Acetylcholine content and viability of cholinergic neurons are influenced by the activity of protein histidine phosphatase.

Background: The first mammalian protein histidine phosphatase (PHP) was discovered in the late 90s of the last century. One of the known substrates of PHP is ATP-citrate lyase (ACL), which is responsible--amongst other functions--for providing acetyl-CoA for acetylcholine synthesis in neuronal tissues. It has been shown in previous studies that PHP downregulates the activity of ACL by dephosphorylation.

Influence of protein histidine phosphatase overexpression and down-regulation on human umbilical-vein endothelial cell viability.

PHP (protein histidine phosphatase) is expressed by mammalian tissues, particularly in blood vessel walls. We investigated whether PHP plays a significant role in endothelial cells. By Western blot and immunofluorescence analysis PHP was found in HUVEC (human umbilical-vein endothelial cells).

MALDI-TOF high mass calibration up to 200 kDa using human recombinant 16 kDa protein histidine phosphatase aggregates.

Background: Protein histidine phosphatase (PHP) is an enzyme which removes phosphate groups from histidine residues. It was described for vertebrates in the year 2002. The recombinant human 16 kDa protein forms multimeric complexes in physiological buffer and in the gas phase.

Protein histidine [de]phosphorylation in insulin secretion: abnormalities in models of impaired insulin secretion.

In the majority of cell types, including the islet β-cell, transduction of extracellular signals involves ligand binding to a receptor, often followed by the activation G proteins and their effector modules. The islet β-cell is unusual in that glucose lacks an extracellular receptor. Instead, events consequent to glucose metabolism promote insulin secretion via the generation of diffusible second messengers and mobilization of calcium.

Reversible histidine phosphorylation in mammalian cells: a teeter-totter formed by nucleoside diphosphate kinase and protein histidine phosphatase 1.

Regulation of protein phosphorylation by kinases and phosphatases is involved in many signaling pathways in mammalian cells. In contrast to prokaryotes and lower eukaryotes a role for the reversible phosphorylation of histidine residues is just emerging. The β subunit of heterotrimeric G proteins, the metabolic enzyme adenosine 5'-triphosphate-citrate lyase (ACL), and the Ca2+-activated K+ channel KCa3.

Regulation of glucose- and mitochondrial fuel-induced insulin secretion by a cytosolic protein histidine phosphatase in pancreatic beta-cells.

We report localization of a cytosolic protein histidine phosphatase (PHP; approximately 16 kDa) in INS 832/13 cells, normal rat islets, and human islets. siRNA-mediated knockdown of PHP markedly reduced glucose- or mitochondrial fuel-induced but not KCl-induced insulin secretion. siRNA-mediated knockdown of PHP also attenuated mastoparan-induced insulin secretion, suggesting its participation in G protein-sensitive signaling steps, leading to insulin secretion.

Damage of guinea pig heart and arteries by a trioleate-enriched diet and of cultured cardiomyocytes by oleic acid.

Background: Mono-unsaturated fatty acids (MUFAs) like oleic acid have been shown to cause apoptosis of cultured endothelial cells by activating protein phosphatase type 2C alpha and beta (PP2C). The question arises whether damage of endothelial or other cells could be observed in intact animals fed with a trioleate-enriched diet.

Methodology/principal Findings: Dunkin-Hartley guinea pigs were fed with a trioleate-enriched diet for 5 months.

Binding of ATP to nerve growth factor: characterization and relevance for bioactivity.

Growth factors and their mechanisms of action have been studied extensively. However, it remained widely unrecognized that binding of ATP to growth factors is a prerequisite for their bioactivity. Here we demonstrated the binding of ATP to nerve growth factor (NGF) as well as its relevance for neuroprotection.

Chemical phosphorylation of histidine-containing peptides based on the sequence of histone H4 and their dephosphorylation by protein histidine phosphatase.

Using peptides based on the amino acid sequences surrounding the two histidine residues in histone H4, we have investigated the kinetics of the phosphorylation and dephosphorylation reactions of their histidine residues, when reacted with potassium phosphoramidate, by (1)H NMR. We have been able to estimate rate constants for the reactions and have shown that there are differences in the kinetics between the two peptides. The kinetics of hydrolysis of phosphoramidate was measured by (31)P NMR and protein histidine phosphatase (PHP) was shown to catalyse the reaction.

Relevance of glycine and cysteine residues as well as N- and C-terminals for the activity of protein histidine phosphatase.

There is increasing evidence that reversible phosphorylation of histidine residues regulates numerous important cellular processes. The first protein histidine phosphatase (PHP) from vertebrates was discovered just recently. Here, we report on amino acids and domains essential for activity of PHP.

ATP-dependent stabilization and protection of fibroblast growth factor 2.

Fibroblast growth factor 2 (FGF2) plays a pivotal role in cell proliferation, angiogenesis and neuroprotection. Several clinical trials using this growth factor in bone regeneration, wound healing and cardioprotection are initiated but the inadequate stability of FGF2 after application is one major problem. Binding of ATP to FGF2 and other growth factors has been demonstrated recently.

Reversible phosphorylation of histidine residues in proteins from vertebrates.

Signaling by kinases and phosphatases that act on serine, threonine, and tyrosine residues of proteins is among the most extensively studied regulatory mechanisms in mammalian cells, and research focused in this area is ongoing. We are just beginning to appreciate that such signaling mechanisms are extended and enriched by the reversible phosphorylation of histidine residues. The most exciting developments in this field to date come from studies on the beta subunit of heterotrimeric guanosine triphosphate-binding proteins (G proteins), the enzyme adenosine 5'-triphosphate-citrate lyase, and now the Ca(2+)-activated K(+) channel KCa3.

Role of protein histidine phosphatase for viability of neuronal cells.

We recently found protein histidine phosphatase (PHP) in eukaryotes and identified ATP-citrate lyase (ACL) and the beta-subunit of G-proteins as its substrates. The aim of the present study was to get information on the significance of PHP for cellular function and viability. PHP was overexpressed by a viral vector in SH-SY5Y cells, a human neuroblastoma cell line, and in primary cultures of cortical neurons from embryonic (E19) rats.

Dephosphorylation of Centrins by Protein Phosphatase 2C α and β.

In the present study, we identified protein phosphatases dephosphorylating centrins previously phosphorylated by protein kinase CK2. The following phosphatases known to be present in the retina were tested: PP1, PP2A, PP2B, PP2C, PP5, and alkaline phosphatase. PP2C α and β were capable of dephosphorylating P-Thr(138)-centrin1 most efficiently.

Influence of various fatty acids on the activity of protein phosphatase type 2C and apoptosis of endothelial cells and macrophages.

In previous work we have demonstrated that protein phosphatase type 2C (PP2C) alpha and beta can be activated by mono-unsaturated fatty acids (MUFAs) leading to apoptosis of cultured endothelial cells. In the present paper we could show that saturated fatty acids (SFAs) did not activate PP2C and did not cause apoptosis both in endothelial cells and macrophages. However, long-chain SFAs (>16 C-atoms) were capable of inhibiting both, activation of PP2C as well as apoptosis of human umbilical vein endothelial cells (HUVECs) and macrophages caused by oleic acid.

Reduced viability of neuronal cells after overexpression of protein histidine phosphatase.

Protein histidine phosphatase (PHP) has just recently been discovered in eukaryotes and ATP-citrate lyase (ACL) was shown to be one of its substrates. Since ACL is crucial for cellular energy and fat metabolism we made an attempt to study the influence of PHP on cell viability. Using an adenoviral vector PHP was overexpressed in SN56 cholinergic murine neuroblastoma cells and in primary cultures of hippocampal neurons obtained from embryonic rats (E18).

ATP-binding on fibroblast growth factor 2 partially overlaps with the heparin-binding domain.

Fibroblast growth factor 2 (FGF2), an intensively studied heparin-binding cytokine, is an important modulator of cell growth and differentiation under both physiological and pathophysiological conditions. It has been shown recently that ATP binds to FGF2 and that this binding is crucial for its biological function. In this study we demonstrated that divalent cations were not necessary for binding of ATP to FGF2, but it could be demonstrated that heparin blocked the labelling of FGF2 with ATP indicating an involvement of the heparin-binding domain (aa 128-144) in ATP-binding.

Unsaturated fatty acids liberated from VLDL cause apoptosis in endothelial cells.

Certain unsaturated fatty acids (UFAs), cleaved from lipoproteins, are known to activate the serine/threonine protein phosphatase type 2C (PP2C) alpha- and beta-isoforms. To investigate the role of UFAs in apoptosis of endothelial cells, we cocultured human umbilical vein endothelial cells (HUVECs) with THP-1 monocytes. Phorbol-12-myristic-13-acetate (PMA)-treated THP-1 monocytes differentiated into macrophages and synthesized lipoprotein lipase (LPL), the major enzyme for hydrolysis of triglycerides.

Fast sample preparation and liquid chromatography-tandem mass spectrometry method for assaying cell lysate acetylcholine.

A fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the analysis of acetylcholine (ACh) in cultured cells. [(2)H(4)]Acetylcholine (ACh-d(4)) was used as an internal standard for calibration. ACh was extracted from the cell lysate with acetonitrile (ACN)/water (80/20, v/v) and the crude extract was analyzed without further purification.

Detection of ATP-binding to growth factors.

It was shown in previous work that the interaction of growth factors (GFs) with adenosine triphosphate (ATP) is essential for their neuroprotective effect. Here we investigated the nature of the association of human basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) with ATP. It was demonstrated that this interaction involves the formation of non-covalent ATP-GF complexes that are labile at low pH and that could be selectively purified and subjected to electrospray and MALDI-TOF mass spectrometry.

Effect of hypothalamic proline-rich peptide (PRP-1) on neuronal and bone marrow cell apoptosis.

The AGAPEPAEPAQPGVY proline-rich peptide (PRP-1) was isolated from neurosecretory granules of the bovine neurohypophysis; it is produced by N. supraopticus and N. paraventricularis.

Basic fibroblast growth factor: lysine 134 is essential for its neuroprotective activity.

Basic fibroblast growth factor (bFGF) is a heparin-binding growth factor known to cause cell proliferation, angiogenesis and neuroprotection. We have performed site-directed mutagenesis to identify the amino acids that are essential for heparin/growth factor interaction and for neuroprotection. Binding to heparin-acrylic beads was markedly reduced when lysine in position 134 of bFGF was replaced by alanine.

Expression of protein histidine phosphatase in Escherichia coli, purification, and determination of enzyme activity.

A protein histidine phosphatase (PHP) from vertebrates was first identified in 2002. Here we describe the expression of that PHP in Escherichia coli and purification of the recombinant protein. In addition, a detailed protocol is provided describing determination of PHP activity in vitro.

Protein phosphatase type 2Calpha and 2Cbeta are involved in fatty acid-induced apoptosis of neuronal and endothelial cells.

Unsaturated fatty acids with special structural features have been shown to activate serine/threonine protein phosphatase type 2C (PP2C) isoforms alpha and beta at physiological Mg(2+)-concentrations in vitro. These compounds also induce apoptosis in neuronal and endothelial cells. In this study we further analysed this striking correlation and tried to elucidate whether or not there is a causative relationship between activation of PP2C and induction of apoptosis.

Phosphorylation of the growth factors bFGF, NGF and BDNF: a prerequisite for their biological activity.

The aim of this work was to test whether growth factors such as basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) undergo autophosphorylation and whether this affects their biological activity. Incubation of those growth factors with [gamma-(32)P]ATP resulted in phosphorylation in vitro. The phosphate bond was resistant to alkaline pH, yet acid-labile.