Nerve growth factor and brain-derived neurotrophic factor but not granulocyte colony-stimulating factor, nimodipine and dizocilpine, require ATP for neuroprotective activity after oxygen-glucose deprivation of primary neurons.

In previous work, we have demonstrated by radiolabeling, mass spectrometry and site-directed mutagenesis that nerve growth factor (NGF) as well as brain-derived neurotrophic factor (BDNF) and fibroblast growth factor 2 (FGF2) are capable of ATP-binding and that this binding appears to be essential for their neuroprotective activity. In this study, we attempted to shed some light on the question whether ATP is a general prerequisite for neuroprotection. Therefore, we used the non-ATP-binding granulocyte colony-stimulating factor (GCSF), the calcium antagonist nimodipine and the NMDA antagonist dizocilpine to find out whether they need ATP for neuroprotection comparable to NGF and BDNF. However, ATP was not necessary for the neuroprotective effects of GCSF, nimodipine and dizocilpine on primary cultures of rat cortical neurons damaged by oxygen-glucose deprivation whereas neuroprotection was demonstrable for NGF and BDNF only when ATP was present in the culture medium at a concentration higher than ca. 0.4nmol/l. In circular dichroism studies ATP caused changes of the secondary structure of NGF but not of GCSF. Taken together, we suggest that ATP is not a general prerequisite for neuroprotectivity but some growth factors like NGF and BDNF can stimulate their receptors only if they have bound ATP.

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. According to this our present work focused on the influence of PHP activity on the acetylcholine level in cholinergic neurons. RESULTS: The amount of PHP in SN56 cholinergic neuroblastoma cells was increased after overexpression of PHP by using pIRES2-AcGFP1-PHP as a vector. We demonstrated that PHP overexpression reduced the acetylcholine level and induced cell death. The acetylcholine content of SN56 cells was measured by fast liquid chromatography-tandem mass spectrometry method. Overexpression of the inactive H53A-PHP mutant also induced cell damage, but in a significantly reduced manner. However, this overexpression of the inactive PHP mutant did not change the acetylcholine content of SN56 cells significantly. In contrast, PHP downregulation, performed by RNAi-technique, did not induce cell death, but significantly increased the acetylcholine content in SN56 cells. CONCLUSIONS: We could show for the first time that PHP downregulation increased the acetylcholine level in SN56 cells. This might be a potential therapeutic strategy for diseases involving cholinergic deficits like Alzheimer's disease.

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). Overexpression of PHP by the use of a plasmid vector, pIRES2-AcGFP1-PHP, induced apoptosis in HUVEC. To exclude the possibility that increased cellular protein alone unspecifically caused cell damage, the inactive H53A mutant of PHP was also overexpressed as a control; it did not lead to apoptosis. Down-regulation of PHP by the RNAi (RNA interference) technique did not affect cell viability. In conclusion, HUVEC are damaged by overexpression, but not down-regulation, of PHP, suggesting a pronounced impact of the enzyme on the cells when its activity is increased.

ATP-NGF-complex, but not NGF, is the neuroprotective ligand.

We have shown previously that nerve growth factor (NGF) requires only low nanomolar ATP concentrations in the cell culture medium to protect cortical rat neurons (CRN) from cellular damage induced by staurosporine (STS). We have also demonstrated before that NGF and other growth factors form stable non-covalent complexes with ATP. Here we demonstrated that 8N(1)ATP-NGF, but not NGF, protected CRN against damage. The photo-reactive ATP derivative 8N(3)ATP was incubated with NGF and was trapped in its position by UV irradiation forming a covalent bond. The cross-link with a molar ratio of 1:1 (8N(1)ATP:NGF) was confirmed by mass spectrometry. Circular dichroism experiments revealed that 8N(1)ATP altered the secondary structure of NGF in the same way as ATP did. Covalently bound 8N(1)ATP-NGF was shown to be stable in the presence of the ATP-hydrolyzing enzyme alkaline phosphatase while the non-covalent ATP-NGF-complex dissociated with the removal of free ATP from the solution. 8N(1)ATP-NGF protected CRN against damage by STS independently of free ATP in the culture medium. These results suggest that the ATP-NGF-complex, but not NGF, is the active ligand.

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. High-mass calibration in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has remained a problem due to the lack of suitable standards. Large proteins can hardly be freed of their substructural microheterogeneity by classical purification procedures so that their use as calibrants is limited. A small adduct-forming protein of validated quality is a valuable alternative for that purpose. METHODOLOGY/PRINCIPAL FINDINGS: Three major PHP clusters of ∼113, 209 and >600 kDa were observed in gel filtration analysis. Re-chromatography of the monomer peak showed the same cluster distribution. The tendency to associate was detected also in MALDI-TOF MS measuring regular adducts up to 200 kDa. CONCLUSIONS/SIGNIFICANCE: PHP forms multimers consisting of up to more than 35 protein molecules. In MALDI-TOF MS it generates adduct ions every 16 kDa. The protein can be produced with high quality so that its use as calibration compound for high mass ranges above 100 kDa, where standards are difficult to obtain, is feasible.

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

In the majority of cell types, including the islet ß-cell, transduction of extracellular signals involves ligand binding to a receptor, often followed by the activation G proteins and their effector modules. The islet ß-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. A selective increase in intracellular calcium has been shown to regulate the phosphorylation status key islet proteins thereby facilitating insulin secretion. In addition to classical protein kinases [e.g., protein kinases A and C], recent studies from our laboratory have focused on the expression and function of various forms of NDPK/nm23-like histidine kinases in clonal ß-cells, normal rodent, and human islets. Further, we recently reported localization of a cytosolic protein histidine phosphatase [PHP] in INS 832/13 cells, normal rat islets, and human islets. siRNA-mediated knock down of nm23-H1 and PHP in insulin-secreting INS 832/13 cells significantly attenuated glucose-induced insulin secretion. We also observed significant alterations in the expression and function of nm23-H1/PHP in ß-cells chronically exposed to elevated levels of glucose and saturated fatty acids, such as palmitate (i.e., glucolipotoxicity). Similar changes were also noted in islets from the Goto-Kakizaki and Zucker Diabetic Fatty rats, two known models for type 2 diabetes. It is concluded that protein histidine phosphorylation-dephosphorylation cycles play novel regulatory roles in G protein-mediated physiological insulin secretion and that abnormalities in this signaling axis lead to impaired insulin secretion in glucolipotoxicity and type 2 diabetes.

Binding of ATP to vascular endothelial growth factor isoform VEGF-A165 is essential for inducing proliferation of human umbilical vein endothelial cells.

BACKGROUND: ATP binding is essential for the bioactivity of several growth factors including nerve growth factor, fibroblast growth factor-2 and brain-derived neurotrophic factor. Vascular endothelial growth factor isoform 165 (VEGF-A(165)) induces the proliferation of human umbilical vein endothelial cells, however a dependence on ATP-binding is currently unknown. The aim of the present study was to determine if ATP binding is essential for the bioactivity of VEGF-A(165). RESULTS: We found evidence that ATP binding to VEGF-A(165) induced a conformational change in the secondary structure of the growth factor. This binding appears to be significant at the biological level, as we found evidence that nanomolar levels of ATP (4-8 nm) are required for the VEGF-A(165)-induced proliferation of human umbilical vein endothelial cells. At these levels, purinergic signaling by ATP via P2 receptors can be excluded. Addition of alkaline phosphate to cell culture lowered the ATP concentration in the cell culture medium to 1.8 nM and inhibited cell proliferation. CONCLUSIONS: We propose that proliferation of endothelial cells is induced by a VEGF-A(165)-ATP complex, rather than VEGF-A(165) alone.

["Why is it always the heart which suffers from myocardial infarction?" The "Marburg hypothesis" of the pathogenesis of atherosclerosis]. / "Warum trifft der Herzinfarkt immer das Herz?" Marburger Hypothese zur Pathogenese der Atherosklerose.

There is no question that cholesterol, especially low-density lipoprotein (LDL) cholesterol, represent a major cardiovascular risk factor. The so-called lipid hypothesis has been proven by almost all epidemiologic studies, animal studies and, most importantly, by interventional studies with lipid-lowering drugs, especially statins. However, despite our better understanding of atherogenesis we cannot explain why atherosclerosis occurs most frequently and severely on coronary arteries rather than on other arteries such as those of the hands or feet. In addition, the "lipid hypothesis" is unable to explain the dramatic change in severity of a far more generalized atherosclerosis in patients suffering from diabetes mellitus.Recently, we studied the effects of fatty acids on endothelial integrity and found a dramatic increase in apoptosis under fatty acid exposition. Since it is well known that the heart depends highly on fatty acid delivery to cover its energy demand, we hypothesize that the heart becomes the victim of its energy demand. With the so-called Marburg hypothesis of atherogenesis we can explain the fact why especially the arteries of the heart show early atherosclerotic lesions, but also the fact why patients with diabetes develop more generalized atherosclerosis. Finding mechanisms to reduce the presence of fatty acids within the arterial wall might prevent plaque destabilization and could be a potential target in our fight against atherosclerosis.

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. Functional assays revealed that the beta-cell PHP catalyzes the dephosphorylation of ATP-citrate lyase (ACL). Silencing of PHP expression markedly reduced ACL activity, suggesting functional regulation of ACL by PHP in beta-cells. Coimmunoprecipitation studies revealed modest effects of glucose on the interaction between PHP and ACL. Confocal microscopic evidence indicated that glucose promotes association between ACL and nm23-H1, a known kinase histidine kinase, but not between PHP and ACL. Furthermore, metabolic viability of INS 832/13 cells was resistant to siRNA-PHP, suggesting no regulatory roles of PHP in cell viability. Finally, long-term exposure (24 h) of INS 832/13 cells or rat islets to high glucose (30 mM) increased the expression of PHP. Such increases in PHP expression were also seen in islets derived from the Zucker diabetic fatty rat compared with islets from the lean control animals. Together, these data implicate regulatory roles for PHP in a G protein-sensitive step involved in nutrient-induced insulin secretion. In light of the current debate on putative regulatory roles of ACL in insulin secretion, additional studies are needed to precisely identify the phosphoprotein substrate(s) for PHP in the cascade of events leading to nutrient-induced 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. Advanced atherosclerotic changes of the aorta and the coronary arteries could not be seen but the arteries appeared in a pre-atherosclerotic stage of vascular remodelling. However, the weight and size of the hearts were lower than in controls and the number of apoptotic myocytes increased in the hearts of trioleate-fed animals. To confirm the idea that oleic acid may have caused this apoptosis by activation of PP2C, cultured cardiomyocytes from guinea pigs and mice were treated with various lipids. It was demonstrable that oleic acid dose-dependently caused apoptosis of cardiomyocytes from both species, yet, similar to previous experiments with cultured neurons and endothelial cells, stearic acid, elaidic acid and oleic acid methylester did not. The apoptotic effect caused by oleic acid was diminished when PP2C alpha and beta were downregulated by siRNA showing that PP2C was causally involved in apoptosis caused by oleic acid. CONCLUSIONS/SIGNIFICANCE: The glycerol trioleate diet given to guinea pigs for 5 months did not cause marked atherosclerosis but clearly damaged the hearts by activating PP2C alpha and beta. The diet used with 24% (wt/wt) glycerol trioleate is not comparable to human diets. The detrimental role of MUFAs for guinea pig heart tissue in vivo is shown for the first time. Whether it is true for humans remains to be shown.

Stepchild phosphohistidine: acid-labile phosphorylation becomes accessible by functional proteomics.

Bioanalytical techniques were preferentially developed for the investigation of phosphohydroxyamino acids in the past and there is a wealth of information on the detection of serine, threonine and tyrosine phosphorylation in functional proteomics. However, similarly important for protein regulation and signalling is the phosphorylation of other amino acids such as histidine, but its detection is hampered by the sensitivity to acid. Mass spectrometry in conjunction with chromatographic methods is allowing us to start to get a handle on phosphohistidine. (32)P-labelling and amino acid analysis for phosphorylation site determination is increasingly complemented by typical proteomic approaches based on reversed-phase peptide separation and gas-phase fragmentation. Chemical phosphorylation of peptides is a valuable tool, therefore, for the generation of analytical standards for use in method development.

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. By using mass spectrometry-based methodology we identified one or two molecules of ATP as being bound to NGF. To test neuroprotective activity of NGF we used primary cultures of rat cortical neurons damaged by staurosporine. ATP was indispensable for the neuroprotective effect of NGF. When the ATP concentration in the culture medium was reduced below approximately 2 nM by adding alkaline phosphatase (AP) or ATPase the neuroprotective activity of NGF was abolished. Site-directed mutagenesis within the heparin-binding domain (HBD) of NGF abolished ATP-binding and the neuroprotective effect. Thus, NGF has to bind ATP to be capable of protecting neurons.

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. Here we report that ATP, other nucleoside triphosphates and sodium triphosphate protect FGF2 from trypsin, plasmin and neutrophile elastase digestion in vitro. A molar ratio of 2:1 (ligand/FGF2) is sufficient for these protective effects. ADP shows only little, AMP no stabilizing effect on FGF2 indicating that the number of phosphate residues is important. Protection of FGF2 by ATP can be abolished by the addition of alkaline phosphatase hydrolyzing free and FGF2-bound ATP. The mutant FGF2 (K128A/R129A/K134A/K144A) with strongly reduced ATP-binding capacity revealed no detectable protease resistance after incubation with ATP. Furthermore, a stabilizing effect of ATP on FGF2 could also be demonstrated in cell culture experiments. ATP bound to FGF2 increased FGF2-dependent human umbilical vein endothelial cells proliferation when the growth factor was treated with neutrophile elastase or heat. For the first time these data demonstrate protection of FGF2 by bound ATP, other nucleoside triphosphates or sodium triphosphate from rapid protease digestion. Our data provide new evidence that nucleoside triphosphates are capable of protecting FGF2 and favours such stabilization for various, especially medical applications.

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. Point mutations of conserved residues and deletions of the N- and C-termini of PHP were analyzed using [(32)P-his]ATP-citrate lyase as a substrate. Individual or joint replacement of all cysteine residues by alanine did not affect PHP activity. Deletion of 9 N-terminal amino acids resulted in inactive PHP. Furthermore, only 4 C-terminal residues could be deleted without losing PHP activity. Single or multiple mutations of the glycine-rich domain (Gly(75), Gly(77)) of a putative nucleotide binding site of PHP (GxGxxG/S) caused inactivation of PHP. Wildtype PHP could be labeled with [alpha-(32)P]ATP. Such radiolabeling was not detectable for catalytically inactive PHP-G75A and PHP-G77A. These data suggest further studies on the interaction between PHP and ATP.

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. We have shown that the dephosphorylation of the phosphohistidine of the phosphopeptides is catalysed by PHP. In terms of substrate specificity, there is a small preference for 1-phosphohistidine compared to 3-phosphohistidine, although the rate accelerations for hydrolysis induced by the enzyme were 1100- and 33,333-fold, respectively. The kinetics of both the phosphorylation and dephosphorylation reactions depend on the amino acid sequence surrounding the histidine. PHP shows greater substrate specificity for the peptide whose sequence is similar to that around histidine 18 of histone H4. PHP was unable to catalyse the dephosphorylation of histone H4 that had been phosphorylated with a histone H4 histidine kinase.

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 KCa3.1, all of which are targeted by nucleoside diphosphate kinase (which phosphorylates histidines) and protein histidine phosphatase (which dephosphorylates phosphorylated histidines).

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. Furthermore, PHP was downregulated by siRNA in SH-SY5Y cells. We could demonstrate that overexpression of PHP decreased the phosphorylation state of ACL. Accordingly, the activity of ACL seemed to be reduced and subsequently, the viability of the cells was diminished. On the other hand, downregulation of PHP did not clearly influence phosphorylation and activity of ACL as well as viability of the cells. The results suggest that an increased activity of PHP impairs cellular function whereas downregulation of PHP does not.

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

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 ß were capable of dephosphorylating P-Thr(138)-centrin1 most efficiently. PP2Cδ was inactive and the other retinal phosphatases also had much less or no effect. Similar results were observed for centrins 2 and 4. Centrin3 was not a substrate for CK2. The results suggest PP2C α and ß to play a significant role in regulating the phosphorylation status of centrins in vivo.

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. Interestingly, docosahexaenoic acid (DHA) known to protect arterial vessels against the progression of atherosclerosis caused apoptosis of HUVECs at high concentrations (200-400microM) but inhibited the apoptotic damage of HUVECs at a low, physiologically relevant concentration range (1-10microM). In contrast, oleic acid did not protect HUVECs against damage even at low concentrations (1-25microM). It is supposed that an unbalanced and chronically increased level of MUFAs in blood has an atherosclerotic potential. Furthermore, PP2C activated by MUFAs appears as a new target for drugs to prevent or treat atherosclerosis.

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). Overexpression of PHP in these cells caused a decrease in ACL activity and consequently impaired viability. To be sure that the reduced cellular viability was achieved by overexpression of PHP we also downregulated ACL in SN56 cells using RNAi-technology. Downregulation of ACL was harmful to the cells similar to what was observed upon overexpression of PHP. Taken together, it is concluded that overexpression of PHP results in increased dephosphorylation with concomitant inactivation of ACL, thus finally leading to cell damage.