bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of a rat kinase-deficient phosphoinositide 3-kinase, Vps34p, inhibits cathepsin D maturation
Authors: ROW Paula E. , REAVES Barbara J. , DOMIN Jan , LUZIO J. Paul , DAVIDSON Howard W.
Published: August 2015

Lipid kinases and their phosphorylated products are important regulators of many cellular processes, including intracellular membrane traffic. The best example of this is provided by the class III phosphoinositide 3-kinase (PI-3K), Vps34p, which is required for correct targeting of newly synthesized carboxypeptidase Y to the yeast vacuole. A probable mammalian Vps34p orthologue has been previously identified, but its function in the trafficking of lysosomal enzymes has not been resolved. To investigate the possible role(s) of mammalian Vps34p in protein targeting to lysosomes, we have cloned the rat orthologue and overexpressed a kinase-deficient mutant in HeLa cells. Expression of the mutant protein inhibited both maturation of procathepsin D and basal secretion of the precursor. In contrast wortmannin, which also inhibited maturation, caused hypersecretion of the precursor. We propose that mammalian Vps34p plays a direct role in targeting lysosomal enzyme precursors to the endocytic pathway in an analogous fashion to its role in the fusion of early endocytic vesicles with endosomes. We further suggest that inhibition of a wortmannin-sensitive enzyme, other than mammalian Vps34p, is responsible for the failure to recycle unoccupied mannose 6-phosphate receptors to the trans-Golgi network, and consequent hypersecretion of lysosomal enzyme precursors observed in the presence of this drug.
Recognition sequences and structural elements contribute to shedding susceptibility of membrane proteins
Authors: ALTHOFF Katja , MÜLLBERG Jürgen , AASLAND Dorthe , VOLTZ Nicole , KALLEN Karl-Josef , GRÖTZINGER Joachim , ROSE-JOHN Stefan
Published: August 2015

Although regulated ectodomain shedding affects a large panel of structurally and functionally unrelated proteins, little is known about the mechanisms controlling this process. Despite a lack of sequence similarities around cleavage sites, most proteins are shed in response to the stimulation of protein kinase C by phorbol esters. The signal-transducing receptor subunit gp130 is not a substrate of the regulated shedding machinery. We generated several chimaeric proteins of gp130 and the proteins tumour necrosis factor α (TNF-α), transforming growth factor α (TGF-α) and interleukin 6 receptor (IL-6R), which are known to be subject to shedding. By exchanging small peptide sequences of gp130 for cleavage-site peptides of TNF-α, TGF-α and IL-6R we showed that these short sequences conferred susceptibility to spontaneous and phorbol-ester-induced shedding of gp130. Importantly, these chimaeric gp130 proteins were functional, as shown by the phosphorylation of gp130 and the activation of signal transduction and activators of transcription 3 (‘STAT3’) on stimulation with cytokine. To investigate minimal requirements for shedding, truncated cleavage-site peptides of IL-6R were inserted into gp130. The resulting chimaeras were susceptible to shedding and showed the same cleavage pattern as observed in the chimaeras containing the complete IL-6R cleavage site. Surprisingly, we could also generate cleavable chimaeras by exchanging the juxtamembrane sequence of gp130 for the corresponding region of leukaemia inhibitory factor (‘LIF’) receptor, a protein that like gp130 is not subject to regulated or spontaneous shedding. Thus it seems that there is no minimal consensus shedding sequence. We speculate that structural changes allow the access of the protease to a membrane-proximal region, leading to shedding of the protein.
Prenatal and postnatal development of peroxisomal lipid-metabolizing pathways in the mouse
Authors: HUYGHE Steven , CASTEELS Minne , JANSSEN Anneleen , MEULDERS Liesbeth , MANNAERTS Guy P. , DECLERCQ Peter E. , VELDHOVEN Paul P. VAN , BAES Myriam
Published: August 2015

The ontogeny of the following peroxisomal metabolic pathways was evaluated in mouse liver and brain: α-oxidation, β-oxidation and ether phospholipid synthesis. In mouse embryos lacking functional peroxisomes (PEX5-/- knock-out), a deficiency of plasmalogens and an accumulation of the very-long-chain fatty acid C26:0 was observed in comparison with control littermates, indicating that ether phospholipid synthesis and β-oxidation are already active at mid-gestation in the mouse. Northern analysis revealed that the enzymes required for the β-oxidation of straight-chain substrates are present in liver and brain during embryonic development but that those responsible for the degradation of branched-chain substrates are present only in liver from late gestation onwards. The expression pattern of transcripts encoding enzymes of the α-oxidation pathway suggested that α-oxidation is initiated in the liver around birth and is not active in brain throughout development. Remarkably, a strong induction of the mRNA levels of enzymes involved in α-oxidation and β-oxidation was observed around birth in the liver. In contrast, enzyme transcripts that were expressed in brain were present at rather constant levels throughout prenatal and postnatal development. These results suggest that the defective ether phospholipid synthesis and/or peroxisomal β-oxidation of straight-chain fatty acids might be involved in the pathogenesis of the prenatal organ defects in peroxisome-deficient mice and men.
Regulation of mucin gene expression in human tracheobronchial epithelial cells by thyroid hormone
Authors: GRAY Thomas , NETTESHEIM Paul , BASBAUM Carol , KOO Ja-Seok
Published: August 2015

We reported previously that the expression of the gene encoding MUC5AC mucin in human airway epithelial cells is controlled by retinoic acid via the retinoic acid receptor (RAR)-α and that 3,3′,5-tri-iodothyronine (T3) inhibits the expression of MUC5AC. The purpose of the present study was to identify mechanisms mediating the effect of T3. T3 has been shown to inhibit gene expression via several mechanisms, either by enhancing or repressing the transcription of target genes or by the regulation of post-transcriptional events. Results showed that T3 strongly inhibited MUC5AC-driven luciferase activity in normal human tracheobronchial epithelial cells that had been transiently transfected with a MUC5AC–luciferase reporter construct; however, it did not affect MUC5AC mRNA stability. These results indicate that T3 suppresses MUC5AC expression at the transcriptional level. An analysis of deletion constructs showed that deletion of the region downstream of 3kb resulted in markedly decreased levels of MUC5AC transcription in the absence of T3 (i.e. under control conditions) as well as a loss of responsiveness to the inhibitory effects of T3. This suggests that this region might contain elements important for the activation as well as the repression of MUC5AC transcription. To determine whether T3 modulates retinoic-acid-dependent MUC5AC transcription via an alteration in the abundance of retinoid receptor proteins, we examined the type and abundance of these receptors in nuclear extracts of airway epithelial cells grown in the presence or absence of T3. Western blots showed that T3 markedly decreased several types of retinoid receptor while not affecting T3 receptor proteins. Consistent with this finding were gel-shift assays revealing a decrease in RAR–retinoic acid response element complexes obtained from T3-treated cells. We propose that T3 might inhibit retinoid-dependent MUC5AC expression by decreasing retinoid receptor levels and thereby decreasing the transcriptional activation of this gene for mucins.
Hydrolysis of platelet-activating factor by human serum paraoxonase
Authors: RODRIGO Lourdes , MACKNESS Bharti , DURRINGTON Paul N. , HERNANDEZ Antonio , MACKNESS Michael I.
Published: August 2015

Human serum paraoxonase (human PON1) has been shown to be important in the metabolism of phospholipid and cholesteryl ester hydroperoxides, thereby preventing the oxidation of low-density lipoprotein (LDL) and retarding atherogenesis. However, the exact substrate specificity of PON1 has not been established. In the present study we show that purified PON1 hydrolyses platelet-activating factor (PAF). We could find no evidence for contamination of our preparation with authentic platelet-activating-factor acetylhydrolase (PAFAH) by immunoblotting with a PAFAH monoclonal antibody or by sequencing the purified protein. In addition the specific PAFAH inhibitor SB-222657 did not affect the ability of PON1 to hydrolyse PAF (30.1±;2.8µmol/min per mg of protein with no inhibitor; 31.4±;2.2µmol/min per mg of protein with 100nM inhibitor) or phenyl acetate (242.6±;30.8 versus 240.8±;31.5µmol/min per mg of protein with and without inhibitor respectively). SB-222657 was also unable to inhibit PAF hydrolysis by isolated human high-density lipoprotein (HDL), but completely abolished the activity of human LDL. Ostrich (Struthio camelus) HDL, which does not contain PON1, was unable to hydrolyse PAF. These data provide evidence that PON1 may limit the action of this bioactive pro-inflammatory phospholipid.
Changes in intracellular calpastatin localization are mediated by reversible phosphorylation
Authors: AVERNA Monica , DE TULLIO Roberta , PASSALACQUA Mario , SALAMINO Franca , PONTREMOLI Sandro , MELLONI Edon
Published: August 2015

We have previously reported that, in neuroblastoma LAN-5 cells, calpastatin is in an aggregated state, close to the cell nucleus [De Tullio, Passalacqua, Averna, Salamino, Melloni and Pontremoli (1999) Biochem. J. 343, 467–472]. In the present paper, we demonstrate that aggregated calpastatin is predominantly in a phosphorylated state. An increase in intracellular free [Ca2+] induces both dephosphorylation of calpastatin, through the action of a phosphoprotein phosphatase, and its redistribution as a soluble inhibitor species. cAMP, but not PMA-induced phosphorylation, reverses calpastatin distribution favouring its aggregation. This intracellular reversible mechanism, regulating the level of cytosolic calpastatin, could be considered a strategy through which calpain can escape calpastatin inhibition, especially during earlier steps of its activation process.
Cell hydration controls autophagosome formation in rat liver in a microtubule-dependent way downstream from p38MAPK activation
Authors: VOM DAHL Stephan , DOMBROWSKI Frank , SCHMITT Marcus , SCHLIESS Freimut , PFEIFER Ulrich , HÄUSSINGER Dieter
Published: August 2015

Autophagic proteolysis in rat liver is under the control of the cellular hydration state. Because the morphological site of swelling-dependent proteolysis regulation has not yet been identified, the formation of autophagosomes was investigated with transmission electron microscopy in slices from perfused livers. In livers from fed rats, hypo-osmotic exposure (185mosmol/l) led within 30min to a decrease in fractional cytoplasmic autophagosome volume that was sensitive to colchicine and p38MAPK inhibition. Similarly, the decrease in autophagosome volume, but not the increase in cell volume caused by insulin or glutamine/glycine, was strongly inhibited by colchicine and SB 203580, an inhibition of p38MAPK activation. Immune complex assays from perfused liver showed that hypo-osmotic activation of p38MAPK was not inhibited by colchicine. Further, experiments using confocal laser microscopy in cultivated hepatocytes incubated with mouse-derived anti-(α-tubulin) showed that microtubular structures were not influenced by the inhibition of p38MAPK by SB 203580. It is concluded that the sequestration of autophagic vacuoles is a major site of proteolysis regulation by cell hydration. Swelling-induced activation of p38MAPK is required for this process and occurs upstream of the putative microtubule regulation site.
Crystal structure of a monocotyledon (maize ZMGlu1) β-glucosidase and a model of its complex with p-nitrophenyl β-d-thioglucoside
Authors: CZJZEK Mirjam , CICEK Muzaffer , ZAMBONI Véronique , BURMEISTER Wim P. , BEVAN David R. , HENRISSAT Bernard , ESEN Asim
Published: August 2015

The maize β-glucosidase isoenzymes ZMGlu1 and ZMGlu2 hydrolyse the abundant natural substrate DIMBOAGlc (2-O-β-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one), whose aglycone DIMBOA (2,4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) is the major defence chemical protecting seedlings and young plant parts against herbivores and other pests. The two isoenzymes hydrolyse DIMBOAGlc with similar kinetics but differ from each other and their sorghum homologues with respect to specificity towards other substrates. To gain insights into the mechanism of substrate (i.e. aglycone) specificity between the two maize isoenzymes and their sorghum homologues, ZMGlu1 was produced in Escherichia coli, purified, crystallized and its structure solved at 2.5 Å resolution by X-ray crystallography. In addition, the complex of ZMGlu1 with the non-hydrolysable inhibitor p-nitrophenyl β-d-thioglucoside was crystallized and, based on the partial electron density, a model for the inhibitor molecule within the active site is proposed. The inhibitor is located in a slot-like active site where its aromatic aglycone is held by stacking interactions with Trp-378. Whereas some of the atoms on the non-reducing end of the glucose moiety can be modelled on the basis of the electron density, most of the inhibitor atoms are highly disordered. This is attributed to the requirement of the enzyme to accommodate two different species, namely the substrate in its ground state and in its distorted conformation, for catalysis.
Caveolae/lipid rafts in fibroblast-like synoviocytes: ectopeptidase-rich membrane microdomains
Authors: RIEMANN Dagmar , HANSEN Gert H. , NIELS-CHRISTIANSEN Lise-Lotte , THORSEN Evy , IMMERDAL Lissi , SANTOS Alexander Navarrete , KEHLEN Astrid , LANGNER Jürgen , DANIELSEN E. Michael
Published: August 2015

Membrane peptidases play important roles in cell activation, proliferation and communication. Human fibroblast-like synoviocytes express considerable amounts of aminopeptidase N/CD13, dipeptidyl peptidase IV/CD26, and neprilysin/CD10, transmembrane proteins previously proposed to be involved in the regulation of intra-articular levels of neuropeptides and chemotactic mediators as well as in adhesion and cell–cell interactions. Here, we report these peptidases in synoviocytes to be localized predominantly in glycolipid- and cholesterol-rich membrane microdomains known as ‘rafts’. At the ultrastructural level, aminopeptidase N/CD13 and dipeptidyl peptidase IV/CD26 were found in caveolae, in particular in intracellular yet surface-connected vesicle-like structures and ‘rosettes’ made up of several caveolae. In addition, clusters of peptidases were seen at the cell surface in flat patches ranging in size from about 60 to 160nm. Cholesterol depletion of synoviocytes by methyl-β-cyclodextrin disrupted > 90% of the caveolae and reduced the raft localization of aminopeptidase N/CD13 without affecting Ala-p-nitroanilide-cleaving activity of confluent cell cultures. In co-culture experiments with T-lymphocytes, cholesterol depletion of synoviocytes greatly reduced their capability to induce an early lymphocytic expression of aminopeptidase N/CD13. We propose caveolae/rafts to be peptidase-rich ‘hot-spot’ regions of the synoviocyte plasma membrane required for functional cell–cell interactions with lymphocytes. The peptidases may act in concert with other types of proteins such as receptors and signal transducers localized in these specialized membrane domains.
Sucrose and light regulation of a cold-inducible UDP-glucose pyrophosphorylase gene via a hexokinase-independent and abscisic acid-insensitive pathway in Arabidopsis
Authors: CIERESZKO Iwona , JOHANSSON Henrik , KLECZKOWSKI Leszek A.
Published: August 2015

UDP-glucose pyrophosphorylase (UGPase) is a key enzyme producing UDP-glucose, which is involved in an array of metabolic pathways concerned with, among other functions, the synthesis of sucrose and cellulose. An Arabidopsis thaliana UGPase-encoding gene, Ugp, was profoundly up-regulated by feeding sucrose to the excised leaves and by an exposure of plants to low temperature (5°C). The UGPase activity and its protein content also increased under conditions of sucrose feeding and exposure to cold. The sucrose effect on Ugp was apparently specific and was mimicked by exposure of dark-adapted leaves to light. Drought and O2 deficiency had some down-regulating effects on expression of Ugp. The sugar-signalling pathway for Ugp regulation was independent of hexokinase, as was found by using transgenic plants with increased and decreased expression of the corresponding gene. Subjecting mutants deficient in abscisic acid (ABA) to cold stress conditions had no effect on Ugp expression profiles. Okadaic acid was a powerful inhibitor of Ugp expression, whereas it up-regulated the gene encoding sucrose synthase (Sus1), indicating distinct transduction pathways in transmitting the sugar signal for the two genes in A. thaliana. We suggest that Ugp gene expression is mediated via a hexokinase-independent and ABA-insensitive pathway that involves an okadaic acid-responsive protein phosphatase. The data point towards Ugp as a possible regulatory entity that is closely involved in the homoeostatic readjustment of plant responses to environmental signals.
Membrane recruitment of DOCK180 by binding to PtdIns(3,4,5)P3
Authors: KOBAYASHI Shin , SHIRAI Toshiyuki , KIYOKAWA Etsuko , MOCHIZUKI Naoki , MATSUDA Michiyuki , FUKUI Yasuhisa
Published: August 2015

DOCK180 was originally identified as one of two major proteins bound to the Crk oncogene product and became an archetype of the CDM family of proteins, including Ced-5 of Caenorhabditis elegans and Mbc of Drosophila melanogaster. Further study has suggested that DOCK180 is involved in the activation of Rac by the CrkII–p130Cas complex. With the use of deletion mutants of DOCK180, we found that the C-terminal region containing a cluster of basic amino acids was required for binding to and activation of Rac. This region showed high amino-acid sequence similarity to the consensus sequence of the phosphoinositide-binding site; this led us to examine whether this basic region binds to phosphoinositides. For this purpose we used PtdIns(3,4,5)P3-APB beads, as reported previously [Shirai, Tanaka, Terada, Sawada, Shirai, Hashimoto, Nagata, Iwamatsu, Okawa, Li et al. (1998) Biochim. Biophys. Acta 1402, 292–302]. By using various competitors, we demonstrated the specific binding of DOCK180 to PtdIns(3,4,5)P3. The expression of active phosphoinositide 3-kinase (PI-3K) did not enhance a DOCK180-induced increase in GTP-Rac; however, the expression of PI-3K translocated DOCK180 to the plasma membrane. Thus DOCK180 contained a phosphoinositide-binding domain, as did the other guanine nucleotide exchange factors with a Dbl homology domain, and was translocated to the plasma membrane on the activation of PI-3K.
The molecular chaperone α-crystallin is in kinetic competition with aggregation to stabilize a monomeric molten-globule form of α-lactalbumin
Authors: LINDNER Robyn A. , TREWEEK Teresa M. , CARVER John A.
Published: August 2015

In vivo, α-crystallin and other small heat-shock proteins (sHsps) act as molecular chaperones to prevent the precipitation of ‘substrate’ proteins under stress conditions through the formation of a soluble sHsp–substrate complex. Using a range of different salt conditions, the rate and extent of precipitation of reduced α-lactalbumin have been altered. The interaction of α-crystallin with reduced α-lactalbumin under these various salt conditions was then studied using a range of spectroscopic techniques. Under conditions of low salt, α-lactalbumin aggregates but does not precipitate. α-Crystallin is able to prevent this aggregation, initially by stabilization of a monomeric molten-globule species of α-lactalbumin. It is proposed that this stabilization occurs through weak transient interactions between α-crystallin and α-lactalbumin. Eventually a stable, soluble high-molecular-mass complex is formed between the two proteins. Thus it appears that a tendency for α-lactalbumin to aggregate (but not necessarily precipitate) is the essential requirement for α-crystallin–α-lactalbumin interaction. In other words, α-crystallin interacts with a non-aggregated form of the substrate to prevent aggregation. The rate of precipitation of α-lactalbumin is increased significantly in the presence of Na2SO4 compared with NaCl. However, in the former case, α-crystallin is unable to prevent this aggregation and precipitation except in the presence of a large excess of α-crystallin, i.e. at mass ratios more than 10 times greater than in the presence of NaCl. It is concluded that a kinetic competition exists between aggregation and interaction of unfolding proteins with α-crystallin.
Heparin-binding epidermal-growth-factor-like growth factor gene expression is induced by scrape-wounding epithelial cell monolayers: involvement of mitogen-activated protein kinase cascades
Authors: ELLIS Peter D. , HADFIELD Kathryn M. , PASCALL John C. , BROWN Kenneth D.
Published: August 2015

Peptide growth factors can promote the cell migration and proliferation that is needed to repair epithelia after mechanical or chemical injury. We report here that scrape-wounding rat intestinal epithelial (RIE-1) cell monolayers caused a rapid increase in levels of heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) mRNA, with a maximal response at approx. 1h. Hybridization in situ showed that transcript induction occurred primarily in cells at or near wound borders. The increase in HB-EGF mRNA was preceded by activation of the p42 mitogen-activated protein kinase (MAPK) in the wounded cell cultures. Moreover, the induction of HB-EGF mRNA was blocked by PD098059 and U0126, inhibitors that prevent the activation of p42/p44 MAPKs and extracellular signal-regulated protein kinase 5 (ERK5). Both p42 MAPK activation and HB-EGF mRNA induction were inhibited by genistein, indicating a requirement for an upstream tyrosine kinase activity. In contrast, neither response was affected by inhibition of phosphoinositide 3-kinase activity, down-regulation of protein kinase C, or disruption of the actin cytoskeleton with cytochalasin B. We conclude that scrape-wounding epithelial cell monolayers induces HB-EGF mRNA expression by a mechanism that most probably requires p42/p44 MAPK activation, although we cannot exclude a role for ERK5. Our results suggest a physiological role for locally synthesized HB-EGF in promoting epithelial repair after injury.
Bioactivity of [6R]-5-formyltetrahydrofolate, an unnatural isomer, in humans and Enterococcus hirae, and cytochrome c oxidation of 10-formyltetrahydrofolate to 10-formyldihydrofolate
Authors: BAGGOTT Joseph E. , ROBINSON Constance B. , JOHNSTON Kelly E.
Published: August 2015

The bio-inactive C-6 isomer, [6R]-5-formyl-tetrahydrofolate (5-HCO-H4F), is not found in Nature. An oral dose of 13.5µmol of [6R]-5-HCO-H4F in humans results in the appearance of the naturally occurring [6S]-5-methyl-tetrahydrofolate and relatively large amounts of other bioactive folates in plasma. The removal of the asymmetry at C-6 could account for these results. Two oxidized cytochrome c [cyt c (Fe3+)] molecules oxidize one 10-formyl-tetrahydrofolate (10-HCO-H4F) with second-order kinetics and a rate constant of 1.3×;104 M-1·s-1. The folate product of this oxidation reaction is 10-formyl-dihydrofolate (10-HCO-H2F), which has no C-6 asymmetric centre and is therefore bioactive. The folate-requiring bacterium, Enterococcus hirae, does not normally biosynthesize cytochromes but does so when given an exogenous source of haem (e.g. haemin). E. hirae grown in haemin-supplemented media for 3 days utilizes both [6R]- and [6S]-5-HCO-H4F in contrast to that grown in control medium, which utilizes only the [6S] isomer. Since known chemical reactions form 10-HCO-H4F from 5-HCO-H4F, the unusually large rate constant for the oxidation of 10-HCO-H4F by cyt c (Fe3+) may account for the unexpected bioactivity of [6R]-5-HCO-H4F in humans and in E. hirae grown in haemin-containing media. We used an unnatural C-6 folate isomer as a tool to reveal the possible in vivo oxidation of 10-HCO-H4F to 10-HCO-H2F; however, nothing precludes this oxidation from occurring in vivo with the natural C-6 isomer.
Gene dose effect reveals no Gs-coupled A2A adenosine receptor reserve in murine T-lymphocytes: studies of cells from A2A-receptor-gene-deficient mice
Authors: ARMSTRONG John M. , CHEN Jiang Fan , SCHWARZSCHILD Michael A. , APASOV Sergey , SMITH Patrick T. , CALDWELL Charles , CHEN Pearl , FIGLER Heidi , SULLIVAN Gail , FINK Steven , LINDEN Joel , SITKOVSKY Michail
Published: August 2015

Agonist binding to extracellular A2A adenosine receptors (A2ARs) inhibits the activation of virtually all tested functions of T-cells and can induce apoptosis in thymocytes. The evaluation of levels of expression of these immunosuppressive receptors is expected to clarify whether the absence of spare A2ARs (no ‘receptor reserve’) might be one of the mechanisms of attenuation of the effects of extracellular adenosine on T-cells. A2A transcript is found in T-cells and functional receptors can be demonstrated, but the density of receptor on T-cells is too low to be detected by radioligand binding. Studies of direct radioligand binding to murine brain with the selective A2AR agonist [3H]CGS21680 (2-{4-[(2-carboxyethyl)-phenyl]ethylamino}-5′-N-ethylcarboxamidoadenosine) established that striata levels of A2AR are virtually absent from A2A knock-out mice. Mice that are heterozygous (A2AR+/-) for the A2AR express significantly decreased levels of A2AR. To test for the presence of spare receptors in T-cells we took advantage of this gene dose effect and examined whether the decrease in the number of receptors in thymocytes from A2AR+/- mice was proportionately reflected in a decrease in the functional cAMP response of T-cells to adenosine. cAMP accumulation and apoptosis induced by adenosine and by A2AR agonist are of a lower magnitude in T-cells from A2AR+/- heterozygous mice than in T-cells from A2AR+/+ littermate control mice. These results indicate that there is no A2AR reserve in murine T-cells. Strongly decreased adenosine-triggered cAMP increases were detected in thymocytes from A2AR-/- mice, suggesting that A2B adenosine receptors cannot fully compensate for the loss of A2ARs in murine T-cells. We conclude that the number of A2ARs is the limiting factor in determining the maximal cAMP response of T-lymphocytes to extracellular adenosine, thereby minimizing the immunosuppressive effects of extracellular adenosine.
LIM-kinase 2 induces formation of stress fibres, focal adhesions and membrane blebs, dependent on its activation by Rho-associated kinase-catalysed phosphorylation at threonine-505
Authors: AMANO Toru , TANABE Kazuyo , ETO Tomonori , NARUMIYA Shuh , MIZUNO Kensaku
Published: August 2015

LIM-kinase 1 and 2 (LIMK1 and LIMK2) phosphorylate cofilin and induce actin cytoskeletal reorganization. LIMK1 is activated by Rho-associated, coiled-coil-forming protein kinase (ROCK) and p21-activated kinase 1 (PAK1), but activation mechanisms and cellular functions of LIMK2 have remained to be determined. We report here that LIMK1 and LIMK2 phosphorylate both cofilin and actin-depolymerizing factor (ADF) specifically at Ser-3 and exhibit partially distinct substrate specificity when tested using site-directed cofilin mutants as substrates. We also show that LIMK2 is activated by ROCK by phosphorylation at Thr-505 within the activation loop. Wild-type LIMK2, but not its mutant (T505V) with replacement of Thr-505 by Val, was activated by ROCK in vitro and in vivo. LIMK2 mutants with replacement of Thr-505 by one or two Glu residues (T505E or T505EE) increased the kinase activity about 3.6-fold but were not further activated by ROCK. When expressed in HeLa cells, wild-type LIMK2, but not the T505V mutant, induced the formation of stress fibres, focal adhesions and membrane blebs. Furthermore, inhibitors of Rho and ROCK significantly suppressed LIMK2-induced stress fibres and membrane blebs. These results suggest that LIMK2 functions downstream of the Rho-ROCK signalling pathway and plays a role in reorganization of actin filaments and membrane structures, by phosphorylating cofilin/ADF proteins.
Roles of aggrecan domains in biosynthesis, modification by glycosaminoglycans and product secretion
Authors: KIANI Chris , LEE Vivian , CAO Liu , CHEN Liwen , WU Yaojiong , ZHANG Yaou , ADAMS Mark E. , YANG Burton B.
Published: August 2015

Aggrecan is a member of the chondroitin sulphate (CS) proteoglycan family, which also includes versican/PG-M, neurocan and brevican. Members of this family exhibit structural similarity: a G1 domain at the N-terminus and a G3 domain at the C-terminus, with a central sequence for modification by CS chains. A unique feature of aggrecan is the insertion of three additional domains, an inter-globular domain (IGD), a G2 domain and a keratan sulphate (KS) domain (sequence modified by KS chains), between the G1 domain and the CS domain (sequence modified by CS chains). The G1 and G3 domains have been implicated in product secretion, but G2, although structurally similar to the tandem repeats of G1, performs an unknown function. To define the functions of each aggrecan domain in product processing, we cloned and expressed these domains in various combinations in COS-7 cells. The results indicated that the G3 domain enhanced product secretion, alone or in combination with the KS or CS domain, and promoted glycosaminoglycan (GAG) chain attachment. Constructs containing the G1 domain were not secreted. Addition of a CS domain sequence to G1 reduced this inhibition, but GAG chain attachment was still decreased. The potential GAG chain attachment site in the IGD was occupied by GAGs, and IGD product was secreted efficiently. The KS domain was modified by GAG chains and secreted. Finally, the G2 domain was expressed but not secreted, and inhibited secretion of the IGD when expressed as an IGD–G2 combination.
Characterization of the Mycobacterium tuberculosis H37Rv alkyl hydroperoxidase AhpC points to the importance of ionic interactions in oligomerization and activity
Authors: CHAUHAN Radha , MANDE Shekhar C.
Published: August 2015

An alkyl hydroperoxidase (AhpC) has been found frequently to be overexpressed in isoniazid-resistant strains of Mycobacterium tuberculosis. These strains have an inactivated katG gene encoding a catalase peroxidase, which might render mycobacteria susceptible to the toxic peroxide radicals, thus leading to the concomitant overexpression of the AhpC. Although the overexpressed AhpC in isoniazid-resistant strains of M. tuberculosis may not directly participate in isoniazid action, AhpC might still assist M. tuberculosis in combating oxidative damage in the absence of the catalase. Here we have attempted to characterize the AhpC protein biochemically and report its functional and oligomerization properties. The alkyl hydroperoxidase of M. tuberculosis is unique in many ways compared with its well-characterized homologues from enteric bacteria. We show that AhpC is a decameric protein, composed of five identical dimers held together by ionic interactions. Dimerization of individual subunits takes place through an intersubunit disulphide linkage. The ionic interactions play a significant role in enzymic activity of the AhpC protein. The UV absorption spectrum and three-dimensional model of AhpC suggest that interesting conformational changes may take place during oxidation and reduction of the intersubunit disulphide linkage. In the absence of the partner AhpF subunit in M. tuberculosis, the mycobacterial AhpC might use small-molecule reagents, such as mycothiol, for completing its enzymic cycle.
Activity of hepatocyte nuclear factor 1α and hepatocyte nuclear factor 1β isoforms is differently affected by the inhibition of protein phosphatases 1/2A
Authors: CARRIÈRE Véronique , LACASA Michel , ROUSSET Monique
Published: August 2015

Phosphorylation/dephosphorylation processes are known to control the activity of several transcription factors. The nutrition-dependent expression of sucrase–isomaltase and Na+/glucose co-transporter 1, two proteins implicated in the intestinal absorption of glucose, has been shown to be closely related to modifications of hepatocyte nuclear factor 1 (HNF1) activity. This study was conducted to determine whether phosphorylation/dephosphorylation processes could control HNF1 activity. We show that expression of the gene encoding sucrase–isomaltase is inhibited in the enterocytic Caco-2 clone TC7 by okadaic acid at a concentration that is known to inhibit protein phosphatases 1/2A and that does not affect cell viability. At the same concentration, phosphorylation of the HNF1α and HNF1β isoforms is greatly enhanced and their DNA-binding capacity is decreased. The phosphorylation state of HNF1β isoforms directly affects their DNA-binding capacity. In contrast, the decreased DNA-binding activity of the HNF1α isoforms, which was observed after the inhibition of protein phosphatases 1/2A, is due to a net decrease in their total cellular and nuclear amounts. Such an effect results from a decrease in both the HNF1α mRNA levels and the half-life of the protein. This is the first evidence for the implication of protein phosphatases 1/2A in the control of the activity of HNF1 isoforms. Moreover, these results emphasize a physiological role for the balance between phosphatases and kinases in the nutrition-dependent regulation of HNF1-controlled genes.
Bi-directional effects of the elevation of intracellular calcium on the expression of inducible nitric oxide synthase in J774 macrophages exposed to low and to high concentrations of endotoxin
Authors: KORHONEN Riku , KANKAANRANTA Hannu , LAHTI Aleksi , LÄHDE Mari , KNOWLES Richard G. , MOILANEN Eeva
Published: August 2015

Nitric oxide produced through the action of inducible nitric oxide synthase (iNOS) is an important mediator in immune responses of the host. Various extracellular factors, including inflammatory stimuli, affect intracellular free Ca2+ levels ([Ca2+]i), modulating cellular signalling and gene expression. In the present study we investigated the effects of increased [Ca2+]i on NO production through the iNOS pathway in J774 macrophages. Thapsigargin (TG), a Ca2+-ATPase inhibitor, and the Ca2+ ionophore A23187 were used as tools to induce an increase in [Ca2+]i in the cytosol. This increase was confirmed by the fura 2 method. The production of NO was measured as accumulated nitrite in the cell culture medium; iNOS protein and iNOS mRNA were detected by Western blotting and reverse-transcriptase-mediated PCR respectively. The activation of nuclear factor κB (NF-κB) was investigated by electrophoretic mobility-shift assay. TG (100nM) induced a marked synthesis of iNOS mRNA, iNOS protein and NO in cells primed with a low concentration of endotoxin [lipopolysaccharide (LPS) 1ng/ml], which on its own induced barely detectable NO synthesis. Stimulation by a high concentration of LPS (100ng/ml) induced a marked expression of iNOS and NO production. Under these conditions, treatment with TG hindered the synthesis of iNOS protein and NO production by accelerating the degradation of iNOS mRNA. Treatment with TG (100nM) did not affect the NF-κB activity induced by low (1ng/ml) or high (100ng/ml) concentrations of LPS. Viability of the cells was confirmed by the 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyaniline (‘XTT’) method; apoptosis was ruled out by propidium iodide staining and flow cytometry. A23187 (1µM) also transiently increased [Ca2+]i and had opposite effects on NO production depending on the LPS concentration. Our results show that increased [Ca2+]i induced the stimulation or suppression of NO production through iNOS in macrophages depending on the state of cell activation. These findings suggest that the receptor-mediated increase in [Ca2+]i might be an important factor in the control of the balance between the up-regulation and down-regulation of inflammatory genes, including that encoding iNOS, depending on the phase of the inflammatory response.
Interaction of phospholipase D1 with a casein-kinase-2-like serine kinase
Authors: GANLEY Ian G. , WALKER Stephanie J. , MANIFAVA Maria , LI Donxia , BROWN H. Alex , KTISTAKIS Nicholas T.
Published: August 2015

Phospholipase D (PLD)1 was phosphorylated invivo and by an associated kinase invitro following immunoprecipitation. Both phosphorylation events were greatly reduced in a catalytically inactive point mutant in which the serine residue at position 911 was converted into alanine (S911A). The kinase could be enriched from detergent-extracted brain membranes and bind and phosphorylate PLD1 that was immunoprecipitated from COS-7 cells. Using in-gel kinase assays we determined that the size of the kinase is approximately 40kDa and that PLD1 is more effective than S911A in binding the kinase. Preliminary analysis of the phosphorylation sites on PLD1 suggested that the kinase belongs to the casein kinase 2 (CK2) family. Consistent with this, we found that the kinase could utilize GTP, and could be inhibited by heparin and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB). Membrane fractions from Chinese hamster ovary (CHO) cell lines that inducibly express PLD1 contained an endogenous kinase activity that phosphorylated PLD1 using GTP and was inhibited by DRB. Direct evidence that the kinase is CK2 came from observations that immunoprecipitates using PLD1 antibodies contained immunoreactive CK2α, and immunoprecipitates using CK2α antibodies contained immunoreactive PLD1. Co-expression of PLD1 in COS-7 cells with the two recombinant CK2 subunits, α or β, suggests that the association of PLD1 with the kinase is through the β subunit. Supporting this, phosphorylation of PLD1 by purified recombinant CK2α was enhanced by purified recombinant CK2β. Assays measuring PLD1 catalytic activity following phosphorylation by CK2 suggest that this phosphorylation event does not influence PLD1-mediated hydrolysis of phosphatidylcholine invitro.
Site-directed mutations in the mitochondrially encoded subunits I and III of yeast cytochrome oxidase
Authors: MEUNIER Brigitte
Published: August 2015

Since yeast is amenable to mitochondrial transformation, designed mutations can be introduced in the mitochondrially encoded subunits of the respiratory complexes. In the present work, six mutations have been introduced by the biolistic method into yeast (Saccharomyces cerevisiae) cytochrome oxidase subunits I and III. The effects of these mutations on respiratory growth competence, cytochrome oxidase activity and optical properties were then characterized. Firstly, the conserved glutamate Glu-243 in the D-channel of subunit I was replaced by an asparagine or an aspartate residue. The effects of the mutations showed that Glu-243, which is essential for proton movement in bacterial oxidases, is also required for the activity of the eukaryotic enzyme. Secondly, four mutations associated with human disease were introduced in yeast, allowing detailed analysis of their deleterious effects on cytochrome oxidase function: Met-273 → Thr, Ile-280 → Thr and Gly-317 → Ser, affecting residues located in or near the K-channel in subunit I, and a short in-frame deletion comprising residues Phe-102 to Phe-106 in subunit III (∆F102–F106). The subunit III mutation was highly deleterious and abolished enzyme assembly. The change Gly-317 → Ser had no effect on respiratory function. However, mutations Met-273 → Thr and Ile-280 → Thr were mildly deleterious, decreased cytochrome oxidase activity and slightly perturbed the properties of the binuclear centre.
Epicatechin and its in vivo metabolite, 3′-O-methyl epicatechin, protect human fibroblasts from oxidative-stress-induced cell death involving caspase-3 activation
Authors: SPENCER Jeremy P. E. , SCHROETER Hagen , KUHNLE Gunter , SRAI S. Kaila S. , TYRRELL Rex M. , HAHN Ulrich , RICE-EVANS Catherine
Published: August 2015

There is considerable current interest in the cytoprotective effects of natural antioxidants against oxidative stress. In particular, epicatechin, a major member of the flavanol family of polyphenols with powerful antioxidant properties in vitro, has been investigated to determine its ability to attenuate oxidative-stress-induced cell damage and to understand the mechanism of its protective action. We have induced oxidative stress in cultured human fibroblasts using hydrogen peroxide and examined the cellular responses in the form of mitochondrial function, cell-membrane damage, annexin-V binding and caspase-3 activation. Since one of the major metabolites of epicatechin in vivo is 3′-O-methyl epicatechin, we have compared its protective effects with that of epicatechin. The results provide the first evidence that 3′-O-methyl epicatechin inhibits cell death induced by hydrogen peroxide and that the mechanism involves suppression of caspase-3 activity as a marker for apoptosis. Furthermore, the protection elicited by 3′-O-methyl epicatechin is not significantly different from that of epicatechin, suggesting that hydrogen-donating antioxidant activity is not the primary mechanism of protection.
The pancreas-specific protein disulphide-isomerase PDIp interacts with a hydroxyaryl group in ligands
Authors: KLAPPA Peter , FREEDMAN Robert B. , LANGENBUCH Martina , LAN Michael S. , ROBINSON Gary K. , RUDDOCK Lloyd W.
Published: August 2015

Using a cross-linking approach, we have recently demonstrated that radiolabelled model peptides or misfolded proteins specifically interact in vitro with two members of the protein disulphide- isomerase family, namely PDI and PDIp, in a crude extract from sheep pancreas microsomes. In addition, we have shown that tyrosine and tryptophan residues within a peptide are the recognition motifs for the binding to PDIp. Here we examine non-peptide ligands and present evidence that a hydroxyaryl group is a structural motif for the binding to PDIp; simple constructs containing this group and certain xenobiotics and phytoestrogens, which contain an unmodified hydroxyaryl group, can all efficiently inhibit peptide binding to PDIp. To our knowledge this is the first time that the recognition motif of a molecular chaperone or folding catalyst has been specified as a simple chemical structure.
Thyrotropin modifies activation of nuclear factor κB by tumour necrosis factor α in rat thyroid cell line
Authors: KIKUMORI Toyone , KAMBE Fukushi , NAGAYA Takashi , FUNAHASHI Hiroomi , SEO Hisao
Published: August 2015

We have recently demonstrated that nuclear factor κB (NF-κB) mediates the tumour necrosis factor α (TNF-α)-dependent expression of the gene encoding interleukin 6 (IL-6) in rat thyroid FRTL-5 cells cultured in the presence of thyrotropin (TSH). In the present study we investigated how TSH is involved in the activation of NF-κB by TNF-α in the cells. Electrophoretic mobility-shift assay revealed that, in the absence of TSH, TNF-α activated a single protein–DNA complex containing the p50 subunit but not other NF-κB subunits such as p65. In contrast, two distinct protein–DNA complexes were activated in the presence of TSH: the faster-migrating complex contained only p50 subunit; the slower-migrating complex consisted of p65–p50heterodimer. This TSH effect was mimicked by forskolin and thyroid-stimulating antibodies obtained from patients with Graves's disease, suggesting that an increase in intracellular cAMP is responsible for the induction of different NF-κBs by TNF-α. A transient transfection study with a luciferase reporter gene driven by multimerized NF-κB sites demonstrated that TNF-α increased the luciferase activities only in the presence of TSH, and that this increase was inhibited by the co-transfection of mutant p65, which prevented the function of wild-type p65 in a dominant-negative manner. Accordingly, TNF-α activated the expression of the IL-6 gene in the presence of TSH but not in its absence. Although the expression of the p105 gene, another known target for NF-κB, was increased by TNF-α in the absence of TSH, the presence of TSH further increased the mRNA level. Taken together, these observations indicate that the presence of TSH is crucial for the NF-κB-mediated actions of TNF-α on thyroid follicular cells.
Cyclosporin A reduces canalicular membrane fluidity and regulates transporter function in rats
Authors: YASUMIBA Shigeyuki , TAZUMA Susumu , OCHI Hidenori , CHAYAMA Kazuaki , KAJIYAMA Goro
Published: August 2015

Changes of the biliary canalicular membrane lipid content can affect membrane fluidity and biliary lipid secretion in rats. The immunosuppressant cyclosporin A is known to cause intrahepatic cholestasis. This study investigated whether cyclosporin A influenced canalicular membrane fluidity by altering membrane phospholipids or transporter expression. In male Sprague–Dawley rats, a bile-duct cannula was inserted to collect bile, and sodium taurocholate was infused (100nmol/min per 100g) for 60min. During steady-state taurocholate infusion, cyclosporin A (20mg/kg) or vehicle was injected intravenously and then bile was collected for 80min. After killing the rats, canalicular membrane vesicles were prepared. Expression of canalicular membrane transporters was assessed by Western blotting and canalicular membrane vesicle fluidity was estimated by fluorescence polarization. Cyclosporin A reduced biliary lipid secretion along with a disproportionate reduction of lipids relative to bile acids. Cyclosporin A significantly decreased canalicular membrane fluidity along with an increase of the cholesterol/phospholipid molar ratio. Only expression of the transporter P-glycoprotein was increased by cyclosporin A. Because canalicular membrane transporter expression was largely unchanged by cyclosporin A despite a marked decrease of biliary lipid secretion, transporter activity may partly depend upon canalicular membrane fluidity.
Identification of Cdc6 protein domains involved in interaction with Mcm2 protein and Cdc4 protein in budding yeast cells
Authors: JANG Sung-Wuk , ELSASSER Suzanne , CAMPBELL Judith L. , KIM Jiyoung
Published: August 2015

The Cdc6 protein (Cdc6p) has essential roles in regulating initiation of DNA replication. Cdc6p is recruited to origins of replication by the origin recognition complex (ORC) late in mitosis; Cdc6p in turn recruits minichromosome maintenance (Mcm) proteins to form the pre-replicative complex. Cdc6p is thought to interact with one or more Mcm proteins but this point has not yet been demonstrated. In the present study we observed that Cdc6p interacted significantly only with Mcm2p out of six Mcm proteins in yeast two-hybrid cells. Our results indicate that the interaction of Cdc6p with Mcm2p is specific, although we cannot exclude the possibility that the interaction might not be direct. In attempts to identify domains of Cdc6p important for interaction with Mcm2p, we tested interactions of various deleted versions of Cdc6p with Mcm2p and also with Cdc4p, which was previously known to interact with Cdc6p. The portion of Cdc6p from amino acid residues 51 to 394 was able to interact with Mcm2p. During the course of the studies we also discovered a previously undetected Cdc4p interaction domain between residues 51 and 394. Interestingly, when all six putative Cdc28 phosphorylation sites in Cdc6p were changed to alanine, a 6–7-fold increase in binding to Mcm2p was observed. This result suggests that unphosphorylated Cdc6p has higher affinity than phosphorylated Cdc6p for Mcm2p; this might partly explain the previous observation that Cdc6p failed to load Mcm proteins on replication origins during S phase when the cyclin-dependent protein kinase was active, thus helping to prevent the reinitiation of activated replicons.