bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional co-operation between the subunits in heterodimeric platelet-derived growth factor receptor complexes
Authors: EMADUDDIN Muhammad , EKMAN Simon , RÖNNSTRAND Lars , HELDIN Carl-Henrik
Published: August 2015

To determine the importance of the phosphorylation capacity of receptor kinase as well as the ability to serve as docking sites for SH2-domain-containing signal transduction molecules, we established pig aortic endothelial cell lines stably expressing kinase-active platelet-derived growth factor (PDGF) α-receptors together with kinase-inactive β-receptors, and vice versa. After stimulation with PDGF-AB, heterodimeric receptor complexes were formed in which the kinase-inactive receptor was phosphorylated by the kinase-active receptor, although less efficiently than in heterodimers of wild-type receptors. The kinase-active receptor was only minimally phosphorylated. Thus the phosphorylation within the receptor dimer occurred in trans between the components. Analyses of the abilities of heterodimeric receptor complexes of one kinase-active and one kinase-inactive receptor to mediate mitogenicity, chemotaxis and activation of mitogen-activated protein kinase revealed less efficient effects than those of heterodimers of wild-type receptors. Importantly, however, the fact that signalling capacities were retained illustrates a functional co-operation between the two receptor molecules in the dimer, where one receptor provides a functional kinase and the other acts as a substrate and provides docking sites for downstream signalling molecules.
The cartilage-specific (V+C)− fibronectin isoform exists primarily in homodimeric and monomeric configurations
Authors: BURTON-WURSTER Nancy , GENDELMAN Rina , CHEN Hao , GU Da-Nian , TETREAULT Jonathan W. , LUST George , SCHWARZBAUER Jean E. , MACLEOD James N.
Published: August 2015

Fibronectin is an extracellular-matrix glycoprotein encoded by a single gene, but with significant protein heterogeneity introduced through alternative RNA splicing and post-translational modifications. The (V+C)- splice variant, in which nucleotides encoding protein segments III-15 and I-10 are deleted along with the entire variable region, is unique in that expression is restricted to cartilaginous tissues. All known fibronectin splice variants retain the two C-terminal cysteine residues essential for dimerization, but cellular and/or structural constraints appear to influence homo- and heterodimerization patterns. Dimerization patterns of the (V+C)- isoform were studied under native conditions within canine articular cartilage and experimentally in COS-7, NIH-3T3 and CHO-K1 cell cultures. In all systems, (V+C)- fibronectin secretion was predominantly in a homodimeric configuration. Lower levels of (V+C)- monomers were also present. Heterodimers of (V+C)- with V+,C+ (V120) isoforms were not detected. Heterodimers of (V+C)- with V-,C+ (V0) subunits were detected only at low levels. Functional properties may differ significantly among monomers, homodimers and heterodimers. The unique dimerization pattern of (V+C)- fibronectin is consistent with this isoform having specialized functional properties in situ that are important for either the structural organization and biomechanical properties of cartilage matrix or regulation of a chondrocytic phenotype.
Two moles of O2 consumption and one mole of H2O2 formation during cholesterol peroxidation with cholesterol oxidase from Pseudomonas sp. strain ST-200
Authors: DOUKYU Noriyuki , AONO Rikizo
Published: August 2015

Cholesterol oxidase from Pseudomonas sp. strain ST-200 oxidized cholesterol and cholestanol to 6β-hydroperoxycholest-4-en-3-one and 5α-cholestan-3-one respectively. The former was converted spontaneously to several oxysteroids such as 6-hydroxycholest-4-en-3-one and cholest-4-ene-3,6-dione, with the consumption of 2 mol of O2 and the formation of 1 mol of H2O2 for each mole of cholesterol oxidized. An oxidized form of the cholesterol oxidase dehydrogenates cholesterol, probably to the 5-en-3-one derivative. A reduced form of the enzyme, yielded from the cholesterol dehydrogenation reaction, dioxygenated cholest-5-en-3-one to 6β-hydroperoxycholest-4-en-3-one.
Specific inhibition of skeletal α-actin gene transcription by applied mechanical forces through integrins and actin
Authors: LEW April M. , GLOGAUER Michael , MCCULLOCH Christopher A. G.
Published: August 2015

Skeletal α-actin (skA), a prominent fetal actin isoform that is re-expressed by adult cardiac myocytes after chronic overload in vivo, provides a model for studying cytoskeletal gene regulation by mechanical forces in vitro. We have determined the mechanisms by which perpendicular applied forces acting through integrins and the actin cytoskeleton regulate the expression of skA. Rat-2 fibroblasts were transiently transfected with plasmids containing 5′-regulatory regions of the skA gene fused to luciferase coding sequences. A constant, perpendicular force (0.2 pN/μm2) was applied by using a collagen-magnetic bead model; a 25% deformation was obtained on the dorsal cell surface. In this system, force is applied through focal adhesion integrins and strongly induces actin assembly [Glogauer, Arora, Yao, Sokholov, Ferrier and McCulloch (1997) J. Cell Sci. 110, 11-21]. skA promoter activity was inhibited by 68% in cells subjected to 4 h of applied force, whereas Rous sarcoma virus promoter activity was unaffected. In cells transiently transfected with a skA expression vector there was also a parallel 40% decrease in skA protein levels by force, as shown by Western blotting. In L8 cells, constitutive skA expression was decreased by more than 50%. Analyses of specific motifs in the skA promoter revealed that transcriptional enhancer factor 1 and Yin and Yang 1 sites, but not serum response factor and Sp1 sites, mediated inhibitory responses to force. In cells treated with cycloheximide the force-induced inhibition was abrogated, indicating a dependence on new protein synthesis. Inhibition of actin filament assembly with either cytochalasin D or Ca2+-depleted medium blocked the inhibitory effect induced by the applied force, suggesting that actin filaments are required for the regulation of skA promoter activity. Western blot analysis showed that p38 kinase, but not Jun N-terminal kinase or extracellular signal-regulated protein kinase 1/2, was activated by force; indeed, the p38 kinase inhibitor SB203580 relieved the force-induced inhibition of skA. We conclude that the force-induced inhibition of skA promoter activity requires an intact actin cytoskeleton and can be mapped to two different response elements. This inhibition might be mediated through the p38 kinase.
The final step of pantothenate biosynthesis in higher plants: cloning and characterization of pantothenate synthetase from Lotus japonicus and Oryza sativum (rice)
Authors: GENSCHEL Ulrich , POWELL Corinna A. , ABELL Chris , SMITH Alison G.
Published: August 2015

We have isolated a Lotus japonicus cDNA for pantothenate (vitamin B5) synthetase (PS) by functional complementation of an Escherichia coli panCmutant (AT1371). A rice (Oryza sativum) expressed sequence tag, identified by sequence similarity to PS, was also able to complement the E. coli auxotroph, as was an open reading frame from Saccharomyces cerevisiae(baker's yeast). The Lotus and rice cDNAs encode proteins of approx. 34 kDa, which are 65% similar at the amino acid level and do not appear to encode N-terminal extensions by comparison with PS sequences from other organisms. Furthermore, analysis of genomic sequence flanking the coding sequence for PS in Lotus suggests the original cDNA is full-length. The Lotus and rice PSs are therefore likely to be cytosolic. Southern analysis of Lotus genomic DNA indicates that there is a single gene for PS. Recombinant PS from Lotus, overexpressed in E. coli AT1371, is a dimer. The enzyme requires D-pantoate, β-alanine and ATP for activity and has a higher affinity for pantoate (Km 45 μM) than for β-alanine (Km 990 μM). Uncompetitive substrate inhibition becomes significant at pantoate concentrations above 1 mM. The enzyme displays optimal activity at about 0.5 mM pantoate (kcat 0.63 s-1) and at pH 7.8. Neither oxopantoate nor pantoyl-lactone can replace pantoate as substrate. Antibodies raised against recombinant PS detected a band of 34 kDa in Western blots of Lotus proteins from both roots and leaves. The implications of these findings for pantothenate biosynthesis in plants are discussed.
Extracellular signal-regulated protein kinase (ERK)-dependent and ERK-independent pathways target STAT3 on serine-727 in human neutrophils stimulated by chemotactic factors and cytokines
Authors: KUROKI Mitsuyuki , O'FLAHERTY Joseph T.
Published: August 2015

STAT3 (signal transducer and activator of transcription 3) is a latent transcription factor that is activated by tyrosine phosphorylation (Tyr-705) in cells stimulated with cytokines or growth factors. Recent studies suggest that one or more cytoplasmic serine kinases also phosphorylate STAT3 and are necessary for maximal gene activation. Here we demonstrate, with a site-specific antibody, that STAT3 is phosphorylated on Ser-727 in human neutrophils stimulated with chemotactic factors (N-formyl-methionyl-leucyl-phenylalanine and complement C5a), cytokines [granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF)], or a protein kinase C activator (PMA). (2-Amino-3′-methoxyphenyl)oxanaphthalen-4-one (PD 98059), an inhibitor of extracellular signal-regulated protein kinase (ERK) activation, blocked the serine phosphorylation of STAT3 induced by chemotactic factors or PMA. The drug was less effective on cytokines: it virtually abolished the response to GM-CSF that occurred 5 min after stimulation but only partly decreased those at 15-30 min and did not appreciably alter responses to G-CSF regardless of incubation time. 1-(5-Isoquinolinylsulphonyl)-2-methylpiperazine dihydrochloride (H7), an inhibitor of a putative STAT3 serine kinase, and 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl) 1H-imidazole (SB 203580), an inhibitor of p38 mitogen-activated protein (MAP) kinase, did not dampen any of these serine phosphorylation responses. We propose that neutrophils use both ERK-dependent and ERK-independent pathways to phosphorylate Ser-727 on STAT3. The former pathway is recruited by all ERK-activating stimuli, whereas the latter pathway uses an undefined serine kinase and is recruited selectively by cytokines.
The purified and reconstituted ornithine/citrulline carrier from rat liver mitochondria catalyses a second transport mode: ornithine+/H+ exchange
Authors: INDIVERI Cesare , TONAZZI Annamaria , STIPANI Italo , PALMIERI Ferdinando
Published: August 2015

The mechanism of unidirectional transport of ornithine (i.e. in the absence of a counter-metabolite) has been investigated in proteoliposomes reconstituted with the ornithine carrier purified from rat liver mitochondria. The efflux of [3H]ornithine from proteoliposomes was stimulated by the addition of H+ (but not of other cations) to the incubation medium . On keeping the pH in the compartment containing ornithine constant at 8.0, the flux of ornithine into or out of the proteoliposomes increased on decreasing the pH in the opposite compartment from 8.0 to 6.0. Ornithine influx was also stimulated when a higher H+ concentration was generated inside the vesicles relative to the outside by the K+/H+ exchanger nigericin in the presence of an outwardly directed K+ gradient. A valinomycin-induced electrogenic flux of K+ did not affect ornithine transport in the absence of a counter-metabolite. Furthermore, changes in fluorescence of the pH indicator pyranine, included inside the proteoliposomes, showed that the flux of ornithine is accompanied by translocation of H+ in the opposite direction. It is concluded that the mitochondrial ornithine carrier catalyses an electroneutral exchange of ornithine+ for H+, in addition to the well-known 1:1 exchange of metabolites. Lysine+, but not citrulline, can also be exchanged for H+ by the ornithine carrier. The ornithine+/H+ transport mode of the exchanger is an essential step in the catabolism of excess arginine.
Fibrinogen up-regulates the expression of monocyte chemoattractant protein 1 in human saphenous vein endothelial cells
Authors: HARLEY Suzanne L. , POWELL Janet T.
Published: August 2015

High concentrations of fibrinogen in plasma have been associated with an increased risk of saphenous vein graft pathology. We have investigated the ability of fibrinogen to up-regulate the expression of monocyte chemoattractant protein 1 (MCP-1) in cultured human saphenous vein endothelial cells (HSVEC) isolated from saphenous vein. Increasing concentrations of fibrinogen (0-4 μM) stimulated a 20-fold increase in MCP-1 secretion within 4 h. Incubation of HSVEC with 2 μM fibrinogen for 4 h also caused a 2-fold increase in the MCP-1-to-glyceraldehyde-3-phosphate dehydrogenase mRNA ratio. The fibrinogen-mediated MCP-1 secretion fell to basal levels after preincubation of HSVEC with the complex of fibrinogen fragments D and E but remained unchanged after preincubation of HSVEC with either fibrinogen fragment E, s-ICAM-1 or the pentapeptide GRGDV. In contrast, fibrinogen fragment D acted as a potent inhibitor of fibrinogen-mediated MCP-1 secretion. Labelled fibrinogen fragment D bound to HSVEC with a Kd of 6.5 μM. These findings indicate that fibrinogen, at physiological concentrations, uses an epitope on the fibrinogen D domain to bind to a receptor on HSVEC to up-regulate MCP-1 expression and secretion. This receptor seems to be distinct from intercellular adhesion molecule 1 and the integrins previously recognized as fibrinogen receptors.
Novel cell lines for the analysis of preprotachykinin A gene expression identify a repressor domain 3′ of the major transcriptional start site
Authors: FISKERSTRAND Carolyn E. , NEWEY Paul , EBRAHIMI Bahram , GERRARD Lesley , HARRISON Patrick , MCGREGOR Gerard P. , QUINN John P.
Published: August 2015

Until now, no clonal cells have been identified that support the expression of a marker gene expressed from the rat preprotachykinin A (rPPT) promoter. We have analysed recently available cell lines that are candidates for supporting reporter gene expression directed by the rPPT promoter. These are the neuronal-derived cell line NF2C and the pancreatic cell lines RINm5F and a derivative RIN-1027-B2. The NF2C line was derived from the brain homogenate of a transgenic animal in which a temperature-sensitive simian virus 40 large T antigen was expressed from a neurofilament promoter. All three lines are able to support expression of a reporter gene directed by a fragment of the 5′ rPPT promoter. Analysis of reporter gene expression supported by various fragments of the rPPT promoter demonstrated that although -865 to +92 bp supported expression, the addition of fragments between +92 and +447 bp led to repression of expression. Subsequent analysis of reporter gene constructs microinjected into primary cultures of dorsal root ganglion neurons (DRG) confirmed the existence of this repressor domain. This repression could be relieved totally in both RIN cell lines and partly in NF2C cells by mutating residues between +373 and +396 bp. This indicates that these cell lines support PPT promoter activity similar to that observed in DRG and determines a novel repressor domain within the promoter.
Mapping of the RNA-binding and endoribonuclease domains of NIPP1, a nuclear targeting subunit of protein phosphatase 1
Authors: JIN Qiming , BEULLENS Monique , JAGIELLO Izabela , EYNDE Aleyde VAN , VULSTEKE Veerle , STALMANS Willy , BOLLEN Mathieu
Published: August 2015

NIPP1 (351 residues) is a major regulatory and RNA-anchoring subunit of protein phosphatase 1 in the nucleus. Using recombinant and synthetic fragments of NIPP1, the RNA-binding domain was mapped to the C-terminal residues 330-351. A synthetic peptide encompassing this sequence equalled intact NIPP1 in RNA-binding affinity and could be used to dissociate NIPP1 from the nuclear particulate fraction. An NIPP1 fragment consisting of residues 225-351 (Ard1/NIPP1γ), that may be encoded by an alternatively spliced transcript in transformed B-lymphocytes, displayed a single-strand Mg2+-dependent endoribonuclease activity. However, full-length NIPP1 and NIPP1143-351 were not able to cleave RNA, indicating that the endoribonuclease activity of NIPP1 is restrained by its central domain. The endoribonuclease activity was also recovered in the RNA-binding domain, NIPP1330-351, but with a 30-fold lower specific activity. Thus, the endoribonuclease catalytic site and the RNA-binding site both reside in the C-terminal 22 residues of NIPP1. The latter domain does not conform to any known nucleic-acid binding motif.
Cloning and expression of murine liver phosphatidylserine synthase (PSS)-2: differential regulation of phospholipid metabolism by PSS1 and PSS2
Authors: STONE Scot J. , VANCE Jean E.
Published: August 2015

Phosphatidylserine (PtdSer) is synthesized in mammalian cells by two base-exchange enzymes: PtdSer synthase (PSS)-1 primarily uses phosphatidylcholine as a substrate for exchange with serine, whereas PSS2 uses phosphatidylethanolamine (PtdEtn). We previously expressed murine PSS1 in McArdle hepatoma cells. The activity of PSS1 in vitro and the synthesis of PtdSer and PtdSer-derived PtdEtn were increased, whereas PtdEtn synthesis from the CDP-ethanolamine pathway was inhibited [Stone, Cui and Vance (1998) J. Biol. Chem. 273, 7293-7302]. We have now cloned and stably expressed a murine PSS2 cDNA in McArdle cells and M.9.1.1 cells [which are ethanolamine-requiring mutant Chinese hamster ovary (CHO) cells defective in PSS1]. Expression of the PSS2 in M.9.1.1 cells reversed the ethanolamine auxotrophy. However, the PtdEtn content was not normalized unless the culture medium was supplemented with ethanolamine. In both M.9.1.1 and hepatoma cells transfected with PSS2 cDNA the rate of synthesis of PtdSer and PtdSer-derived PtdEtn did not exceed that in parental CHO cells or control McArdle cells respectively, in contrast to cells expressing similar levels of murine PSS1. These observations suggest that PtdSer synthesis via murine PSS2, but not PSS1, is regulated by end-product inhibition. Moreover, expression of murine PSS2 in McArdle cells did not inhibit PtdEtn synthesis via the CDP-ethanolamine pathway, whereas expression of similar levels of PSS1 activity inhibited this pathway by approx. 50%. We conclude that murine PSS1 and PSS2, which are apparently derived from different genes, independently modulate phospholipid metabolism. In addition, mRNAs encoding the two synthases are differentially expressed in several murine tissues, supporting the idea that PSS1 and PSS2 might perform unique functions.
A collagen-related peptide regulates phospholipase Cγ2 via phosphatidylinositol 3-kinase in human platelets
Authors: PASQUET Jean-Max , BOBE Régis , GROSS Barbara , GRATACAP Marie-Pierre , TOMLINSON Michael G. , PAYRASTRE Bernard , WATSON Steve P.
Published: August 2015

The collagen receptor glycoprotein VI (GPVI) induces platelet activation through a similar pathway to that used by immune receptors. In the present study we have investigated the role of phosphatidylinositol 3-kinase (PI 3-kinase) in GPVI signalling. Our results show that collagen-related peptide {CRP: [GCP*(GPP*)10GCP*G]n; P* = hydroxyproline}, which is selective to GPVI, induces formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] in platelets. The increase in the two 3-phosphorylated lipids is inhibited completely by wortmannin and by LY294002, two structurally unrelated inhibitors of PI 3-kinase. The formation of inositol phosphates and phosphatidic acid (PA), two markers of phospholipase C (PLC) activation, by CRP are inhibited by between 50 and 85% in the presence of wortmannin and LY294002. This is associated with inhibition of elevation of intracellular Ca2+ ([Ca2+]i) and aggregation. Wortmannin and LY294002 also partially inhibit elevation of Ca2+ by CRP in murine megakaryocytes. Microinjection of the pleckstrin-homology PH domain of Bruton's tyrosine kinase, which binds selectively to PI(3,4,5)P3, but not the R28C (Arg28 → Cys) mutant which binds to PI(3,4,5)P3 with low affinity, also inhibits elevation of [Ca2+]i in megakaryocytes, suggesting that it is this lipid species which mediates the action of the PI 3-kinase pathway. Studies in platelets show that the action of wortmannin and LY294002 is not mediated through an alteration in tyrosine phosphorylation of PLCγ2. These results demonstrate that PI 3-kinase is required for full activation of PLCγ2 by GPVI in platelets and megakaryocytes.
Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo
Authors: CASAMAYOR Antonio , MORRICE Nick A. , ALESSI Dario R.
Published: August 2015

3-Phosphoinositide-dependent protein kinase-1 (PDK1) expressed in unstimulated 293 cells was phosphorylated at Ser-25, Ser-241, Ser-393, Ser-396 and Ser-410 and the level of phosphorylation of each site was unaffected by stimulation with insulin-like growth factor-1. Mutation of Ser-241 to Ala abolished PDK1 activity, whereas mutation of the other phosphorylation sites individually to Ala did not affect PDK1 activity. Ser-241, unlike the other phosphorylation sites on PDK1, was resistant to dephosphorylation by protein phosphatase 2A1. Ser-241 lies in the activation loop of the PDK1 kinase domain between subdomains VII and VIII in the equivalent position to the site that PDK1 phosphorylates on its protein kinase substrates. PDK1 expressed in bacteria was active and phosphorylated at Ser-241, suggesting that PDK1 can phosphorylate itself at this site, leading to its own activation.
Overexpression of the FAD-binding domain of the sulphite reductase flavoprotein component from Escherichia coli and its inhibition by iodonium diphenyl chloride
Authors: COVÈS Jacques , LEBRUN Colette , GERVASI Gaspard , DALBON Pascal , FONTECAVE Marc
Published: August 2015

SiR-FP43, the NADPH- and FAD-binding domain of the Escherichia colisulphite reductase flavoprotein component (SiR-FP), has been overexpressed and characterized. It folds independently, retaining FAD as a cofactor and the catalytic properties associated with the presence of this cofactor. Iodonium diphenyl chloride (IDP) was shown to be a very efficient inhibitor of SiR-FP43 and SiR-FP60, the monomeric form of SiR-FP, containing both FMN and FAD as cofactors (Ki = 18.5±5 μM, maximal inactivation rate = 0.053±0.005 s-1). In both cases, inactivation was shown to result from covalent binding of a phenyl group to FAD exclusively, in marked contrast with previous results obtained with cytochrome P450 reductase (CPR), where FMN and a tryptophan were phenylated, but not FAD. However, our kinetic analyses are in agreement with the inhibition mechanism demonstrated with CPR [Tew (1993) Biochemistry 32, 10209-10215]. Nine different FAD phenylated adducts were isolated and, for the first time, two FAD phenylated adducts were identified directly after extraction from a protein. Taken together, our results have shown that flavoprotein inactivation by IDP is not a reliable indicator for a flavin radical intermediate in catalysis.
Salicylate-enhanced activation of transcription factors induced by interferon-γ
Authors: CHEN Li-Chun , KEPKA-LENHART Diane , WRIGHT Timothy M. , MORRIS Sidney M.
Published: August 2015

Salicylate enhanced the interferon-γ-dependent activation of two transcription factors in a murine macrophage cell line: signal transducer and activator of transcription (STAT)1 and interferon-γ-responsive factor 1. Salicylate alone did not activate these transcription factors. This enhancement was reflected by increased DNA-binding activities and was the consequence of prolonged tyrosine phosphorylation of these transcription factors following interferon-γ treatment. However, salicylate did not directly inhibit protein-tyrosine phosphatase activity in nuclear extracts of interferon-γ-treated cells. The enhanced activation of STAT1 resulted in increased induction of mRNA encoding interferon regulatory factor-1. These results not only demonstrate that aspirin and its metabolite salicylate may have pro-inflammatory as well as anti-inflammatory effects but also raise the possibility that new cellular targets may be identified for modulating the Janus kinase-STAT signalling pathway.
Production and functional activity of a recombinant von Willebrand factor-A domain from human complement factor B
Authors: WILLIAMS Samantha C. , HINSHELWOOD Justin , PERKINS Stephen J. , SIM Robert B.
Published: August 2015

Factor B is a five-domain 90 kDa serine protease proenzyme which is part of the human serum complement system. It binds to other complement proteins C3b and properdin, and is activated by the protease factor D. The fourth domain of factor B is homologous to the type A domain of von Willebrand Factor (vWF-A). A full-length human factor B cDNA clone was used to amplify the region encoding the vWF-A domain (amino acids 229-444 of factor B). A fusion protein expression system was then used to generate it in high yield in Escherichia coli, where thrombin cleavage was used to separate the vWF-A domain from its fusion protein partner. A second vWF-A domain with improved stability and solubility was created using a Cys267 → Ser mutation and a four-residue C-terminal extension of the first vWF-A domain. The recombinant domains were investigated by analytical gel filtration, sucrose density centrifugation and analytical ultracentrifugation, in order to show that both domains were monomeric and possessed compact structures that were consistent with known vWF-A crystal structures. This expression system and its characterization permitted the first investigation of the function of the isolated vWF-A domain. It was able to inhibit substantially the binding of 125I-labelled factor B to immobilized C3b. This demonstrated both the presence of a C3b binding site in this portion of factor B and a ligand-binding property of the vWF-A domain. The site at which factor D cleaves factor B is close to the N-terminus of both recombinant vWF-A domains. Factor D was shown to cleave the vWF-A domain in the presence or absence of C3b, whereas the cleavage of intact factor B under the same conditions occurs only in the presence of C3b.
GA2/GM2/GD2 synthase localizes to the trans-Golgi network of CHO-K1 cells
Authors: GIRAUDO Claudio G. , FRITZ Víctor M. ROSALES , MACCIONI Hugo J. F.
Published: August 2015

U D P - G a l N A c: l a c t o s y l c e r a m i d e / G M 3 / G D 3 β-1,4-N-a c e t y lgalactosaminyltransferase (GalNAc-T) transforms its acceptors into the gangliosides GA2, GM2 and GD2. It is well established that it is a Golgi-located glycosyltransferase, but its sub-Golgi localization is still unclear. We addressed this question in Chinese hamster ovary K1 cell clones stably transfected with a c-myc-tagged version of GalNAc-T which express the enzyme at different levels of activity. In these cell clones we examined the effect of brefeldin A (BFA) on the synthesis of glycolipids (in metabolic-labelling experiments) and on the sub-Golgi localization of the GalNAc-T (by immunocytochemistry). We found that in cell clones expressing moderate levels of activity, GalNAc-T immunoreactivity behaved as thetrans-Golgi network (TGN) marker mannose-6-P receptor (M6PR) both in BFA-treated and untreated cells, and that BFA completely blocked the synthesis of GM2, GM1 and GD1a. On the other hand, in cell clones expressing high levels of activity and treated with BFA, most GalNAc-T immunoreactivity redistributed to the endoplasmic reticulum, as did the medial-Golgi marker mannosidase II, and the synthesis of GM2, GM1 and GD1a was not completely blocked. These results indicate that GalNAc-T is a TGN-located enzyme and that the mechanism that localizes it to this compartment involves steps that, when saturated, lead to its mislocalization to the cis-, medial- or trans-Golgi. Changes of Golgi membrane properties by modification of local glycolipid composition due to the activity of the expressed enzyme were not the main cause of mislocalization, since it persists when glycolipid synthesis is inhibited with D,L-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCl.
Iron-dependent regulation of transferrin receptor expression in Trypanosoma brucei
Authors: FAST Beate , KREMP Katrin , BOSHART Michael , STEVERDING Dietmar
Published: August 2015

Transferrin is an essential growth factor for African trypanosomes. Here we show that expression of the trypanosomal transferrin receptor, which bears no structural similarity with mammalian transferrin receptors, is regulated by iron availability. Iron depletion of bloodstream forms of Trypanosoma brucei with the iron chelator deferoxamine resulted in a 3-fold up-regulation of the transferrin receptor and a 3-fold increase of the transferrin uptake rate. The abundance of expression site associated gene product 6 (ESAG6) mRNA, which encodes one of the two subunits of the trypanosome transferrin receptor, is regulated 5-fold by a post-transcriptional mechanism. In mammalian cells the stability of transferrin receptor mRNA is controlled by iron regulatory proteins (IRPs) binding to iron-responsive elements (IREs) in the 3′-untranslated region (UTR). Therefore, the role of a T. brucei cytoplasmic aconitase (TbACO) that is highly related to mammalian IRP-1 was investigated. Iron regulation of the transferrin receptor was found to be unaffected in δaco::NEO/δaco::HYG null mutants generated by targeted disruption of the TbACO gene. Thus, the mechanism of post-transcriptional transferrin receptor regulation in trypanosomes appears to be distinct from the IRE/IRP paradigm. The transferrin uptake rate was also increased when trypanosomes were transferred from medium supplemented with foetal bovine serum to medium supplemented with sera from other vertebrates. Due to varying binding affinities of the trypanosomal transferrin receptor for transferrins of different species, serum change can result in iron starvation. Thus, regulation of transferrin receptor expression may be a fast compensatory mechanism upon transmission of the parasite to a new host species.
Calcium-dependent properties of CIB binding to the integrin αIIb cytoplasmic domain and translocation to the platelet cytoskeleton
Authors: SHOCK David D. , NAIK Ulhas P. , BRITTAIN Julia E. , ALAHARI Suresh K. , SONDEK John , PARISE Leslie V.
Published: August 2015

The αIIbβ3 integrin receives signals in agonist-activated platelets, resulting in its conversion to an active conformation that binds fibrinogen, thereby mediating platelet aggregation. Fibrinogen binding to αIIbβ3 subsequently induces a cascade of intracellular signalling events. The molecular mechanisms of this bi-directional αIIbβ3-mediated signalling are unknown but may involve the binding of proteins to the integrin cytoplasmic domains. We reported previously the sequence of a novel 22-kDa, EF-hand-containing, protein termed CIB (calcium- and integrin-binding protein) that interacts specifically with the αIIb cytoplasmic domain in the yeast two-hybrid system. Further analysis of numerous tissues and cell lines indicated that CIB mRNA and protein are widely expressed. In addition, isothermal titration calorimetry indicated that CIB binds to an αIIb cytoplasmic-domain peptide in a Ca2+-dependent manner, with moderate affinity (Kd, 700 nM) and 1:1 stoichiometry. In aggregated platelets, endogenous CIB and αIIbβ3 translocate to the Triton X-100-insoluble cytoskeleton in a parallel manner, demonstrating that the cellular localization of CIB is regulated, potentially by αIIbβ3. Thus CIB may contribute to integrin-related functions by mechanisms involving Ca2+-modulated binding to the αIIb cytoplasmic domain and changes in intracellular distribution.
Tryptophan glycoconjugates in food and human urine
Authors: GUTSCHE Birgit , GRUN Christoph , SCHEUTZOW Dieter , HERDERICH Markus
Published: August 2015

Evaluating the formation of tryptophan glycoconjugates other than well-established Amadori rearrangement products, HPLC-tandem MS (MS/MS) analysis of human urine collected from several healthy individuals proved the presence of one distinct tryptophan C-glycosyl compound [Horiuchi, Yonekawa, Iwahara, Kanno, Kurihara and Fujise (1994) J. Biochem. (Tokyo) 115, 362-366]. After isolation, unambiguous identification of this novel tryptophan metabolite as 2-(α-mannopyranosyl)-L-tryptophan was achieved by tandem MS combined with NMR spectroscopy including homonuclear COSY, heteronuclear multiple-bond connectivity and 1H-detected heteronuclear multiple-quantum coherence experiments. Remarkably, a thorough evaluation of vicinal proton-proton coupling constants in different solvents and nuclear Overhauser effect experiments demonstrate the predominant axial orientation of the hydroxymethyl group of the hexopyranosyl residue. Likewise this spatial arrangement indicates that the respective α-anomeric C-mannosylhexopyranose is preferentially adopting a 1C4 conformation in acidic methanol. Whereas only one distinct tryptophan mannoconjugate could be observed in human urine, HPLC-MS/MS analysis of food samples for the first time led to the identification of numerous N1-(β-D-hexopyranosyl)-L-tryptophan, 2-(β-D-hexopyranosyl)-L-tryptophan and 1-(1,2,3,4,5-p e n t a h y d r o x y p e n t - 1 - yl) - 1, 2, 3, 4 - t e t r a h y d r o - β - c a r b o l i n e - 3 -carboxylic acid derivatives derived from the condensation of tryptophan with aldohexoses. Taking into consideration the significant differences between profiles and configurations of tryptophan glycoconjugates originating from dietary sources and human urine, C-2 mannosylation of tryptophan residues [de Beer, Vliegenthart, Loeffler and Hofsteenge (1995) Biochemistry 34, 11785-11789] represents a novel enzymic pathway in tryptophan metabolism in humans.
Role of Ca2+ in apoptosis evoked by human amylin in pancreatic islet β-cells
Authors: BAI Ji-Zhong , SAAFI Edward L. , ZHANG Shaoping , COOPER Garth J. S.
Published: August 2015

The objective of these studies was to clarify the role of Ca2+ in the mechanism of death evoked by human amylin (hA) in islet β-cells. hA forms fibrils in vitroand islet amyloid in vivo. Here we show that pure synthetic hA aggregated in solution, formed fibrils and evoked death in cultured RINm5F islet β-cells in a time-dependent (0-24 h) and concentration-dependent (0-20 μM) manner. Dying cells underwent shrinkage of the nucleus, with clumping and segregation of chromatin into masses that lay against the nuclear envelope, and internucleosomal DNA fragmentation. These cells therefore show many features of apoptosis, although aspects of the morphology might be characteristic of this particular cell type rather than of a general apoptotic nature. Aurintricarboxylic acid, an inhibitor of both Ca2+-dependent and Ca2+-independent nucleases, suppressed this DNA fragmentation and inhibited apoptosis at concentrations between 25 and 200 μM. Direct measurements of the cytoplasmic free Ca2+ concentration ([Ca2+]i) in fura-2 acetoxymethyl ester (AM)-loaded β-cells showed that neither hA nor its non-cytotoxic homologue, rat amylin were effective in raising [Ca2+]i. Modulators of Ca2+ regulation were tested for their effects on hA-induced β-cell apoptosis. Ca2+ ionophore (A23187) and thapsigargin (an inhibitor of endoplasmic reticular Ca2+-ATPase activity) by themselves evoked apoptosis accompanied by increased [Ca2+]i. Neither the Ca2+ channel blocker verapamil, the extracellular Ca2+ chelator EGTA nor the cytosolic Ca2+ buffer bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (‘BAPTA’)/AM protected β-cells from hA-evoked apoptosis. Prolonged incubation of β-cells with a lethal dose of hA altered neither the basal [Ca2+]i nor the thapsigargin-induced release of Ca2+ from intracellular stores. Furthermore, 45CaCl2 uptake by RINm5F cells did not differ in the presence or absence of hA. These results suggest that, whereas alterations in cytosolic Ca2+ homoeostasis do have a significant role in certain forms of β-cell death, they do not contribute to the pathway of apoptosis evoked by hA in islet β-cells.
Synthesis and shedding of hyaluronan from plasma membranes of human fibroblasts and metastatic and non-metastatic melanoma cells
Authors: LüKE Hans-Joachim , PREHM Peter
Published: August 2015

The regulation of hyaluronan synthesis and shedding was analysed in human fibroblasts and in two melanoma cells that differed in the metastatic potential and proteolysis of the hyaluronan receptor CD44. Dissociation of nascent hyaluronan from plasma membranes isolated from fibroblasts by high salt concentrations led to activation of hyaluronan synthase. Hyaluronan synthesis was also enhanced in plasma membranes from fibroblasts that had been treated with hyaluronidase or trypsin. Hyaluronan oligosaccharides stimulated hyaluronan production in fibroblast cultures. These results indicated that nascent high-molecular-mass hyaluronan inhibited its own chain elongation, if it was retained in the vicinity of the synthase by cell-surface receptors. The results also indicated that increased hyaluronan synthesis and shedding correlated with proteolysis of CD44 on the melanoma cell lines, which has been observed by others.
Poly(ADP-ribosylation) protects maternally derived histones from proteolysis after fertilization
Authors: MORIN Violeta , DIAZ Freddy , MONTECINO Martin , FOTHERGILL-GILMORE Linda , PUCHI Marcia , IMSCHENETZKY Maria
Published: August 2015

Fertilization in sea urchins is followed by the replacement of sperm-specific histones by cleavage-stage histone variants recruited from maternal stores. Such remodelling of zygote chromatin involves a cysteine proteinase that degrades the sperm-specific histones in a selective manner, leaving the maternal cleavage-stage histone variants intact. The mechanism that determines the selectivity of the sperm-histone-selective proteinase (SpH-proteinase) was analysed by focusing on the post-translational modification status of both sets of histones. It has previously been reported that only native cleavage-stage histones are poly(ADP-ribosylated), whereas the sperm-specific histones are not modified. To determine whether the poly(ADP-ribose) moiety afforded protection from degradation, the ADP-ribose polymers were removed from the cleavage-stage histones in vitro; these proteins were then assayed as potential substrates of the SpH-proteinase. Strikingly, the cleavage-stage histone variants were extensively degraded after the enzymic removal of their ADP-ribose moieties. In addition, the SpH cysteine proteinase was not inhibited by isolated poly(ADP-ribose) polymers. Consequently, only poly(ADP-ribosylated) cleavage-stage histone variants are protected from proteolysis. These results demonstrate a novel role for this type of post-translational modification, namely the protection of nuclear proteins against nuclear proteinases after fertilization.
Pathway alignment: application to the comparative analysis of glycolytic enzymes
Authors: DANDEKAR Thomas , SCHUSTER Stefan , SNEL Berend , HUYNEN Martijn , BORK Peer
Published: August 2015

Comparative analysis of metabolic pathways in different genomes yields important information on their evolution, on pharmacological targets and on biotechnological applications. In this study on glycolysis, three alternative ways of comparing biochemical pathways are combined: (1) analysis and comparison of biochemical data, (2) pathway analysis based on the concept of elementary modes, and (3) a comparative genome analysis of 17 completely sequenced genomes. The analysis reveals a surprising plasticity of the glycolytic pathway. Isoenzymes in different species are identified and compared; deviations from the textbook standard are detailed. Several potential pharmacological targets and by-passes (such as the Entner-Doudoroff pathway) to glycolysis are examined and compared in the different species. Archaean, bacterial and parasite specific adaptations are identified and described.
Structure–function analysis of yeast hexokinase: structural requirements for triggering cAMP signalling and catabolite repression
Authors: KRAAKMAN Leon S. , WINDERICKX Joris , THEVELEIN Johan M. , WINDE Johannes H. DE
Published: August 2015

In baker's yeast (Saccharomyces cerevisiae) the hexokinases PI (Hxk1) and PII (Hxk2) are required for triggering of the activation of the Ras-cAMP pathway and catabolite repression. Specifically, Hxk2 is essential for the establishment of glucose repression, whereas either Hxk1 or Hxk2 can sustain fructose repression. Previous studies have suggested that the extent of glucose repression is inversely correlated with hexokinase catalytic activity and hence with an adequate elevation of intracellular sugar phosphate levels. However, several lines of evidence indicate that glucose 6-phosphate is not the trigger of catabolite repression in yeast. In the present study we employed site-directed mutagenesis of amino acids important for the binding of sugar and ATP, for efficient phosphoryl transfer and for the closure of the substrate-binding cleft, to obtain an insight into the structural requirements of Hxk2 for sugar-induced signalling. We show that the ATP-binding Lys-111 is not essential for catalysis in vivo or for signal triggering. Substitution of the catalytic-centre Asp-211 caused loss of catalytic activity, but high-affinity sugar binding was retained. However, this was not sufficient to cause cAMP activation nor catabolite repression. Mutation of Ser-158 abrogated glucose-induced, but not fructose-induced, repression. Moreover, 2-deoxyglucose sustained repression despite an extremely low catalytic activity. We conclude that the establishment of catabolite repression is dependent on the onset of the phosphoryl transfer reaction on hexokinase and is probably related to the stable formation of a transition intermediate and concomitant conformational changes within the enzyme. In contrast, the role of Hxk2 in Ras-cAMP activation seems to be directly connected to its catalytic function. The implications of this model are discussed.
Bacterial lipolytic enzymes: classification and properties
Authors: ARPIGNY Jean Louis , JAEGER Karl-Erich
Published: August 2015

Knowledge of bacterial lipolytic enzymes is increasing at a rapid and exciting rate. To obtain an overview of this industrially very important class of enzymes and their characteristics, we have collected and classified the information available from protein and nucleotide databases. Here we propose an updated and extensive classification of bacterial esterases and lipases based mainly on a comparison of their amino acid sequences and some fundamental biological properties. These new insights result in the identification of eight different families with the largest being further divided into six subfamilies. Moreover, the classification enables us to predict (1) important structural features such as residues forming the catalytic site or the presence of disulphide bonds, (2) types of secretion mechanism and requirement for lipase-specific foldases, and (3) the potential relationship to other enzyme families. This work will therefore contribute to a faster identification and to an easier characterization of novel bacterial lipolytic enzymes.
Aberrant expression of cAMP-response-element-binding protein (‘CREB’) induces apoptosis
Authors: SAEKI Kumiko , YUO Akira , SUZUKI Emiko , YAZAKI Yoshio , TAKAKU Fumimaro
Published: August 2015

We have reported previously that cAMP-response-element-binding protein (CREB) was phosphorylated in a cell-cycle-dependent manner, showing that it was phosphorylated at early S-phase at casein kinase II target sites. To assess the possible involvement of CREB in cell cycle progression, CREB expression vector was transiently transfected into various cells. Unexpectedly we found that transfection with CREB expression vector resulted in an abundance of dead cells. Morphological examination revealed that these cells had undergone apoptosis. The coincidence of CREB overexpression and apoptosis induction at the individual cell level was confirmed by a immunohistochemical study. To confirm that overexpression of CREB was the cause of apoptosis, a dominant-negative mutant of CREB, KCREB, was co-expressed with the wild type. The co-existence of KCREB effectively rescued CREB-mediated apoptosis in a dose-dependent manner, verifying that apoptosis was truly a specific effect of overexpressed CREB and not an artifact of the transfection procedure. Deletion analysis indicates that neither the Q1 transactivation domain, which functions in transcription, nor the kinase-inducible domain, in which a cluster of various kinase targets exists, is necessary; however, the Q2 transactivation domain is required for the induction of apoptosis. A more precise study indicates that the four-residue stretch Glu-Glu-Ala-Ala at the most C-terminal region of the Q2 domain is especially important for the induction of apoptosis. Thus overexpressed CREB induces apoptosis by transmitting certain signals from the C-terminal portion of the Q2 domain. Possible roles of cell-cycle-regulated phosphorylation and also an elevation of the intracellular cAMP level in CREB-induced apoptosis are suggested.
Enzymic characterization of a novel member of the regulatory B-like carboxypeptidase with transcriptional repression function: stimulation of enzymic activity by its target DNA
Authors: MUISE Aleixo M. , RO Hyo-Sung
Published: August 2015

The adipocyte-enhancer binding protein (AEBP) 1 is a novel transcriptional repressor with carboxypeptidase (CP) activity. AEBP1 binds to a regulatory sequence (termed adipocyte enhancer 1, AE-1) located in the proximal promoter region of the adipose P2 (aP2) gene, which encodes the adipocyte fatty-acid binding protein. Sequence comparisons and kinetic studies using known carboxypeptidase substrates, activators and inhibitors have characterized AEBP1 as a member of the regulatory B-like CP family. Significantly, the inherent CP activity of AEBP1 is stimulated by the AE-1 sequence. Our results indicate that AEBP1 is activated by a novel mechanism, wherby the direct binding of DNA enhances its protease activity. These results represent the first demonstration of DNA-mediated regulation of CP activity.
A phosphotyrosine-containing quenched fluorogenic peptide as a novel substrate for protein tyrosine phosphatases
Authors: NISHIKATA Makoto , SUZUKI Kuniaki , YOSHIMURA Yoshitaka , DEYAMA Yoshiaki , MATSUMOTO Akira
Published: August 2015

M c a - G l y - A s p - A l a - G l u - T y r ( P O3H2 ) - A l a - A l a - L y s ( D N P ) - A r g-NH2, where Mca is (7-methoxycoumarin-4-yl)acetyl and DNP is 2,4-dinitrophenyl, was synthesized as a fluorogenic substrate for protein tyrosine phosphatases (PTPs). In the peptide, the fluorescent Mca group is quenched efficiently by the DNP group. Although the fluorescence intensity of the substrate was practically unchanged upon PTP-catalysed dephosphorylation, it increased approx. 120-fold upon subsequent treatment with chymotrypsin. Analysis by HPLC showed that chymotrypsin cleaved only the dephosphorylated substrate at the Tyr-Ala bond. Thus with the aid of chymotrypsin, dephosphorylation of the substrate can be measured fluorometrically. A strictly linear correlation was observed between PTP concentration and dephosphorylation rate. The fluorogenic substrate was dephosphorylated by some PTPs much more rapidly than the corresponding 32P-labelled substrate used for comparison, whereas alkaline phosphatase dephosphorylated the two substrates at similar rates. The fluorogenic substrate is therefore more specific for PTPs than the radiolabelled substrate. The assay with the fluorogenic substrate could be applied to the estimation of kinetc parameters and measurement of PTP activity in crude-enzyme preparations. The lower detection limit of our assay (1 μM substrate in 200 μl of reaction mixture) was estimated to be 0.2-0.4 pmol, whereas it was estimated to be about 1 pmol in the assay that used 32P-labelled peptide (specific radioactivity of approx. 1000 c.p.m./pmol). Our assay is simple, specific, highly sensitive and non-radioisotopic, and hence would contribute greatly to the development of PTP biology.