bluuebunny/crossref_metadata_2025 huggingface dataset:
Early phosphoinositide 3-kinase activity is required for late activation of protein kinase Cε in platelet-derived-growth-factor-stimulated cells: evidence for signalling across a large temporal gap
Authors: BALCIUNAITE Egle , KAZLAUSKAS Andrius
Published: August 2015

At least two signalling systems have the potential to contribute to the activation of protein kinase C (PKC) family members such as PKC∊. One of these is phosphoinositide 3-kinase (PI 3-kinase), whose lipid products activate PKC∊ in vitro and in living cells. The recent observation that there are multiple waves of PI 3-kinase and PKC∊ activity within the G0-to-S phase interval provides a new opportunity to investigate the relationship between these two signalling enzymes in vivo. We have assessed the relative importance of the early and late waves of PI 3-kinase activity for the corresponding waves of PKC∊ activity. Blocking the first phase of PI 3-kinase activity inhibited both early and late activation of PKC∊. In contrast, the second wave of PI 3-kinase activity was dispensable for late activation of PKC∊. These findings suggested that early PI 3-kinase activation induced a stable change in PKC∊, which predisposed it to subsequent activation by lipid cofactors. Indeed, partial proteolysis of PKC∊ indicated that early activation of PI 3-kinase led to a conformation change in PKC∊ that persisted as the activity of PKC∊ cycled. We propose a two-step hypothesis for the activation of PKC∊ in vivo. One step is stable and depends on PI 3-kinase, whereas the other is transient and may depend on the availability of lipid cofactors. Finally, these studies reveal that PI 3-kinase and PKC∊ are capable of communicating over a relatively long time interval and begin to elucidate the mechanism.
Heterogeneous nuclear ribonucleoprotein E1B-AP5 is methylated in its Arg-Gly-Gly (RGG) box and interacts with human arginine methyltransferase HRMT1L1
Authors: KZHYSHKOWSKA Julia , SCHÜTT Holger , LISS Michael , KREMMER Elisabeth , STAUBER Roland , WOLF Hans , DOBNER Thomas
Published: August 2015

The heterogeneous nuclear ribonucleoprotein (hnRNP) family includes predominantly nuclear proteins acting at different stages of mRNA metabolism. A characteristic feature of hnRNPs is to undergo post-translational asymmetric arginine methylation catalysed by different type 1 protein arginine methyltransferases (PRMTs). A novel mammalian hnRNP, E1B-AP5, recently identified by its interaction with adenovirus early protein E1B-55kDa, has been proposed to have a regulatory role in adenoviral and host-cell mRNA processing/nuclear export [Gabler, Schutt, Groitl, Wolf, Shenk and Dobner (1998) J. Virol. 72, 7960–7971]. Here we report that E1B-AP5 is methylated in vivo in its Arg-Gly-Gly (RGG)-box domain, known to mediate protein–RNA interactions. The activity responsible for E1B-AP5 methylation forms a complex with E1B-AP5 in vivo. The predominant mammalian arginine methyltransferase HRMT1L2 (hPRMT1) did not detectably methylate endogenous E1B-AP5 despite efficiently methylating a recombinant RGG-box domain of E1B-AP5. Using yeast two-hybrid screening we identified HRMT1L1 (PRMT2) as one of the proteins interacting with E1B-AP5. By in situ immunofluorescence we demonstrated that E1B-AP5 co-localizes with the nuclear fraction of HRMT1L1. The Src homology 3 (SH3) domain of HRMT1L1 was essential for its interaction with E1B-AP5 in vivo. We suggest that HRMT1L1 is responsible for specific E1B-AP5 methylation in vivo.
Inhibition of the neutrophil NADPH oxidase and associated H+ channel by diethyl pyrocarbonate (DEPC), a histidine-modifying agent: evidence for at least two target sites
Authors: MANKELOW Tosti J. , HENDERSON Lydia M.
Published: August 2015

Diethyl pyrocarbonate (DEPC), a histidine-modifying reagent, has been utilized to demonstrate the importance of histidine residues in the functioning of proteins. In previous studies of the NADPH oxidase, histidine residues have been determined to be important in the ability of gp91phox to function as an H+ pathway and in the binding of haem and FAD. We have investigated the ability of DEPC to inhibit H+ flux and superoxide generation by human neutrophils. Proton flux through the NADPH oxidase-associated H+ channel was inhibited by DEPC only if applied simultaneously with an activator of the channel. This suggested that the site modified by DEPC is not accessible in the closed channel. Superoxide generation by the NADPH oxidase was also inhibited by DEPC when applied after or simultaneously with the activator. Translocation of the NADPH oxidase cytosolic components, p67phox and p47phox, to the membrane was unaffected by DEPC. In a cell-free system, DEPC-treated membranes failed to support superoxide generation or the reduction of Iodonitrotetrazolium Violet and showed a loss of the characteristic cytochrome b558 spectrum. Superoxide generation by DEPC-treated cytosol was inhibited slightly. Therefore it can be concluded that there are two sites within the NADPH oxidase that interact with DEPC, one in the H+ pathway, only accessible in the activated oxidase, and a second accessible prior to activation of the NADPH oxidase. The latter non-proton pathway DEPC site is located within the membrane components of the NADPH oxidase and is associated with the binding of haem in the enzyme complex.
Suicide inactivation of xanthine oxidoreductase during reduction of inorganic nitrite to nitric oxide
Authors: GODBER Ben L. J. , DOEL Justin J. , GOULT Tracey A. , EISENTHAL Robert , HARRISON Roger
Published: August 2015

Xanthine oxidoreductase (XOR) is progressively inactivated while catalysing the reduction of inorganic nitrite to NO by xanthine. Inactivation results from conversion of the enzyme into its desulpho-form. The rate of inactivation increases with nitrite concentration. Similar behaviour was shown when NADH replaced xanthine as reducing substrate. A kinetic model is proposed incorporating a ‘suicide’ inactivation involving an enzyme–substrate (product) complex, rather than inactivation by free NO. The model provides a good fit to progress curves of the reaction of xanthine or NADH with nitrite in the presence of the oxidase or dehydrogenase forms of the enzyme. Inorganic nitrate, like nitrite, was shown to be reduced at the molybdenum site of XOR. With xanthine as reducing substrate, nitrite was produced in essentially a 1:1 stoichiometric ratio with respect to urate. Unlike the case of nitrite, the enzyme was not significantly inactivated, implying that inactivation during nitrite reduction depends on the presence of nascent NO in its enzyme complex.
Ubiquitination is essential for human cytomegalovirus US11-mediated dislocation of MHC class I molecules from the endoplasmic reticulum to the cytosol
Authors: KIKKERT Marjolein , HASSINK Gerco , BAREL Martine , HIRSCH Christian , van der WAL Fimme Jan , WIERTZ Emmanuel
Published: August 2015

Human cytomegalovirus encodes two glycoproteins, US2 and US11, which cause rapid degradation of MHC class I molecules, thus preventing recognition of virus-infected cells by the immune system. This degradation process involves retrograde transport or ‘dislocation’ of MHC class I molecules from the endoplasmic reticulum (ER) to the cytosol, where they are deglycosylated by an N-glycanase and degraded by the proteasome. At present it is unknown whether ubiquitination is required for US2- and US11-mediated dislocation and degradation of MHC class I molecules. Here, we show that in E36ts20 hamster cells, which contain a temperature-sensitive mutation in the E1 ubiquitin-activating enzyme, US11-mediated degradation of MHC class I molecules is strongly impaired at the non-permissive temperature, indicating the necessity for ubiquitination in this process. We next addressed the question of whether ubiquitination is a condition for the retrograde movement of MHC class I molecules from the ER to the cytosol, or whether ubiquitination is merely required for recognition of dislocated MHC class I molecules by the proteasome. In the absence of a functional ubiquitin system, complexes of US11 and MHC class I molecules accumulate in the ER. In this state the membrane topology of MHC class I molecules does not significantly change, as judged from proteinase K digestions. Thus the results indicate that a functional ubiquitin system is essential for dislocation of MHC class I molecules from the ER to the cytosol.
Kinetic competence of the cADP-ribose–CD38 complex as an intermediate in the CD38/NAD+ glycohydrolase-catalysed reactions: implication for CD38 signalling
Authors: CAKIR-KIEFER Céline , MULLER-STEFFNER Hélène , OPPENHEIMER Norman , SCHUBER Francis
Published: August 2015

CD38/NAD+ glycohydrolase is a type II transmembrane glycoprotein widely used to study T- and B-cell activation and differentiation. CD38 is endowed with two different activities: it is a signal transduction molecule and an ectoenzyme that converts NAD+ into ADP-ribose (NAD+ glycohydrolase activity) and small proportions of cADP-ribose (cADPR; ADP-ribosyl cyclase activity), a calcium-mobilizing metabolite, which, ultimately, can also be hydrolysed (cADPR hydrolase activity). The relationship between these two properties, and strikingly the requirement for signalling in the formation of free or enzyme-complexed cADPR, is still ill-defined. In the present study we wanted to test whether the CD38–cADPR complex is kinetically competent in the conversion of NAD+ into the reaction product ADP-ribose. In principle, such a complex could be invoked for cross-talk, via conformational changes, with neighbouring partner(s) of CD38 thus triggering the signalling phenomena. Analysis of the kinetic parameters measured for the CD38/NAD+ glycohydrolase-catalysed hydrolysis of 2′-deoxy-2′-aminoribo-NAD+ and ADP-cyclo[N1,C1′]-2′-deoxy-2′-aminoribose (slowly hydrolysable analogues of NAD+ and cADPR respectively) ruled out that the CD38–cADPR complex can accumulate under steady-state conditions. This was borne out by simulation of the prevalent kinetic mechanism of CD38, which involve the partitioning of a common E·ADP-ribosyl intermediate in the formation of the enzyme-catalysed reaction products. Using this mechanism, microscopic rate conditions were found which transform a NAD+ glycohydrolase into an ADP-ribosyl cyclase. Altogether, the present work shows that if the cross-talk with a partner depends on a conformational change of CD38, this is most probably not attributable to the formation of the CD38–cADPR complex. In line with recent results on the conformational change triggered by CD38 ligands [Berthelier, Laboureau, Boulla, Schuber and Deterre (2000) Eur. J. Biochem. 267, 3056–3064], we believe that the Michaelis CD38–NAD+ complex could play such a role instead.
Glycosylation by Pichia pastoris decreases the affinity of a family 2a carbohydrate-binding module from Cellulomonas fimi: a functional and mutational analysis
Authors: BORASTON Alisdair B. , WARREN R. Antony J. , KILBURN Douglas G.
Published: August 2015

When produced by Pichia pastoris, three of the five Asn-Xaa-Ser/Thr sequences (corresponding to Asn-24, Asn-73 and Asn-87) in the carbohydrate-binding module CBM2a of xylanase 10A from Cellulomonas fimi are glycosylated. The glycans are of the high-mannose type, ranging in size from GlcNAc2Man8 to GlcNAc2Man14. The N-linked glycans block the binding of CBM2a to cellulose. Analysis of mutants of CBM2a shows that glycans on Asn-24 decrease the association constant (Ka) for the binding of CBM2a to bacterial microcrystalline cellulose approx. 10-fold, whereas glycans on Asn-87 destroy binding. The Ka of a mutant of CBM2a lacking all three N-linked glycosylation sites is the same when the polypeptide is produced by either Escherichia coli or P. pastoris and is approx. half that of wild-type CBM2a produced by E. coli.
Colony-stimulating factor-1 (CSF-1) receptor-mediated macrophage differentiation in myeloid cells: a role for tyrosine 559-dependent protein phosphatase 2A (PP2A) activity
Authors: McMAHON Kerrie-Ann , WILSON Nicholas J. , MARKS Denese C. , BEECROFT Tina L. , WHITTY Genevieve A. , HAMILTON John A. , CSAR Xavier F.
Published: August 2015

M1 myeloid cells transfected with the wild-type (WT) colony-stimulating factor-1 (CSF-1) receptor (CSF-1R; M1/WT cells) undergo CSF-1-dependent macrophage differentiation. By mutation studies, we have provided prior evidence that tyrosine 559 in the CSF-1R cytoplasmic domain governs the Src-dependent differentiation pathway. Further components of this pathway were then sought. We report that the extent of CSF-1-mediated tyrosine phosphorylation of protein phosphatase 2A (PP2A), and the associated loss of its activity were reduced in M1 cells transfected with the CSF-1R with a tyrosine-to-phenylalanine mutation at position 559 (M1/559 cells), compared with the corresponding responses in CSF-1-treated M1/WT cells. This evidence for an involvement of a reduction in PP2A activity in the differentiation process was supported by the restoration of the defect in the CSF-1-mediated differentiation of M1/559 cells by the addition of the PP2A inhibitor, okadaic acid. It was also found that the degree of activation of extracellular-signal-regulated kinase (ERK) activities by CSF-1 was reduced in M1/559 cells, suggesting their involvement in the differentiation process. These data suggest that PP2A and ERK form part of the Src-dependent signal-transduction cascade governing CSF-1-mediated macrophage differentiation in M1 cells.
Human glycosaminoglycan glucuronyltransferase I gene and a related processed pseudogene: genomic structure, chromosomal mapping and characterization
Authors: KITAGAWA Hiroshi , TAOKA Masateru , TONE Yuko , SUGAHARA Kazuyuki
Published: August 2015

Here we describe the characterization of the human glycosaminoglycan glucuronyltransferase I gene (GlcAT-I) and a related pseudogene. The GlcAT-I gene was localized to human chromosome 11q12–q13 by in situ hybridization of metaphase chromosomes. GlcAT-I spanned 7kb of human genomic DNA and was divided into five exons. Northern blot analysis showed that GlcAT-I exhibited ubiquitous but markedly different expressions in the human tissues examined. The GlcAT-I promoter was approx. 3-fold more active in a melanoma cell line than in a hepatoma cell line, providing evidence for the differential regulation of the gene's expression. Stepwise 5′ deletions of the promoter identified a strong enhancer element between −303 and −153bp that included binding motifs for Ets, CREB (cAMP-response-element-binding protein) and STAT (signal transducers and activators of transcription). Screening of a human genomic library identified one additional distinct genomic clone containing an approx. 1.4kb sequence region that shared an overall 95.3% nucleotide identity with exons 1–5 of GlcAT-I. However, a lack of intron sequences, as well as the presence of several nucleotide mutations, insertions and deletions that disrupted the potential GlcAT-I reading frame, suggested that the clone contained a processed pseudogene. The pseudogene was localized to chromosome 3. The human genome therefore contains two related GlcAT-I genes that are located on separate chromosomes.
Flavonoids protect neurons from oxidized low-density-lipoprotein-induced apoptosis involving c-Jun N-terminal kinase (JNK), c-Jun and caspase-3
Authors: SCHROETER Hagen , SPENCER Jeremy P. E. , RICE-EVANS Catherine , WILLIAMS Robert J.
Published: August 2015

Oxidative stress has been associated with neuronal loss in neurodegenerative diseases and during age-associated cognitive decline. Flavonoids have been proposed to play a useful role in protecting the central nervous system against oxidative and excitotoxic stress, although the mechanism of action is unknown. Using oxidized low-density lipoprotein (oxLDL) as the oxidative insult we investigated the mechanism of neurotoxicity and attempted to identify possible sites of action of two of the most potent protective flavonoids, epicatechin and kaempferol, in cultured primary neurons. Using cultured striatal neurons and selective phosphospecific antibodies we addressed the potential role of extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). OxLDL stimulated a Ca2+-dependent activation of both ERK1/2 and JNK that was strongly inhibited by pre-treatment with low micromolar concentrations of epicatechin. Neurotoxicity induced by oxLDL, however, was neither reduced nor enhanced by inhibiting ERK1/2 activation with mitogen-activated protein kinase kinase (MEK) inhibitors, suggesting that this cascade is unlikely to be involved in either oxLDL toxicity or the protective effects of flavonoids. oxLDL caused a sustained activation of JNK that resulted in the phosphorylation of the transcription factor c-Jun, which was abolished in neurons pre-treated with flavonoids. Furthermore, oxLDL induced the cleavage of procaspase-3 and increased caspase-3-like protease activity in neurons, an effect which was strongly inhibited by pre-exposure to either epicatechin or kaempferol. In addition, a caspase-3 inhibitor reduced oxLDL-induced neuronal death, implicating an apoptotic mechanism. A major in vivo metabolite of epicatechin, 3′-O-methyl-epicatechin was as effective as epicatechin in protecting neurons. Thus dietary flavonoids might have potential as protective agents against neuronal apoptosis through selective actions within stress-activated cellular responses, including protein kinase signalling cascades.
Lactate supply as a determinant of the distribution of intracellular pH within the hepatic lobule
Authors: BURNS Shamus P. , MURPHY Helena C. , ILES Richard A. , COHEN Robert D.
Published: August 2015

When isolated livers from starved rats are perfused with lactate at constant perfusate pH and Pco2, there is a marked gradient of cell pH (pHi) along the length of the lobular radius, with periportal cells being substantially more alkaline than perivenous cells. In the present studies, the perivenous 21% of the lobular volume was destroyed by retrograde digitonin perfusion, and antegrade perfusion restored. pHi was determined by 31P-NMR. The remaining periportal cells, the site of gluconeogenesis from lactate, had a substantially higher mean pHi (7.42) than did the intact liver (7.23). When lactate was removed from the perfusate, mean pHi decreased to 7.25. The corresponding concentration of cell bicarbonate fell with a half-time of approximately 5min. When lactate was re-introduced mean pHi rose to 7.34. We conclude that a major contributor to periportal alkalinity under these conditions is proton consumption during gluconeogenesis from lactate ions.
Pseudomonas cellulosa expresses a single membrane-bound glycoside hydrolase family 51 arabinofuranosidase
Authors: BEYLOT Marie-Helene , EMAMI Kaveh , McKIE Vincent A. , GILBERT Harry J. , PELL Gavin
Published: August 2015

In the accompanying paper [Beylot, McKie, Voragen, Doeswijk-Voragen and Gilbert (2001) Biochem. J. 358, 607–614] the chromosome of Pseudomonas cellulosa was shown to contain two genes, abf51A and abf62A, that encode arabinofuranosidases belonging to glycoside hydrolase families 51 and 62, respectively. In this report we show that expression of Abf51A is induced by arabinose and arabinose-containing polysaccharides. Northern-blot analysis showed that abf51A was efficiently transcribed, whereas no transcript derived from abf62A was detected in the presence of arabinose-containing polysaccharides. Zymogram and Western-blot analyses revealed that Abf51A was located on the outer membrane of P. cellulosa. To investigate the importance of Abf51A in the release of arabinose from poly- and oligosaccharides, transposon mutagenesis was used to construct an abf51A-inactive mutant of P. cellulosa (Δabf51A). The mutant did not grow on linear arabinan or sugar beet arabinan, and utilized arabinoxylan much more slowly than the wild-type bacterium. Arabinofuranosidase activity in Δabf51A against aryl-α-arabinofuranosides, arabinan and α1,5-linked arabino-oligosaccharides was approx. 1% of the wild-type bacterium. The mutant bacterium did not exhibit arabinofuranosidase activity against arabinoxylan, supporting the view that abf62A is not expressed in P. cellulosa. These data indicate that P. cellulosa expresses a membrane-bound glycoside hydrolase family 51 arabinofuranosidase that plays a pivotal role in releasing arabinose from polysaccharides and arabino-oligosaccharides.
The Pseudomonas cellulosa glycoside hydrolase family 51 arabinofuranosidase exhibits wide substrate specificity
Authors: BEYLOT Marie-Helene , McKIE Vincent A. , VORAGEN Alphons G. J. , DOESWIJK-VORAGEN Chantal H. L. , GILBERT Harry J.
Published: August 2015

To investigate the mechanism by which Pseudomonas cellulosa releases arabinose from polysaccharides and oligosaccharides, a gene library of P. cellulosa genomic DNA was screened for 4-methylumbelliferyl-α-l-arabinofuranosidase (MUAase) activity. A single MUAase gene (abf51A) was isolated, which encoded a non-modular glycoside hydrolase family (GH) 51 arabinofuranosidase (Abf51A) of 57000Da. The substrate specificity of the Abf51A showed that it preferentially removed α1,2- and α1,3-linked arabinofuranose side chains from either arabinan or arabinoxylan, and hydrolysed α1,5-linked arabino-oligosaccharides, although at a much lower rate. The activity of Abf51A against arabinoxylan was similar to a GH62 arabinofuranosidase encoded by a P. cellulosa gene. Glu-194 and Glu-321 of Abf51A are conserved in GH51 enzymes, and it has been suggested that these amino acids comprise the key catalytic acid/base and nucleophile residues, respectively. To evaluate this hypothesis the biochemical properties of E194A and E321A mutants of Abf51A were evaluated. The data were consistent with the view that Glu-194 and Glu-321 comprise the key catalytic residues of Abf51A. These data, in conjunction with the results presented in the accompanying paper [Beylot, Emami, McKie, Gilbert and Pell (2001) Biochem. J. 358, 599–605], indicate that P. cellulosa expresses a membrane-bound GH51 arabinofuranosidase that plays a pivotal role in releasing arabinose from a range of polysaccharides and oligosaccharides.
Analysis of aggrecan in human knee cartilage and synovial fluid indicates that aggrecanase (ADAMTS) activity is responsible for the catabolic turnover and loss of whole aggrecan whereas other protease activity is required for C-terminal processing in vivo
Authors: SANDY John D. , VERSCHAREN Christie
Published: August 2015

Studies of aggrecan proteolysis in human joints have implicated both the aggrecanase [ADAMTS, a disintegrin-like and metalloprotease (reprolysin-type) with thrombospondin type 1 motif] and matrix metalloproteinase (MMP) families. We have analysed the aggrecan core protein species present in vivo in both articular cartilage and synovial fluids from normal, acutely injured and osteoarthritic joints. Normal cartilage contains at least seven major G1 domain (the N-terminal globular domain of aggrecan)-bearing species, of which three (full-length core, G1-NITEGE373 and G1-VDIPEN341) have been identified. The C-terminals of the others are unknown but digestion of fetal human aggrecan with MMP-3 and crude aggrecanase suggests that they are products of MMP-like activity in vivo. Normal synovial fluids contain at least 10 species, of which nine result from ADAMTS-dependent cleavage, and this cleavage occurs at all of the five known aggrecanase sites. Aggrecan fragments in the cartilage and synovial fluids of acutely injured joints are generally similar to normal, but all contain a markedly increased ratio of G1-NITEGE to G1-VDIPEN. Aggrecan from the cartilage of late-stage osteoarthritis patients is remarkably similar to normal, whereas the synovial fluid aggrecan is more fragmented than that from normal or injured knees. The analyses suggest that the role of the ADAMTS and these MMP-like activities in human cartilage are distinctly different. Excessive ADAMTS activity in vivo is destructive to cartilage matrix, since the bulk of the glycosaminoglycan (GAG)-bearing products are released from the tissue into the synovial fluid following cleavage of the Glu373–Ala374 bond. In contrast, the MMP-like activity appears to be essentially non-destructive, since much of the GAG-bearing product is retained in the tissue following cleavages that are in the more C-terminal regions of the molecule.
Urokinase-catalysed cleavage of the urokinase receptor requires an intact glycolipid anchor
Authors: HØYER-HANSEN Gunilla , PESSARA Ulrich , HOLM Arne , PASS Jesper , WEIDLE Ulrich , DANØ Keld , BEHRENDT Niels
Published: August 2015

Urokinase (uPA) has the striking ability to cleave its receptor, uPAR, thereby inactivating the binding potential of this molecule. Here we demonstrate that the glycosylphosphatidylinositol (GPI) anchor of uPAR, which is attached to the third domain, is an important determinant in governing this reaction, even though the actual cleavage occurs between the first and second domains. Purified full-length GPI-anchored uPAR (GPI-uPAR) proved much more susceptible to uPA-mediated cleavage than recombinant truncated soluble uPAR (suPAR), which lacks the glycolipid anchor. This was not a general difference in proteolytic susceptibility since GPI-uPAR and suPAR were cleaved with equal efficiency by plasmin. Since the amino acid sequences of GPI-uPAR and suPAR are identical except for the C-terminal truncation, the different cleavage patterns suggest that the two uPAR variants differ in the conformation or the flexibility of the linker region between domains 1 and 2. This was supported by the fact that an antibody to the peptide AVTYSRSRYLE, amino acids 84–94 in the linker region, recognizes GPI-uPAR but not suPAR. This difference in the linker region is thus caused by a difference in a remote hydrophobic region. In accordance with this model, when the hydrophobic lipid moiety was removed from the glycolipid anchor by phospholipase C, low concentrations of uPA could no longer cleave the modified GPI-uPAR and the reactivity to the peptide antibody was greatly decreased. Naturally occurring suPAR, purified from plasma, was found to have a similar resistance to uPA cleavage as phospholipase C-treated GPI-uPAR and recombinant suPAR.
Parameters affecting in vitro oxidation/folding of maurotoxin, a four-disulphide-bridged scorpion toxin
Authors: Di LUCCIO Eric , AZULAY David-Olivier , REGAYA Imed , FAJLOUN Ziad , SANDOZ Guillaume , MANSUELLE Pascal , KHARRAT Ryad , FATHALLAH Mohamed , CARREGA Louis , ESTÈVE Eric , ROCHAT Hervé , De WAARD Michel , SABATIER Jean-Marc
Published: August 2015

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K+ channel subtypes. MTX adopts a disulphide bridge organization of the type C1–C5, C2–C6, C3–C4 and C7–C8, and folds according to the common α/β scaffold reported for other known scorpion toxins. Here we have investigated the process and kinetics of the in vitro oxidation/folding of reduced synthetic L-MTX (L-sMTX, where L-MTX contains only l-amino acid residues). During the oxidation/folding of reduced L-sMTX, the oxidation intermediates were blocked by iodoacetamide alkylation of free cysteine residues, and analysed by MS. The L-sMTX intermediates appeared sequentially over time from the least (intermediates with one disulphide bridge) to the most oxidized species (native-like, four-disulphide-bridged L-sMTX). The mathematical formulation of the diffusion-collision model being inadequate to accurately describe the kinetics of oxidation/folding of L-sMTX, we have formulated a derived mathematical description that better fits the experimental data. Using this mathematical description, we have compared for the first time the oxidation/folding of L-sMTX with that of D-sMTX, its stereoisomer that contains only d-amino acid residues. Several experimental parameters, likely to affect the oxidation/folding process, were studied further; these included temperature, pH, ionic strength, redox potential and concentration of reduced toxin. We also assessed the effects of some cellular enzymes, peptidylprolyl cis–trans isomerase (PPIase) and protein disulphide isomerase (PDI), on the folding pathways of reduced L-sMTX and D-sMTX. All the parameters tested affect the oxidative folding of sMTX, and the kinetics of this process were indistinguishable for L-sMTX and D-sMTX, except when stereospecific enzymes were used. The most efficient conditions were found to be: 50mM Tris/HCl/1.4mM EDTA, pH7.5, supplemented by 0.5mM PPIase and 50units/ml PDI for 0.1mM reduced compound. These data represent the first report of potent stereoselective effects of cellular enzymes on the oxidation/folding of a scorpion toxin.
Targeting of the transcription factor Max during apoptosis: phosphorylation-regulated cleavage by caspase-5 at an unusual glutamic acid residue in position P1
Authors: KRIPPNER-HEIDENREICH Anja , TALANIAN Robert V. , SEKUL Renate , KRAFT Regine , THOLE Hubert , OTTLEBEN Holger , LÜSCHER Bernhard
Published: August 2015

Max is the central component of the Myc/Max/Mad network of transcription factors that regulate growth, differentiation and apoptosis. Whereas the Myc and Mad genes and proteins are highly regulated, Max expression is constitutive and no post-translational regulation is known. We have found that Max is targeted during Fas-induced apoptosis. Max is first dephosphorylated and subsequently cleaved by caspases. Two specific cleavage sites for caspases in Max were identified, one at IEVE10↓S and one at SAFD135↓G near the C-terminus, which are cleaved in vitro by caspase-5 and caspase-7 respectively. Mutational analysis indicates that both sites are also used in vivo. Thus Max represents the first caspase-5 substrate. The unusual cleavage after a glutamic acid residue is observed only with full-length, DNA-binding competent Max protein but not with corresponding peptides, suggesting that structural determinants might be important for this activity. Furthermore, cleavage by caspase-5 is inhibited by the protein kinase CK2-mediated phosphorylation of Max at Ser-11, a previously mapped phosphorylation site in vivo. These findings suggest that Fas-mediated dephosphorylation of Max is required for cleavage by caspase-5. The modifications that occur on Max in response to Fas signalling affect the DNA-binding activity of Max/Max homodimers. Taken together, our findings uncover three distinct processes, namely dephosphorylation and cleavage by caspase-5 and caspase-7, that target Max during Fas-mediated apoptosis, suggesting the regulation of the Myc/Max/Mad network through its central component.
A diacylglycerol-activated Ca2+ channel in PC12 cells (an adrenal chromaffin cell line) correlates with expression of the TRP-6 (transient receptor potential) protein
Authors: TESFAI Yordanos , BRERETON Helen M. , BARRITT Greg J.
Published: August 2015

The structures, and mechanisms of activation, of plasma membrane intracellular-messenger-activated, non-selective cation channels in animal cells are not well understood. The PC12 adrenal chromaffin cell line is a well-characterized example of a nerve cell. In PC12 cells, 1-oleolyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol, initiated the inflow of Ca2+, Mn2+ and Sr2+. Acetylcholine and thapsigargin initiated the inflow of Ca2+ and Mn2+, but not of Sr2+. The activation of bivalent cation inflow by OAG: (i) was mimicked by another membrane-permeant diacylglycerol analogue, 1,2-dioctanoyl-sn-glycerol, but not by the membrane-impermeant analogue 1-stearoyl-2-arachidonyl-sn-glycerol; (ii) was not blocked by staurosporin or chelerythrine, inhibitors of protein kinase C; (iii) was enhanced by RHC80267 and R50922, inhibitors of diacylglycerol lipase and diacylglycerol kinase respectively; and (iv) was inhibited by extracellular Ca2+. When OAG was added after acetylcholine, the effect of OAG on Ca2+ inflow was over-and-above that induced by acetylcholine. 2-Aminoethyl diphenylborate (2-APB) inhibited Ca2+ inflow initiated by either acetylcholine or thapsigargin, but not that initiated by OAG. Flufenamic acid inhibited OAG-initiated, but not acetylcholine-initiated, Ca2+ and Mn2+ inflow. OAG-initiated Ca2+ inflow was less sensitive to inhibition by SK&F96365 than acetylcholine-initiated Ca2+ inflow. In polyadenylated RNA prepared from PC12 cells, mRNA encoding TRP (transient receptor potential) proteins 1–6 was detected by reverse transcriptase (RT)-PCR, and in lysates of PC12 cells the endogenous TRP-6 protein was detected by Western blot analysis. It is concluded that PC12 cells express a diacylglycerol-activated, non-selective cation channel. Expression of this channel function correlates with expression of the TRP-3 and TRP-6 proteins, which have been shown previously to be activated by diacylglycerol when expressed heterologously in animal cells [Hofmann, Obukhov, Schaefer, Harteneck, Gudermann, and Schultz (1999) Nature (London) 397, 259–263].
Molecular cloning, gene organization and expression of rainbow trout (Oncorhynchus mykiss) inducible nitric oxide synthase (iNOS) gene
Authors: WANG Tiehui , WARD Mike , GRABOWSKI Peter , SECOMBES Christopher J.
Published: August 2015

A full-length inducible nitric oxide synthase (iNOS) gene has been sequenced for the first time outside the mammals, and the gene organization compared with that already determined for human iNOS. While there are some differences from the human gene, overall the exons show remarkable conservation in sequence and organization. As in human, the trout iNOS gene has 27 exons, with 18 of the trout exons being identical in size with the equivalent human exons. The cofactor-binding domains are found in the same exons and in some cases are absolutely conserved. Differences include the start of the ORF in exon 3 instead of exon 2, resulting in a deletion at the 5′ end of the trout iNOS protein. Exon 27 also shows a large difference in size and although the trout exon is larger this is due to the length of the 3′-UTR. Several non-mammalian features are notable, and include a conserved potential glycosylation site in chicken and fish, and an insertion at the boundary of exons 20 and 21 in fish. The intron sizes in trout were generally much smaller than in human iNOS, making the trout iNOS gene approximately half the size of the human gene. Analysis of RNA secondary structure revealed two regions with complementarity, which could interfere with reverse transcription. Using a trout fibroblast cell line (RTG-2 cells), it was shown by reverse transcriptase (RT)-PCR that virus infection was a good inducer of iNOS expression. However, when using a combination of Superscript™ II for reverse transcription and primers at the 5′ end of the gene only very weak products were amplified, in contrast with the situation when primers at the 3′ end of the gene were used, or ThermoScript™-derived cDNA was used. The impact of such results on RT-PCR analysis of iNOS expression in trout is discussed.
Two starch-branching-enzyme isoforms occur in different fractions of developing seeds of kidney bean
Authors: HAMADA Shigeki , NOZAKI Kouichi , ITO Hiroyuki , YOSHIMOTO Yasushi , YOSHIDA Hironori , HIRAGA Susumu , ONODERA Shuichi , HONMA Mamoru , TAKEDA Yasuhito , MATSUI Hirokazu
Published: August 2015

The nature and enzymic properties of starch-branching enzyme (SBE) are two of the dominant factors influencing the fine structure of starch. To understand the role of this enzyme's activity in the formation of starch in kidney bean (Phaseolus vulgaris L.), a study was undertaken to identify the major SBE sequences expressed during seed development and to characterize the enzymic properties of the coded recombinant enzymes. Two SBE cDNA species (designated pvsbe2 and pvsbe1) that displayed significant similarity (more than 70%) to other family A and B SBEs respectively were isolated. Northern blot analysis revealed that pvsbe1 and pvsbe2 were differentially expressed during seed development. pvsbe2 showed maximum steady-state transcript levels at the mid-stage of seed maturation, whereas pvsbe1 reached peak levels at a later stage. Western blot analysis with antisera raised against both recombinant proteins (rPvSBE1 and rPvSBE2) showed that these two SBEs were located in different amyloplast fractions of developing seeds of kidney bean. PvSBE2 was present in the soluble fraction, whereas PvSBE1 was associated with the starch granule fraction. The differences in location suggest that these two SBE isoenzymes have different roles in amylopectin synthesis in kidney bean seeds. rPvSBE1 and rPvSBE2 were purified from Escherichia coli and their kinetic properties were determined. The affinity of rPvSBE2 for amylose (Km 1.27mg/ml) was lower than that of rPvSBE1 (0.46mg/ml). The activity of rPvSBE2 was stimulated more than 3-fold in the presence of 0.3M citrate, whereas rPvSBE1 activity was not affected. The implications of the enzymic properties and the distribution of SBEs and amylopectin structure are discussed.
Eicosanoids participate in the regulation of cardiac glucose transport by contribution to a rearrangement of actin cytoskeletal elements
Authors: DRANSFELD Olaf , RAKATZI Irini , SASSON Shlomo , GRUZMAN Arie , SCHMITT Marcus , HÄUSSINGER Dieter , ECKEL Jürgen
Published: August 2015

Intact actin microfilaments are required for insulin-regulated glucose transporter isoform 4 (GLUT4) translocation to the plasma membrane. Lipoxygenase (LO) metabolites have recently been shown to contribute to the regulation of actin cytoskeleton rearrangement. In the present investigation, ventricular cardiomyocytes were used to study the effects of two structurally different LO inhibitors (esculetin and nordihydroguaiaretic acid) on insulin signalling events, glucose uptake, GLUT4 translocation and the actin network organization. Insulin stimulation increased glucose uptake 3-fold in control cells, whereas LO inhibition completely blocked this effect. This was paralleled by a slight reduction in the insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2. However, inhibition of 12-LO did not affect the association of phosphatidylinositol 3-kinase with IRS-1 and the phosphorylation of Akt/protein kinase B in response to insulin. Addition of 12(S)-hydroxyeicosatetraenoic acid almost completely restored the insulin action in cells exposed to nordihydroguaiaretic acid. Insulin stimulation increased cell surface GLUT4 2-fold in control cells, whereas LO inhibition abrogated the insulin-stimulated GLUT4 translocation. LO inhibition induced a prominent disassembly of actin fibres compared with control cells. In conclusion, we show here that 12(S)-hydroxyeicosatetraenoic acid plays a role in the organization of the actin network in cardiomyocytes. LO inhibition blocks GLUT4 translocation without affecting downstream insulin signalling. These data suggest that LO metabolites participate in the regulation of glucose transport by contributing to a rearrangement of actin cytoskeletal elements.
A role for the perlecan protein core in the activation of the keratinocyte growth factor receptor
Authors: GHISELLI Giancarlo , EICHSTETTER Inge , IOZZO Renato V.
Published: August 2015

Perlecan, a widespread heparan sulphate (HS) proteoglycan, is directly involved in the storing of angiogenic growth factors, mostly members of the fibroblast growth factor (FGF) gene family. We have previously shown that antisense targeting of the perlecan gene causes a reduced growth and responsiveness to FGF7 [also known as keratinocyte growth factor (KGF)] in human cancer cells, and that the perlecan protein core interacts specifically with FGF7. In the present paper, we have investigated human colon carcinoma cells in which the perlecan gene was disrupted by targeted homologous recombination. After screening over 1000 clones, we obtained two clones heterozygous for the null mutation with no detectable perlecan, indicating that the other allele was non-functioning. The perlecan-deficient cells grew more slowly, did not respond to FGF7 with or without the addition of heparin, and were less tumorigenic than control cells. Paradoxically, the perlecan-deficient cells displayed increased FGF7 surface binding. However, the perlecan protein core was required for functional activation of the KGF receptor and downstream signalling. Because heparin could not substitute for perlecan, the HS chains are not critical for FGF7-mediated signalling in this cell system. These results provide the first genetic evidence that the perlecan protein core is a molecular entity implicated in FGF7 binding and activation of its receptor.
Regulatory elements in the FBP1 promoter respond differently to glucose-dependent signals in Saccharomyces cerevisiae
Authors: ZARAGOZA Oscar , VINCENT Olivier , GANCEDO Juana M.
Published: August 2015

In Saccharomyces cerevisiae expression of the fructose-1,6-bisphosphatase-encoding gene, FBP1, is controlled by glucose through the upstream activating sequences UAS1 and UAS2 and the upstream repressing sequence URS1 in its promoter. We have studied the regulation of the proteins that could bind to these elements. We have investigated the role of the putative transcription factors Cat8 and Sip4 in the formation of specific DNA–protein complexes with UAS1 and UAS2, and in the expression of UAS1-lacZ and UAS2-lacZ. The expression of CAT8-lacZ and SIP4-lacZ has been also measured in mig1, tup1 or hxk2 mutants, partially refractory to catabolite repression. We conclude that there is no strict correlation between Cat8 and Sip4 expression or in vitro formation of DNA–protein complexes and expression of UAS1-lacZ and UAS2-lacZ. The URS1 element binds the regulatory protein Mig1, which blocks transcription by recruiting the proteins Cyc8 and Tup1. The pattern of complexes of URS1 with nuclear extracts was dependent on the carbon source and on Cyc8, but not on Tup1; it was also affected by the protein kinase Snf1 and by the exportin Msn5. The repression caused by URS1 in a fusion gene was dependent on Mig1, Cyc8 and Tup1, and on the carbon source in the medium; in a snf1 strain the repression observed was independent of the carbon source. Expression of Mig1 could occur in the absence of Snf1 and was moderately sensitive to glucose. We present data showing that different elements of the regulatory system controlling FBP1 responded differently to the concentration of glucose in the medium.
Regulation of phospholipase D isoenzymes by transforming Ras and atypical protein kinase C-ι
Authors: MWANJEWE James , SPITALER Martin , EBNER Maria , WINDEGGER Michaela , GEIGER Markus , KAMPFER Sonja , HOFMANN Johann , ÜBERALL Florian , GRUNICKE Hans H.
Published: August 2015

The activation of phospholipase D (PLD) by transforming Ras is well documented. Although two distinct PLD isoforms, PLD1 and PLD2, have been cloned from mammalian cells, it has remained unclear whether both isoenzymes are activated by Ras and, if this is the case, whether they are stimulated by a common mechanism. In the present study we show that expression of transforming Ras in HC11 mouse mammary epithelial cells enhanced the activity of endogenous PLD. Co-expression of Ras with either PLD1b or PLD2 resulted in elevated activities of both PLD isoenzymes in HC11 cells, indicating that transforming Ras was capable of activating both PLD isoforms in vivo. Ras-induced activation of PLD was resistant to the protein kinase C (PKC) inhibitor GF109203X, which preferentially affects conventional- and novel-type PKCs, but sensitive to Ro-31-8220, which inhibits atypical PKCs more effectively. Co-transfection of atypical PKC-ι with either PLD1b or PLD2 led to a selective activation of PLD2 by PKC-ι, whereas PLD1b was not affected. PLD1b, however, was found to be a potent activator of PKC-ι, whereas PLD2 was less effective in this respect. The data suggest that PKC-ι acts upstream of PLD2 and that PLD1b is implicated in the activation of PKC-ι. The data are discussed as indicating a putative signalling cascade comprising Ras → PLD1b →PKC-ι → PLD2. Evidence for the implication of this pathway in the transcriptional regulation of cyclin D1 is also presented.
Effects of environment on flavin reactivity in morphinone reductase: analysis of enzymes displaying differential charge near the N-1 atom and C-2 carbonyl region of the active-site flavin
Authors: CRAIG Daniel H. , BARNA Terez , MOODY Peter C. E. , BRUCE Neil C. , CHAPMAN Stephen K. , MUNRO Andrew W. , SCRUTTON Nigel S.
Published: August 2015

The side chain of residue Arg238 in morphinone reductase (MR) is located close to the N-1/C-2 carbonyl region of the flavin isoalloxazine ring. During enzyme reduction negative charge develops in this region of the flavin. The positioning of a positively charged side chain in the N-1/C-2 carbonyl region of protein-bound flavin is common to many flavoprotein enzymes. To assess the contribution made by Arg238 in stabilizing the reduced flavin in MR we isolated three mutant forms of the enzyme in which the position of the positively charged side chain was retracted from the N-1/C-2 carbonyl region (Arg238 → Lys), the positive charge was removed (Arg238 → Met) or the charge was reversed (Arg238 → Glu). Each mutant enzyme retains flavin in its active site. Potentiometric studies of the flavin in the wild-type and mutant forms of MR indicate that the flavin semiquinone is not populated to any appreciable extent. Reduction of the flavin in each enzyme is best described by a single Nernst function, and the values of the midpoint reduction potentials (E12) for each enzyme fall within the region of −247±10mV. Stopped-flow studies of NADH binding to wild-type and mutant MR enzymes reveal differences in the kinetics of formation and decay of an enzyme–NADH charge-transfer complex, reflecting small perturbations in active-site geometry. Reduced rates of hydride transfer in the mutant enzymes are attributed to altered geometrical alignment of the nicotinamide coenzyme with FMN rather than major perturbations in reduction potential, and this is supported by an observed entropy–enthalpy compensation effect on the hydride transfer reaction throughout the series of enzymes. The data indicate, in contrast with dogma, that the presence of a positively charged side chain close to the N-1/C-2 carbonyl region of the flavin in MR is not required to stabilize the reduced flavin. This finding may have general implications for flavoenzyme catalysis, since it has generally been assumed that positive charge in this region has a stabilizing effect on the reduced form of flavin.
Calmodulin inhibitors trigger the proteolytic processing of membrane type-1 matrix metalloproteinase, but not its shedding in glioblastoma cells
Authors: ANNABI Borhane , PILORGET Anthony , BOUSQUET-GAGNON Nathalie , GINGRAS Denis , BÉLIVEAU Richard
Published: August 2015

Most transmembrane proteins are subjected to limited proteolysis by cellular proteases, and stimulation of cleavage of membrane proteins by calmodulin (CaM) inhibitors was recently shown. The present study investigated the ability of several CaM inhibitors to induce the proteolytic cleavage of the membrane type-1 matrix metalloproteinase (MT1-MMP) from the cell surface of highly invasive U-87 glioblastoma cells. Although no shedding of a soluble MT1-MMP form was induced by CaM inhibitors in the conditioned media, we showed that these inhibitors induced MT1-MMP proteolytic processing to the 43kDa membrane-bound inactive form that was not correlated with an increase in proMMP-2 activation but rather with an increase in tissue inhibitor of MMPs (TIMP)-2 expression levels. Moreover, this proteolytic processing was sensitive to marimastat suggesting the involvement of MMPs. Interestingly, CaM inhibitors antagonized concanavalin A- and cytochalasin D-induced proMMP-2 activation, and affected the cytoskeletal actin organization resulting in the loss of migratory potential of U-87 glioblastoma cells. Cytoplasmic tail-truncated MT1-MMP constructs expressed in COS-7 cells were also affected by CaM inhibitors suggesting that these inhibitors stimulated MT1-MMP proteolytic processing by mechanisms independent of the CaM–substrate interaction. We also propose that TIMP-2 acts as a negative regulator of MT1-MMP-dependent activities promoted by the action of CaM inhibitors in U-87 glioblastoma cells.