bluuebunny/crossref_metadata_2025 huggingface dataset:
Cysteine residues of SNAP-25 are required for SNARE disassembly and exocytosis, but not for membrane targeting
Authors: WASHBOURNE Philip , CANSINO Victor , MATHEWS James R. , GRAHAM Margaret , BURGOYNE Robert D. , WILSON Michael C.
Published: August 2015

The release of neurotransmitter at a synapse occurs via the regulated fusion of synaptic vesicles with the plasma membrane. The fusion of the two lipid bilayers is mediated by a protein complex that includes the plasma membrane target soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (t-SNAREs), syntaxin 1A and synaptosome-associated protein of 25kDa (SNAP-25), and the vesicle SNARE (v-SNARE), vesicle-associated membrane protein (VAMP). Whereas syntaxin 1A and VAMP are tethered to the membrane by a C-terminal transmembrane domain, SNAP-25 has been suggested to be anchored to the membrane via four palmitoylated cysteine residues. We demonstrate that the cysteine residues of SNAP-25 are not required for membrane localization when syntaxin 1A is present. Analysis of the 7S and 20S complexes formed by mutants that lack cysteine residues demonstrates that the cysteines are required for efficient SNARE complex dissociation. Furthermore, these mutants are unable to support exocytosis, as demonstrated by a PC12 cell secretion assay. We hypothesize that syntaxin 1A serves to direct newly synthesized SNAP-25 through the Golgi transport pathway to the axons and synapses, and that palmitoylation of cysteine residues is not required for targeting, but to optimize interactions required for SNARE complex dissociation.
Molecular modelling and experimental studies of mutation and cell-adhesion sites in the fibronectin type III and whey acidic protein domains of human anosmin-1
Authors: ROBERTSON Alexis , MacCOLL Gavin S. , NASH Julia A.B. , BOEHM Mark K. , PERKINS Stephen J. , BOULOUX Pierre-Marc G.
Published: August 2015

Anosmin-1, the gene product of the KAL gene, is implicated in the pathogenesis of X-linked Kallmann's syndrome. Anosmin-1 protein expression is restricted to the basement membrane and interstitial matrix of tissues affected in this syndrome during development. The anosmin-1 sequence indicates an N-terminal cysteine-rich domain, a whey acidic protein (WAP) domain, four fibronectin type III (FnIII) domains and a C-terminal histidine-rich region, and shows similarity with cell-adhesion molecules, such as neural cell-adhesion molecule, TAG-1 and L1. We investigated the structural and functional significance of three loss-of-function missense mutations of anosmin-1 using comparative modelling of the four FnIII and the WAP domains based on known NMR and crystal structures. Three missense mutation-encoded amino acid substitutions, N267K, E514K and F517L, were mapped to structurally defined positions on the GFCC′ β-sheet face of the first and third FnIII domains. Electrostatic maps demonstrated large basic surfaces containing clusters of conserved predicted heparan sulphate-binding residues adjacent to these mutation sites. To examine these modelling results anosmin-1 was expressed in insect cells. The incorporation of the three mutations into recombinant anosmin-1 had no effect on its secretion. The removal of two dibasic motifs that may constitute potential physiological cleavage sites for anosmin-1 had no effect on cleavage. Peptides based on the anosmin-1 sequences R254–K285 and P504–K527 were then synthesized in order to assess the effect of the three mutations on cellular adhesion, using cell lines that represented potential functional targets of anosmin-1. Peptides (10μg/ml) incorporating the N267K and E514K substitutions promoted enhanced adhesion to 13.S.1.24 rat olfactory epithelial cells and canine MDCK1 kidney epithelial cells (P<0.01) compared with the wild-type peptides. This result was attributed to the introduction of a lysine residue adjacent to the large basic surfaces. We predict that two of the three missense mutants increase the binding of anosmin-1 to an extracellular target, possibly by enhancing heparan sulphate binding, and that this critically affects the function of anosmin-1.
Identification and characterization of the STIM (stromal interaction molecule) gene family: coding for a novel class of transmembrane proteins
Authors: WILLIAMS Richard T. , MANJI Shehnaaz S. M. , PARKER Nigel J. , HANCOCK Manuela S. , van STEKELENBURG Leonie , EID Jean-Pierre , SENIOR Paul V. , KAZENWADEL Janette S. , SHANDALA Tetyana , SAINT Robert , SMITH Peter J. , DZIADEK Marie A.
Published: August 2015

STIM1 (where STIM is stromal interaction molecule) is a candidate tumour suppressor gene that maps to human chromosome 11p15.5, a region implicated in a variety of cancers, particularly embryonal rhabdomyosarcoma. STIM1 codes for a transmembrane phosphoprotein whose structure is unrelated to that of any other known proteins. The precise pathway by which STIM1 regulates cell growth is not known. In the present study we screened gene databases for STIM1-related sequences, and have identified and characterized cDNA sequences representing a single gene in humans and other vertebrates, which we have called STIM2. We identified a single STIM homologue in Drosophila melanogaster (D-Stim) and Caenorhabditis elegans, but no homologues in yeast. STIM1, STIM2 and D-Stim have a conserved genomic organization, indicating that the vertebrate family of two STIM genes most probably arose from a single ancestral gene. The three STIM proteins each contain a single SAM (sterile α-motif) domain and an unpaired EF hand within the highly conserved extracellular region, and have coiled-coil domains that are conserved in structure and position within the cytoplasmic region. However, the STIM proteins diverge significantly within the C-terminal half of the cytoplasmic domain. Differential levels of phosphorylation appear to account for two molecular mass isoforms (105 and 115kDa) of STIM2. We demonstrate by mutation analysis and protein sequencing that human STIM2 initiates translation exclusively from a non-AUG start site in vivo. STIM2 is expressed ubiquitously in cell lines, and co-precipitates with STIM1 from cell lysates. This association into oligomers in vivo indicates a possible functional interaction between STIM1 and STIM2. The structural similarities between STIM1, STIM2 and D-STIM suggest conserved biological functions.
Measurement of proteases using chemiluminescence-resonance-energy-transfer chimaeras between green fluorescent protein and aequorin
Authors: WAUD Jonathan P. , BERMÚDEZ FAJARDO Alexandra , SUDHAHARAN Thankiah , TRIMBY Andrew R. , JEFFERY Jinny , JONES Ann , CAMPBELL Anthony K.
Published: August 2015

Homogeneous assays, without a separation step, are essential for measuring chemical events in live cells and for drug discovery screens, and are desirable for making measurements in cell extracts or clinical samples. Here we demonstrate the principle of chemiluminescence resonance energy transfer (CRET) as a homogeneous assay system, using two proteases as models, one extracellular (α-thrombin) and the other intracellular (caspase-3). Chimaeras were engineered with aequorin as the chemiluminescent energy donor and green fluorescent protein (GFP) or enhanced GFP as the energy acceptors, with a protease linker (6 or 18 amino acid residues) recognition site between the donor and acceptor. Flash chemiluminescent spectra (20–60 s) showed that the spectra of chimaeras matched GFP, being similar to that of luminous jellyfish, justifying their designation as ‘Rainbow’ proteins. Addition of the protease shifted the emission spectrum to that of aequorin in a time- and dose-dependent manner. Separation of the proteolysed fragments showed that the ratio of green to blue light matched the extent of proteolysis. The caspase-3 Rainbow protein was able to provide information on the specificity of caspases in vitro and in vivo. It was also able to monitor caspase-3 activation in cells provoked into apoptosis by staurosporine (1 or 2μM). CRET can also monitor GFP fluor formation. The signal-to-noise ratio of our Rainbow proteins is superior to that of fluorescence resonance energy transfer, providing a potential platform for measuring agents that interact with the reactive site between the donor and acceptor.
Two splice variants of Golgi-microtubule-associated protein of 210 kDa (GMAP-210) differ in their binding to the cis-Golgi network
Authors: RAMOS-MORALES Francisco , VIME Carmen , BORNENS Michel , FEDRIANI Concepción , RIOS Rosa M.
Published: August 2015

GMAP-210 (Golgi-microtubule-associated protein of 210kDa) is a peripheral Golgi protein that interacts with the minus end of microtubules through its C-terminus and with cis-Golgi network membranes through its N-terminus; it participates in the maintenance of the structural integrity of the Golgi apparatus [Infante, Ramos-Morales, Fedriani, Bornens and Rios (1999) J. Cell Biol. 145, 83–98]. We report here the cloning of a new isoform of GMAP-210 that lacks amino acid residues 105–196. On the basis of the analysis of the gmap-210 genomic sequence, we propose that the small isoform, GMAP-200, arises from alternative splicing of exon 4 of the primary transcript. Overexpression of GMAP-200 induces perturbations in both the Golgi apparatus and the microtubule network that are similar to those previously reported for GMAP-210 overexpression. We show that both isoforms are able to oligomerize under overexpression conditions. Analysis in vitro and in vivo, with the green fluorescent protein as a marker, reveals that the binding of the N-terminal domain of GMAP-200 to the cis-Golgi network membranes is lower than that of the N-terminal domain of GMAP-210. Implications for the regulation of interaction between the cis-Golgi network and microtubules are discussed.
Purification, molecular cloning and mechanism of action of graminelysin I, a snake-venom-derived metalloproteinase that induces apoptosis of human endothelial cells
Authors: WU Wen Bin , CHANG Shin C. , LIAU Ming-Yi , HUANG Tur-Fu
Published: August 2015

Apoptosis, a programmed, physiological mode of cell death, is important in tissue homoeostasis. Here we report that a new metalloproteinase, graminelysin I, purified from Trimeresurus gramineus venom, induced apoptosis of human endothelial cells as examined by electrophoresis and flow cytometry. Graminelysin I contains only a metalloproteinase domain. It is a single-chain proteinase with a molecular mass of 27020Da. cDNA sequence analysis revealed that the disintegrin-like and cysteine-rich domains of the putative precursor protein of graminelysin I are likely to be processed post-translationally, producing the proteinase domain (graminelysin I). Graminelysin I cleaved the α chain of fibrinogen preferentially and cleaved the β chain either on longer incubation or at higher concentration. Graminelysin I inhibited the adhesion of human umbilical-vein endothelial cells (HUVECs) to immobilized fibrinogen and induced HUVECs detachment in a dose-dependent manner. These effects on HUVECs were abolished when graminelysin I was pretreated with EDTA. However, graminelysin I did not inhibit the adhesion of HUVECs to immobilized collagen. HUVECs were susceptible to death after treatment with graminelysin I when they were cultured on immobilized fibrinogen. In contrast, HUVECs were rather resistant to treatment with graminelysin I if they were cultured on immobilized collagen. Furthermore, graminelysin I induced apoptosis of HUVECs in a dose-dependent manner. Similarly, its apoptosis-inducing activity was blocked if it was treated with EDTA. These results suggest that the catalytic activity of graminelysin I on matrix proteins contributes to its apoptosis-inducing activity.
Regulation of yeast acetohydroxyacid synthase by valine and ATP
Authors: PANG Siew Siew , DUGGLEBY Ronald G.
Published: August 2015

The first step in the common pathway for the biosynthesis of branched-chain amino acids is catalysed by acetohydroxyacid synthase (AHAS; EC 4.1.3.18). The enzyme is found in plants, fungi and bacteria, and is regulated by controls on transcription and translation, and by allosteric modulation of catalytic activity. It has long been known that the bacterial enzyme is composed of two types of subunit, and a similar arrangement has been found recently for the yeast and plant enzymes. One type of subunit contains the catalytic machinery, whereas the other has a regulatory function. Previously, we have shown [Pang and Duggleby (1999) Biochemistry 38, 5222–5231] that yeast AHAS can be reconstituted from its separately purified subunits. The reconstituted enzyme is inhibited by valine, and ATP reverses this inhibition. In the present work, we further characterize the structure and the regulatory properties of reconstituted yeast AHAS. High phosphate concentrations are required for reconstitution and it is shown that these conditions are necessary for physical association between the catalytic and regulatory subunits. It is demonstrated by CD spectral changes that ATP binds to the regulatory subunit alone, most probably as MgATP. Neither valine nor MgATP causes dissociation of the regulatory subunit from the catalytic subunit. The specificity of valine inhibition and MgATP activation are examined and it is found that the only effective analogue of either regulator of those tested is the non-hydrolysable ATP mimic, adenosine 5′-[β,γ-imido]triphosphate. The kinetics of regulation are studied in detail and it is shown that the activation by MgATP depends on the valine concentration in a complex manner that is consistent with a proposed quantitative model.
Selenium regulation of transcript abundance and translational efficiency of glutathione peroxidase-1 and −4 in rat liver
Authors: WEISS SACHDEV Sherri , SUNDE Roger A.
Published: August 2015

Glutathione peroxidase (GPX)1 mRNA in rat liver falls dramatically during Se deficiency to levels that are approx. 10% of Se-adequate levels. This regulation is mediated by mRNA stability, and is hypothesized to involve nonsense-mediated mRNA decay. mRNA levels for GPX4 and other selenoproteins are much less regulated by Se status. To evaluate the relative contribution of mRNA abundance versus translational efficiency to overall regulation of GPX1 expression, we quantified GPX1, GPX4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts per cell in rat liver. Surprisingly, we found that GPX1 transcripts in Se deficiency are moderately abundant and similar in abundance to GAPDH and other selenoprotein mRNAs; Se supplementation increases GPX1 mRNA so that it is 30-fold higher than GAPDH mRNA. Translational efficiency of GPX1 mRNA is half of that of GPX4. Translational efficiency of GPX1 mRNA increases approx. 20-fold with Se supplementation and appears to switch GPX1 mRNA from nonsense-mediated degradation to translation. This regulatory switch can explain why GPX1 expression is an excellent parameter for assessment of Se status.
Treatment of PC12 cells with nerve growth factor increases iron uptake
Authors: MWANJEWE James , HUI Betsy King , COUGHLIN Michael D. , GROVER Ashok K.
Published: August 2015

Phaeochromocytoma PC12 cells treated with nerve growth factor (NGF) differentiate into a neuronal phenotype. Here we compare the uptake of transferrin-bound and non-transferrin-bound iron in NGF-treated (neuronal phenotype) and control (proliferating) PC12 cells. The non-transferrin-bound iron uptake was greater in the NGF-treated cells than in the control, independently of the uptake time, the iron-chelating agents used, the oxidation state of iron (Fe2+ or Fe3+) and the iron concentration tested. The NGF-treated cells expressed L-type and N-type voltage-operated Ca2+ channels. Nitrendipine (an L-type inhibitor) and possibly ω-conotoxin (an N-type inhibitor) inhibited the iron uptake by 20%. Thapsigargin inhibits the endoplasmic reticulum Ca2+ pump and allowed Mn2+ entry into cells. Preincubating PC12 cells with thapsigargin increased the iron uptake. The rate of transferrin-bound iron uptake was less than 1% of the non-transferrin-bound iron uptake and the maximum transferrin-bound iron uptake was also very low. We conclude that an increase in the iron uptake by multiple pathways accompanies the transition of PC12 cells from the proliferating to the neuronal phenotype.
Sensitivity of respiratory chain activities to lipid peroxidation: effect of vitamin E deficiency
Authors: RAFIQUE Roozina , SCHAPIRA Anthony H. V. , COOPER J. Mark
Published: August 2015

Rats fed a vitamin E-depleted diet for 48 weeks had undetectable levels of vitamin E in the gastrocnemius muscle and liver, leading to elevated malondialdehyde levels in both tissues and an elevated GSH level in muscle. Skeletal-muscle mitochondria showed decreased mitochondrial respiratory chain (MRC) activities, whereas liver MRC activities were increased. Exposure of normal rat liver submitochondrial particles (SMPs) to an in vitro NADPH-dependent lipid peroxidation system resulted in a dose-dependent increase in lipid peroxidation and inhibition of complex I and complex IV activities. Complex I exhibited greater sensitivity to lipid peroxidation than complex IV. At low and high NADPH concentrations, the rate of lipid peroxidation and the level of enzyme inhibition were essentially the same in liver SMPs from both vitamin E-deficient and control rats, suggesting that under these conditions, the loss of vitamin E did not exacerbate the effects of either lipid peroxidation or enzyme inhibition. These results indicate that normal vitamin E levels in liver mitochondria are not required for protection against lipid peroxidation and are consistent with the normal liver mitochondrial function in vitamin E-deficient animals. This suggests other antioxidants, such as ubiquinol and GSH, may be more important in protecting liver mitochondria and MRC from lipid peroxidation.
Ca2+ stabilizes the semiquinone radical of pyrroloquinoline quinone
Authors: SATO Akihiro , TAKAGI Kazuyoshi , KANO Kenji , KATO Nobuo , DUINE Johannis A. , IKEDA Tokuji
Published: August 2015

Spectroelectrochemical studies were performed on the interaction between Ca2+ and pyrroloquinoline quinone (PQQ) in soluble glucose dehydrogenase (sGDH) and in the free state by applying a mediated continuous-flow column electrolytic spectroelectrochemical technique. The enzyme forms used were holo-sGDH (the holo-form of sGDH from Acinetobacter calcoaceticus) and an incompletely reconstituted form of this, holo-X, in which the PQQ-activating Ca2+ is lacking. The spectroelectrochemical and ESR data clearly demonstrated the generation of the semiquinone radical of PQQ in holo-sGDH and in the free state in the presence of Ca2+. In contrast, in the absence of Ca2+ no semiquinone was observed, either for PQQ in the free state (at pH7.0) or in the enzyme (holo-X). Incorporation of Ca2+ into the active site of holo-X, yielding holo-sGDH, caused not only stabilization of the semiquinone form of PQQ but also a negative shift (of 26.5mV) of the two-electron redox potential, indicating that the effect of Ca2+ is stronger on the oxidized than on the reduced PQQ. Combining these data with the observations on the kinetic and chemical mechanisms, it was concluded that the strong stimulating effect of Ca2+ on the activity of sGDH can be attributed to facilitation of certain kinetic steps, and not to improvement of the thermodynamics of substrate oxidation. The consequences of this conclusion are discussed for the oxidative as well as for the reductive part of the reaction of sGDH.
Stimulation of ATP secretion in the liver by therapeutic bile acids
Authors: NATHANSON Michael H. , BURGSTAHLER Angela D. , MASYUK Anatoly , LaRUSSO Nicholas F.
Published: August 2015

ATP receptors are ubiquitously expressed and are potential targets for the therapy of a number of disorders. However, delivery of ATP or other nucleotides to specific tissues is problematic, and no pharmacological means to stimulate the release of endogenous ATP has been described. We examined the effects of the bile acid ursodeoxycholic acid (UDCA) on ATP release into bile, since this bile acid is the only agent known to be of therapeutic benefit in secretory disorders of the liver, and since its mechanism of action is not established. Both UDCA and its taurine conjugate stimulated secretion of ATP by isolated rat hepatocytes, and produced measurable increases in ATP in bile of isolated rat liver. Perfusion of ATP into microdissected bile-duct segments induced Ca2+ signalling in bile-duct epithelia, while perfusion of bile acid did not. Thus UDCA may promote bile flow by inducing hepatocytes to release ATP into bile, which then stimulates fluid and electrolyte secretion by bile-duct epithelia downstream via changes in cytosolic Ca2+. Moreover, these findings demonstrate the feasibility of using pharmacological means to induce secretion of endogenous ATP. Since the liver and other epithelial organs express luminal ATP receptors, these findings more generally suggest that a mechanism exists for pharmacological activation of this paracrine signalling pathway. This strategy may be useful for treatment of cystic fibrosis and other secretory disorders of the liver and other epithelial tissues.
Assembly of protein kinase CK2: investigation of complex formation between catalytic and regulatory subunits using a zinc-finger-deficient mutant of CK2β
Authors: CANTON David A. , ZHANG Cunjie , LITCHFIELD David W.
Published: August 2015

Protein kinase CK2 is a tetrameric enzyme comprised of two regulatory subunits (CK2β) and two catalytic subunits (CK2α and/or CK2α′). The crystal structure of dimeric CK2β demonstrated that a zinc finger mediates CK2β dimerization, therefore we constructed a mutant in which cysteine residues 109 and 114 were mutated to serine. Our objectives were to examine the effects of disrupting the zinc finger of the regulatory CK2β subunit on CK2 tetramer assembly. Examination of this zinc-finger-deficient mutant of CK2β using a yeast two-hybrid assay demonstrates that the mutant fails to form CK2β homodimers. In order to extend these studies, we co-transfected COS-7 cells with epitope-tagged constructs and performed co-immunoprecipitation assays. The results from these studies demonstrate that the mutant fails to form CK2β homodimers and fails to interact with catalytic CK2 subunits. Furthermore, we demonstrate that the mutant CK2β is not appreciably phosphorylated in cells. Using in vitro binding assays, we demonstrated that the mutant CK2β protein fails to interact with glutathione S-transferase–CK2α′. Finally, we demonstrate that the mutant is translated at an equivalent rate to wild-type CK2β, but is degraded much more rapidly. Overall, our results are consistent with the model that β–β dimerization precedes incorporation of catalytic subunits into tetrameric CK2 complexes, and that β–β dimerization is a prerequisite for the stable incorporation of catalytic subunits into CK2 complexes.
Structural and kinetic modifications of aldose reductase by S-nitrosothiols
Authors: SRIVASTAVA Sanjay , DIXIT Bharat L. , RAMANA Kota V. , CHANDRA Animesh , CHANDRA Deepak , ZACARIAS Albert , PETRASH J. Mark , BHATNAGAR Aruni , SRIVASTAVA Satish K.
Published: August 2015

Modification of aldose reductase (AR) by the nitrosothiolsS-nitroso-N-acetyl penicillamine (SNAP) and N-(β-glucopyranosyl)-N2-acetyl-S-nitrosopenicillamide (glyco-SNAP) resulted in a 3–7-fold increase in its kcat and a 25–40-fold increase in its Km for glyceraldehyde. In comparison with the native protein, the modified enzyme was less sensitive to inhibition by sorbinil and was not activated by SO2−4 anions. The active-site residue, Cys-298, was identified as the main site of modification, because the site-directed mutant in which Cys-298 was replaced by serine was insensitive to glyco-SNAP. The extent of modification was not affected by Pi or O2, indicating that it was not due to spontaneous release of nitric oxide (NO) by the nitrosothiols. Electrospray ionization MS revealed that the modification reaction proceeds via the formation of an N-hydroxysulphenamide-like adduct between glyco-SNAP and AR. In time, the adduct dissociates into either nitrosated AR (AR-NO) or a mixed disulphide between AR and glyco-N-acetylpenicillamine (AR-S-S-X). Removal of the mixed-disulphide form of the protein by lectin-column chromatography enriched the preparation in the high-Km–high-kcat form of the enzyme, suggesting that the kinetic changes are due to the formation of AR-NO, and that the AR-S-S-X form of the enzyme is catalytically inactive. Modification of AR by the non-thiol NO donor diethylamine NONOate (DEANO) increased enzyme activity and resulted in the formation of AR-NO. However, no adducts between AR and DEANO were formed. These results show that nitrosothiols cause multiple structural and functional changes in AR. Our observations also suggest the general possibility that transnitrosation reactions can generate both nitrosated and thiolated products, leading to non-unique changes in protein structure and function.
Kinetics of CO binding to the haem domain of murine inducible nitric oxide synthase: differential effects of haem domain ligands
Authors: STEVENSON Thirza H. , GUTIERREZ Aldo F. , ALDERTON Wendy K. , LIAN Lu-yun , SCRUTTON Nigel S.
Published: August 2015

The binding of CO to the murine inducible nitric oxide synthase (iNOS) oxygenase domain has been studied by laser flash photolysis. The effect of the (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4) cofactor l-arginine and several Type I l-arginine analogues/ligands on the rates of CO rebinding has been evaluated. The presence of BH4 in the iNOS active site has little effect on the rebinding of protein-caged haem–CO pairs (geminate recombination), but decreases the bimolecular association rates 2-fold. Addition of l-arginine to the BH4-bound complex completely abolishes geminate recombination and results in a further 80-fold decrease in the overall rate of bimolecular association. Three of the Type I ligands, S-ethylisothiourea, l-canavanine and 2,5-lutidine, displaced the CO from the haem iron upon addition to the iNOS oxygenase domain. The Type I ligands significantly decreased the rate of bimolecular binding of CO to the haem iron after photolysis. Most of these ligands also completely abolished geminate recombination. These results are consistent with a relatively open distal pocket that allows CO to bind unhindered in the active site of murine iNOS in the absence of l-arginine or BH4. In the presence of BH4 and l-arginine, however, the enzyme adopts a more closed structure that can greatly reduce ligand access to the haem iron. These observations are discussed in terms of the known structure of iNOS haem domain and solution studies of ligand binding in iNOS and neuronal NOS isoenzymes.
Endoplasmic reticulum Ca2+-ATPase pump is up-regulated in calcium-transporting dental enamel cells: a non-housekeeping role for SERCA2b
Authors: FRANKLIN Isobel K. , WINZ Robert A. , HUBBARD Michael J.
Published: August 2015

Dental enamel-forming cells face a major challenge to avoid the cytotoxic effects of excess calcium. We have characterized sarcoplasmic/endoplasmic reticulum calcium-ATPase pumps (SERCA) in rat enamel cells to address the proposal that non-mitochondrial calcium stores play a dominant role in transcellular calcium transport. A single major isoform, SERCA2b, was detected during the protein-secretory and calcium-transport stages of enamel formation using reverse-transcriptase PCR, cDNA cloning, Northern analysis and immunoblotting. Most importantly, SERCA2b exhibited a specific 3-fold up-regulation to high expression levels during calcium transport, as determined by quantitative immunoblotting and ATPase assays. Sensitivity of the calcium-dependent ATPase to thapsigargin and three other SERCA inhibitors was characterized. These findings indicate that enamel cells are well-equipped to sequester calcium in endoplasmic reticulum stores and so protect against calcium toxicity, associate SERCA with transcellular calcium transport for the first time, and establish SERCA2b as a molecular and pharmacological target for future investigations of calcium transcytosis. The observed physiological regulation in enamel cells contradicts the widespread perception that SERCA2b is restricted to general housekeeping duties.
Semicarbazide-sensitive amine oxidase activation promotes adipose conversion of 3T3-L1 cells
Authors: MERCIER Nathalie , MOLDES Marthe , EL HADRI Khadija , FÈVE Bruno
Published: August 2015

Semicarbazide-sensitive amine oxidase (SSAO) is an amine oxidase related to the copper-containing amine oxidase family. The tissular form of SSAO is located at the plasma membrane, and is mainly expressed in vascular smooth muscle cells and adipocytes. Recent studies have suggested that SSAO could activate glucose transport in fat cells. In the present work, we investigated the potential role of a chronic SSAO activation on adipocyte maturation of the 3T3-L1 pre-adipose cell line. Exposure of post-confluent 3T3-L1 pre-adipocytes to methylamine, a physiological substrate of SSAO, promoted adipocyte differentiation in a time- and dose-dependent manner. This effect could be related to SSAO activation, since it was antagonized in the presence of the SSAO inhibitor semicarbazide, but not in the presence of the monoamine oxidase inhibitor pargyline. In addition, methylamine-induced adipocyte maturation was mimicked by 3T3-L1 cell treatment with other SSAO substrates. Finally, the large reversion of methylamine action by catalase indicated that hydrogen peroxide generated by SSAO was involved, at least in part, in the modulation of adipocyte maturation. Taken together, our results suggest that SSAO may contribute to the control of adipose tissue development.
Cell-cycle-dependent association of protein phosphatase 1 and focal adhesion kinase
Authors: FRESU Marco , BIANCHI Mariarita , PARSONS J. Thomas , VILLA-MORUZZI Emma
Published: August 2015

Immunofluorescence studies with protein phosphatase-1 (PP1) isoforms-specific antibodies detected PP1δ, but not α or γ1, at focal adhesions. PP1δ also co-immunoprecipitated with the focal adhesion kinase (FAK) and the αv-integrin. In the present study glutathione S-transferase (GST)–PP1δ pulled-down FAK from fibroblasts extract and the interaction domain localized between residues 159 and 295 of δ. The association was confirmed by the ability to GST–FAK-related non-kinase (FRNK) to pull-down PP1δ from fibroblasts extract. GST–FRNK also pulled-down purified muscle PP1 catalytic subunit, thus indicating direct interaction between FAK and PP1. FAK displays consensus sequences for phosphorylation by cell division cycle kinase-2–cyclin B, and might be a PP1 substrate. In fact, FAK immunoprecipitated from metabolically-labelled mitotic HeLa cells without tyrosine phosphatase inhibitors was phosphorylated on Ser only and was dephosphorylated in vitro by purified muscle PP1, with loss of phospho-Ser. No PP1 was associated with FAK immunoprecipitated from mitotic HeLa cells. However, progressively more PP1 activity was assayed in FAK-immunoprecipitates obtained from cells released from mitosis. The associated activity was maximal at 2h from the mitotic release (when 85–90% of the cells remained round) and decreased to basal level by 8h (when cells were all polygonal). At the same time FAK underwent dephosphorylation, which was completed by 4h. FAK obtained from cells at 1.5h was Ser-phosphorylated, and underwent dephosphorylation during in vitro incubation, with loss of phospho-Ser, indicating the presence of active FAK-bound phosphatase. The only FAK-associated PP1 isoform between 1 and 8h was PP1δ. The results suggest that FAK dephosphorylation by PP1δ occurs in cells released from mitosis, and confirmed the specific association of PP1δ, as detected previously in adherent cells.
Analysis of sucrose accumulation in the sugar cane culm on the basis of in vitro kinetic data
Authors: ROHWER Johann M. , BOTHA Frederik C.
Published: August 2015

Sucrose accumulation in developing sugar cane (Saccharum officinarum) is accompanied by a continuous synthesis and cleavage of sucrose in the storage tissues. Despite numerous studies, the factors affecting sucrose accumulation are still poorly understood, and no consistent pattern has emerged which pinpoints certain enzyme activities as important controlling steps. Here, we develop an approach based on pathway analysis and kinetic modelling to assess the biochemical control of sucrose accumulation and futile cycling in sugar cane. By using the concept of elementary flux modes, all possible routes of futile cycling of sucrose were enumerated in the metabolic system. The available kinetic data for the pathway enzymes were then collected and assembled in a kinetic model of sucrose accumulation in sugar cane culm tissue. Although no data were fitted, the model agreed well with independent experimental results: in no case was the difference between calculated and measured fluxes and concentrations greater than 2-fold. The model thus validated was then used to assess different enhancement strategies for increasing sucrose accumulation. First, the control coefficient of each enzyme in the system on futile cycling of sucrose was calculated. Secondly, the activities of those enzymes with the numerically largest control coefficients were varied over a 5-fold range to determine the effect on the degree of futile cycling, the conversion efficiency from hexoses into sucrose, and the net sucrose accumulation rate. In view of the modelling results, overexpression of the fructose or glucose transporter or the vacuolar sucrose import protein, as well as reduction of cytosolic neutral invertase levels, appear to be the most promising targets for genetic manipulation. This offers a more directed improvement strategy than cumbersome gene-by-gene manipulation. The kinetic model can be viewed and interrogated on the World Wide Web at http://jjj.biochem.sun.ac.za.
Biochemical characterization of the β-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis A.T.C.C. 31461
Authors: VIDEIRA Paula , FIALHO Arsénio , GEREMIA Roberto A. , BRETON Christelle , SÁ-CORREIA Isabel
Published: August 2015

Biosynthesis of bacterial polysaccharide-repeat units proceeds by sequential transfer of sugars, from the appropriate sugar donor to an activated lipid carrier, by committed glycosyltransferases (GTs). Few studies on the mechanism of action for this type of GT are available. Sphingomonas paucimobilis A.T.C.C. 31461 produces the industrially important polysaccharide gellan gum. We have cloned the gelK gene from S. paucimobilis A.T.C.C. 31461. GelK belongs to family 1 of the GT classification [Campbell, Davies, Bulone, Henrissat (1997) Biochem. J. 326, 929–939]. Sequence similarity studies suggest that GelK consists of two protein modules corresponding to the -NH2 and -CO2H halves, the latter possibly harbouring the GT activity. The gelK gene and the open reading frames coding for the -NH2 (GelKNH2) and -CO2H (GelKCOOH) halves were overexpressed in Escherichia coli. GelK and GelKNH2 were present in both the soluble and membrane fractions of E. coli, whereas GelKCOOH was only present in the soluble fraction. GelK catalysed the transfer of [14C]glucuronic acid from UDP-[14C]glucuronic acid into a glycolipid extracted from S. paucimobilis or E. coli, even in the presence of EDTA, and the radioactive sugar was released from the glycolipid by β-1,4-glucuronidase. GelK was not able to use synthetic glucosyl derivatives as acceptors, indicating that the PPi-lipid moiety is needed for enzymic activity. Recombinant GelKNH2 and GelKCOOH did not show detectable activity. Based on the biochemical characteristics of GelK and on sequence similarities with N-acetylglucosaminyltransferase, we propose that GT families 1 and 28 form a superfamily.
Phosphoinositide 3-kinase accelerates necrotic cell death during hypoxia
Authors: AKI Toshihiko , MIZUKAMI Yoichi , OKA Yoshitomo , YAMAGUCHI Kazuhito , UEMURA Koichi , FUJIMIYA Tatsuya , YOSHIDA Ken-ichi
Published: August 2015

Using H9c2 cells derived from rat cardiomyocytes, we investigated the mechanism of cell death during hypoxia in the presence of serum and glucose. Hypoxic cell death is by necrosis and is accompanied by metabolic acidosis. Moreover, hypoxic cell death is inhibited by Hepes buffer as well as by 2-deoxyglucose, an inhibitor of glycolysis, indicating that metabolic acidosis should play an essential role in hypoxic injury. The involvement of phosphoinositide 3-kinase (PI 3-kinase), which is known to activate glucose metabolism, was examined using its inhibitor, LY290042, or adenovirus-mediated gene transfer. Hypoxic cell death was inhibited by LY294002 in a dose-dependent manner. Overexpression of dominant negative PI 3-kinase was found to reduce cell death, whereas wild-type PI 3-kinase enhanced it. Dominant negative PI 3-kinase also reduced glucose consumption and acidosis, but this was stimulated by wild-type PI 3-kinase. The data indicate that PI 3-kinase stimulates cell death by enhancing metabolic acidosis. LY294002 significantly reduced glucose uptake, showing that PI 3-kinase regulates glycolysis at the step of glucose transport. These findings indicate the pivotal role of glucose metabolism in hypoxic cell death, and reveal a novel death-promoting effect of PI 3-kinase during hypoxia, despite this enzyme being considered to be a survival-promoting factor.
Kinetic properties of the acylneuraminate cytidylyltransferase from Pasteurella haemolytica A2
Authors: BRAVO Ignacio G. , BARRALLO Sofía , FERRERO Miguel A. , RODRÍGUEZ-APARICIO Leandro B. , MARTÍNEZ-BLANCO Honorina , REGLERO Ángel
Published: August 2015

Neuroinvasive and septicaemia-causing pathogens often display a polysialic acid capsule that is involved in invasive behaviour. N-Acetylneuraminic acid (NeuAc) is the basic monomer of polysialic acid. The activated form, CMP-Neu5Ac, is synthesized by the acylneuraminate cytidylyltransferase (ACT; EC 2.7.7.43). We have purified this enzyme from Pasteurella haemolytica A2 to apparent homogeneity (522-fold). The protein behaved homogeneously on SDS/PAGE as a 43kDa band, a size similar to that of Escherichia coli, calf, mouse and rat. Specific activity in crude lysate displayed one of the highest values cited in the literature (153m-units/mg). We have studied the steady-state kinetic mechanism of the enzyme by using normalized plot premises. The catalysis proceeds through a Ping Pong Bi Bi mechanism, with CTP as the first substrate and CMP-NeuAc as the last product. The true Km values were 1.77mM for CTP and 1.82mM for NeuAc. The nucleotides CDP, UTP, UDP and TTP, and the modified sialic acid N-glycolylneuraminic acid were also substrates of the ACT activity. The enzyme is inhibited by cytidine nucleotides through binding to a second cytidyl-binding site. This inhibition is greater with nucleotides that display a long phosphate tail, and the genuine inhibitor is the substrate CTP. At physiological concentrations, ATP is an activator, and AMP an inhibitor, of the ACT activity. The activated sugar UDP-N-acetylglucosamine acts as an inhibitor, thus suggesting cross-regulation of the peptidoglycan and polysialic acid pathways. Our findings provide new mechanistic insights into the nature of sialic acid activation and suggest new targets for the approach to the pathogenesis of encapsulated bacteria.
Pulmonary lipid phosphate phosphohydrolase in plasma membrane signalling platforms
Authors: NANJUNDAN Meera , POSSMAYER Fred
Published: August 2015

Lipid phosphate phosphohydrolase (LPP) has recently been proposed to have roles in signal transduction, acting sequentially to phospholipase D (PLD) and in attenuating the effects of phospholipid growth factors on cellular proliferation. In this study, LPP activity is reported to be enriched in lipid-rich signalling platforms isolated from rat lung tissue, isolated rat type II cells and type II cell-mouse lung epithelial cell lines (MLE12 and MLE15). Lung and cell line caveolin-enriched domains (CEDs), prepared on the basis of their detergent-insolubility in Triton X-100, contain caveolin-1 and protein kinase C isoforms. The LPP3 isoform was predominantly localized to rat lung CEDs. These lipid-rich domains, including those from isolated rat type II cells, were enriched both in phosphatidylcholine plus sphingomyelin (PC+SM) and cholesterol. Saponin treatment of MLE15 cells shifted the LPP activity, cholesterol, PC+SM and caveolin-1 from lipid microdomains to detergent-soluble fractions. Elevated LPP activity and LPP1/1a protein are present in caveolae from MLE15 cells prepared using the cationic-colloidal-silica method. In contrast, total plasma membranes had a higher abundance of LPP1/1a protein with low LPP activity. Phorbol ester treatment caused a 3.8-fold increase in LPP specific activity in MLE12 CEDs. Thus the activated form of LPP1/1a may be recruited into caveolae/rafts. Transdifferentiation of type II cells into a type I-like cell demonstrated enrichment in caveolin-1 levels and LPP activity. These results indicate that LPP is localized in caveolae and/or rafts in lung tissue, isolated type II cells and type II cell lines and is consistent with a role for LPP in both caveolae/raft signalling and caveolar dynamics.
Cloning and characterization of the pseudonajatoxin b precursor
Authors: GONG NanLing , ARMUGAM Arunmozhiarasi , MIRTSCHIN Peter , JEYASEELAN Kandiah
Published: August 2015

An Australian common brown snake, Pseudonaja textilis, is known to contain highly lethal neurotoxins. Among them, a long-chain α-neurotoxin, pseudonajatoxin b, has been identified. In this report, while presenting evidence for the presence of at least four such long-chain α-neurotoxins in the venom of P. textilis, we describe the characteristics of both the mRNA and the gene responsible for the synthesis of these neurotoxins. A precursor toxin synthesized from the gene has been identified as being capable of producing the isoforms possibly by post-translational modifications at its C-terminal end. Recombinant toxins corresponding to the precursor and its product have been found to possess similar binding affinities for muscular acetylcholine receptors (IC50 = 3×10−8 M) and a lethality, LD50, of 0.15μg/g in mice.
Crystal structure of the wild-type and D30A mutant thioredoxin h of Chlamydomonas reinhardtii and implications for the catalytic mechanism
Authors: MENCHISE Valeria , CORBIER Catherine , DIDIERJEAN Claude , SAVIANO Michele , BENEDETTI Ettore , JACQUOT Jean-Pierre , AUBRY André
Published: August 2015

Thioredoxins are ubiquitous proteins which catalyse the reduction of disulphide bridges on target proteins. The catalytic mechanism proceeds via a mixed disulphide intermediate whose breakdown should be enhanced by the involvement of a conserved buried residue, Asp-30, as a base catalyst towards residue Cys-39. We report here the crystal structure of wild-type and D30A mutant thioredoxin h from Chlamydomonas reinhardtii, which constitutes the first crystal structure of a cytosolic thioredoxin isolated from a eukaryotic plant organism. The role of residue Asp-30 in catalysis has been revisited since the distance between the carboxylate OD1 of Asp-30 and the sulphur SG of Cys-39 is too great to support the hypothesis of direct proton transfer. A careful analysis of all available crystal structures reveals that the relative positioning of residues Asp-30 and Cys-39 as well as hydrophobic contacts in the vicinity of residue Asp-30 do not allow a conformational change sufficient to bring the two residues close enough for a direct proton transfer. This suggests that protonation/deprotonation of Cys-39 should be mediated by a water molecule. Molecular-dynamics simulations, carried out either in vacuo or in water, as well as proton-inventory experiments, support this hypothesis. The results are discussed with respect to biochemical and structural data.
Tumour necrosis factor-α activation of protein kinase B in WEHI-164 cells is accompanied by increased phosphorylation of Ser473, but not Thr308
Authors: O'TOOLE Alison , MOULE S. Kelly , LOCKYER Peter J. , HALESTRAP Andrew P.
Published: August 2015

Tumour necrosis factor-α (TNF-α) may activate both cell survival and cell death pathways. In the murine fibrosarcoma cell line WEHI-164, physiological concentrations (1ng/ml) of TNF-α induced wortmannin-sensitive cell ruffling characteristic of the phosphoinositide 3-kinase (PI3-kinase) activation associated with cell survival. Wortmannin also enhanced cell death induced by TNF-α in the presence of actinomycin D, confirming that TNF-α activates a transcription-independent survival pathway requiring PI3-kinase activity. Both TNF-α and insulin-like growth factor 1 (IGF-1) caused a 6–10-fold wortmannin-sensitive increase in protein kinase B (PKB) activity within 5min. For IGF-1, this was associated with an increase in phosphorylation of both Thr308 and Ser473, whereas for TNF-α only phosphorylation of Ser473 was increased, even in the presence of okadaic acid to inhibit protein phosphatases 1 and 2A. TNF-α did not decrease the phosphorylation of Thr308 induced by IGF-1, implying that TNF-α neither inhibits phosphoinositide-dependent kinase 1 (PDK1) nor activates an opposing phosphatase. In WEHI cells overexpressing a form of PKB, IGF-1 increased phosphorylation of Ser473 on PKB, but not its kinase activity, whereas TNF-α failed to induce Ser473 phosphorylation or kinase activation of either overexpressed T308A or wild-type PKB (where T308A is the mutant bearing the substitution Thr308 → A). IGF-1 caused translocation of green-fluorescent-protein-tagged ADP-ribosylation factor nucleotide-binding site opener (ARNO) to the plasma membrane of WEHI cells, but this was not detected with TNF-α. We conclude that, at physiological concentrations, TNF-α activates endogenous PKB by stimulating PDK2 (increase in Ser473 phosphorylation) in a PI3-kinase-dependent (wortmannin-sensitive) manner, without causing detectable stimulation of PDK1 (no increase in Thr308 phosphorylation) or ARNO translocation. Possible explanations of these observations are discussed.
Nitric oxide inhibits mitochondrial NADH:ubiquinone reductase activity through peroxynitrite formation
Authors: RIOBÓ Natalia A. , CLEMENTI Emilio , MELANI Mariana , BOVERIS Alberto , CADENAS Enrique , MONCADA Salvador , PODEROSO Juan J.
Published: August 2015

This study was aimed at assessing the effects of long-term exposure to NO of respiratory activities in mitochondria from different tissues (with different ubiquinol contents), under conditions that either promote or prevent the formation of peroxynitrite. Mitochondria and submitochondrial particles isolated from rat heart, liver and brain were exposed either to a steady-state concentration or to a bolus addition of NO. NO induced the mitochondrial production of superoxide anions, hydrogen peroxide and peroxynitrite, the latter shown by nitration of mitochondrial proteins. Long-term incubation of mitochondrial membranes with NO resulted in a persistent inhibition of NADH:cytochrome c reductase activity, interpreted as inhibition of NADH:ubiquinone reductase (Complex I) activity, whereas succinate:cytochrome c reductase activity, including Complex II and Complex III electron transfer, remained unaffected. This selective effect of NO and derived species was partially prevented by superoxide dismutase and uric acid. In addition, peroxynitrite mimicked the effect of NO, including tyrosine nitration of some Complex I proteins. These results seem to indicate that the inhibition of NADH:ubiquinone reductase (Complex I) activity depends on the NO-induced generation of superoxide radical and peroxynitrite and that Complex I is selectively sensitive to peroxynitrite. Inhibition of Complex I activity by peroxynitrite may have critical implications for energy supply in tissues such as the brain, whose mitochondrial function depends largely on the channelling of reducing equivalents through Complex I.