The role of the active site residues in human galactokinase: implications for the mechanisms of GHMP kinases.

Galactokinase catalyses the phosphorylation of galactose at the expense of ATP. Like other members of the GHMP family of kinases it is postulated to function through an active site base mechanism in which Asp-186 abstracts a proton from galactose. This asparate residue was altered to alanine and to asparagine by site-directed mutagenesis of the corresponding gene. This resulted in variant enzyme with no detectable galactokinase activity. Alteration of Arg-37, which lies adjacent to Asp-186 and is postulated to assist the catalytic base, to lysine resulted in an active enzyme. However, alteration of this residue to glutamate abolished activity. All the variant enzymes, except the arginine to lysine substitution, were structurally unstable (as judged by native gel electrophoresis in the presence of urea) compared to the wild type. This suggests that the lack of activity results from this structural instability, in addition to any direct effects on the catalytic mechanism. Computational estimations of the pK(a) values of the arginine and aspartate residues, suggest that Arg-37 remains protonated throughout the catalytic cycle whereas Asp-186 has an abnormally high pK(a) value (7.18). Quantum mechanics/molecular mechanics (QM/MM) calculations suggest that Asp-186 moves closer to the galactose molecule during catalysis. The experimental and theoretical studies presented here argue for a mechanism in which the C-1-OH bond in the sugar is weakened by the presence of Asp-186 thus facilitating nucleophilic attack by the oxygen atom on the gamma-phosphorus of ATP.

Donepezil attenuates hippocampal neuronal damage and cognitive deficits after global cerebral ischemia in gerbils.

Decreased cerebral blood flow causes cognitive impairments and neuronal injury in vascular dementia. In the present study, we reported that donepezil, a cholinesterase inhibitor, improved transient global cerebral ischemia-induced spatial memory impairment in gerbils. Treatment with 5mg/kg of donepezil for 21 consecutive days following a 10-min period of ischemia significantly inhibited delayed neuronal death in the hippocampal CA1 region. In Morris water maze test, memory impairment was significantly improved by donepezil treatment. Western blot analysis showed that donepezil treatment prevented reductions in p-CaMKII and p-CREB protein levels in the hippocampus. These results suggest that donepezil attenuates the memory deficit induced by transient global cerebral ischemia and this neuroprotection may be associated with the phosphorylation of CaMKII and CERB in the hippocampus.

N-acetylgalactosamine kinase: a naturally promiscuous small molecule kinase

N-acetylgalactosamine kinase is a member of the GHMP family of small molecule kinases which catalyses the ATP-dependent phosphorylation of N-acetylgalactosamine. It is highly similar in structure and sequence to galactokinase. Alteration of galactokinase at a key tyrosine residue (Tyr-379 in the human enzyme) has been shown to dramatically enhance the substrate range of this enzyme. Here, we investigated the substrate specificity of the wild type N-acetylgalactosamine kinase and demonstrated that it can also catalyse the phosphorylation of N-acetylglucosamine and N-acetylmannosamine. In human N-acetylgalactosamine kinase, the equivalent residue to Tyr-379 in galactokinase is Phe-444. Alteration of this residue did not result in dramatic changes to the specificity of the enzyme. The more relaxed substrate specificity of N-acetylgalactosamine kinase, compared to galactokinase, can be explained by the greater flexibility of a glycine rich loop in the active site of the enzyme. These results suggest that N-acetylgalactosamine kinase is a potential biocatalyst for the phosphorylation of N-acetyl sugars. However, it is unlikely that it will be possible to further broaden the substrate range by alteration of Phe-444.

Fasciolicides: Efficacy, actions, resistance and its management

The modes of action of fasciolicides are described. Closantel and other salicylanilides interfere with energy metabolism by uncoupling oxidative phosphorylation in the fluke. Other fasciolicides are believed to have a metabolic action-halogenated phenols (via uncoupling) and clorsulon (via inhibition of glycolysis)-but direct evidence is lacking. Benzimidazoles (in particular, riclabendazole) bind to fluke tubulin and disrupt microtubule-based processes. Diamphenethide inhibits protein synthesis in the fluke. Other potential drug actions may contribute to overall drug efficacy. In particular, a number of fasciolicides-salicylanilides, phenols, diamphenethide-induce a rapid paralysis of the fluke, so their action may have a neuromuscular basis, although the actions remain ill-defined. Resistance to salicylanilides and triclabendazole has been detected in the field, although drug resistance does not appear to be a major problem yet. Strategies to minimize the development of resistance include the use of synergistic drug combinations, together with the design of integrated management programmes and the search for alternatives to drugs, in particular, vaccines. (C) 1999 Harcourt Publishers Ltd.

Non-radial pulsation, rotation and outburst in the Be star omega Orionis from the MuSiCoS 1998 campaign

omega Ori (HD37490, HR1934) is a Be star known to have presented variations. In order to investigate the nature and origin of its short-term and mid-term variability, a study is performed of several spectral lines (Halpha, Hdelta, HeI 4471, 4713, 4921, 5876, 6678, CII 4267, 6578, 6583, Mg II 4481, Si III 4553 and Si II 6347), based on 249 high signal-to-noise high-resolution spectra taken with 8 telescopes over 22 consecutive nights during the MuSiCoS (Multi SIte COntinuous Spectroscopy) campaign in November-December 1998. The stellar parameters are revisited and the projected rotational velocity (v sin i = 179 km s(-1)) is redetermined using several methods. With the MuSiCoS 98 dataset, a time series analysis of line-profile variations (LPVs) is performed using the Restricted Local Cleanest (RLC) algorithm and a least squares method. The behaviour of the velocity of the centroid of the lines, the equivalent widths and the apparent vsini for several lines, as well as Violet and Red components of photospheric lines affected by emission (red He i lines, Si II 6347, CII 6578, 6583) are analyzed. The non-radial pulsation (NRP) model is examined using phase diagrams and the Fourier-Doppler Imaging (FDI) method. The LPVs are consistent with a NRP mode with l = 2 or 3, \m\ = 2 with frequency 1.03 cd(-1). It is shown that an emission line outburst occurred in the middle of the campaign. Two scenarios are proposed to explain the behaviour of a dense cloud, temporarily orbiting around the star with a frequency 0.46 c d(-1), in relation to the outburst.

Local DNA damage by proton microbeam irradiation induces poly(ADP-ribose) synthesis in mammalian cells

Cellular recovery from ionizing radiation (IR)-induced damage involves poly(ADP-ribose) polymerase (PARP-1 and PARP-2) activity, resulting in the induction of a signalling network responsible for the maintenance of genomic integrity. In the present work, a charged particle microbeam delivering 3.2 MeV protons from a Van de Graaff accelerator has been used to locally irradiate mammalian cells. We show the immediate response of PARPs to local irradiation, concomitant with the recruitment of ATM and Rad51 at sites of DNA damage, both proteins being involved in DNA strand break repair. We found a co-localization but no connection between two DNA damage-dependent post-translational modifications, namely poly(ADP-ribosyl)ation of nuclear proteins and phosphorylation of histone H2AX. Both of them, however, should be considered and used as bona fide immediate sensitive markers of IR damage in living cells. This technique thus provides a powerful approach aimed at understanding the interactions between the signals originating from sites of DNA damage and the subsequent activation of DNA strand break repair mechanisms.

Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo

Despite a clear link between ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using GO-G, confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser(15) (p53(Ser15)), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologicafly distinct subpool of p53(Ser15) is ATM dependent and resistant to 26S-proteasomal degradation. p53(Ser15) colocalizes and coimmunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear micro-beam irradiation studies confirm p53 S,,15 is recruited to sites of DNA damage containing gamma-H2AX, ATM(Ser1981), and DNA-PKcs(Thr2609) in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser(15) or Ser(18) phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21(WAF) decreased gamma-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis. (Cancer Res 2005; 65(23): 10810-21).

Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion

Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described. We have identified a cell non-autonomous role of Rb, using three-dimensional cultures, where depletion of Rb in stromal fibroblasts enhances invasive potential of transformed epithelia. In part, this is mediated by upregulation of keratinocyte growth factor (KGF), which is produced by the depleted fibroblasts. KGF drives invasion of epithelial cells through induction of MMP1 expression in an AKT- and Ets2-dependent manner. Our data identify that stromal fibroblasts can alter the invasive behaviour of the epithelium, and we show that altered expression of KGF can mediate these functions. © European Molecular Biology Organization.

Akt-Mediated Proinflammatory Response of Mononuclear Phagocytes Infected with Burkholderia cenocepacia Occurs by a Novel GSK3 beta-Dependent, I kappa B Kinase-Independent Mechanism

The environmental bacterium Burkholderia cenocepacia causes opportunistic lung infections in immunocompromised individuals, particularly in patients with cystic fibrosis. Infections in these patients are associated with exacerbated inflammation leading to rapid decay of lung function, and in some cases resulting in cepacia syndrome, which is characterized by a fatal acute necrotizing pneumonia and sepsis. B. cenocepacia can survive intracellularly in macrophages by altering the maturation of the phagosome, but very little is known on macrophage responses to the intracellular infection. In this study, we have examined the role of the PI3K/Akt signaling pathway in B. cenocepacia-infected monocytes and macrophages. We show that PI3K/Akt activity was required for NF-kappa B activity and the secretion of proinflammatory cytokines during infection with B. cenocepacia. In contrast to previous observations in epithelial cells infected with other Gram-negative bacteria, Akt did not enhance I kappa B kinase or NF-kappa B p65 phosphorylation, but rather inhibited GSK3 beta, a negative regulator of NF-kappa B transcriptional activity. This novel mechanism of modulation of NF-kappa B activity may provide a unique therapeutic target for controlling excessive inflammation upon B. cenocepacia infection. The Journal of Immunology, 2011, 187: 635-643.

Structural and kinetic characterization of the LPS biosynthetic enzyme D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) from Escherichia coli

Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.

Undecaprenyl phosphate recycling comes out of age

The recycling of the lipid carrier undecaprenyl-phosphate (Und-P) requires the dephosphorylation of Und-PP, a reaction proposed to occur at the external or periplasmic side of the bacterial cell membrane. In this issue of Molecular Microbiology, experiments based on the analysis of lipopolysaccharide modifications in Escherichia coli demonstrate that the phosphorylation of lipid A at position 1 is catalysed by the membrane enzyme LpxT (formerly YeiU). This enzyme specifically transfers the distal phosphate group from Und-PP to lipid A 1-phosphate to produce lipid A 1-diphosphate. Furthermore, this reaction requires a functionally intact MsbA protein, which catalyses the transfer of lipid A across the membrane, confirming that the LpxT-mediated lipid A modification occurs on the periplasmic side of the membrane. These observations provide a novel and unexpected link between periplasmic lipid A modifications and the Und-PP recycling pathway.

A plasma membrane Ca2+-ATPase (PMCA) from the liver fluke, Fasciola hepatica

Fasciolosis is a parasitic infection by the liver fluke Fasciola hepatica, which costs the global agricultural community over US $2 billion per year. Its prevalence is rising due to factors such as climate change and drug resistance. ATP-dependent membrane transporters are considered good potential drug targets as they are essential for cellular processes and are in an exposed, accessible position in the cell. Immunolocalisation studies demonstrated that a plasma membrane calcium ATPase (PMCA) was localised to the parenchymal tissue in F. hepatica. The coding sequence for a F. hepatica PMCA (FhPMCA) has been obtained. This sequence encodes a 1,163 amino acid protein which contains motifs which are commonly conserved in PMCAs. Molecular modelling predicted that the protein has 10 transmembrane segments which include a potential calcium ion binding site and phosphorylation motif. FhPMCA interacts with the calmodulin-like protein FhCaM1, but not the related proteins FhCaM2 or FhCaM3, in a calcium-ion dependent manner. This interaction occurs through a region in the C-terminal region of FhPMCA which most likely adopts an a-helical conformation. When FhPMCA was heterologously expressed in a budding yeast strain deleted for its PMCA (Pmc1p), it restored viability. Microsomes prepared from these yeast cells had calcium ion stimulated ATPase activity which was inhibited by the known PMCA inhibitors, bisphenol and eosin. The potential of FhPMCA as a new drug target is discussed.

Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

When it is difficult to develop selective ligands within a family of related G-protein-coupled receptors (GPCRs), chemically engineered receptors activated solely by synthetic ligands (RASSLs) are useful alternatives for probing receptor function. In the present work, we explored whether a RASSL of the free fatty acid receptor 2 (FFA2) could be developed on the basis of pharmacological variation between species orthologs. For this, bovine FFA2 was characterized, revealing distinct ligand selectivity compared with human FFA2. Homology modeling and mutational analysis demonstrated a single mutation in human FFA2 of C4.57G resulted in a human FFA2 receptor with ligand selectivity similar to the bovine receptor. This was exploited to generate human FFA2-RASSL by the addition of a second mutation at a known orthosteric ligand interaction site, H6.55Q. The resulting FFA2-RASSL displayed a >100-fold loss of activity to endogenous ligands, while responding to the distinct ligand sorbic acid with pEC(50) values for inhibition of cAMP, 5.83 ± 0.11; Ca(2+) mobilization, 4.63 ± 0.05; ERK phosphorylation, 5.61 ± 0.06; and dynamic mass redistribution, 5.35 ± 0.06. This FFA2-RASSL will be useful in future studies on this receptor and demonstrates that exploitation of pharmacological variation between species orthologs is a powerful method to generate novel chemically engineered GPCRs.-Hudson, B. D., Christiansen, E., Tikhonova, I. G., Grundmann, M., Kostenis, E., Adams, D. R., Ulven, T., Milligan, G. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs.

IHG-1 amplifies TGF-beta1 signaling and is increased in renal fibrosis

Induced in high glucose-1 (IHG-1) is an evolutionarily conserved gene transcript upregulated by high extracellular glucose concentrations, but its function is unknown. Here, it is reported that the abundance of IHG-1 mRNA is nearly 10-fold higher in microdissected, tubule-rich renal biopsies from patients with diabetic nephropathy compared with control subjects. In the diabetic nephropathy specimens, in situ hybridization localized IHG-1 to tubular epithelial cells along with TGF-beta1 and activated Smad3, suggesting a possible role in the development of tubulointerstitial fibrosis. Supporting this possibility, IHG-1 mRNA and protein expression also increased with unilateral ureteral obstruction. In the HK-2 proximal tubule cell line, overexpression of IHG-1 increased TGF-beta1-stimulated expression of connective tissue growth factor and fibronectin. IHG-1 was found to amplify TGF-beta1-mediated transcriptional activity by increasing and prolonging phosphorylation of Smad3. Conversely, inhibition of endogenous IHG-1 with small interference RNA suppressed transcriptional responses to TGF-beta1. In summary, IHG-1, which increases in diabetic nephropathy, may enhance the actions of TGF-beta1 and contribute to the development of tubulointerstitial fibrosis.

Dysregulated intracellular signaling impairs CTGF-stimulated responses in human mesangial cells exposed to high extracellular glucose

High ambient glucose activates intracellular signaling pathways to induce the expression of extracellular matrix and cytokines such as connective tissue growth factor (CTGF). Cell responses to CTGF in already glucose-stressed cells may act to transform the mesangial cell phenotype leading to the development of glomerulosclerosis. We analyzed cell signaling downstream of CTGF in high glucose-stressed mesangial cells to model signaling in the diabetic milieu. The addition of CTGF to primary human mesangial cells activates cell migration which is associated with a PKC-zeta-GSK3beta signaling axis. In high ambient glucose basal PKC-zeta and GSK3beta phosphorylation levels are selectively increased and CTGF-stimulated PKC-zeta and GSK3beta phosphorylation was impaired. These effects were not induced by osmotic changes. CTGF-driven profibrotic cell signaling as determined by p42/44 MAPK and Akt phosphorylation was unaffected by high glucose. Nonresponsiveness of the PKC-zeta-GSK3beta signaling axis suppressed effective remodeling of the microtubule network necessary to support cell migration. However, interestingly the cells remain plastic: modulation of glucose-induced PKC-beta activity in human mesangial cells reversed some of the pathological effects of glucose damage in these cells. We show that inhibition of PKC-beta with LY379196 and PKC-beta siRNA reduced basal PKC-zeta and GSK3beta phosphorylation in human mesangial cells exposed to high glucose. CTGF stimulation under these conditions again resulted in PKC-zeta phosphorylation and human mesangial cell migration. Regulation of PKC-zeta by PKC-beta in this instance may establish PKC-zeta as a target for constraining the progression of mesangial cell dysfunction in the pathogenesis of diabetic nephropathy.