bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and characterization of mutations in housefly (Musca domestica) acetylcholinesterase involved in insecticide resistance
Authors: WALSH Sinéad B. , DOLDEN Tracey A. , MOORES Graham D. , KRISTENSEN Michael , LEWIS Terence , DEVONSHIRE Alan L. , WILLIAMSON Martin S.
Published: August 2015

Acetylcholinesterase (AChE) insensitive to organophosphate and carbamate insecticides has been identified as a major resistance mechanism in numerous arthropod species. However, the associated genetic changes have been reported in the AChE genes from only three insect species; their role in conferring insecticide insensitivity has been confirmed, using functional expression, only for those in Drosophila melanogaster. The housefly, Musca domestica, was one of the first insects shown to have this mechanism; here we report the occurrence of five mutations (Val-180 → Leu, Gly-262 → Ala, Gly-262 → Val, Phe-327 → Tyr and Gly-365 → Ala) in the AChE gene of this species that, either singly or in combination, confer different spectra of insecticide resistance. The baculovirus expression of wild-type and mutated housefly AChE proteins has confirmed that the mutations each confer relatively modest levels of insecticide insensitivity except the novel Gly-262 → Val mutation, which results in much stronger resistance (up to 100-fold) to certain compounds. In all cases the effects of mutation combinations are additive. The mutations introduce amino acid substitutions that are larger than the corresponding wild-type residues and are located within the active site of the enzyme, close to the catalytic triad. The likely influence of these substitutions on the accessibility of the different types of inhibitor and the orientation of key catalytic residues are discussed in the light of the three-dimensional structures of the AChE protein from Torpedo californica and D. melanogaster.
Internal ribosome entry segment-mediated initiation of c-Myc protein synthesis following genotoxic stress
Authors: SUBKHANKULOVA Tatiana , MITCHELL Sally A. , WILLIS Anne E.
Published: August 2015

Initiation of translation of the proto-oncogene c-myc can occur by either the cap-dependent scanning mechanism or by internal ribosome entry. The latter mechanism requires a complex RNA structural element that is located in the 5′ untranslated region of c-myc, termed an internal ribosome entry segment (IRES). Recent work has shown that IRESs are used to maintain protein expression under conditions when cap-dependent translation initiation is compromised; for example, during mitosis, apoptosis and under conditions of cell stress, such as hypoxia or heat shock. Induction of genotoxic stress also results in a large reduction in global protein synthesis rates and therefore we investigated whether the c-myc IRES was active following DNA damage. As expected, in cells treated with either ethylmethane sulphonate or mitomycin C there was a large reduction in protein synthesis, although this was brought about by two different mechanisms. However, in each case the c-myc IRES was active and c-Myc protein expression was maintained. Finally we showed that the proteins required for this process are downstream of the p38 mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated protein kinase (ERK)/MEK(MAPK/ERK kinase) signalling pathways, since pre-treatment of cells with inhibitors of these pathways before DNA damage is initiated inhibits both c-myc IRES activity and expression of c-Myc protein.
Survival-factor-induced phosphorylation of Bad results in its dissociation from Bcl-xL but not Bcl-2
Authors: HIRAI Itaru , WANG Hong-Gang
Published: August 2015

The pro-apoptotic Bcl-2-family protein Bad heterodimerizes with Bcl-2 and Bcl-xL in the outer mitochondrial membranes, nullifying their anti-apoptotic activities and promoting cell death. We report that interleukin-3 (IL-3) stimulation induces Bad phosphorylation and triggers its translocation from mitochondria to cytoplasm in cells expressing Bcl-xL but not Bcl-2. Overexpression of Bad sensitized Bcl-xL-expressing FL5.12 cells to apoptosis induced by IL-3 deprivation, but had no effect on the viability of cells expressing Bcl-2. IL-3 stimulation induced Bad phosphorylation at Ser-112, impairing its binding to Bcl-xL and resulting in its association with 14-3-3 proteins in the cytosol. However, Ser-112 phosphorylation could not trigger Bad dissociation from mitochondria in FL5.12 cells expressing Bcl-2. In 293T cells expressing Bcl-xL, Bad was phosphorylated at three serines, 112, 136 and 155, and was largely localized in the cytosolic fraction. In contrast, overexpression of Bcl-2 prevented phosphorylation of Bad at Ser-136 and Ser-155, sequestering this protein in the mitochondrial membranes. When the N-terminal regions of Bcl-2 and Bcl-xL were swapped with each other, the Bcl-xL(N)–Bcl-2 chimaeric protein (containing the N-terminal region of Bcl-xL) failed to prevent Bad phosphorylation in cells and was unable to block the cytosolic distribution of this pro-apoptotic protein. Additional experiments with the Bcl-2(N)–Bcl-xL chimaeric protein (containing the N-terminal region of Bcl-2) indicated that, although the N-terminal region of Bcl-2 is necessary, it is not sufficient for sequestering Bad in the mitochondrial membranes. These observations suggest that growth-factor-mediated phosphorylation of Bad contributes to the cytoprotective function of Bcl-xL but not Bcl-2.
Cloning and expression of a single-chain catalytic antibody that acts as a glutathione peroxidase mimic with high catalytic efficiency
Authors: REN Xiaojun , GAO Shujuan , YOU Delin , HUANG Hualiang , LIU Zi , MU Ying , LIU Junqiu , ZHANG Yan , YAN Ganglin , LUO Guimin , YANG Tongshu , SHEN Jiacong
Published: August 2015

Glutathione peroxidase (GPX) has a powerful role in scavenging reactive oxygen species. In previous papers we have developed a new strategy for generating abzymes: the monoclonal antibody with a substrate-binding site is first prepared, then a catalytic group is incorporated into the monoclonal antibody's binding site by using chemical mutation [Luo, Zhu, Ding, Gao, Sun, Liu, Yang and Shen (1994) Biochem. Biophys. Res. Commun. 198, 1240–1247; Ding, Liu, Zhu, Luo, Zhao and Ni (1998) Biochem. J. 332, 251–255]. Since then we have established a series of catalytic antibodies capable of catalysing the decomposition of hydroperoxides by GSH. The monoclonal antibody 2F3 was raised against GSH-S-2,4-dinitrophenyl t-butyl ester and exhibited high catalytic efficiency, exceeding that of rabbit liver GPX, after chemical mutation. To produce pharmaceutical proteins and to study the reason why it exhibits high catalytic efficiency, we sequenced, cloned and expressed the variable regions of 2F3 antibody as a single-chain Fv fragment (2F3-scFv) in different bacterial strains. The amounts of 2F3-scFv proteins expressed from JM109 (DE3), BL21 (DE3), and BL21 (coden plus) were 5–10%, 15–20% and 25–30% of total bacterial proteins respectively. The 2F3-scFv was expressed as inclusion bodies, purified in the presence of 8M urea by Co2+-immobilized metal-affinity chromatography (IMAC) and renatured to the active form in vitro by gel filtration. The binding constants of the active 2F3-scFv for GSH and GSSG were 2.46×105M−1 and 1.03×105M−1 respectively, which were less by one order of magnitude than that of the intact 2F3 antibody. The active 2F3-scFv was converted into selenium-containing 2F3-scFv (Se-2F3-scFv) by chemical modification of the reactive serine; the GPX activity of the Se-2F3-scFv was 3394units/μmol, which approaches the activity of rabbit liver GPX.
LIM-domain protein cysteine- and glycine-rich protein 2 (CRP2) is a novel marker of hepatic stellate cells and binding partner of the protein inhibitor of activated STAT1
Authors: WEISKIRCHEN Ralf , MOSER Markus , WEISKIRCHEN Sabine , ERDEL Martin , DAHMEN Sandra , BUETTNER Reinhard , GRESSNER Axel M.
Published: August 2015

Activation of hepatic stellate cells is considered to be the main step in the development of liver fibrosis, which is characterized by the transition of quiescent vitamin-A-rich cells to proliferative, fibrogenic and contractile myofibroblasts. The identification of regulatory genes during early cell activation and transdifferentiation is essential to extend our knowledge of hepatic fibrogenesis. In liver, the gene CSRP2 is exclusively expressed by stellate cells, whereas no transcripts are detectable in hepatocytes, sinusoidal endothelial cells or Kupffer cells. The early activation of stellate cells induced by platelet-derived growth factor is accompanied by an enhanced expression of CSRP2. During later stages of transdifferentiation, the expression of CSRP2 in these cells is suppressed in vitro and in vivo. The CSRP2-encoded cysteine- and glycine-rich double-LIM-domain protein (CRP)2 is proposed to function as a molecular adapter, arranging two or more as yet unidentified protein constituents into a macromolecular complex. To identify these proteins and assign a cellular function to CRP2, a human cDNA library was screened with full-length CRP2 as bait in a yeast two-hybrid screen. The protein inhibitor of activated STAT1 (‘PIAS1’) was shown to associate selectively with the C-terminal LIM domain of CRP2. Physical interaction of both proteins in the cellular environment was confirmed by co-localization experiments with confocal laser scanning microscopy and co-immunoprecipitation analysis. These results establish CRP2 as a potential new factor in the JAK/STAT-signalling pathway and suggest that the suppression of CSRP2 might be a prerequisite for the myofibroblastic transition of hepatic stellate cells.
PtdIns(4,5)P2 and phospholipase C-independent Ins(1,4,5)P3 signals induced by a nitrogen source in nitrogen-starved yeast cells
Authors: BERGSMA Jan C. T. , KASRI Nael Nadif , DONATON Monica C. V. , De WEVER Veerle , TISI Renata , de WINDE Johannes H. , MARTEGANI Enzo , THEVELEIN Johan M. , WERA Stefaan
Published: August 2015

Addition of ammonium sulphate to nitrogen-depleted yeast cells resulted in a transient increase in Ins(1,4,5)P3, with a maximum concentration reached after 7–8min, as determined by radioligand assay and confirmed by chromatography. Surprisingly, the transient increase in Ins(1,4,5)P3 did not trigger an increase in the concentration of intracellular calcium, as determined in vivo using the aequorin method. Similar Ins(1,4,5)P3 signals were also observed in wild-type cells treated with the phospholipase C inhibitor 3-nitrocoumarin and in cells deleted for the only phospholipase C-encoding gene in yeast, PLC1. This showed clearly that Ins(1,4,5)P3 was not generated by phospholipase C-dependent cleavage of PtdIns(4,5)P2. Apart from a transient increase in Ins(1,4,5)P3, we observed a transient increase in PtdIns(4,5)P2 after the addition of a nitrogen source to nitrogen-starved glucose-repressed cells. Inhibition by wortmannin of the phosphatidylinositol 4-kinase, Stt4, which is involved in PtdIns(4,5)P2 formation, did not affect the Ins(1,4,5)P3 signal, but significantly delayed the PtdIns(4,5)P2 signal. Moreover, wortmannin addition inhibited the nitrogen-induced activation of trehalase and the subsequent mobilization of trehalose, suggesting a role for PtdIns(4,5)P2 in nitrogen activation of the fermentable-growth-medium-induced signalling pathway.
Type II tumour necrosis factor-α receptor (TNFR2) activates c-Jun N-terminal kinase (JNK) but not mitogen-activated protein kinase (MAPK) or p38 MAPK pathways
Authors: JUPP Orla J. , McFARLANE Shona M. , ANDERSON Helen M. , LITTLEJOHN Alison F. , MOHAMED Ahmed A. A. , MACKAY Ruth H. , VANDENABEELE Peter , MACEWAN David J.
Published: August 2015

The pleitropic actions of tumour necrosis factor-α (TNF) are transmitted by the type I 55kDa TNF receptor (TNFR1) and type II 75kDa TNF receptor (TNFR2), but the signalling mechanisms elicited by these two receptors are not fully understood. In the present study, we report for the first time subtype-specific differential kinase activation in cell models that respond to TNF by undergoing apoptotic cell death. KYM-1human rhabdomyosarcoma cells and HeLa human cervical epithelial cells, engineered to overexpress TNFR2, displayed c-Jun N-terminal kinase (JNK) activation by wild-type TNF, a TNFR1-specific TNF mutant and a TNFR2-specific mutant TNF in combination with an agonistic TNFR2-specific monoclonal antiserum. A combination of the TNFR2-specific mutant and agonistic antiserum elicited maximal endogenous or exogenous TNFR2 responsiveness. Moreover, alternative expression of a TNFR2 deletion mutant lacking its cytoplasmic domain rendered the cells unable to activate JNK activity through this receptor subtype. The profile of JNK activation by TNFR1 was more transient than that of TNFR2, with TNFR2-induced JNK activity also being more sensitive to the caspase inhibitor, benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone. Conversely, only activation of the TNFR1 could stimulate mitogen-activated protein kinase (MAPK) or p38 MAPK activities in a time-dependent manner. The role of TNFR2 activation in enhanced apoptotic cell death was confirmed with agonistic monoclonal antisera in cells expressing high levels of TNFR2. Activation of TNFR2 alone elicited cell death, but full TNF-induced death required stimulation of both receptor types. These findings indicate that efficient activation of TNFR2 by soluble TNFs is achievable with co-stimulation by antisera, and that both receptors differentially modulate extracellular signal-regulated kinases contributing to the cytokine's cytotoxic response.
Regulation of mammalian phospholipase D2: interaction with and stimulation by GM2 activator
Authors: SARKAR Sukumar , MIWA Noriko , KOMINAMI Hiroaki , IGARASHI Nobuaki , HAYASHI Shun , OKADA Taro , JAHANGEER Saleem , NAKAMURA Shun-ichi
Published: August 2015

We have previously reported that a heat-stable activator for ganglioside metabolism, GM2 activator, potently stimulates ADP-ribosylation factor (ARF)-dependent phospholipase D (PLD) activity (presumably PLD1) in an in vitro system [Nakamura, Akisue, Jinnai, Hitomi, Sarkar, Miwa, Okada, Yoshida, Kuroda, Kikkawa and Nishizuka (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 12249–12253]. However, little is known about the regulation of PLD2. In the present studies we have investigated the regulation of PLD2 by GM2 activator and various other regulators including ARF. PLD2 was potently stimulated in vitro by GM2 activator in a time- and dose-dependent manner. Neither ARF nor protein kinase C caused any significant changes in PLD2 activity. Importantly, PLD2 responsiveness to ARF was greatly enhanced by GM2 activator, suggesting a possible role for GM2 activator as a coupling factor. GM2 activator was also demonstrated to physically associate with PLD2 in a stoichiometric manner. Further, PMA stimulation of COS-7 cells overexpressing both GM2 activator and PLD2 resulted in a marked increase in the association of the two molecules. Interestingly, ARF association with PLD2 was greatly increased by GM2 activator. Moreover, GM2 activator enhanced PMA-induced PLD activity in a synergistic manner with ARF in streptolysin-O-permeabilized, cytosol-depleted HL-60 cells, suggesting that GM2 activator may regulate PLD in a concerted manner with other factors, including ARF, inside the cells.
Bile-salt hydrophobicity is a key factor regulating rat liver plasma-membrane communication: relation to bilayer structure, fluidity and transporter expression and function
Authors: ASAMOTO Yasumasa , TAZUMA Susumu , OCHI Hidenori , CHAYAMA Kazuaki , SUZUKI Hiroshi
Published: August 2015

Bile-salt hydrophobicity regulates biliary phospholipid secretion and subselection. The aim of this study was to determine whether bile salts can influence liver plasma membrane phospholipids and fluidity in relation to the ATP-dependent transporter. Rats were depleted of bile salts by overnight biliary diversion and then sodium taurocholate was infused intravenously at a constant rate (200nmol/min per 100g of body weight), followed by infusion of bile salts with various hydrophobicities (taurochenodeoxycholate, tauroursodeoxycholate, tauro-β-muricholate, tauro-α-muricholate at 200nmol/min per 100g of body weight). The hydrophobicity of the infused bile salts correlated with that of biliary phospholipids, but was inversely related to that of the canalicular membrane bilayer. Canalicular membrane fluidity (estimated by 1,6-diphenyl-1,3,5-hexatriene fluorescence depolarization) and expression of multidrug-resistance proteins (Mrp2, Mrp3) and apical Na+-dependent bile-salt transporter (ASBT) were increased by hydrophilic bile salts, although there was no marked change in the expression of P-glycoprotein subfamilies (Mdr2). Bile-salt export pump (Bsep) expression was increased along with increasing bile-salt hydrophobicity. Bile salts modulate canalicular membrane phospholipids and membrane fluidity, as well as the ATP-dependent transporter expression and function, and these actions are associated with their hydrophobicity. The cytoprotective effect of hydrophilic bile salts seems to be associated with induction of Mrp2, Mrp3 and ASBT.
γ-Glutamyl transpeptidase in the yeast Saccharomyces cerevisiae and its role in the vacuolar transport and metabolism of glutathione
Authors: MEHDI Karim , THIERIE Jacques , PENNINCKX Michel J.
Published: August 2015

In the yeast Saccharomyces cerevisiae, the enzyme γ-glutamyl transpeptidase (γ-GT; EC 2.3.2.2) is a glycoprotein that is bound to the vacuolar membrane. The kinetic parameters of GSH transport into isolated vacuoles were measured using intact vacuoles isolated from the wild-type yeast strain Σ1278b, under conditions of γ-GT synthesis (nitrogen starvation) and repression (growth in the presence of ammonium ions). Vacuoles devoid of γ-GT displayed a Km (app) of 18±2mM and a Vmax (app) of 48.5±5nmol of GSH/min per mg of protein. Vacuoles containing γ-GT displayed practically the same Km, but a higher Vmax (app) (150±12nmol of GSH/min per mg of protein). Vacuoles prepared from a disruptant lacking γ-GT showed no increase in Vmax (app) with nitrogen starvation. From a comparison of the transport data obtained for vacuoles isolated from various reference and mutant strains, it appears that the yeast cadmium factor 1 (YCF1) transport system accounts for approx. 70% of the GSH transport capacity of the vacuoles, the remaining 30% being due to a vacuolar (H+) ATPase-coupled system. The Vmax (app)-increasing effect of γ-GT concerns only the YCF1 system. γ-GT in the vacuolar membrane activates the Ycf1p transporter, either directly or indirectly. Moreover, GSH accumulating in the vacuolar space may exert a feedback effect on its own entry. Excretion of glutamate from radiolabelled GSH in isolated vacuoles containing γ-GT was also measured. It is proposed that γ-GT and a l-Cys-Gly dipeptidase catalyse the complete hydrolysis of GSH stored in the central vacuole of the yeast cell, prior to release of its constitutive amino acids l-glutamate, l-cysteine and glycine into the cytoplasm. Yeast appears to be a useful model for studying γ-GT physiology and GSH metabolism.
Cysteine-directed cross-linking localizes regions of the human erythrocyte anion-exchange protein (AE1) relative to the dimeric interface
Authors: TAYLOR Andrew M. , ZHU Quansheng , CASEY Joseph R.
Published: August 2015

The human erythrocyte anion-exchanger isoform 1 (AE1) is a dimeric membrane protein that exchanges chloride for bicarbonate across the erythrocyte plasma membrane. Crystallographic studies suggest that the transmembrane anion channel lies at the interface between the two monomers, whereas kinetic analysis provides evidence that each monomer contains an anion channel. We have studied the structure–function relationship of residues at the dimeric interface of AE1 by cysteine-directed cross-linking. Single cysteine mutations were introduced in 16 positions of putative loop regions throughout AE1. The ability of these residues to be chemically cross-linked to their partner within the dimeric protein complex was assessed by mobility of the protein on immunoblots. Introduced cysteine residues in extracellular loops (ECs) 1–4 and intracellular loop 1 formed disulphide cross-linked dimers. Treatment with homobifunctional maleimide cross-linkers of different lengths (6, 10 and 16 Å; 1Å≡0.1nm) also cross-linked AE1 with introduced cysteines in EC5 and close to the start of transmembrane segment (TM) 1. On the basis of these data, tentative positional constraints of TMs 1–4 and 6 relative to the dimeric interface are proposed. Neither disulphide- nor maleimide-mediated cross-linking perturbed AE1 transport function, suggesting that loop–loop contacts across the dimeric interface are not primarily responsible for allosteric interactions between monomers within the functional dimeric protein complex.
Multiple stimuli induce tyrosine phosphorylation of the Crk-binding sites of paxillin
Authors: SCHALLER Michael D. , SCHAEFER Erik M.
Published: August 2015

Paxillin is a focal-adhesion-associated, tyrosine-phosphorylated protein. In cells transformed by the src, crk or BCR-Abl oncogenes, the phosphotyrosine content of paxillin is elevated. In normal cells paxillin functions in signalling following integrin-dependent cell adhesion or exposure to a number of stimuli, including growth factors and neuropeptides. These stimuli induce tyrosine phosphorylation of paxillin, regulating the association of Src homology 2 domain-containing signalling molecules with paxillin. There are multiple sites of tyrosine phosphorylation on paxillin. To elucidate the role of paxillin in transducing signals in response to various stimuli, it is essential to identify all of the sites of phosphorylation on paxillin and to define which residues are phosphorylated in response to distinct stimuli. We describe two new sites of tyrosine phosphorylation on paxillin, residues at positions 40 and 88. Using paxillin variants with phenylalanine substitutions at phosphorylation sites and phospho-specific paxillin antibodies, tyrosine phosphorylation of paxillin in response to distinct stimuli was examined. The results demonstrate that Tyr31 and Tyr118, which are binding sites for Crk, are major sites of tyrosine phosphorylation following cell adhesion or stimulation with platelet-derived growth factor or angiotensin II. Thus multiple stimuli may elicit similar signalling events downstream of paxillin.
Src-family tyrosine kinases, phosphoinositide 3-kinase and Gab1 regulate extracellular signal-regulated kinase 1 activation induced by the type A endothelin-1 G-protein-coupled receptor
Authors: BISOTTO Sandra , FIXMAN Elizabeth D.
Published: August 2015

The multisubstrate docking protein, growth-factor-receptor-bound protein 2-associated binder 1 (Gab1), which is phosphorylated on tyrosine residues following activation of receptor tyrosine kinases and cytokine receptors, regulates cell proliferation, survival and epithelial morphogenesis. Gab1 is also tyrosine phosphorylated following activation of G-protein-coupled receptors (GPCRs) where its function is poorly understood. To elucidate the role of Gab1 in GPCR signalling, we investigated the mechanism by which the type A endothelin-1 (ET-1) GPCR induced tyrosine phosphorylation of Gab1. Tyrosine phosphorylation of Gab1 induced by endothelin-1 was inhibited by PP1, a pharmacological inhibitor of Src-family tyrosine kinases. ET-1-induced Gab1 tyrosine phosphorylation was also inhibited by LY294002, which inhibits phosphoinositide 3-kinase (PI 3-kinase) enzymes. Inhibition of Src-family tyrosine kinases or PI 3-kinase also inhibited ET-1-induced activation of the mitogen activated protein kinase family member, extracellular signal-regulated kinase (ERK) 1. Thus we determined whether Gab1 regulated ET-1-induced ERK1 activation. Overexpression of wild-type Gab1 potentiated ET-1-induced activation of ERK1. Structure–function analyses of Gab1 indicated that mutant forms of Gab1 that do not bind the Src homology (SH) 2 domains of the p85 adapter subunit of PI 3-kinase or the SH2-domain-containing protein tyrosine phosphatase 2 (SHP-2) were impaired in their ability to potentiate ET-1-induced ERK1 activation. Taken together, our data indicate that PI 3-kinase and Src-family tyrosine kinases regulate ET-1-induced Gab1 tyrosine phosphorylation, which, in turn, induces ERK1 activation via PI 3-kinase- and SHP-2-dependent pathways.
Structure, properties and enhanced expression of galactose-binding C-type lectins in mucous cells of gills from freshwater Japanese eels (Anguilla japonica)
Authors: MISTRY Abinash Chandra , HONDA Shinji , HIROSE Shigehisa
Published: August 2015

Using a Japanese-eel (Anguilla japonica) gill cDNA subtraction library, two novel β-d-galactose-binding lectins were identified that belong to group VII of the animal C-type lectin family. The eel C-type lectins, termed eCL-1 and eCL-2, are simple lectins composed of 163 amino acid residues, including a 22-residue signal peptide for secretion and a single carbohydrate-recognition domain (CRD) of ∼ 130 residues typical of C-type lectins. The galactose specificity of the CRD was suggested by the presence of a QPD motif and confirmed by a competitive binding assay. Using Ruthenium Red staining, the lectins were shown to bind Ca2+ ions. SDS/PAGE showed that native eCL-1 and eCL-2have an SDS-resistant octameric structure (a tetramer of disulphide-linked dimers). Northern and Western blot analyses demonstrated high-level expression of eCL-1 and eCL-2 mRNAs and their protein products in gills from freshwater eels, which decreased markedly when the eels were transferred from freshwater to seawater. Immunohistochemistry showed that the eel lectins are localized in the exocrine mucous cells of the gill.
Identification of N-acetyl-d-glucosamine-specific lectins from rat liver cytosolic and nuclear compartments as heat-shock proteins
Authors: LEFEBVRE Tony , CIENIEWSKI Caroline , LEMOINE Jérôme , GUERARDEL Yann , LEROY Yves , ZANETTA Jean-Pierre , MICHALSKI Jean-Claude
Published: August 2015

Cytosolic and nuclear O-linked N-acetylglucosaminylation has been proposed to be involved in the nuclear transport of cytosolic proteins. We have isolated nuclear and cytosolic N-acetyl-d-glucosamine (GlcNAc)-specific lectins from adult rat liver by affinity chromatography on immobilized GlcNAc and identified these lectins, by a proteomic approach, as belonging to the heat-shock protein (HSP)-70 family (one of them being heat-shock cognate 70 stress protein). Two-dimensional electrophoresis indicated that the HSP-70 fraction contained three equally abundant proteins with molecular masses of 70, 65 and 55kDa. The p70 and p65 proteins are phosphorylated and are themselves O-linked GlcNAc (O-GlcNAc)-modified. The HSP-70 associated into high molecular mass complexes that dissociated in the presence of reductive and chaotropic agents. The lectin(s) present in this complex was (were) able to recognize cytosolic and nuclear ligands, which have been isolated using wheat germ agglutinin affinity chromatography. These ligands are O-GlcNAc glycosylated as demonstrated by [3H]galactose incorporation and analysis of the products released by reductive β-elimination. The isolated lectins specifically recognized ligands present in both the cytosol and the nucleus of human resting lymphocytes. These results demonstrated the existence of endogenous GlcNAc-specific lectins, identified as HSP-70 proteins, which could act as a shuttle for the nucleo-cytoplasmic transport of O-GlcNAc glycoproteins between the cytosol and the nucleus.
Activation of phosphodiesterase 5 and inhibition of guanylate cyclase by cGMP-dependent protein kinase in smooth muscle
Authors: MURTHY Karnam S.
Published: August 2015

The regulation of cGMP-specific phosphodiesterase (PDE) 5 and soluble guanylate cyclase (GC) by cGMP- and cAMP-dependent protein kinases (PKG and PKA respectively) was examined in gastric smooth muscle. The NO donor, sodium nitroprusside (SNP), stimulated PDE5 phosphorylation and activity, which was blocked by the selective PKG inhibitor, KT5823, resulting in an elevation of cGMP levels. Activation of PKA either directly by Sp-5,6-dichloro-1-β-d-ribofuranosyl benzimidazole 3′,5′-cyclic monophosphothioate, or via isoproterenol- and forskolin-dependent increase in cAMP, also caused an increase in PDE5 phosphorylation and activity, but only in the presence of cGMP; consistent with the dependence of PDE5 phosphorylation and activity on cGMP binding to allosteric sites in the regulatory domain of PDE5. The selective PKA inhibitors, myristoylated protein kinase inhibitor and H-89, blocked the increase in PDE5 phosphorylation and activity induced by PKA. SNP also stimulated soluble GC phosphorylation and activity. KT5823 abolished phosphorylation and augmented soluble GC activity, implying feedback inhibition of soluble GC by PKG-dependent phosphorylation. Phosphorylation by PKG was direct and could be induced in vitro. Activation of PKA had no effect on soluble GC. Thus cGMP levels are regulated by PKG- and PKA-dependent activation of PDE5 and PKG-specific inhibition of soluble GC.
Functional regulation of glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1) of Drosophila melanogaster in a UDP-N-acetylglucosamine and cAMP-dependent manner
Authors: GRAACK Hanns-Rüdiger , CINQUE Ursula , KRESS Horst
Published: August 2015

Glutamine:fructose-6-phosphate aminotransferase (GFAT; EC 2.6.1.16) expression is tightly regulated in the context of amino sugar synthesis in many organisms from yeast to humans by transcriptional and post-translational processes. We have cloned the cDNA of the GFAT1 of Drosophila melanogaster (Dmel/Gfat1). One of the two putative protein kinase A (PKA) phosphorylation sites proposed for the regulation of human GFAT1 [Zhou, Huynh, Hoffmann, Crook, Daniels, Gulve and McClain (1998) Diabetes 47, 1836–1840] is conserved in Dmel/GFAT1. In the other one the reactive serine has been converted to a cysteine, making further access by PKA unlikely. The Dmel/Gfat1 gene is localized at position 81F on the right arm of chromosome 3. By whole-mount in situ hybridization specific expression of Dmel/GFAT1 was detected in embryonic chitin-synthesizing tissues and in the corpus cells of salivary glands from late third larval instar. Expressing Dmel/GFAT1 in yeast we showed that Dmel/GFAT1 activity is controlled by UDP-N-acetylglucosamine and PKA in the yeast total protein extract system. We propose a model for the independent regulation of the Dmel/GFAT1 enzyme by feedback inhibition and PKA.
A constitutive 70 kDa heat-shock protein is localized on the fibres of spindles and asters at metaphase in an ATP-dependent manner: a new chaperone role is proposed
Authors: AGUELI Cecilia , GERACI Fabiana , GIUDICE Giovanni , CHIMENTI Laura , CASCINO Domenico , SCONZO Gabriella
Published: August 2015

In the present study, double immunofluorescence and immunoblot analysis have been used to show that centrosomes, isolated from Paracentrotus lividus sea urchin embryos at the first mitotic metaphase, contain the constitutive chaperone, heat-shock protein (HSP) 70. More specifically, we demonstrate that centrosomes contain only the HSP70-d isoform, which is one of the four isoforms identified in P. lividus. We also provide evidence that p34(cell division control kinase-2) and t complex polypeptide-1 (TCP-1) α, a subunit of the TCP-1 complex, are localized on the centrosomes. Furthermore, inhibition of TCP-1 in vivo, via microinjecting an anti-(TCP-1α) antibody into P. lividus eggs before fertilization, either impaired mitosis or induced severe malformations in more than 50% of embryos. In addition, we have isolated the whole mitotic apparatus and shown that HSP70 localizes on the fibres of spindles and asters, and binds them in an ATP-dependent manner. These observations suggest that HSP70has a chaperone role in assisting the TCP-1 complex in tubulin folding, when localized on centrosomes, and during the assembling and disassembling of the mitotic apparatus, when localized on the fibres of spindles and asters.
Localization of p24 putative cargo receptors in the early secretory pathway depends on the biosynthetic activity of the cell
Authors: KUIPER Roland P. , BOUW Gerrit , JANSSEN Karel P. C. , RÖTTER Jutta , van HERP François , MARTENS Gerard J. M.
Published: August 2015

Members of the p24 family of putative cargo receptors (subdivided into p24-α, −β, −γ and −δ) are localized in the intermediate-and cis-Golgi compartments of the early secretory pathway, and are thought to play an important role in protein transport. In the present study, we wondered what effect increased biosynthetic cell activity with resulting high levels of protein transport would have on the subcellular localization of p24. We examined p24 localization in Xenopus intermediate pituitary melanotrope cells, which in black- and white-adapted animals are biosynthetically highly active and virtually inactive respectively. In addition, p24 localization was studied in Xenopus anterior pituitary cells whose activity is not changed during background adaptation. Using organelle fractionation, we found that in the inactive melanotropes and moderately active anterior pituitary cells of white-adapted animals, the p24-α, −β, −γ and −δ proteins are all located in the Golgi compartment. In the highly active melanotropes, but not in the anterior cells of black-adapted animals, the steady-state distribution of all four p24 members changed towards the intermediate compartment and subdomains of the endoplasmic reticulum (ER), most probably the ER exit sites. In the active melanotropes, the major cargo protein pro-opiomelanocortin was mostly localized to ER subdomains and partially co-localized with the p24 proteins. Furthermore, in the active cells, in vitro blocking of protein biosynthesis by cycloheximide or dispersion of the Golgi complex by brefeldin A led to a redistribution of the p24 proteins, indicating their involvement in ER-to-Golgi protein transport and extensive cycling in the early secretory pathway. We conclude that the subcellular localization of p24 proteins is dynamic and depends on the biosynthetic activity of the cell.
Expression and characterization of the Plasmodium falciparum haemoglobinase falcipain-3
Authors: SIJWALI Puran S. , SHENAI Bhaskar R. , GUT Jiri , SINGH Ajay , ROSENTHAL Philip J.
Published: August 2015

In the malaria parasite Plasmodium falciparum, erythrocytic trophozoites hydrolyse haemoglobin to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block parasite haemoglobin hydrolysis and development, indicating that cysteine proteases are required for these processes. Three papain-family cysteine protease sequences have been identified in the P. falciparum genome, but the specific roles of their gene products and other plasmodial proteases in haemoglobin hydrolysis are uncertain. Falcipain-2 was recently identified as a principal trophozoite cysteine protease and potential drug target. The present study characterizes the related P. falciparum cysteine protease falcipain-3. As is the case with falcipain-2, falcipain-3 is expressed by trophozoites and appears to be located within the food vacuole, the site of haemoglobin hydrolysis. Both proteases require a reducing environment and acidic pH for optimal activity, and both prefer peptide substrates with leucine at the P2 position. The proteases differ, however, in that falcipain-3 undergoes efficient processing to an active form only at acidic pH, is more active and stable at acidic pH, and has much lower specific activity against typical papain-family peptide substrates, but has greater activity against native haemoglobin. Thus falcipain-3 is a second P. falciparum haemoglobinase that is particularly suited for the hydrolysis of native haemoglobin in the acidic food vacuole. The redundancy of cysteine proteases may offer optimized hydrolysis of both native haemoglobin and globin peptides. Consideration of both proteases will be necessary to evaluate cysteine protease inhibitors as antimalarial drugs.
Implication of protein S thrombin-sensitive region with membrane binding via conformational changes in the γ-carboxyglutamic acid-rich domain
Authors: BORGEL Delphine , GAUSSEM Pascale , GARBAY Christiane , BACHELOT-LOZA Christilla , KAABACHE Tahar , LIU Wang-Qing , BROHARD-BOHN Brigitte , LE BONNIEC Bernard , AIACH Martine , GANDRILLE Sophie
Published: August 2015

In the vitamin K-dependent protein family, only protein S (PS) contains a thrombin-sensitive region (TSR), located between the domain containing the γ-carboxyglutamic acid and the first epidermal growth factor-like domain. To better define the role of TSR in the PS molecule, we expressed a recombinant human PS (rHPS) and its analogue lacking TSR (rTSR-less), and prepared factor Xa- and thrombin-cleaved rHPS. A peptide reproducing TSR (TSR-peptide) was also synthesized in an attempt to obtain direct evidence of the domain involvement in PS anticoagulant activity. In a coagulation assay, both rTSR-less and factor Xa-cleaved PS were devoid of activated protein C cofactor activity. The TSR-peptide did not inhibit rHPS activity, showing that TSR must be embedded in the native protein to promote interaction with activated protein C. The binding of rHPS to activated platelets and to phospholipid vesicles was not modified after factor Xa- or thrombin-mediated TSR cleavage, whereas the binding of rTSR-less was markedly reduced. This suggested a role for TSR in conferring to PS a strong affinity for phospholipid membranes. TSR-peptide did not directly bind to activated platelets or compete with rHPS for phospholipid binding. The results of the present study show that TSR may not interact directly with membranes, but probably constrains the γ-carboxyglutamic acid-rich domain in a conformation allowing optimal interaction with phospholipids.
Bacterial peptide methionine sulphoxide reductase: co-induction with glutathione S-transferase during chemical stress conditions
Authors: TAMBURRO Antonio , ALLOCATI Nerino , MASULLI Michele , ROTILIO Domenico , DI ILIO Carmine , FAVALORO Bartolo
Published: August 2015

Peptide methionine sulphoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous enzyme catalysing the reduction of methionine sulphoxide to methionine in proteins, while the glutathione S-transferases (GSTs) are a major family of detoxification enzymes. A gene homologous to MsrA was identified in a chromosomal fragment from the bacterium Ochrobactrum anthropi, and this gene is located just downstream of a GST gene identified previously (OaGST) [Favaloro, Tamburro, Angelucci, De Luca, Melino, Di Ilio and Rotilio (1998) Biochem. J. 335, 573–579]. This raises the question of whether the products of these two genes may be involved in a common cellular protection function. To test this hypothesis, the hypothetical MsrA protein has been overexpressed in Escherichia coli as a functional 51kDa GST fusion protein. Following cleavage with thrombin and purification, the soluble 24kDa protein showed MsrA activity with N-acetylmethionine sulphoxide as substrate, as well as with other sulphoxide compounds. Therefore polyclonal antibodies were raised against the recombinant protein, and the modulation of MsrA in this bacterium, grown in the presence of different stimulants simulating several stress conditions, was investigated. The level of expression of MsrA was detected both by measuring the mRNA level and by immunoblotting experiments, in addition to measuring its catalytic activity. MsrA is a constitutive enzyme which is also inducible by chemical stress involving phenolic compounds such as phenol and 4-chlorophenol. Recently we reported that the GST of this bacterium, like MsrA, is only modulated by toxic chemical compounds [Favaloro, Tamburro, Trofino, Bologna, Rotilio and Heipieper (2000) Biochem. J. 346, 553–559]; therefore this is the first indication of a co-induction of the MsrA and GST enzymes during chemical stress.
Proprotein convertase PACE4 is down-regulated by the basic helix–loop–helix transcription factor hASH-1 and MASH-1
Authors: YOSHIDA Ichiro , KOIDE Shizuyo , HASEGAWA Shin-ichi , NAKAGAWARA Akira , TSUJI Akihiko , MATSUDA Yoshiko
Published: August 2015

PACE4 is a mammalian subtilisin-like proprotein convertase that activates transforming growth factor (TGF)-β-related proteins such as bone morphogenetic protein 2 (BMP2), BMP4 and Nodal and exhibits a dynamic expression pattern during embryogenesis. We recently determined that the 1kb 5′-upstream region of the PACE4 gene contains 12 E-box (E1–E12) elements and that an E-box cluster (E4–E9) acts as a negative regulator [Tsuji, Yoshida, Hasegawa, Bando, Yoshida, Koide, Mori and Matsuda (1999) J. Biochem. (Tokyo) 126, 494–502]. It is known that the mammalian achaete–scute homologue 1 (MASH-1) binds specifically to an E-box (CACCTG) sequence in collaboration with E47, a ubiquitously expressed basic helix–loop–helix (bHLH) factor. To identify the roles of the bHLH factor and E-box elements in regulating PACE4 gene expression in neural development, we analysed the effects of human achaete–scute homologue 1 (hASH-1) on PACE4 gene expression with various neuroblastoma cell lines. The expressions of PACE4 and hASH-1 are correlated inversely in these cell lines. The overexpression of hASH-1 or MASH-1 causes a marked decrease in endogenous PACE4 gene expression but has no effect on the expression of other subtilisin-like proprotein convertases such as furin, PC5/6 and PC7/8. In contrast, other neural bHLH factors (MATH-1, MATH-2, neurogenin 1, neurogenin 2, neurogenin 3 and E47) did not affect PACE4 gene expression. Furthermore, an E-box cluster was a negative regulatory element for the promoter activity in NBL-S cells expressing hASH-1 at high level as determined by a luciferase assay. Binding of hASH-1 to the E-box cluster was confirmed by gel mobility-shift assay. In the present study we identified the PACE4 gene as one of the targets of hASH-1, which is a key factor in the initiation of neural differentiation. These results suggest that the alteration of PACE4 gene expression by hASH-1 causes rapid changes in the biological activities of TGF-β-related proteins via post-translational modification of these proteins.
Determination of the native form of FadD, the Escherichia coli fatty acyl-CoA synthetase, and characterization of limited proteolysis by outer membrane protease OmpT
Authors: YOO Jae-Ho , CHENG Oscar H. , GERBER Gerhard E.
Published: August 2015

Several studies have described FadD, the Escherichia coli fatty acyl-CoA synthetase [also known as fatty acid:CoA ligase (AMP-forming); EC 6.2.1.3], as a 42–50kDa enzyme. Based on sequencing and expression data from the fadD gene, other reports have suggested that FadD is a 62kDa protein and represents the sole fatty acyl-CoA synthetase in E. coli. We report that the 62kDa FadD enzyme is a substrate for the outer membrane protease OmpT in vitro, producing a 43kDa C-terminal fragment and a 19kDa N-terminal fragment. Immunoblotting with a FadD antibody revealed that only the 62kDa form of the enzyme is present in vivo, but we utilized the proteolytic sensitivity of FadD to investigate its structure. Photoaffinity labelling experiments revealed that both intact FadD and the 43kDa fragment bound a long-chain fatty acid. Intact and cleaved FadD were also purified to determine the effect of cleavage on function. When using oleate as a substrate, cleaved FadD displayed 2-fold higher Km and Vmax values compared with intact FadD, but the catalytic efficiencies (kcat/Km) of the two forms were similar. This indicated that cleavage did not adversely affect enzyme activity. Proteolysis of FadD by OmpT was altered by the presence of oleate or ATP, both of which are ligands for the fatty acyl-CoA synthetase. This suggested that FadD undergoes ligand-induced conformational changes and implies that the region surrounding the cleavage site is mobile, a common characteristic of linker domains.
Phospholipase D1b and D2a generate structurally identical phosphatidic acid species in mammalian cells
Authors: PETTITT Trevor R. , McDERMOTT Mark , SAQIB Khalid M. , SHIMWELL Neil , WAKELAM Michael J. O.
Published: August 2015

Mammalian cells contain different phospholipase D enzymes (PLDs) whose distinct physiological roles are poorly understood and whose products have not been characterized. The development of porcine aortic endothelial (PAE) cell lines able to overexpress PLD-1b or −2a under the control of an inducible promoter has enabled us to characterize both the substrate specificity and the phosphatidic acid (PtdOH) product of these enzymes under controlled conditions. Liquid chromatography–MS analysis showed that PLD1b- and PLD2a-transfected PAE cells, as well as COS7 and Rat1 cells, generate similar PtdOH and, in the presence of butan-1-ol, phosphatidylbutanol (PtdBut) profiles, enriched in mono- and di-unsaturated species, in particular 16:0/18:1. Although PtdBut mass increased, the species profile did not change in cells stimulated with ATP or PMA. Overexpression of PLD made little difference to basal or stimulated PtdBut formation, indicating that activity is tightly regulated in vivo and that factors other than just PLD protein levels limit hydrolytic function. In vitro assays using PLD-enriched lysates showed that the enzyme could utilize both phosphatidylcholine and, much less efficiently, phosphatidylethanolamine, with slight selectivity towards mono- and di-unsaturated species. Phosphatidylinositol was not a substrate. Thus PLD1b and PLD2a hydrolyse a structurally similar substrate pool to generate an identical PtdOH product enriched in mono- and di-unsaturated species that we propose to function as the intracellular messenger forms of this lipid.
A+U-rich-element RNA-binding factor 1/heterogeneous nuclear ribonucleoprotein D gene expression is regulated by oestrogen in the rat uterus
Authors: ARAO Yukitomo , KIKUCHI Atsumi , IKEDA Kazuhiro , NOMOTO Satoshi , HORIGUCHI Hyogo , KAYAMA Fujio
Published: August 2015

Oestrogen-mediated gene expression is regulated at both the transcriptional and post-transcriptional levels. The molecular mechanism of transcriptional regulation has been well characterized. On the other hand, there is little understanding of the mechanism of post-transcriptional regulation. To clarify the mechanism of oestrogen-mediated post-transcriptional regulation, we focused on A+U-rich-element RNA-binding factor 1/heterogeneous nuclear ribonucleoprotein D (AUF1/hnRNP D), which is known as a regulator of cytosolic mRNA degradation and nuclear pre-mRNA maturation. However, little is known about the expression levels and the regulation of AUF1/hnRNP D mRNA in tissues. We further investigated the expression levels of AUF1/hnRNP D isoform mRNAs to determine whether AUF1/hnRNP D gene expression is regulated by oestrogen in the ovariectomized adult female rat uterus. Uterine AUF1/hnRNP D mRNA was induced by a single subcutaneous injection (1μg/kg) of 17β-oestradiol (E2), reaching a peak level within 6h. Furthermore, we observed that the E2-induced AUF1/hnRNP D isoform mRNAs are p45 and p40 transcripts, and that E2-mediated induction is suppressed by the oestrogen receptor antagonist ICI 182,780. Finally, using the transcriptional inhibitor actinomycin D, we confirmed that the E2-mediated increase in AUF1/hnRNP D mRNA is caused by E2-dependent AUF1/hnRNP D mRNA stabilization.