Regulation of early steps of GPVI signal transduction by phosphatases: a systems biology approach

We present a data-driven mathematical model of a key initiating step in platelet activation, a central process in the prevention of bleeding following Injury. In vascular disease, this process is activated inappropriately and causes thrombosis, heart attacks and stroke. The collagen receptor GPVI is the primary trigger for platelet activation at sites of injury. Understanding the complex molecular mechanisms initiated by this receptor is important for development of more effective antithrombotic medicines. In this work we developed a series of nonlinear ordinary differential equation models that are direct representations of biological hypotheses surrounding the initial steps in GPVI-stimulated signal transduction. At each stage model simulations were compared to our own quantitative, high-temporal experimental data that guides further experimental design, data collection and model refinement. Much is known about the linear forward reactions within platelet signalling pathways but knowledge of the roles of putative reverse reactions are poorly understood. An initial model, that includes a simple constitutively active phosphatase, was unable to explain experimental data. Model revisions, incorporating a complex pathway of interactions (and specifically the phosphatase TULA-2), provided a good description of the experimental data both based on observations of phosphorylation in samples from one donor and in those of a wider population. Our model was used to investigate the levels of proteins involved in regulating the pathway and the effect of low GPVI levels that have been associated with disease. Results indicate a clear separation in healthy and GPVI deficient states in respect of the signalling cascade dynamics associated with Syk tyrosine phosphorylation and activation. Our approach reveals the central importance of this negative feedback pathway that results in the temporal regulation of a specific class of protein tyrosine phosphatases in controlling the rate, and therefore extent, of GPVI-stimulated platelet activation.

Electronic Structure and Spectro-Structural Correlations of Fe(III)Zn(II) Biomimetics for Purple Acid Phosphatases: Relevance to DNA Cleavage and Cytotoxic Activity

Purple acid phosphatases (PAPs) are a group of metallohydrolases that contain a dinuclear Fe(II)M(II) center (M(II) = Fe, Mn, Zn) in the active site and are able to catalyze the hydrolysis of a variety of phosphoric acid esters. The dinuclear complex [(H(2)O)Fe(III)(mu-OH)Zn(II)(L-H)](CIO(4))(2) (2) with the ligand 2-[N-bis(2-pyridylmethyl)aminomethyl]-4-methyl-6-[N-(2-pyridylmethyl)(2-hydroxybenzyl) aminomethyl]phenol (H(2)L-H) has recently been prepared and is found to closely mimic the coordination environment of the Fe(III)Zn(II) active site found in red kidney bean PAP (Neves et al. J. Am. Chem. Soc. 2007, 129, 7486). The biomimetic shows significant catalytic activity in hydrolytic reactions. By using a variety of structural, spectroscopic, and computational techniques the electronic structure of the Fe(III) center of this biomimetic complex was determined. In the solid state the electronic ground state reflects the rhombically distorted Fe(III)N(2)O(4) octahedron with a dominant tetragonal compression align ad along the mu-OH-Fe-O(phenolate) direction. To probe the role of the Fe-O(phenolate) bond, the phenolate moiety was modified to contain electron-donating or -withdrawing groups (-CH(3), -H, -Br, -NO(2)) in the 5-position. Tie effects of the substituents on the electronic properties of the biomimetic complexes were studied with a range of experimental and computational techniques. This study establishes benchmarks against accurate crystallographic struck ral information using spectroscopic techniques that are not restricted to single crystals. Kinetic studies on the hydrolysis reaction revealed that the phosphodiesterase activity increases in the order -NO(2)<- Br <- H <- CH(3) when 2,4-bis(dinitrophenyl)phosphate (2,4-bdnpp) was used as substrate, and a linear free energy relationship is found when log(k(cat)/k(0)) is plotted against the Hammett parameter a. However, nuclease activity measurements in the cleavage of double stranded DNA showed that the complexes containing the electron-withdrawing -NO(2) and electron-donating CH3 groups are the most active while the cytotoxic activity of the biomimetics on leukemia and lung tumoral cells is highest for complexes with electron-donating groups.

Flexibility and inhibitor binding in Cdc25 phosphatases

Cdc25 phosphatases involved in cell cycle checkpoints are now active targets for the development of anti-cancer therapies. Rational drug design would certainly benefit from detailed structural information for Cdc25s. However, only apo- or sulfate-bound crystal structures of the Cdc25 catalytic domain have been described so far. Together with previously available crystalographic data, results from molecular dynamics simulations, bioinformatic analysis, and computer-generated conformational ensembles shown here indicate that the last 30-40 residues in the C-terminus of Cdc25B are partially unfolded or disordered in solution. The effect of C-terminal flexibility upon binding of two potent small molecule inhibitors to Cdc25B is then analyzed by using three structural models with variable levels of flexibility, including an equilibrium distributed ensemble of Cdc25B backbone conformations. The three Cdc25B structural models are used in combination with flexible docking, clustering, and calculation of binding free energies by the linear interaction energy approximation to construct and validate Cdc25B-inhibitor complexes. Two binding sites are identified on top and beside the Cdc25B active site. The diversity of interaction modes found increases with receptor flexibility. Backbone flexibility allows the formation of transient cavities or compact hydrophobic units on the surface of the stable, folded protein core that are unexposed or unavailable for ligand binding in rigid and densely packed crystal structures. The present results may help to speculate on the mechanisms of small molecule complexation to partially unfolded or locally disordered proteins.

Thermostable conidial and mycelial alkaline phosphatases from the thermophilic fungus Scytalidium thermophilum

An extracellular (conidial) and an intracellular (mycelial) alkaline phosphatase from the thermophilic fungus Scytalidium thermophilum were purified by DEAE-cellulose and Concanavalin A-Sepharose chromatography. These enzymes showed allosteric behavior either in the presence or absence of MgCl2, BaCl2, CuCl2, and ZnCl2. All of these ions increased the maximal velocity of both enzymes. The molecular masses of the conidial and mycelial enzymes, estimated by gel filtration, were 162 and 132 kDa, respectively. Both proteins migrated on SDS-PAGE as a single polypeptide of 63 and 58.5 kDa, respectively, suggesting that these enzymes were dimers of identical subunits. The best substrate for the conidial and mycelial phosphatases was p-nitrophenylphosphate, but,beta -glycerophosphate and other phosphorylated compounds also served as substrates. The optimum pH for the conidial and mycelial alkaline phosphatases was 10.0 and 9.5 in the presence of AMPOL buffer, and their carbohydrate contents were about 54% and 63%, respectively. The optimum temperature was 70-75 degreesC for both activities. The enzymes were fully stable up to 1 h at 60 degreesC. These and other properties suggested that the alkaline phosphatases of S. thermophilum might be suitable for biotechnological applications.

Distribution of acid phosphatases in the hypopharyngeal glands from workers, queens and males of a Brazilian stingless bee Scaptotrigona postica Latreille: An ultrastructural cytochemical study

The present study reports the localization of acid phosphatase in the hypopharyngeal gland cells from workers (newly-emerged, nurse and forager), queens (newly-emerged and laying) and males (newly-emerged and mature for mating) of the Brazilian stingless bee, Scaptotrigona postica. The phosphatase activity varied in intensity and localization depending on the individual class, physiological age and the substrate used. In newly-emerged workers, the phosphatase-positive sites suggest the involvement of the enzyme with cellular differentiation that occurs in the presecretory phase, in nurse workers with protein synthesis and in forager workers with changes in cellular activity or glandular regression. In males mature for mating and laying queens, the positive sites are related to secretory activity, showing that the gland maintains some activity in spite of the regressive aspect. Of the substrates used, β-glycerophosphate gave the least specific localization.

Synthesis, Biological Evaluation, And Molecular Modeling of Chalcone Derivatives As Potent Inhibitors of Mycobacterium tuberculosis Protein Tyrosine Phosphatases (PtpA and PtpB)

Tuberculosis (TB) is a major infectious disease caused by Mycobacterium tuberculosis (Mtb). According to the World Health Organization (WHO), about 1.8 million people die from TB and 10 million new cases are recorded each year. Recently, a new series of naphthylchalcones has been identified as inhibitors of Mtb protein tyrosine phosphatases (PTPs). In this work, 100 chalcones were designed, synthesized, and investigated for their inhibitory properties against MtbPtps. Structure-activity relationships (SAR) were developed, leading to the discovery of new potent inhibitors with IC50 values in the low-micromolar range. Kinetic studies revealed competitive inhibition and high selectivity toward the Mtb enzymes. Molecular modeling investigations were carried out with the aim of revealing the most relevant structural requirements underlying the binding affinity and selectivity of this series of inhibitors as potential anti-TB drugs.

Functional characterisation of the non-essential protein kinases and phosphatases regulating Aspergillus nidulans hydrolytic enzyme production

Abstract Background Despite recent advances in the understanding of lignocellulolytic enzyme regulation, less is known about how different carbon sources are sensed and the signaling cascades that result in the adaptation of cellular metabolism and hydrolase secretion. Therefore, the role played by non-essential protein kinases (NPK) and phosphatases (NPP) in the sensing of carbon and/or energetic status was investigated in the model filamentous fungus Aspergillus nidulans. Results Eleven NPKs and seven NPPs were identified as being involved in cellulase, and in some cases also hemicellulase, production in A. nidulans. The regulation of CreA-mediated carbon catabolite repression (CCR) in the parental strain was determined by fluorescence microscopy, utilising a CreA: GFP fusion protein. The sensing of phosphorylated glucose, via the RAS signalling pathway induced CreA repression, while carbon starvation resulted in derepression. Growth on cellulose represented carbon starvation and derepressing conditions. The involvement of the identified NPKs in the regulation of cellulose-induced responses and CreA derepression was assessed by genome-wide transcriptomics (GEO accession 47810). CreA:GFP localisation and the restoration of endocellulase activity via the introduction of the ∆creA mutation, was assessed in the NPK-deficient backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses, including the expression of hydrolytic enzymes and transporters. The mechanism by which these two NPKs controlled gene transcription was identified, as the NPK-deficient mutants were not able to unlock CreA-mediated carbon catabolite repression under derepressing conditions, such as carbon starvation or growth on cellulose. Conclusions Collectively, this study identified multiple kinases and phosphatases involved in the sensing of carbon and/or energetic status, while demonstrating the overlapping, synergistic roles of schA and snfA in the regulation of CreA derepression and hydrolytic enzyme production in A. nidulans. The importance of a carbon starvation-induced signal for CreA derepression, permitting transcriptional activator binding, appeared paramount for hydrolase secretion.

Improving adoptive T cell therapies of cancer by ectopic expression of miR181a to repress inhibitory phosphatases

Adoptive T cell therapy using antigen-specific T lymphocytes is a powerful immunotherapeutic approach against cancer. Nevertheless, many T cells against tumor-antigens exhibit only weak anti-tumoral response. To overcome this barrier it is necessary to improve the potency and anti-tumoral efficacy of these T cells. Activation and activity of T cells are tightly controlled to inhibit unwanted T cell responses and to reduce the risk of autoimmunity. Both are regulated by extrinsic signals and intrinsic mechanisms which suppress T cell activation. The intrinsic mechanisms include the expression of phosphatases that counteract the activation-inducing kinases. Modifying the expression of these phosphatases allows the targeted modulation of T cell reactivity. MicroRNAs (miRNAs) are regulatory small noncoding RNA molecules that control gene expression by targeting messenger RNAs in a sequence specific manner. Gene-specific silencing plays a key role in diverse biological processes, such as development, differentiation, and functionality. miR181a has been shown to be highly expressed in immature T cells that recognize low-affinity antigens.rnThe present study successfully shows that ectopic expression of miR181a is able to enhance the sensitivity of both murine and human T cells. In CD4+ T helper cells as well as in CD8+ cytotoxic T cells the overexpression of miR181a leads to downregulation of multiple phosphatases involved in the T cell receptor signaling pathway. Overexpression of miR181a in human T cells achieves a co-stimulatory independent activation and has an anti-apoptotic effect on CD4+ T helper cells. Additionally, increasing the amount of miR181a enhances the cytolytic activity of murine CD8+ TCRtg T cells in an antigen-specific manner.rnTo test miR181a overexpressing T cells in vivo, a mouse tumor model using a B cell lymphoma cell line (A20-HA) expressing the Influenza hemagglutinin (Infl.-HA) antigen was established. The expression of model antigens in tumor cell lines enables targeted elimination of tumors using TCRtg T cells. The transfer of miR181a overexpressing Infl.-HA TCRtg CD8+ T cells alone has no positive effect neither on tumor control nor on survival of A20-HA tumor-bearing mice. In contrast, the co-transfer of miR181a overexpressing Infl.-HA TCRtg CD8+ and CD4+ T cells leads to improved tumor control and prolongs survival of A20-HA tumor-bearing mice. This effect is characterized by higher amounts of effector T cells and the expansion of Infl.-HA TCRtg CD8+ T cells.rnAll effects were achieved by changes in expression of several genes including molecules involved in T cell differentiation, activation, and regulation, cytotoxic effector molecules, and receptors important for the homing process of T cells in miR181a overexpressing T cells. The present study demonstrates that miR181a is able to enhance the anti-tumoral response of antigen-specific T cells and is a promising candidate for improving adoptive cell therapy.

The type II secretion system (Xcp) of Pseudomonas putida is active and involved in the secretion of phosphatases.

The genome of the Gram-negative bacterium Pseudomonas putida harbours a complete set of xcp genes for a type II protein secretion system (T2SS). This study shows that expression of these genes is induced under inorganic phosphate (Pi ) limitation and that the system enables the utilization of various organic phosphate sources. A phosphatase of the PhoX family, previously designated UxpB, was identified, which was produced under low Pi conditions and transported across the cell envelope in an Xcp-dependent manner demonstrating that the xcp genes encode an active T2SS. The signal sequence of UxpB contains a twin-arginine translocation (Tat) motif as well as a lipobox, and both processing by leader peptidase II and Tat dependency were experimentally confirmed. Two different tat gene clusters were detected in the P.?putida genome, of which one, named tat-1, is located adjacent to the uxpB and xcp genes. Both Tat systems appeared to be capable of transporting the UxpB protein. However, expression of the tat-1 genes was strongly induced by low Pi levels, indicating a function of this system in survival during Pi starvation.

The type II secretion system (Xcp) of Pseudomonas putida is active and involved in the secretion of phosphatases.

The genome of the Gram-negative bacterium Pseudomonas putida harbours a complete set of xcp genes for a type II protein secretion system (T2SS). This study shows that expression of these genes is induced under inorganic phosphate (Pi ) limitation and that the system enables the utilization of various organic phosphate sources. A phosphatase of the PhoX family, previously designated UxpB, was identified, which was produced under low Pi conditions and transported across the cell envelope in an Xcp-dependent manner demonstrating that the xcp genes encode an active T2SS. The signal sequence of UxpB contains a twin-arginine translocation (Tat) motif as well as a lipobox, and both processing by leader peptidase II and Tat dependency were experimentally confirmed. Two different tat gene clusters were detected in the P.?putida genome, of which one, named tat-1, is located adjacent to the uxpB and xcp genes. Both Tat systems appeared to be capable of transporting the UxpB protein. However, expression of the tat-1 genes was strongly induced by low Pi levels, indicating a function of this system in survival during Pi starvation.

A peptide export–import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay

The phosphorelay signal transduction system activates developmental transcription in sporulation of Bacillus subtilis by phosphorylation of aspartyl residues of the Spo0F and Spo0A response regulators. The phosphorylation level of these response regulators is determined by the opposing activities of protein kinases and protein aspartate phosphatases that interpret positive and negative signals for development in a signal integration circuit. The RapA protein aspartate phosphatase of the phosphorelay is regulated by a peptide that directly inhibits its activity. This peptide is proteolytically processed from an inactive pre-inhibitor protein encoded in the phrA gene. The pre-inhibitor is cleaved by the protein export apparatus to a putative pro-inhibitor that is further processed to the active inhibitor peptide and internalized by the oligopeptide permease. This export–import circuit is postulated to be a mechanism for timing phosphatase activity where the processing enzymes regulate the rate of formation of the active inhibitor. The processing events may, in turn, be controlled by a regulatory hierarchy. Chromosome sequencing has revealed several other phosphatase–prepeptide gene pairs in B. subtilis, suggesting that the use of this mechanism may be widespread in signal transduction.

Identification and characterization of a conserved family of protein serine/threonine phosphatases homologous to Drosophila retinal degeneration C (rdgC)

The Drosophila retinal degeneration C (rdgC) gene encodes an unusual protein serine/threonine phosphatase in that it contains at least two EF-hand motifs at its carboxy terminus. By a combination of large-scale sequencing of human retina cDNA clones and searches of expressed sequence tag and genomic DNA databases, we have identified two sequences in mammals [Protein Phosphatase with EF-hands-1 and 2 (PPEF-1 and PPEF-2)] and one in Caenorhabditis elegans (PPEF) that closely resemble rdgC. In the adult, PPEF-2 is expressed specifically in retinal rod photoreceptors and the pineal. In the retina, several isoforms of PPEF-2 are predicted to arise from differential splicing. The isoform that most closely resembles rdgC is localized to rod inner segments. Together with the recently described localization of PPEF-1 transcripts to primary somatosensory neurons and inner ear cells in the developing mouse, these data suggest that the PPEF family of protein serine/threonine phosphatases plays a specific and conserved role in diverse sensory neurons.

Distinct, Constitutively Active MAPK Phosphatases Function in Xenopus Oocytes: Implications for p42 MAPK Regulation In Vivo

Xenopus oocyte maturation requires the phosphorylation and activation of p42 mitogen-activated protein kinase (MAPK). Likewise, the dephosphorylation and inactivation of p42 MAPK are critical for the progression of fertilized eggs out of meiosis and through the first mitotic cell cycle. Whereas the kinase responsible for p42 MAPK activation is well characterized, little is known concerning the phosphatases that inactivate p42 MAPK. We designed a microinjection-based assay to examine the mechanism of p42 MAPK dephosphorylation in intact oocytes. We found that p42 MAPK inactivation is mediated by at least two distinct phosphatases, an unidentified tyrosine phosphatase and a protein phosphatase 2A–like threonine phosphatase. The rates of tyrosine and threonine dephosphorylation were high and remained constant throughout meiosis, indicating that the dramatic changes in p42 MAPK activity seen during meiosis are primarily attributable to changes in MAPK kinase activity. The overall control of p42 MAPK dephosphorylation was shared among four partially rate-determining dephosphorylation reactions, with the initial tyrosine dephosphorylation of p42 MAPK being the most critical of the four. Our findings provide biochemical and kinetic insight into the physiological mechanism of p42 MAPK inactivation.

Cell-cell communication regulates the effects of protein aspartate phosphatases on the phosphorelay controlling development in Bacillus subtilis.

Rap phosphatases are a recently discovered family of protein aspartate phosphatases that dephosphorylate the Spo0F--P intermediate of the phosphorelay, thus preventing sporulation of Bacillus subtilis. They are regulators induced by physiological processes that are antithetical to sporulation. The RapA phosphatase is induced by the ComP-ComA two-component signal transduction system responsible for initiating competence. RapA phosphatase activity was found to be controlled by a small protein, PhrA, encoded on the same transcript as RapA. PhrA resembles secreted proteins and the evidence suggests that it is cleaved by signal peptidase I and a 19-residue C-terminal domain is secreted from the cell. The sporulation deficiency caused by the uncontrolled RapA activity of a phrA mutant can be complemented by synthetic peptides comprising the last six or more of the C-terminal residues of PhrA. Whether the peptide controls RapA activity directly or by regulating its synthesis remains to be determined. Complementation of the phrA mutant can also be obtained in mixed cultures with a wild-type strain, suggesting the peptide may serve as a means of communication between cells. Importation of the secreted peptide required the oligopeptide transport system. The sporulation deficiency of oligopeptide transport mutants can be suppressed by mutating the rapA and rapB genes or by introduction of a spo0F mutation Y13S that renders the protein insensitive to Rap phosphatases. The data indicate that the sporulation deficiency of oligopeptide transport mutants is due to their inability to import the peptides controlling Rap phosphatases.