevated phosphorus content serves as evidence for the successful incorporation of phosphate groups through the functionalization process. The corresponding PT and T were employed as adsorbents for methylene blue (MB) removal from aqueous solutions. The results revealed that the Langmuir isotherm model effectively represents the adsorption isotherms. Additionally, the pseudo-second-order model indicates that chemisorption predominantly controls the adsorption mechanism. This finding also supports the fact that the introduced phosphate groups via the phosphorylation process significantly contributed to the improved adsorption capacity. Under neutral pH conditions and at room temperature, the material achieved an impressive adsorption capacity of 339.26 mg·g-1 in about 2 h.

Spectral Library Based Analysis of Arginine Phosphorylations in Staphylococcus aureus

Reversible protein phosphorylation is one of the major mechanisms in the regulation of protein expression and protein activity, controlling physiological functions of the important human pathogen Staphylococcus aureus Phosphorylations at serine, threonine and tyrosine are known to influence for example protein activity in central metabolic pathways and the more energy-rich phosphorylations at histidine, aspartate or cysteine can be found as part of two component system sensor domains or mediating bacterial virulence. In addition to these well-known phosphorylations, the phosphorylation at arginine residues plays an essential role. Hence, the deletion mutant S. aureus COL ΔptpB (protein tyrosine phosphatase B) was studied because the protein PtpB is assumed to be an arginine phosphatase. A gel-free approach was applied to analyze the changes in the phosphoproteome of the deletion mutant ΔptpB and the wild type in growing cells, thereby focusing on the occurrence of phosphorylation on arginine residues. In order to enhance the reliability of identified phosphorylation sites at arginine residues, a subset of arginine phosphorylated peptides was chemically synthesized. Combined spectral libraries based on phosphoenriched samples, synthetic arginine phosphorylated peptides and classical proteome samples provide a sophisticated tool for the analysis of arginine phosphorylations. This way, 212 proteins phosphorylated on serine, threonine, tyrosine or arginine residues were identified within the mutant ΔptpB and 102 in wild type samples. Among them, 207 arginine phosphosites were identified exclusively within the mutant ΔptpB, widely distributed along the whole bacterial metabolism. This identification of putative targets of PtpB allows further investigation of the physiological relevance of arginine phosphorylations and provides the basis for reliable quantification of arginine phosphorylations in bacteria.

Molecular mechanisms of asymmetric RAF dimer activation.

Protein phosphorylation is one of the most common post-translational modifications in cell regulatory mechanisms. Dimerization plays also a crucial role in the kinase activity of many kinases, including RAF, CDK2 (cyclin-dependent kinase 2) and EGFR (epidermal growth factor receptor), with heterodimers often being the most active forms. However, the structural and mechanistic details of how phosphorylation affects the activity of homo- and hetero-dimers are largely unknown. Experimentally, synthesizing protein samples with fully specified and homogeneous phosphorylation states remains a challenge for structural biology and biochemical studies. Typically, multiple changes in phosphorylation lead to activation of the same protein, which makes structural determination methods particularly difficult. It is also not well understood how the occurrence of phosphorylation and dimerization processes synergize to affect kinase activities. In the present article, we review available structural data and discuss how MD simulations can be used to model conformational transitions of RAF kinase dimers, in both their phosphorylated and unphosphorylated forms.

Role of NPxY motif in Draper-mediated apoptotic cell clearance in Drosophila

Draper, a receptor responsible for the phagocytosis of apoptotic cells in Drosophila, possesses atypical epidermal growth factor (EGF)-like sequences in the extracellular region and the two phosphorylatable motifs NPxY and YxxL in the intracellular portion. We previously suggested that Pretaporter, a ligand for Draper, binds to the EGF-like repeat and augments the tyrosine phosphorylation of Draper. In this study, we first tested the binding of Pretaporter to various parts of the extracellular region of Draper and found that a single EGF-like sequence is sufficient for the binding. We next determined roles of the two intracellular motifs by forcedly expressing Draper proteins, in which tyrosine residues within the motifs had been substituted with phenylalanine, in hemocytes of Draper-lacking flies. We found that Draper proteins with Y-to-F substitution in either motif still underwent tyrosine phosphorylation, suggesting the occurrence of phosphorylation at both motifs. The Draper protein with substitution in the YxxL motif rescued a defect of phagocytosis, as did intact Draper, but the Draper protein with substitution in the NPxY motif did not, indicating a role of the motif NPxY, but not YxxL, in Draper-mediated phagocytosis. This coincides with our previous finding that Ced-6, an NPxY-binding signaling adaptor, is required for Draper's actions in apoptotic cell clearance. In summary, we demonstrated that Draper binds to its ligand Pretaporter using EGF-like sequences, and that the NPxY motif in the intracellular region of Draper plays an essential role in its actions as an engulfment receptor.

Protein Kinase A Activity And Protein Phosphorylation During The Mouse Sperm Acrosomal Reaction

Using two-dimensional gel electrophoresis, changes in protein phosphorylation caused by cyclic nucleotide-dependent protein kinases were analyzed with or without exposure to a protein kinase inhibitor, H-8, during the mouse sperm acrosomal reaction. The acrosomal reaction, induced by the treatment of sperm with dibutyryl cyclic AMP or dibutyryl cyclic GMP, was inhibited by H-8. The activities of cyclic AMP-dependent protein kinase (PKA) and cyclic GMP-dependent protein kinase induced by the sperm extract were also inhibited by H-8. When endogenous PKA in sperm was activated by the addition of cyclic AMP, a 45-kDa protein spot identified by electrophoresis indicated the occurrence of phosphorylation in vivo. Furthermore, the enhanced phosphorylation of the 45-kDa protein spot was inhibited by H-8. These results suggest that the PKA-catalyzed phosphorylation of the 45-kDa protein may be involved in the regulation of the mouse sperm acrosomal reaction.

Mass spectrometric approach for the analysis of food proteins.

In the study of food proteins, the need for accurate protein structural analysis has been acknowledged because of the fact that nucleotide sequencing alone is of limited analytical value if not combined with relevant information regarding the specific protein expressed and the occurrence of phosphorylation, glycosylation and disulphide bridges, and with the modification induced by the technological treatment. Mass spectrometry, whether used alone or to complement the traditional molecular-based techniques has become fundamental to the structural analysis of proteins. It is, moreover, virtually irreplaceable in determining post-translational modifications as conventional methods cannot deliver reliable data. What lies at the root of this methodological breakthrough is the combination of high-resolution separation techniques such as two-dimensional electrophoresis or capillary reverse- phase high-performance liquid chromatography with mass spectrometric analysis, what is termed "proteomic" analysis. Thus, it appears appropriate to state that the new mass spectrometric techniques have been established as a valuable and efficient tool for protein and peptide analysis in complex mixtures, like those from food matrices, enabling us therefore to provide accurate information on molecular weight and also to put forth a structural assessment at a low-picomole level of material. Thus, a series of alternative approaches have been developed based on advanced mass spectrometric analysis in conjunction with classic protein chemistry in order to provide an in-depth view of food protein structure. This review outlines several of these novel methodologies as they apply to structural characterization of food products.

Photophosphorylation accompanying the hill reaction with ferricyanide

A study of the dependence upon pH of ferricyanide photoreduction revealed that the reaction was most rapid but least stable at high pH (above pH 8) and that inadequate buffering could readily lead to differences in rate due to pH changes. Since the occurrence of phosphorylation may be associated with a pH change, precautions are necessary to prevent artifacts due to pH when determining the effect of phosphorylation on Hill reaction rates. Depending upon circumstances one may overestimate or underestimate the effect. For this reason a reappraisal has been made of the relationship between photophosphorylation and the photoreduction of ferricyanide as catalyzed by spinach chloroplasts. A study of the stoichiometry of photophosphorylation in relation to the Hill reaction revealed that the quantity of ATP formed approached a maximum of 1 mole for 2 moles of ferricyanide reduced and for 0.5 mole of O 2 evolved, in agreement with previous reports from other laboratories. The effects of concomitant phosphorylation upon rates of ferricyanide photoreduction were smaller than those reported previously by other workers. They may best be described as an inhibition of ferricyanide photoreduction by added ADP and the relief of this inhibition by added phosphate. When phosphate was added to the ADP-inhibited system the extra ferricyanide reduced was equal to the amount of phosphate consumed. A scheme for the mechanism of the Hill reaction which can accommodate all of the observed stoichiometry is presented and discussed. It is pointed out that the only mechanism for the Hill reaction which can easily be brought into congruence with this scheme appears to be that proposed by Warburg.