bluuebunny/crossref_metadata_2025 huggingface dataset:
Importance of post-transcriptional regulation of chemokine genes by oxidative stress
Authors: JOSSE Claire , BOELAERT Johan R. , BEST-BELPOMME Martin , PIETTE Jacques
Published: August 2015

The transcription factor, nuclear factor κB (NF-κB), is activated by various stimuli including cytokines, radiation, viruses and oxidative stress. Here we show that, although induction with H2O2 gives rise to NF-κB nuclear translocation in both lymphocyte (CEM) and monocyte (U937) cells, it leads only to the production of mRNA species encoding interleukin-8 (IL-8) and macrophage inflammatory protein 1α in U937 cells. Under similar conditions these mRNA species are not observed in CEM cells. With the use of a transient transfection assay of U937 cells transfected with reporter constructs of the IL-8 promoter and subsequently treated with H2O2, we show that (1) IL-8-promoter-driven transcription is stimulated in both U937 and CEM cells and (2) the NF-κB site is crucial for activation because its deletion abolishes activation by H2O2. The production of IL-8 mRNA in U937 cells is inhibited by the NF-κB inhibitors clasto-lactacystin-β-lactone and E-64D (l-3-trans-ethoxycarbonyloxirane-2-carbonyl-l-leucine-3-methyl amide) but requires protein synthesis de novo. Moreover, inhibition of the p38 mitogen-activated protein kinase also decreases the IL-8 mRNA up-regulation mediated by H2O2. Taken together, these results show the importance of post-transcriptional events controlled by a p38-dependent pathway in the production of IL-8 mRNA in U937. The much lower activation of p38 in CEM cells in response to H2O2 could explain the lack of stabilization of IL-8 mRNA in these cells.
The p85 subunit of phosphoinositide 3-kinase is associated with β-catenin in the cadherin-based adhesion complex
Authors: WOODFIELD Richard J. , HODGKIN Matthew N. , AKHTAR Nasreen , MORSE Mary A. , FULLER Kerensa J. , SAQIB Khalid , THOMPSON Neil T. , WAKELAM Michael J.O.
Published: August 2015

Cell adhesion is fundamental to establishing and maintaining the discrete tissues in multicellular organisms. Adhesion must be sufficiently strong to preserve tissue architecture, whilst having the capacity to readily dissociate to permit fundamental processes, such as wound repair, to occur. However, very little is known about the signalling mechanisms involved in temporary down-regulation of cell adhesion to facilitate such processes. Cadherins are the principal mediators of cell–cell adhesion in a wide variety of tissues and species and form multi-protein complexes with cytosolic and cytoskeletal proteins to express their full adhesive capacity. In the present study we report that the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase) is associated with the cadherin-based adhesion complex in human epithelial cells. The interaction of p85 with the complex is via β-catenin. We also show that the interaction of p85 and β-catenin is direct, involves the N-terminal Src homology domain 2 of p85 and is regulated by tyrosine phosphorylation. These data suggest that PI 3-kinase may play a role in the functional regulation of the cadherin-based adhesion complex.
Microsomal glutathione S-transferase A1-1 with glutathione peroxidase activity from sheep liver: molecular cloning, expression and characterization
Authors: PRABHU K. Sandeep , REDDY Padala V. , GUMPRICHT Eric , HILDENBRANDT George R. , SCHOLZ Richard W. , SORDILLO Lorriane M. , REDDY C. Channa
Published: August 2015

A 25kDa subunit of glutathione S-transferase (GST) from sheep liver microsomes (microsomal GSTA1-1) with a significant selenium-independent glutathione peroxidase activity has been isolated and characterized. Several analytical criteria, including EDTA stripping, protease protection assay and extraction with alkaline Na2CO3, indicate that the microsomal GSTA1-1 is associated with the inner microsomal membrane. The specific cDNA nucleotide sequence reveals that the enzyme is made up of 222 amino acid residues and shares approx. 73–83% sequence similarity to Alpha-class GSTs from different species. The molecular mass, as determined by electrospray mass ionization, is 25611.3Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Guthenberg and DePierre (1982) Eur. J. Biochem. 128, 243–248]. The microsomal GSTA1-1 differs from the sheep liver cytosolic GSTs, reported previously from this laboratory, in its substrate specificity profile and molecular mass [Reddy, Burgess, Gong, Massaro and Tu (1983) Arch. Biochem. Biophys. 224, 87–101]. In addition to catalysing the conjugation of 4-hydroxynonenal with GSH, the enzyme also exhibits significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides, such as linoleic and arachidonic acid hydroperoxides, as well as phosphatidylcholine hydroperoxide, but not with H2O2. Thus the microsomal GSTA1-1 isoenzyme might have an important role in the protection of biological membranes against oxidative damage.
The influence of oxidation of membrane thiol groups on lysosomal proton permeability
Authors: WAN Feng-Yi , WANG Yi-Nan , ZHANG Guo-Jiang
Published: August 2015

The influence of oxidation of membrane thiol groups on lysosomal proton permeability was studied by measuring lysosomal pH with FITC-conjugated dextran, determining the membrane potential with 3,3′-dipropylthiadicarbocyanine iodide and monitoring their proton leakage with p-nitrophenol. Residual membrane thiol groups were measured with 5,5′-dithiobis-(2-nitrobenzoic acid). The lysosomal membrane thiol groups were modified by treatment with diamide and dithiothreitol. SDS/PAGE revealed aggregations of the membrane proteins induced by the treatment of lysosomes with diamide. The cross-linkage of proteins could be abolished by subsequent treatment with dithiothreitol, indicating that the proteins were linked via disulphide bonds. Treating the lysosomes with diamide decreased their membrane thiol groups and caused increases in lysosomal pH, membrane potential and proton leakage, which could be reversed by treatment of the lysosomes with dithiothreitol. This indicates that the lysosomal proton permeability can be increased by oxidation of the membrane thiol groups and restored to the normal level by reduction of the groups. Treatment of the lysosomes with N-ethylmaleimide reduced their membrane thiol groups but did not change the lysosomal pH or their degree of proton leakage. It suggests that protein aggregation may be an important mechanism for the increase in lysosomal proton permeability. The results raise the possibility that the proton permeability of lysosomes in vivo may be affected by the redox states of their membrane thiol groups.
Diverse effects of inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase on the expression of VCAM-1 and E-selectin in endothelial cells
Authors: RASMUSSEN Lars M. , HANSEN Peter R. , NABIPOUR Mehdi T. , OLESEN Ping , KRISTIANSEN Mikkel T. , LEDET Thomas
Published: August 2015

The expression of monocyte adhesion molecules, such as VCAM-1 (vascular cell adhesion molecule-1) and E-selectin, on the surface of the endothelium is an important step in the initiation and progression of atherosclerotic lesions. We hypothesized that the inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in endothelial cells could influence the expression of VCAM-1 and E-selectin. Using cultured human umbilical vein endothelial cells, we found that mevastatin (0.1–1μM) significantly reduced the expression of VCAM-1 protein in cells activated by tumour necrosis factor-α (TNF-α) for 7h. In contrast, TNF-α-induced E-selectin protein expression was augmented after mevastatin treatment. Mevastatin inhibited the mRNA expression of both VCAM-1 and E-selectin in TNF-α-stimulated endothelial cells. The activity of the transcription factor nuclear factor-κB, which is known to regulate the transcription of VCAM-1 and E-selectin, was significantly reduced after incubation with mevastatin. Analysis of the time-dependent variation in the TNF-α-induced expression of E-selectin, and estimation of the rate of surface disappearance of E-selectin together with measurement of the amounts of E-selectin molecules secreted, indicated that mevastatin inhibited the surface removal of E-selectin. This is compatible with the observed increase in E-selectin expression after statin treatment. All observed effects of mevastatin were reversed by mevalonate, the product of the HMG-CoA reductase reaction. In conclusion, inhibition of HMG-CoA reductase in endothelial cells attenuates VCAM-1 expression, but increases E-selectin expression, after cytokine induction. These diverse effects are associated with changes in the transcriptional regulation of the two adhesion molecule genes and modulation of the surface removal of E-selectin.
The N-terminal cysteine cluster is essential for membrane targeting of B/K protein
Authors: FUKUDA Mitsunori , MIKOSHIBA Katsuhiko
Published: August 2015

B/K protein belongs to a family of C-terminal-type (C-type) tandem C2 proteins that contain two C2 Ca2+-binding motifs at the C-terminus. Although other C-type tandem C2 proteins have been found to have a unique N-terminal domain that is involved in membrane anchoring (e.g. synaptotagmin) or specific ligand binding (e.g. rabphilin-3A and Doc2), no research has been conducted on the function of the N-terminal domain of B/K protein. In this study we showed that despite lacking a transmembrane domain, both native and recombinant B/K proteins are tightly bound to the membrane fraction, which was completely resistant to 0.1M Na2CO3, pH11, or 1M NaCl treatment. Deletion and mutation analyses indicated that the cysteine cluster at the N-terminal domain (consisting of seven cysteine residues, Cys-19, Cys-23, Cys-26, Cys-27, Cys-30, Cys-35 and Cys-36) is essential for the membrane localization of B/K protein. When wild-type B/K was expressed in PC12 cells, B/K proteins were localized mainly in the perinuclear region (trans-Golgi network), whereas mutant B/K proteins carrying Cys-to-Ala substitutions were present in the cytosol. Based on our findings, we propose that the N-terminal domain of B/K protein contains a novel cysteine-based protein motif that may allow B/K protein to localize in the trans-Golgi network.
Purification and characterization of acyl-acyl carrier protein synthetase from oleaginous yeast and its role in triacylglycerol biosynthesis
Authors: GANGAR Akanksha , KARANDE Anjali A. , RAJASEKHARAN Ram
Published: August 2015

Fatty acids are activated in an ATP-dependent manner before they are utilized. We describe here how the 10S triacylglycerol biosynthetic multienzyme complex from Rhodotorula glutinis is capable of activating non-esterified fatty acids for the synthesis of triacylglycerol. The photolabelling of the complex with [32P]azido-ATP showed labelling of a 35kDa polypeptide. The labelled polypeptide was identified as acyl-acyl carrier protein (ACP) synthetase, which catalyses the ATP-dependent ligation of fatty acid with ACP to form acyl-ACP. The enzyme was purified by successive PAGE separations to apparent homogeneity from the soluble fraction of oleaginous yeast and its apparent molecular mass was 35kDa under denaturing and reducing conditions. Acyl-ACP synthetase was specific for ATP. The Km values for palmitic, stearic, oleic and linoleic acids were found to be 42.9, 30.4, 25.1 and 22.7μM, respectively. The antibodies to acyl-ACP synthetase cross-reacted with Escherichia coli acyl-ACP synthetase. Anti-ACP antibodies showed no cross-reactivity with the purified acyl-ACP synthetase, indicating no bound ACP with the enzyme. Immunoprecipitations with antibodies to acyl-ACP synthetase revealed that this enzyme is a part of the 10S triacylglycerol biosynthetic complex. These results demonstrate that the soluble acyl-ACP synthetase plays a novel role in activating fatty acids for triacylglycerol biosynthesis in oleaginous yeast.
Identification of a new polyphosphoinositide in plants, phosphatidylinositol 5-monophosphate (PtdIns5P), and its accumulation upon osmotic stress
Authors: MEIJER Harold J. G. , BERRIE Christopher P. , IURISCI Cristiano , DIVECHA Nullin , MUSGRAVE Alan , MUNNIK Teun
Published: August 2015

Polyphosphoinositides play an important role in membrane trafficking and cell signalling. In plants, two PtdInsP isomers have been described, PtdIns3P and PtdIns4P. Here we report the identification of a third, PtdIns5P. Evidence is based on the conversion of the endogenous PtdInsP pool into PtdIns(4,5)P2 by a specific PtdIns5P 4-OH kinase, and on in vivo32P-labelling studies coupled to HPLC head-group analysis. In Chlamydomonas, 3–8% of the PtdInsP pool was PtdIns5P, 10–15% was PtdIns3P and the rest was PtdIns4P. In seedlings of Vicia faba and suspension-cultured tomato cells, the level of PtdIns5P was about 18%, indicating that PtdIns5P is a general plant lipid that represents a significant proportion of the PtdInsP pool. Activating phospholipase C (PLC) signalling in Chlamydomonas cells with mastoparan increased the turnover of PtdIns(4,5)P2 at the cost of PtdIns4P, but did not affect the level of PtdIns5P. This indicates that PtdIns(4,5)P2 is synthesized from PtdIns4P rather than from PtdIns5P during PLC signalling. However, when cells were subjected to hyperosmotic stress, PtdIns5P levels rapidly increased, suggesting a role in osmotic-stress signalling. The potential pathways of PtdIns5P formation are discussed.
The Phox homology (PX) domain, a new player in phosphoinositide signalling
Authors: XU Yue , SEET Li-Fong , HANSON Brendon , HONG Wanjin
Published: August 2015

Phosphoinositides are key regulators of diverse cellular processes. The pleckstrin homology (PH) domain mediates the action of PtdIns(3,4)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3, while the FYVE domain relays the pulse of PtdIns3P. The recent establishment that the Phox homology (PX) domain interacts with PtdIns3P and other phosphoinositides suggests another mechanism by which phosphoinositides can regulate/integrate multiple cellular events via a spectrum of PX domain-containing proteins. Together with the recent discovery that the epsin N-terminal homologue (ENTH) domain interacts with PtdIns(4,5)P2, it is becoming clear that phosphoinositides regulate diverse cellular events through interactions with several distinct structural motifs present in many different proteins.
Hydrogen peroxide-induced production of a 40 kDa immunoreactive thyroglobulin fragment in human thyroid cells: the onset of thyroid autoimmunity?
Authors: DUTHOIT Christine , ESTIENNE Valérie , GIRAUD Annie , DURAND-GORDE Josée-Martine , RASMUSSEN Åse Krogh , FELDT-RASMUSSEN Ulla , CARAYON Pierre , RUF Jean
Published: August 2015

We recently reported that, during in vitro thyroid-hormone synthesis, H2O2 stress cleaved thyroglobulin (Tg) into C-terminal peptides. These peptides were found to contain the immunodominant region of Tg recognized by Tg autoantibodies from patients with an autoimmune thyroid disease. To test the hypothesis that Tg fragmentation is an early upstream initiating event involved in Tg autoimmune response and the consequence of oxidative injuries, we studied the effect of H2O2 stress on human thyroid cells. In culture conditions allowing Tg synthesis and iodine organification by the cells, we found that bolus addition of increasing millimolar doses of H2O2 induced a dose–response appearance of floating cells in the culture medium. These cells apparently resulted from a necrotic process, and they bore iodinated Tg fragments. These fragments were found to be similar to those previously obtained in vitro from purified Tg. In both cases, Tg peptides were recognized by a well-defined monoclonal antibody directed to the immunodominant region of Tg. The smallest immunoreactive Tg peptide had a molecular mass of 40kDa and entered human thyrocytes more efficiently than the entire Tg. These data suggest that thyrocytes exposed to locally increased H2O2 doses accumulate fragmented Tg for further delivery into surrounding living thyrocytes in the course of an autoimmune response.
Peroxynitrite-induced nitration of tyrosine-34 does not inhibit Escherichia coli iron superoxide dismutase
Authors: SOULÈRE Laurent , CLAPAROLS Catherine , PÉRIÉ Jacques , HOFFMANN Pascal
Published: August 2015

The peroxynitrite anion is a potent oxidizing agent, formed by the diffusion-limited combination of nitric oxide and superoxide, and its production under physiological conditions is associated with the pathologies of a number of inflammatory and neurodegenerative diseases. Nitration of Escherichia coli iron superoxide dismutase (Fe-SOD) by peroxynitrite was investigated, and demonstrated by spectral changes and electrospray mass spectroscopic analysis. HPLC and mass studies of the tryptic digests of the mono-nitrated Fe-SOD indicated that tyrosine-34 was the residue most susceptible to nitration by peroxynitrite. Exclusive nitration of this residue occurred when Fe-SOD was exposed to a cumulative dose of 0.4mM peroxynitrite. Unlike with human Mn-SOD, this single modification did not inactivate E. coli Fe-SOD at pH7.4. When Fe-SOD was exposed to higher concentrations of peroxynitrite (7mM), eight tyrosine residues per subunit of the protein, of the nine available, were nitrated without loss of catalytic activity of the enzyme. The pKa of nitrated tyrosine-34 was determined to be 7.95±0.15, indicating that the peroxynitrite-modified enzyme appreciably maintains its protonation state under physiological conditions.
RB2/p130 ectopic gene expression in neuroblastoma stem cells: evidence of cell-fate restriction and induction of differentiation
Authors: JORI Francesco P. , GALDERISI Umberto , PIEGARI Elena , PELUSO Gianfranco , CIPOLLARO Marilena , CASCINO Antonio , GIORDANO Antonio , MELONE Mariarosa A. B.
Published: August 2015

The activity of the RB2/p130 gene, which is a member of the retinoblastoma gene family, is cell-cycle-regulated and plays a key role in growth inhibition and differentiation. We used neuroblastoma cell lines as a model for studies on neural crest progenitor cell differentiation. We show that Rb2/p130 ectopic protein expression induces morphological and molecular modifications, promoting differentiation of intermediate (I) phenotype SK-N-BE(2)-C neuroblastoma cells towards a neuroblastic (N) rather than a Schwann/glial/melanocytic (S) phenotype. These modifications are stable as they persist even after treatment with an S-phenotype inducer. Rb2/p130 ectopic expression also induces a more differentiated phenotype in N-type SH-SY-5Y cells. Further, this function appears to be independent of cell-cycle withdrawal. The data reported suggest that the Rb2/p130 protein is able to induce neuronal lineage specification and differentiation in neural crest stem and committed neuroblastoma cells, respectively. Thus, the Rb2/p130 protein seems to be required throughout the full neural maturation process.
Lysine164α of protein farnesyltransferase is important for both CaaX substrate binding and catalysis
Authors: HIGHTOWER Kendra E. , DE Smita , WEINBAUM Carolyn , SPENCE Rebecca A. , CASEY Patrick J.
Published: August 2015

Protein farnesyltransferase (FTase) catalyses the formation of a thioether linkage between proteins containing a C-terminal CaaX motif and a 15-carbon isoprenoid. The involvement of substrates such as oncogenic Ras proteins in tumour formation has led to intense efforts in targeting this enzyme for development of therapeutics. In an ongoing programme to elucidate the mechanism of catalysis by FTase, specific residues of the enzyme identified in structural studies as potentially important in substrate binding and catalysis are being targeted for mutagenesis. In the present study, the role of the positive charge of Lys164 of the α subunit of FTase in substrate binding and catalysis was investigated. Comparison of the wild-type enzyme with enzymes that have either an arginine or alanine residue substituted at this position revealed unexpected roles for this residue in both substrate binding and catalysis. Removal of the positive charge had a significant effect on the association rate constant and the binding affinity of a CaaX peptide substrate, indicating that the positive charge of Lys164α is involved in formation of the enzyme (E)·farnesyl diphosphate (FPP)·peptide ternary complex. Furthermore, mutation of Lys164α resulted in a substantial decrease in the observed rate constant for product formation without alteration of the chemical mechanism. These and additional studies provide compelling evidence that both the charge on Lys164α, as well as the positioning of the charge, are important for overall catalysis by FTase.
HstK, a cyanobacterial protein with both a serine/threonine kinase domain and a histidine kinase domain: implication for the mechanism of signal transduction
Authors: PHALIP Vincent , LI Jian-Hong , ZHANG Cheng-Cai
Published: August 2015

Two distinct families of protein kinases are involved in signal transduction: Ser, Thr and Tyr kinases, which are predominantly found among eukaryotes, and His kinases, as part of bacterial two-component signalling systems. Genetic studies in Arabidopsis and Saccharomyces have demonstrated that bacterial-type two-component systems may act upstream of Ser/Thr kinases in the same signalling pathway, but how this coupling is accomplished remains unclear. In the present study, we report the characterization of a protein kinase, HstK, from the N2-fixing cyanobacterium Anabaena sp. PCC 7120, that possesses both a Ser/Thr kinase domain and a His kinase domain. Proteins with a structural architecture similar to that of HstK can be found in the eukaryote, Schizosaccharomyces pombe, and the bacterium, Rhodococcus sp. M5. HstK was present in cells grown with NH4+ or N2 as the nitrogen source, but was absent in cells grown with NO3−. The hstK gene was inactivated and the mutant phenotype was characterized. The catalytic domain of the Ser/Thr kinase of HstK functionally replaced that of Hog1p, a well-characterized protein kinase required for the response to high osmolarity in the S. cerevisiae heterologous system. The unusual multidomain structure of HstK suggests that a two-component system could be directly coupled to Ser/Thr kinases in the same signal transduction pathway.
Allosteric behaviour of 1:5 hybrids of mutant subunits of Clostridium symbiosum glutamate dehydrogenase differing in their amino acid specificity
Authors: GOYAL Arun , WANG Xing-Guo , ENGEL Paul C.
Published: August 2015

Hybrid hexamers were made by refolding mixtures of two mutant forms of clostridial glutamate dehydrogenase. Mutant Cys320Ser (C320S) has a similar activity to the wild-type enzyme, but is unreactive with Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoate) (DTNB). The triple mutant Lys89Leu/Ala163Gly/Ser380Ala (K89L/A163G/S380A), active with norleucine but not glutamate, is inactivated by DTNB, since the amino acid residue at position 320 is a cysteine residue. The chosen ratio favoured 1:5 hybrids of the triple mutant and C320S. The renatured mixture was treated with DTNB and separated on an NAD+–agarose column to which only C320S subunits bind tightly. Fractions were monitored for glutamate and norleucine activity and for releasable thionitrobenzoate to establish subunit stoichiometry. A fraction highly enriched in the 1:5 hybrid was identified. Homohexamers (C320S with 40mM glutamate and 1mM NAD+ at pH8.8, or K89L/A163G/S380A with 70mM norleucine and 1mM NAD+ at pH8.5) showed allosteric activation; succinate activated C320S approx. 50-fold (EC50 = 70mM, h = 2.4), and glutarate gave approx. 30-fold activation (EC50 = 35mM, h = 2.3). For the triple mutant, corresponding values were 80mM and 2.2 for succinate, and 75mM and 1.7 for glutarate, but maximal activation was only about 2-fold. In the 1:5 hybrid, with only one norleucine-active subunit per hexamer, responses to glutarate and succinate were still co-operative, and activation was more extensive than in the triple mutant homohexamer. A single norleucine-active subunit can thus respond co-operatively to a substrate analogue at the other five inactive sites. On the other hand, similar hyperbolic dependence on the norleucine concentration for the hybrid and the triple mutant homohexamer reflected the inability of C320S subunits to bind norleucine. With glutamate at pH8.8, an h value of 3.6 was obtained for the 1:5 hybrid, in contrast with an h value of 5.2 for the C320S homohexamer. The ‘foreign’ subunit evidently impedes inter-subunit communication to some extent.
UDP-glucose pyrophosphorylase: up-regulation in hypertrophic cartilage and role in hyaluronan synthesis
Authors: MAGEE Cordula , NURMINSKAYA Maria , LINSENMAYER Thomas F.
Published: August 2015

Previously, we have performed subtractive hybridization to identify genes up-regulated in hypertrophic chondrocytes of the avian epiphyseal growth plate. In the present study, we report the identification of one of the clones as UDP-glucose pyrophosphorylase (UDPG-PPase) and propose a possible function for this enzyme in regulating hyaluronan (HA) synthesis in hypertrophic cartilage. We have cloned the 2.6kb full-length cDNA for avian UDPG-PPase and confirmed its up-regulation in hypertrophic versus non-hypertrophic cartilage by Northern-blot analysis. The 6-fold increase in mRNA was paralleled by an equivalent increase in enzymic activity. The enzyme catalyses the conversion of glucose 1-phosphate into UDP-glucose, which is used to synthesize a number of cellular components, including HA. Overexpression of enzymically active UDPG-PPase in non-hypertrophic chondrocytes resulted in a 2–3-fold increase in total HA, as determined by a competitive binding assay and immunohistochemistry. In the developing growth plate, HA synthesis was elevated in the hypertrophic zone along with the up-regulation of the HA synthase (HAS)-2 gene. Our data suggest that an increase in both activities, UDPG-PPase and HAS-2, is required for non-hypertrophic chondrocytes to synthesize an amount of HA comparable with that in hypertrophic chondrocytes. Therefore we conclude that HA synthesis during chondrocyte differentiation is regulated at the level of the substrate-provider gene, UDPG-PPase, as well as the HAS genes.
Phosphoinositide 3-kinase-γ induces Xenopus oocyte maturation via lipid kinase activity
Authors: HEHL Solveig , STOYANOV Borislav , OEHRL Wolf , SCHÖNHERR Roland , WETZKER Reinhard , HEINEMANN Stefan H.
Published: August 2015

Type-I phosphoinositide 3-kinases (PI3Ks) were characterized as a group of intracellular signalling proteins expressing both protein and lipid kinase activities. Recent studies implicate PI3Ks as mediators of oocyte maturation, but the molecular mechanisms are poorly defined. Here we used the Xenopus oocyte expression system as a model to investigate a possible contribution of the γ-isoform of PI3K (PI3Kγ) in the different pathways leading to cell-cycle progression by monitoring the time course of germinal vesicle breakdown (GVBD). Expression of a constitutive active PI3Kγ (PI3Kγ-CAAX) induced GVBD and increased the levels of phosphorylated Akt/protein kinase B and mitogen-activated protein kinase (MAPK). Furthermore, PI3Kγ-CAAX accelerated progesterone-induced GVBD, but had no effect on GVBD induced by insulin. The effects of PI3Kγ-CAAX could be suppressed by pre-incubation of the oocytes with LY294002, PD98059 or roscovitine, inhibitors of PI3K, MEK (MAPK/extracellular-signal-regulated protein kinase kinase) and cdc2/cyclin B kinase, respectively. Mutants of PI3Kγ-CAAX, in which either lipid kinase or both lipid and protein kinase activities were altered or eliminated, did not induce significant GVBD. Our data demonstrate that expression of PI3Kγ in Xenopus oocytes accelerates their progesterone-induced maturation and that lipid kinase activity is required to induce this effect.
Influence of electrochemical properties in determining the sensitivity of [4Fe-4S] clusters in proteins to oxidative damage
Authors: TILLEY Gareth J. , CAMBA Raul , BURGESS Barbara K. , ARMSTRONG Fraser A.
Published: August 2015

Interconversion between [4Fe-4S] cubane and [3Fe-4S] cuboidal states represents one of the simplest structural changes an iron–sulphur cluster can undertake. This reaction is implicated in oxidative damage and in modulation of the activity and regulation of certain enzymes, and it is therefore important to understand the factors governing cluster stability and the processes that activate cluster conversion. In the present study, protein film voltammetry has been used to induce and monitor the oxidative conversion of [4Fe-4S] into [3Fe-4S] clusters in different variants of Azotobacter vinelandii ferredoxin I (AvFdI; the 8Fe form of the native protein), and ΔThr14/ΔAsp15, Thr14 → Cys (T14C) and C42D mutants. The electrochemical results have been correlated with the differing oxygen sensitivities of [4Fe-4S] clusters, and comparisons have been drawn with other ferredoxins (Desulfovibrio africanus FdIII, Clostridium pasteurianum Fd, Thauera aromatica Fd and Pyrococcus furiosus Fd). In contrast with high-potential iron–sulphur proteins (HiPIPs) for which the oxidized species [4Fe-4S]3+ is inert to degradation and can be isolated, the hypervalent state in these ferredoxins (most obviously the 3+ level) is very labile, and the reduction potential at which this is formed is a key factor in determining the cluster's resistance to oxidative damage.
α-Retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors
Authors: NIDETZKY Bernd , EIS Christian
Published: August 2015

Fungal trehalose phosphorylase is classified as a family 4 glucosyltransferase that catalyses the reversible phosphorolysis of α,α-trehalose with net retention of anomeric configuration. Glucosyl transfer to and from phosphate takes place by the partly rate-limiting interconversion of ternary enzyme–substrate complexes formed from binary enzyme–phosphate and enzyme–α-d-glucopyranosyl phosphate adducts respectively. To advance a model of the chemical mechanism of trehalose phosphorylase, we performed a steady-state kinetic study with the purified enzyme from the basidiomycete fungus Schizophyllum commune by using alternative substrates, inhibitors and combinations thereof in pairs as specific probes of substrate-binding recognition and transition-state structure. Orthovanadate is a competitive inhibitor against phosphate and α-d-glucopyranosyl phosphate, and binds 3×104-fold tighter (Ki≈ 1μM) than phosphate. Structural alterations of d-glucose at C-2 and O-5 are tolerated by the enzyme at subsite +1. They lead to parallel effects of approximately the same magnitude (slope = 1.14; r2 = 0.98) on the reciprocal catalytic efficiency for reverse glucosyl transfer [log (Km/kcat)] and the apparent affinity of orthovanadate determined in the presence of the respective glucosyl acceptor (log Ki). An adduct of orthovanadate and the nucleophile/leaving group bound at subsite +1 is therefore the true inhibitor and displays partial transition state analogy. Isofagomine binds to subsite −1 in the enzyme–phosphate complex with a dissociation constant of 56μM and inhibits trehalose phosphorylase at least 20-fold better than 1-deoxynojirimycin. The specificity of the reversible azasugars inhibitors would be explained if a positive charge developed on C-1 rather than O-5 in the proposed glucosyl cation-like transition state of the reaction. The results are discussed in the context of α-retaining glucosyltransferase mechanisms that occur with and without a β-glucosyl enzyme intermediate.
Direct interactions between molecular chaperones heat-shock protein (Hsp) 70 and Hsp40: yeast Hsp70 Ssa1 binds the extreme C-terminal region of yeast Hsp40 Sis1
Authors: QIAN Xinguo , HOU Wenbo , ZHENGANG Li , SHA Bingdong
Published: August 2015

Heat-shock protein 40 (Hsp40) enables Hsp70 to play critical roles in a number of cellular processes, such as protein folding, assembly, degradation and translocation in vivo. Hsp40 recognizes and binds non-native polypeptides and delivers them to Hsp70. Then Hsp40 stimulates the ATPase activity of Hsp70 to fold the polypeptides. By using yeast Hsp40 Sis1 and yeast Hsp70 Ssa1 as our model proteins, we found that the Sis1 peptide-binding fragment interacts directly with the full-length Ssa1 in vitro. Further studies showed that the C-terminal lid domain of Ssa1 could interact with Sis1 peptide-binding domain physically in vitro. The Sis1 peptide-binding fragment forms a stable complex with the Ssa1 C-terminal lid domain in solution. The interactions between these two proteins appear to be charge–charge interactions because high-ionic-strength buffer can dissociate the complex. Further mapping studies showed that the Sis1 peptide-binding fragment binds the extreme C-terminal 15 amino acid residues of Ssa1. A flexible glycine-rich region is followed by these 15 residues in the Ssa1 primary sequence. Atomic force microscopy of the Sis1–Ssa1 complex showed that only one end of the Ssa1 lid domain binds the Sis1 peptide-binding-fragment dimer at the upper level of the huge groove within the Sis1 dimer. Based on the data, we propose an ‘anchoring and docking’ model to illustrate the mechanisms by which Hsp40 interacts with Hsp70 and delivers the non-native polypeptide to Hsp70.
Identification and glucan-binding properties of a new carbohydrate-binding module family
Authors: BORASTON Alisdair B. , GHAFFARI Mazyar , WARREN R. Antony J. , KILBURN Douglas G.
Published: August 2015

The C-terminal 191-residue module of Cel5A from the alkalophilic Bacillus sp. 1139 comprises a carbohydrate-binding module (CBM) belonging to a previously unidentified family that we have classified as CBM family 28. This example, called CBM28, bound specifically to cello-oligosaccharides and mixed β-(1,3)(1,4)-glucans (barley β-glucan) with association constants of approximately (1–4)×104 M−1. Its binding to barley β-glucan was remarkably insensitive to pH between 7.0 and 10.9, in keeping with its alkalophilic source. CBM28 bound to cellulose having a significant non-crystalline content with an association constant similar to that for its binding to soluble glucans. CBM17 (CBM family 17) and CBM28 modules naturally occur as tandems. The CBM17/CBM28 tandem from Cel5A bound with apparent co-operativity to barley β-glucan. The association of CBM28 with cello-oligosaccharides was driven enthalpically and marked by the different thermodynamic contribution of three putative binding subsites that accommodate a cellohexaose molecule.
Artifactual uncoupling by uncoupling protein 3 in yeast mitochondria at the concentrations found in mouse and rat skeletal-muscle mitochondria
Authors: HARPER James A. , STUART Jeff A. , JEKABSONS Mika B. , ROUSSEL Damien , BRINDLE Kevin M. , DICKINSON Keith , JONES Robert B. , BRAND Martin D.
Published: August 2015

Western blots detected uncoupling protein 3 (UCP3) in skeletal-muscle mitochondria from wild-type but not UCP3 knock-out mice. Calibration with purified recombinant UCP3 showed that mouse and rat skeletal muscle contained 0.14μg of UCP3/mg of mitochondrial protein. This very low UCP3 content is 200–700-fold less than the concentration of UCP1 in brown-adipose-tissue mitochondria from warm-adapted hamster (24–84μg of UCP1/mg of mitochondrial protein). UCP3 was present in brown-adipose-tissue mitochondria from warm-adapted rats but was undetectable in rat heart mitochondria. We expressed human UCP3 in yeast mitochondria at levels similar to, double and 7-fold those found in rodent skeletal-muscle mitochondria. Yeast mitochondria containing UCP3 were more uncoupled than empty-vector controls, particularly at concentrations that were 7-fold physiological. However, uncoupling by UCP3 was not stimulated by the known activators palmitate and superoxide; neither were they inhibited by GDP, suggesting that the observed uncoupling was a property of non-native protein. As a control, UCP1 was expressed in yeast mitochondria at similar concentrations to that of UCP3 and at up to 50% of the physiological level of UCP1. Low levels of UCP1 gave palmitate-dependent and GDP-sensitive proton conductance but higher levels of UCP1 caused an additional GDP-insensitive uncoupling artifact. We conclude that the uncoupling of yeast mitochondria by high levels of UCP3 expression is entirely an artifact and provides no evidence for any native uncoupling activity of the protein.
Tolerance of glycosylphosphatidylinositol (GPI)-specific phospholipase D overexpression by Chinese hamster ovary cell mutants with aberrant GPI biosynthesis
Authors: DU Xiaohan , CAI Jiewei , ZHOU Jian-zhong , STEVENS Victoria L. , LOW Martin G.
Published: August 2015

Mammalian glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is capable of releasing GPI-anchored proteins by cleavage of the GPI moiety. A previous study indicated that overexpression of GPI-PLD in mouse RAW 264.7 monocytes/macrophages could be cytotoxic, since survivors of stable transfections had enzymic activity no higher than untransfected cells [Du and Low (2001) Infect. Immun. 69, 3214–3223]. We investigated this phenomenon by transfecting bovine GPI-PLD cDNA stably into Chinese hamster ovary (CHO) cells using a bi-cistronic expression system. The surviving transfectants showed an unchanged cellular level of GPI-PLD, supporting the cytotoxicity hypothesis. However, when using a CHO mutant defective in the second step of GPI biosynthesis as host, the expression level of GPI-PLD in stable transfectants was increased by 2.5-fold compared with untransfected or empty-vector-transfected cells. To identify the mechanism, we studied another CHO cell mutant (G9PLAP.D5), which seems to be defective at a later stage in GPI biosynthesis. In sharp contrast with wild-type cells, GPI-PLD activity in G9PLAP.D5 transfected with bovine GPI-PLD cDNA was 100-fold higher than untransfected or empty-vector-transfected cells. This was accompanied by a significant release of alkaline phosphatase into the medium and a decrease in membrane-associated alkaline phosphatase. Taken together, our results indicate that overexpression of GPI-PLD is lethal to wild-type cells, possibly by catalysing the overproduction of GPI-derived toxic substances. We propose that cells with abnormal GPI biosynthesis/processing can escape the toxic effect of these substances.
Characterization of the Saccharomyces cerevisiae YMR318C (ADH6) gene product as a broad specificity NADPH-dependent alcohol dehydrogenase: relevance in aldehyde reduction
Authors: LARROY Carol , FERNÁNDEZ M. Rosario , GONZÁLEZ Eva , PARÉS Xavier , BIOSCA Josep A.
Published: August 2015

YMR318C represents an open reading frame from Saccharomyces cerevisiae with unknown function. It possesses a conserved sequence motif, the zinc-containing alcohol dehydrogenase (ADH) signature, specific to the medium-chain zinc-containing ADHs. In the present study, the YMR318C gene product has been purified to homogeneity from overexpressing yeast cells, and found to be a homodimeric ADH, composed of 40kDa subunits and with a pI of 5.0–5.4. The enzyme was strictly specific for NADPH and was active with a wide variety of substrates, including aliphatic (linear and branched-chain) and aromatic primary alcohols and aldehydes. Aldehydes were processed with a 50-fold higher catalytic efficiency than that for the corresponding alcohols. The highest kcat/Km values were found with pentanal>veratraldehyde > hexanal > 3-methylbutanal >cinnamaldehyde. Taking into consideration the substrate specificity and sequence characteristics of the YMR318C gene product, we have proposed this gene to be called ADH6. The disruption of ADH6 was not lethal for the yeast under laboratory conditions. Although S. cerevisiae is considered a non lignin-degrading organism, the catalytic activity of ADHVI can direct veratraldehyde and anisaldehyde, arising from the oxidation of lignocellulose by fungal lignin peroxidases, to the lignin biodegradation pathway. ADHVI is the only S. cerevisiae enzyme able to significantly reduce veratraldehyde in vivo, and its overexpression allowed yeast to grow under toxic concentrations of this aldehyde. The enzyme may also be involved in the synthesis of fusel alcohols. To our knowledge this is the first NADPH-dependent medium-chain ADH to be characterized in S. cerevisiae.
Regulation of epithelial and lymphocyte cell adhesion by adenosine deaminase–CD26 interaction
Authors: GINÉS Silvia , MARIÑO Marta , MALLOL Josefa , CANELA Enric I. , MORIMOTO Chikao , CALLEBAUT Christian , HOVANESSIAN Ara , CASADÓ Vicent , LLUIS Carmen , FRANCO Rafael
Published: August 2015

The extra-enzymic function of cell-surface adenosine deaminase (ADA), an enzyme mainly localized in the cytosol but also found on the cell surface of monocytes, B cells and T cells, has lately been the subject of numerous studies. Cell-surface ADA is able to transduce co-stimulatory signals in T cells via its interaction with CD26, an integral membrane protein that acts as ADA-binding protein. The aim of the present study was to explore whether ADA—CD26 interaction plays a role in the adhesion of lymphocyte cells to human epithelial cells. To meet this aim, different lymphocyte cell lines (Jurkat and CEM T) expressing endogenous, or overexpressing human, CD26 protein were tested in adhesion assays to monolayers of colon adenocarcinoma human epithelial cells, Caco-2, which express high levels of cell-surface ADA. Interestingly, the adhesion of Jurkat and CEM T cells to a monolayer of Caco-2 cells was greatly dependent on CD26. An increase by 50% in the cell-to-cell adhesion was found in cells containing higher levels of CD26. Incubation with an anti-CD26 antibody raised against the ADA-binding site or with exogenous ADA resulted in a significant reduction (50–70%) of T-cell adhesion to monolayers of epithelial cells. The role of ADA—CD26 interaction in the lymphocyte—epithelial cell adhesion appears to be mediated by CD26 molecules that are not interacting with endogenous ADA (ADA-free CD26), since SKW6.4 (B cells) that express more cell-surface ADA showed lower adhesion than T cells. Adhesion stimulated by CD26 and ADA is mediated by T cell lymphocyte function-associated antigen. A role for ADA—CD26 interaction in cell-to-cell adhesion was confirmed further in integrin activation assays. FACS analysis revealed a higher expression of activated integrins on T cell lines in the presence of increasing amounts of exogenous ADA. Taken together, these results suggest that the ADA—CD26 interaction on the cell surface has a role in lymphocyte—epithelial cell adhesion.
abf-1 and abf-2, ASABF-type antimicrobial peptide genes in Caenorhabditis elegans
Authors: KATO Yusuke , AIZAWA Tomoyasu , HOSHINO Hirokazu , KAWANO Keiichi , NITTA Katsutoshi , ZHANG Hong
Published: August 2015

Two genes encoding the ASABF (Ascarissuumantibacterial factor)-type antimicrobial peptide, abf-1 and abf-2, were identified in Caenorhabditis elegans. Recombinant ABF-2 exhibited potent microbicidal activity against Gram-positive and Gram-negative bacteria, and yeasts. The tissue-specific distribution estimated by immunofluorescence staining and transgenic analysis of a gfp fusion gene (where GFP corresponds to green fluorescent protein) suggested that ABF-2 contributes to surface defence in the pharynx. abf-1 contains a single intron at a conserved position, suggesting that asabf and abf originated from a common ancestor. Both transcripts for abf-1 and abf-2 were detected as two distinct forms, i.e. spliced leader (SL)1-trans-spliced with a long 5′-untranslated region (UTR) and SL-less with a short 5′-UTR. A polycistronic precursor RNA encoding ABF-1 and ABF-2 was detected, suggesting that these genes form an operon. An ‘opportunistic operon’ model for regulation of abf genes, including the generation of short SL-less transcripts, is proposed. In conclusion, C. elegans should have an immune defence system due to the antimicrobial peptides. C. elegans can be a novel model for innate immunity. Furthermore, the combination of biochemical identification in Ascaris suum and homologue hunting in C. elegans should be a powerful method of finding rapidly evolved proteins, such as some immune-related molecules in C. elegans.
Promoter activity of the 5′-flanking regions of medaka fish soluble guanylate cyclase α1 and β1 subunit genes
Authors: YAMAMOTO Takehiro , SUZUKI Norio
Published: August 2015

We examined the spatial expression pattern of medaka fish (Oryzias latipes) soluble guanylate cyclase α1 and β1 subunit genes, OlGCS-α1 and OlGCS-β1, and characterized the 5′-flanking region required for expression of both genes by introducing various promoter-luciferase fusion-gene constructs into COS-1 cells and medaka fish embryos. The OlGCS-α1 and OlGCS-β1 gene transcripts were detected in whole brain and kidney in 7-day and 9-day embryos. Primer-extension analysis demonstrated that there were no differences among various adult organs (brain, eye, kidney, ovary and testis) in the transcription start site of the OlGCS-α1 and OlGCS-β1 genes. Neither gene contained the functional TATA box within its 5′-flanking region, and the basal promoter activity was found between nucleotides +33 and +42 in the OlGCS-α1 gene and between nucleotides +146 and +155 in the OlGCS-β1 gene. In the assay of medaka fish embryos, the 5′-flanking region of the OlGCS-β1 gene exhibited lower promoter activity than that of the OlGCS-α1 gene. In the experiments on dual-luciferase fusion-gene constructs, the 5′-flanking region of the OlGCS-α1 gene connected to the 5′-flanking region of the OlGCS-β1 gene was introduced into medaka fish embryos, and the 5′-flanking regions of both subunit genes were shown to mutually influence each other's promoter activity.
Pre-steady-state phosphorylation and dephosphorylation of detergent-purified plasma-membrane Ca2+-ATPase
Authors: BREDESTON Luis M. , REGA Alcides F.
Published: August 2015

Pre-steady-state phosphorylation and dephosphorylation of purified and phospholipid-depleted plasma-membrane Ca2+-ATPase (PMCA) solubilized in the detergent polyoxyethylene 10 lauryl ether were studied at 25°C. The time course of phosphorylation with ATP of the enzyme associated with Ca2+, probably the true phosphorylation reaction, showed a fast phase (kapp near 400s−1) followed by a slow phase (kapp = 23s−1). With asolectin or acidic phosphatidylinositol, the concentration of phosphoenzyme (EP) increased at as high a rate as before, passed through a maximum at 4ms and stabilized at a steady level that was approx. half that without lipids. Calmodulin (CaM) did not change the rate of the fast phase, accelerated the slow phase (kapp = 93s−1) and increased [EP] with small changes in the shape of the time course. Dephosphorylation was slow (kapp = 30s−1) and insensitive to CaM. Asolectin accelerated dephosphorylation, which followed biexponential kinetics with fast (kapp = 220s−1) and slow (kapp = 20s−1) components. CaM stimulated the fast component by nearly 50%. The results show that the behaviour of the PMCA is complex, and suggest that acidic phospholipids and CaM activate PMCA through different mechanisms. Acceleration of dephosphorylation seems relevant during activation of the PMCA by acidic phospholipids.