bluuebunny/crossref_metadata_2025 huggingface dataset:
A role for smooth endoplasmic reticulum membrane cholesterol ester in determining the intracellular location and regulation of sterol-regulatory-element-binding protein-2
Authors: IDDON Christopher R. , WILKINSON Jane , BENNETT Andrew J. , BENNETT Julie , SALTER Andrew M. , HIGGINS Joan A.
Published: August 2015

Cellular cholesterol homoeostasis is regulated through proteolysis of the membrane-bound precursor sterol-regulatory-element-binding protein (SREBP) that releases the mature transcription factor form, which regulates gene expression. Our aim was to identify the nature and intracellular site of the putative sterol-regulatory pool which regulates SREBP proteolysis in hamster liver. Cholesterol metabolism was modulated by feeding hamsters control chow, or a cholesterol-enriched diet, or by treatment with simvastatin or with the oral acyl-CoA:cholesterol acyltransferase inhibitor C1-1011 plus cholesterol. The effects of the different treatments on SREBP activation were confirmed by determination of the mRNAs for the low-density lipoprotein receptor and hydroxymethylglutaryl-CoA (HMG-CoA) reductase and by measurement of HMG-CoA reductase activity. The endoplasmic reticulum was isolated from livers and separated into subfractions by centrifugation in self-generating iodixanol gradients. Immunodetectable SREBP-2 accumulated in the smooth endoplasmic reticulum of cholesterol-fed animals. Cholesterol ester levels of the smooth endoplasmic reticulum membrane (but not the cholesterol levels) increased after cholesterol feeding and fell after treatment with simvastatin or C1-1011. The results suggest that an increased cellular cholesterol load causes accumulation of SREBP-2 in the smooth endoplasmic reticulum and, therefore, that membrane cholesterol ester may be one signal allowing exit of the SREBP-2/SREBP-cleavage-regulating protein complex to the Golgi.
Direct identification of a major autophosphorylation site on vascular endothelial growth factor receptor Flt-1 that mediates phosphatidylinositol 3′-kinase binding
Authors: YU Ying , HULMES Jeffrey D. , HERLEY Mark T. , WHITNEY Ronald G. , CRABB John W. , SATO J. Denry
Published: August 2015

Progress has been made in our understanding of the mechanism by which the binding of vascular endothelial growth factor (VEGF) to cognate receptors induces a range of biological responses, but it is far from complete. Identification of receptor autophosphorylation sites will allow us to determine how activated VEGF receptors are coupled to specific downstream signalling proteins. In the present study, we have expressed human VEGF receptors in insect cells using the baculovirus expression system, identified a major autophosphorylation site on the VEGF receptor fms-like tyrosine kinase-1 (Flt-1) by HPLC–electrospray ionization (ESI)–MS, and characterized in vitro interactions between Flt-1 and phosphatidylinositol 3′-kinase (PI3-kinase). Infection of High 5 insect cells with Flt-1 recombinant virus resulted in the expression of a 170kDa glycoprotein, which bound VEGF with a Kd of 2×10−10M in intact insect cells. The overexpressed recombinant Flt-1 receptors exhibited tyrosine kinase activity and were constitutively phosphorylated. Analysis of Flt-1 tryptic peptides by HPLC–ESI–MS with selective phosphate ion monitoring identified a hexapeptide (YVNAFK; where single-letter amino-acid code has been used) containing a phosphotyrosine (pTyr) residue at position 1213. Using synthetic phosphopeptides, this pTyr residue was found to be directly involved in the binding of PI3-kinase in vitro even though it did not fall within a consensus pYM/VXM PI3-kinase binding motif. These results suggest that phosphorylated Flt-1 associates with PI3-kinase at pTyr1213 to mediate the activation of this pathway in VEGF signalling.
Carbohydrate specificity of a galectin from chicken liver (CG-16)
Authors: WU Albert M. , WU June H. , TSAI Ming-Sung , KALTNER Herbert , GABIUS Hans-J.
Published: August 2015

Owing to the expression of more than one type of galectin in animal tissues, the delineation of the functions of individual members of this lectin family requires the precise definition of their carbohydrate specificities. Thus, the binding properties of chicken liver galectin (CG-16) to glycoproteins (gps) and Streptococcus pneumoniae type 14 polysaccharide were studied by the biotin/avidin-mediated microtitre-plate lectin-binding assay and by the inhibition of lectin–glycan interactions with sugar ligands. Among 33 glycans tested for lectin binding, CG-16 reacted best with human blood group ABO (H) precursor gps and their equivalent gps, which contain a high density of d-galactopyranose(β1–4)2-acetamido-2-deoxy-d-glucopyranose [Gal(β1–4)GlcNAc] and Gal(β1–3)GlcNAc residues at the non-reducing end, but this lectin reacted weakly or not at all with A-,H-type and sialylated gps. Among the oligosaccharides tested by the inhibition assay, the tri-antennary Gal(β1–4)GlcNAc (Tri-II) was the best. It was 2.1×103 nM and 3.0 times more potent than Gal and Gal(β1–4)GlcNAc (II)/Gal(β1–3)GlcNAc(β1–3)Gal(β1–4)Glc (lacto-N-tetraose) respectively. CG-16has a preference for the β-anomer of Gal at the non-reducing end of oligosaccharides with a Gal(β1–4) linkage >Gal(β1–3)Gal(β1–6). From the results, it can be concluded that the combining site of this agglutinin should be a cavity type, and that a hydrophobic interaction in the vicinity of the binding site for sugar accommodation increases the affinity. The binding site of CG-16 is as large as a tetrasaccharide of the β-anomer of Gal, and is most complementary to lacto-N-tetraose and Gal(β1–4)GlcNAc related sequences.
Dimethyl sulphoxide enhances the effects of Pi in myofibrils and inhibits the activity of rabbit skeletal muscle contractile proteins
Authors: MARIANO Ana C. , ALEXANDRE Giani M. C. , SILVA Leonardo C. , ROMEIRO Alexandre , CAMERON Luiz Claudio , CHEN Ying , CHASE P. Bryant , SORENSON Martha M.
Published: August 2015

In the catalytic cycle of skeletal muscle, myosin alternates between strongly and weakly bound cross-bridges, with the latter contributing little to sustained tension. Here we describe the action of DMSO, an organic solvent that appears to increase the population of weakly bound cross-bridges that accumulate after the binding of ATP, but before Pi release. DMSO (5–30%, v/v) reversibly inhibits tension and ATP hydrolysis in vertebrate skeletal muscle myofibrils, and decreases the speed of unregulated F-actin in an in vitro motility assay with heavy meromyosin. In solution, controls for enzyme activity and intrinsic tryptophan fluorescence of myosin subfragment 1 (S1) in the presence of different cations indicate that structural changes attributable to DMSO are small and reversible, and do not involve unfolding. Since DMSO depresses S1 and acto–S1MgATPase activities in the same proportions, without altering acto–S1 affinity, the principal DMSO target apparently lies within the catalytic cycle rather than with actin–myosin binding. Inhibition by DMSO in myofibrils is the same in the presence or the absence of Ca2+ and regulatory proteins, in contrast with the effects of ethylene glycol, and the Ca2+ sensitivity of isometric tension is slightly decreased by DMSO. The apparent affinity for Pi is enhanced markedly by DMSO (and to a lesser extent by ethylene glycol) in skinned fibres, suggesting that DMSO stabilizes cross-bridges that have ADP·Pi or ATP bound to them.
Lipid modification of the Cu,Zn superoxide dismutase from Mycobacterium tuberculosis
Authors: D'ORAZIO Melania , FOLCARELLI Silvia , MARIANI Francesca , COLIZZI Vittorio , ROTILIO Giuseppe , BATTISTONI Andrea
Published: August 2015

The leader sequence of Mycobacterium tuberculosis Cu,Zn superoxide dismutase (Cu,ZnSOD) contains a prokaryotic membrane lipoprotein attachment site. In the present study, we have found that the protein, which exhibits detectable SOD activity, is lipid-modified and associated with the bacterial membrane when expressed either in M. tuberculosis or in Escherichia coli. These results provide the first demonstration of lipid modification of a Cu,ZnSOD. An analysis of the sodC genes present in available databases indicates that the same signal for lipid modification is also present in the sodC gene products from other mycobacteria and Gram-positive bacteria and, uniquely, in two distinct sodC gene products from the Gram-negative bacterium Salmonella typhimurium. Evidence is also provided for an up-regulation of M. tuberculosis sodC in response to phagocytosis by human macrophages, suggesting that Cu,ZnSOD is involved in the mechanisms that facilitate mycobacterial intracellular growth.
A Drosophila haemocyte-specific protein, hemolectin, similar to human von Willebrand factor
Authors: GOTO Akira , KUMAGAI Takeshi , KUMAGAI Chino , HIROSE Junko , NARITA Hiroshi , MORI Hitoshi , KADOWAKI Tatsuhiko , BECK Konrad , KITAGAWA Yasuo
Published: August 2015

We identified a novel Drosophila protein of ≈ 400kDa, hemolectin (d-Hml), secreted from haemocyte-derived Kc167 cells. Its 11.7kbp cDNA contains an open reading frame of 3843 amino acid residues, with conserved domains in von Willebrand factor (VWF), coagulation factor V/VIII and complement factors. The d-hml gene is located on the third chromosome (position 70C1-5) and consists of 26 exons. The major part of d-Hml consists of well-known motifs with the organization: CP1-EG1-CP2-EG2-CP3-VD1-VD2-VD′-VD3-VC1-VD′′-VD′′′-FC1-FC2-VC2-LA1-VD4-VD5-VC3-VB1-VB2-VC4-VC5-CK1 (CP, complement-control protein domain; EG, epidermal-growth-factor-like domain; VB, VC, VD, VWF type B-, C- and D-like domains; VD′, VD′′, VD′′′, truncated C-terminal VDs; FC, coagulation factor V/VIII type C domain; LA, low-density-lipoprotein-receptor class A domain; CK, cysteine knot domain). The organization of VD1-VD2-VD′-VD3, essential for VWF to be processed by furin, to bind to coagulation factor VIII and to form interchain disulphide linkages, is conserved. The 400kDa form of d-Hml was sensitive to acidic cleavage near the boundary between VD2 and VD′, where the cleavage site of pro-VWF is located. Agarose-gel electrophoresis of metabolically radiolabelled d-Hml suggested that it is secreted from Kc167 cells mainly as dimers. Resembling VWF, 7.9% (305 residues) of cysteine residues on the d-Hml sequence had well-conserved positions in each motif. Coinciding with the development of phagocytic haemocytes, d-hml transcript was detected in late embryos and larvae. Its low-level expression in adult flies was induced by injury at any position on the body.
Ligand-independent activation of oestrogen receptor α by caveolin-1
Authors: SCHLEGEL Amnon , WANG Chenguang , PESTELL Richard G. , LISANTI Michael P.
Published: August 2015

Expression of caveolin-1 in the human mammary adenocarcinoma cell line MCF-7 causes ligand-independent concentration of oestrogen receptor α (ERα) in the nucleus, and potentiates ligand-independent and ligand-dependent transcription from an oestrogen response element-driven reporter gene. Furthermore, caveolin-1 co-immunoprecipitates with ERα [Schlegel, Wang, Katzenellenbogen, Pestell and Lisanti (1999) J. Biol. Chem. 274, 33551–33556]. In the present study we show that caveolin-1 binds directly to ERα. This interaction is mediated by residues 82–101 of caveolin-1 (i.e. the caveolin scaffolding domain) and residues 1–282 of ERα. The caveolin-binding domain of ERα includes the ligand-independent transactivation domain, activation function (AF)-1, but lacks the hormone-binding domain and the ligand-gated transactivation domain, AF-2. In co-transfection studies, caveolin-1 potentiates the transcriptional activation of ERα(1–282), a truncation mutant that has intact AF-1 and DNA-binding domains. Since AF-1 activity is regulated largely by phosphorylation we determined that co-expression with caveolin-1 increased the basal phosphorylation of ERα(1–282), but blocked the epidermal growth factor-dependent increase in phosphorylation. Indeed, caveolin-1 interacted with and potentiated the transactivation of an ERα mutant that cannot be phosphorylated by extracellular signal-regulated kinase (ERK)1/2 [ERα(Ser118 → Ala)]. Thus caveolin-1 is a novel ERα regulator that drives ERK1/2-independent phosphorylation and activation of AF-1.
Human salivary agglutinin binds to lung surfactant protein-D and is identical with scavenger receptor protein gp-340
Authors: LIGTENBERG Toon J. M. , BIKKER Floris J. , GROENINK Jasper , TORNOE Ida , LETH-LARSEN Rikke , VEERMAN Enno C. I. , NIEUW AMERONGEN Arie V. , HOLMSKOV Uffe
Published: August 2015

Salivary agglutinin is a 300–400kDa salivary glycoprotein that binds to antigen B polypeptides of oral streptococci, thereby playing a role in their colonization and the development of caries. A mass spectrum was recorded of a trypsin digest of agglutinin. A dominant peak of 1460Da was sequenced by quadrupole time-of-flight (Q-TOF) tandem MS. The sequence showed 100% identity with part of the scavenger receptor cysteine-rich (‘SRCR’) domain found in gp-340/DMBT1 (deleted in malignant brain tumours-1). The mass spectrum revealed 11 peaks with an identical mass as a computer-simulated trypsin digest of gp-340. gp-340 is a 340kDa glycoprotein isolated from bronchoalveolar lavage fluid that binds specifically to lung surfactant protein-D. DMBT1 is a candidate tumour suppressor gene. A search in the human genome revealed only one copy of this gene. The molecular mass, as judged from SDS/PAGE and the amino acid composition of agglutinin, was found to be nearly identical with that of gp-340. It was shown by Western blotting that monoclonal antibodies against gp-340 reacted with salivary agglutinin, and monoclonals against agglutinin reacted with gp-340. It was demonstrated that gp-340 and agglutinin bound in a similar way to Streptococcus mutans and surfactant protein-D. Histochemically, the distribution of gp-340 in the submandibular salivary glands was identical with the agglutinin distribution, as shown in a previous paper [Takano, Bogert, Malamud, Lally and Hand (1991) Anat. Rec. 230, 307–318]. We conclude that agglutinin is identical with gp-340, and that this molecule interacts with S. mutans and surfactant protein-D.
Conformation of heparin pentasaccharide bound to antithrombin III
Authors: HRICOVÍNI Miloš , GUERRINI Marco , BISIO Antonella , TORRI Giangiacomo , PETITOU Maurice , CASU Benito
Published: August 2015

The interaction, in aqueous solution, of the synthetic pentasaccharide AGA∗IAM (GlcN,6-SO3α1–4GlcAβ1–4GlcN,3,6-SO3α1–4IdoA,2-SO3α1–4GlcN,6-SO3αOMe; where GlcN,6-SO3 is 2-deoxy-2-sulphamino-α-d-glucopyranosyl 6-sulphate, IdoA is l-iduronic acid and IdoA2-SO3 is l-iduronic acid 2-sulphate), which exactly reproduces the structure of the specific binding sequence of heparin and heparan sulphate for antithrombin III, has been studied by NMR. In the presence of antithrombin there were marked changes in the chemical shifts and nuclear Overhauser effects (NOEs), compared with the free state. On the basis of the optimized geometry of the pentasaccharide the transferred NOEs were interpreted with full relaxation and conformational exchange matrix analysis. An analysis of the three-dimensional structures of the pentasaccharide in the free state, and in the complex, revealed the binding to be accompanied by dihedral angle variation at the A–G and I–AM (where G, I, A and AM are β-d-glucuronic acid, 2-O-sulphated α-l-iduronic acid, N,6-O-sulphated α-d-glucosamine and the α-methyl-glycoside of A respectively) glycosidic linkages. Evidence is also provided that the protein drives the conformation of the 2-O-sulphated iduronic acid residue towards the skewed 2S0 form.
S-phase-specific expression of the Mad3 gene in proliferating and differentiating cells
Authors: FOX Elizabeth J. , WRIGHT Stephanie C.
Published: August 2015

The Myc/Max/Mad transcription factor network plays a central role in the control of cellular proliferation, differentiation and apoptosis. In order to elucidate the biological function of Mad3, we have analysed the precise temporal patterns of Mad3 mRNA expression during the cell cycle and differentiation in cultured cells. We show that Mad3 is induced at the G1/S transition in proliferating cells; expression persists throughout S-phase, and then declines as cells pass through G2 and mitosis. The expression pattern of Mad3 is coincident with that of Cdc2 throughout the cell cycle. In contrast, the expression of Mad3 during differentiation of cultured mouse erythroleukemia cells shows two transient peaks of induction. The first of these occurs at the onset of differentiation, and does not correlate with the S-phase of the cell cycle, whereas the second is coincident with the S-phase burst that precedes the terminal stages of differentiation. Our results therefore suggest that Mad3 serves a cell-cycle-related function in both proliferating and differentiating cells, and that it may also have a distinct role at various stages of differentiation.
Study of the interaction of the medium chain μ2 subunit of the clathrin-associated adapter protein complex 2 with cytotoxic T-lymphocyte antigen 4 and CD28
Authors: FOLLOWS Emily R. , McPHEAT Jane C. , MINSHULL Claire , MOORE Nel C. , PAUPTIT Richard A. , ROWSELL Siân , STACEY Claire L. , STANWAY Judith J. , TAYLOR Ian W. F. , ABBOTT W. Mark
Published: August 2015

The medium chain μ2 subunit (AP50) of the clathrin-associated adapter protein complex 2 (AP-2) interacts specifically with the tyrosine-based signals of several integral membrane proteins through the consensus sequence YXXΦ, where X can be any residue and Φ is a large hydrophobic residue. Using surface plasmon resonance combined with structural information, we have analysed the interaction of AP50 with peptides derived from the cytoplasmic tail of cytotoxic T-lymphocyte antigen 4 (CTLA-4). The crystal structure of AP50 in complex with a CTLA-4-derived peptide was determined to 3.6 Å (1 Å = 0.1nm) resolution. The binding domain of AP50 (residues 164–435) was expressed in Escherichia coli and purified. In agreement with previous reports, the AP50 domain bound to residues 152–174 of CTLA-4, but not to the same peptide that was phosphorylated at the single tyrosine residue (position 165). The interaction exhibited fast kinetics with rapid on and off rates and a Kd of 0.7μM. In order to further understand why AP50 binds to CTLA-4, but not to the homologous receptor CD28, a comparison of binding of AP50 with five peptides with single changes in and around the YXXΦ motif to the equivalent residues of CD28 was made. T162H greatly reduced binding, whereas T161L had little effect. Mutations G163S, V164D and K167N all exhibited reduced binding. Modelling of the single amino acid changes using structural information, was in broad agreement with the binding data, demonstrating that residues outside of the YXXΦ motif are also important in the interaction of membrane proteins with AP50.
Characterization of human glucose transporter (GLUT) 11 (encoded by SLC2A11), a novel sugar-transport facilitator specifically expressed in heart and skeletal muscle
Authors: DOEGE Holger , BOCIANSKI Andreas , SCHEEPERS Andrea , AXER Hubertus , ECKEL Jürgen , JOOST Hans-Georg , SCHÜRMANN Annette
Published: August 2015

Human GLUT11 (encoded by the solute carrier 2A11 gene, SLC2A11) is a novel sugar transporter which exhibits significant sequence similarity with the members of the GLUT family. The amino acid sequence deduced from its cDNAs predicts 12 putative membrane-spanning helices and all the motifs (sugar-transporter signatures) that have previously been shown to be essential for sugar-transport activity. The closest relative of GLUT11 is the fructose transporter GLUT5 (sharing 41.7% amino acid identity with GLUT11). The human GLUT11 gene (SLC2A11) consists of 12 exons and is located on chromosome 22q11.2. In human tissues, a 7.2kb transcript of GLUT11 was detected exclusively in heart and skeletal muscle. Transfection of COS-7 cells with GLUT11 cDNA significantly increased the glucose-transport activity reconstituted from membrane extracts as well as the specific binding of the sugar-transporter ligand cytochalasin B. In contrast to that of GLUT4, the glucose-transport activity of GLUT11 was markedly inhibited by fructose. It is concluded that GLUT11 is a novel, muscle-specific transport facilitator that is a member of the extended GLUT family of sugar/polyol-transport facilitators.
Pharmacological characterization of the putative cADP-ribose receptor
Authors: THOMAS Justyn M. , MASGRAU Roser , CHURCHILL Grant C. , GALIONE Antony
Published: August 2015

cADP-ribose (cADPR), a naturally occurring metabolite of NAD+, has been shown to be an important regulator of intracellular Ca2+ release. Considerable evidence suggests that cADPR is the endogenous modulator of the ryanodine receptor (RyR), which mediates Ca2+-induced Ca2+ release (CICR). Indeed, cADPR-mediated Ca2+ release is subject to functional regulation by other modulators of CICR, including Ca2+, caffeine and calmodulin. However, the underlying basis behind the effect of such agents on cADPR activity (in particular whether they regulate cADPR binding), as well as the precise nature of the cADPR receptor remains unclear. In the present study, use of 32P-radiolabelled cADPR has enabled a detailed pharmacological characterization of cADPR-binding sites in sea urchin egg homogenates. We report that cADPR binds specifically to a single class of high affinity receptor. Retainment of binding to membranes after a high-salt wash suggests the involvement of either an integral membrane protein (possibly the RyR itself) or a peripheral protein tightly associated to the membrane. Insensitivity of [32P]cADPR binding to either FK506 or rapamycin suggests that this does not concern the FK506-binding protein. Significantly, binding is highly robust, being relatively insensitive to both endogenous and pharmacological modulators of RyR-mediated CICR. In turn, this suggests that such agents modulate cADPR-mediated Ca2+ release primarily by tuning the ‘gain’ of the CICR system, upon which cADPR acts, rather than influencing the interaction of cADPR with its target receptor. The exception to this is calmodulin, for which our results indicate an additional role in facilitating cADPR binding.
Albumin-derived advanced glycation end-products trigger the disruption of the vascular endothelial cadherin complex in cultured human and murine endothelial cells
Authors: OTERO Karel , MARTÍNEZ Fernando , BELTRÁN Amada , GONZÁLEZ Deyarina , HERRERA Beatriz , QUINTERO Gypsy , DELGADO René , ROJAS Armando
Published: August 2015

Endothelial cell (EC) junctions regulate in large part the integrity and barrier function of the vascular endothelium. Advanced glycation end-products (AGEs), the irreversibly formed reactive derivatives of non-enzymic glucose–protein condensation reactions, are strongly implicated in endothelial dysfunction that distinguishes diabetes- and aging-associated vascular complications. The aim of the present study was to determine whether AGEs affect EC lateral junction proteins, with particular regard to the vascular endothelial cadherin (VE-cadherin) complex. Our results indicate that AGE-modified BSA (AGE-BSA), a prototype of advanced glycated proteins, disrupts the VE-cadherin complex when administered to ECs. AGE-BSA, but not unmodified BSA, was found to induce decreases in the levels of VE-cadherin, β-catenin and γ-catenin in the complex and in total cell extracts, as well as a marked reduction in the amount of VE-cadherin present at the cell surface. In contrast, the level of platelet endothelial cell adhesion molecule-1 (PECAM-1), which is located at lateral junctions, was not altered. Supplementation of the cellular antioxidative defences abolished these effects. Finally, the loss of components of the VE-cadherin complex was correlated with increases in vascular permeability and in EC migration. These findings suggest that some of the AGE-induced biological effects on the endothelium could be mediated, at least in part, by the weakening of intercellular contacts caused by decreases in the amount of VE-cadherin present.
N-terminal truncation affects the kinetics and structure of fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana
Authors: VILLADSEN Dorthe , NIELSEN Tom H.
Published: August 2015

The enzyme fructose-6-phosphate 2-kinase (F6P,2K; 6-phosphofructo-2-kinase)/fructose-2,6-bisphosphatase(F26BPase) catalyses the formation and degradation of the regulatory metabolite fructose 2,6-bisphosphate. A cDNA encoding the bifunctional plant enzyme isolated from Arabidopsis thaliana (AtF2KP) was expressed in yeast, and the substrate affinities and allosteric properties of the affinity-purified enzyme were characterized. In addition to the known regulators 3-phosphoglycerate, dihydroxyacetone phosphate, fructose 6-phosphate and Pi, several metabolites were identified as important new effectors. PPi, phosphoenolpyruvate and 2-phosphoglycerate strongly inhibited F6P,2K activity, whereas fructose 1,6-bisphosphate and 6-phosphogluconate inhibited F26BPase activity. Furthermore, pyruvate was an activator of F6P,2K and an inhibitor of F26BPase. Both kinase and phosphatase activities were rapidly inactivated by mild heat treatment (42°C, 10min), but the presence of phosphate protected both enzyme activities from inactivation. In addition to the catalytic regions, the Arabidopsis enzyme comprises a 345-amino-acid N-terminus of unknown function. The role of this region was examined by the expression of a series of N-terminally truncated enzymes. The full-length and truncated enzymes were analysed by gel-filtration chromatography. The full-length enzyme was eluted as a homotetramer, whereas the truncated enzymes were eluted as monomers. Deletion of the N-terminus decreased the kinase/phosphatase activity ratio by 4-fold, and decreased the affinity for the substrate fructose 6-phosphate. The data show that the N-terminus is important both for subunit assembly and for defining the kinetic properties of the enzyme.
Comparative impacts of glutathione peroxidase-1 gene knockout on oxidative stress induced by reactive oxygen and nitrogen species in mouse hepatocytes
Authors: FU Yangxin , PORRES Jesus M. , LEI Xin Gen
Published: August 2015

Selenium-dependent glutathione peroxidase-1 (GPX1) protects against reactive-oxygen-species (ROS)-induced oxidative stress in vivo, but its role in coping with reactive nitrogen species (RNS) is unclear. Our objective was to compare the protection of GPX1 against cytotoxicity of superoxide generator diquat (DQ), NO donor S-nitroso-N-acetyl-penicillamine (SNAP) and peroxynitrite generator 3-morpholinosydnonimine (SIN-1). Primary hepatocytes were isolated from GPX1-knockout (KO) and wild-type (WT) mice and cultured in complete Williams's medium E with various levels of these agents alone or in combination for up to 12h. While the KO cells were more susceptible to cell death, DNA fragmentation and protein carbonyl formation induced by 0.25–1mM DQ, these cells were as tolerant as the WT cells to cytotoxicity of 0.1–1mM SNAP or 0.5–2mM SIN-1. Treating cells with SNAP (0.1 or 0.25mM) in addition to DQ produced synergistic cytotoxicity that minimized differences in apoptotic cell death and oxidative injuries between the KO and WT cells. Less protein nitrotyrosine was induced by 0.05–0.5mM DQ+0.25mM SNAP in the KO than in the WT cells. Total GPX activity in the WT cells was reduced by 65 and 25% by 0.5mM DQ+0.1mM SNAP and 0.5mM DQ, respectively. Decreases in Cu,Zn-superoxide dismutase (SOD) activity and increases in Mn-SOD activity in response to DQ or DQ+SNAP were greater in the KO cells than in the WT cells. In conclusion, GPX1 was more effective in protecting hepatocytes against oxidative injuries mediated by ROS alone than by ROS and RNS together. Knockout of GPX1 did not enhance cell susceptibility to RNS-associated cytotoxicity. Instead, it attenuated protein nitration induced by DQ+SNAP.
Commitment of apolipoprotein B RNA to the splicing pathway regulates cytidine-to-uridine editing-site utilization
Authors: SOWDEN Mark P. , SMITH Harold C.
Published: August 2015

A tripartite motif located in the centre of the 7.5kb exon 26 of apolipoprotein B (apoB) mRNA directs editosome assembly and site-specific cytidine-to-uridine editing at nucleotide 6666. apoB mRNA editing is a post-transcriptional event, occurring primarily at the time exon 26 is spliced or at a time after splicing, but before nuclear export. We show, through reporter RNA constructs, that RNA splice sites suppress editing of precursor RNAs when placed proximal or distal to the editing site. Processed RNAs were edited more efficiently than precursor RNAs. Mutation of both the splice donor and acceptor sites was necessary for RNAs to be edited efficiently. The results suggested that commitment of pre-mRNA to the splicing and/or nuclear-export pathways may play a role in regulating editing-site utilization. The HIV-1 Rev–Rev response element (‘RRE’) interaction was utilized to uncouple the commitment of precursor RNAs to the spliceosome assembly pathway and associated nuclear-export pathway. Under these conditions, unspliced reporter RNAs were edited efficiently. We propose that pre-mRNA passage through the temporal or spatial restriction point where they become committed to spliceosome assembly contributes regulatory information for subsequent editosome activity.
Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily
Authors: SHEEHAN David , MEADE Gerardene , FOLEY Vivienne M. , DOWD Catriona A.
Published: August 2015

The glutathione transferases (GSTs; also known as glutathione S-transferases) are major phase II detoxification enzymes found mainly in the cytosol. In addition to their role in catalysing the conjugation of electrophilic substrates to glutathione (GSH), these enzymes also carry out a range of other functions. They have peroxidase and isomerase activities, they can inhibit the Jun N-terminal kinase (thus protecting cells against H2O2-induced cell death), and they are able to bind non-catalytically a wide range of endogenous and exogenous ligands. Cytosolic GSTs of mammals have been particularly well characterized, and were originally classified into Alpha, Mu, Pi and Theta classes on the basis of a combination of criteria such as substrate/inhibitor specificity, primary and tertiary structure similarities and immunological identity. Non-mammalian GSTs have been much less well characterized, but have provided a disproportionately large number of three-dimensional structures, thus extending our structure–function knowledge of the superfamily as a whole. Moreover, several novel classes identified in non-mammalian species have been subsequently identified in mammals, sometimes carrying out functions not previously associated with GSTs. These studies have revealed that the GSTs comprise a widespread and highly versatile superfamily which show similarities to non-GST stress-related proteins. Independent classification systems have arisen for groups of organisms such as plants and insects. This review surveys the classification of GSTs in non-mammalian sources, such as bacteria, fungi, plants, insects and helminths, and attempts to relate them to the more mainstream classification system for mammalian enzymes. The implications of this classification with regard to the evolution of GSTs are discussed.
Identification of an octamer element required for in vivo expression of the TIE1 gene in endothelial cells
Authors: BOUTET Stephane C. , QUERTERMOUS Thomas , FADEL Bahaa M.
Published: August 2015

TIE1, an endothelial-cell-specific tyrosine kinase receptor, is required for the survival and growth of microvascular endothelial cells during the capillary sprouting phase of vascular development. To investigate the molecular mechanisms that regulate the expression of TIE1 in the endothelium, we analysed transgenic mouse embryos carrying wild-type or mutant TIE1 promoter/LacZ constructs. Our data indicate that an upstream DNA octamer element (5′-ATGCAAAT-3′) is required for the in vivo expression of TIE1 in embryonic endothelial cells. Transgenic embryos carrying the wild-type TIE1 promoter (−466 to +78bp) fused to LacZ and spanning the octamer element demonstrate endothelial-cell-specific expression of the reporter transgene. Point mutations introduced within the octamer element result in a significant decrease of endothelial LacZ expression, suggesting that the octamer site functions as a positive regulator for TIE1 gene expression in endothelial cells. DNA–protein binding studies show that the octamer element exhibits an endothelial-cell-specific pattern of binding via interaction with endothelial-cell-restricted factor(s). Our findings suggest an important role for the octamer element in regulating the expression of the TIE1 receptor in the embryonic endothelium and suggest a common mechanism for the regulation of the angiogenic and cell-specific TIE1 and TIE2 genes during vascular development.
Replication of HIV-1 viruses in the presence of the Portland α1-antitrypsin variant (α1-PDX) inhibitor
Authors: BAHBOUHI Bouchaib , SEIDAH Nabil Georges , BAHRAOUI Elmostafa
Published: August 2015

The Portland α1-antitrypsin variant (α1-PDX) inhibits gp160 cleavage into gp120 and gp41 by different prohormone convertases (PCs) including furin, PC5 and PC7. Jurkat cells stably transfected with this inhibitor (J-PDX cells) and, as controls, Jurkat cells transfected with the empty vector (J-pcDNA3) were tested for their susceptibility to HIV-1 infection. We found that HIV-1 replication was significantly impaired in J-PDX cells. However, the analysis of the infectivity of HIV-1 viruses produced in J-PDX cells on different days during the infection indicated that they recovered infectivity starting from 13 days post-infection. The sequencing of viruses collected earlier and later from J-PDX cells revealed no mutations in envelope-glycoprotein precursor (Env) maturation sites or in the N-terminal sequence of gp41 fusion peptide, which plays a key role in membrane fusion. Although conserved mutations were detected at the C-terminus of the gp41 fusion peptide and ectodomain, the replication of mutant HIV-1 viruses produced on day 20 in J-PDX cells was inhibited at a similar level to wild-type viruses after a second passage in J-PDX cells. We then investigated the expression of the α1-PDX protein, and found that HIV-1 replication activated its proteolysis since the 54kDa cleaved form became predominant later on in the infection. In contrast, the expression of PC7, a protein that is transported through the secretory pathway, was unaltered in HIV-1 infected cells. We conclude that recovered HIV-1 infectivity in J-PDX cells was due to a loss of α1-PDX activity via its extensive processing by intracellular proteases that cleave it through the substrate pathway.
C-terminal heparin-binding domain of fibronectin regulates integrin-mediated cell spreading but not the activation of mitogen-activated protein kinase
Authors: KIM Jungyean , HAN Innoc , KIM Yeonhee , KIM Seungin , OH Eok-Soo
Published: August 2015

Fibronectin (FN) stimulates multiple signalling events including mitogen-activated protein kinase (MAPK) activation. During cell spreading, both the cell-binding domain and the C-terminal heparin-binding domain (HepII) of FN co-operatively regulate cytoskeleton organization. However, in comparison with the large number of studies on the functions of cell-binding domain, there is little information about the role of HepII. We therefore investigated the effect of HepII on integrin-mediated cell spreading and adhesion on FN and MAPK activation. In contrast with cells on FN substrates, rat embryo fibroblasts on FN120, which lacks HepII, were less spread, had weaker adhesion to FN and failed to form focal adhesions and actin stress fibres. Phosphotyrosine was present in the focal contacts of rat embryo fibroblasts on FN within 30min but was absent from cells on FN120. Overall, tyrosine phosphorylation was much less in cell lysates from cells on FN120, with decreased phosphorylation of focal adhesion kinase (‘pp125FAK’) on tyrosine-397, implying additional regulation of tyrosine phosphorylation by HepII. Nevertheless, adhesion-mediated MAPK activity was similar in cells on FN and on FN120. Furthermore, cells spread on FN and on FN120 substrates showed similar MAPK activation in response to treatment with epidermal growth factor and with platelet-derived growth factor. Consistently, overexpression of syndecan-4, which binds to HepII, enhanced cell spreading and adhesion on FN but did not affect integrin-mediated MAPK activation. We therefore conclude that both HepII and syndecan-4 regulate integrin-mediated cell spreading but not MAPK activation.
Src mediates stimulation by vascular endothelial growth factor of the phosphorylation of focal adhesion kinase at tyrosine 861, and migration and anti-apoptosis in endothelial cells
Authors: ABU-GHAZALEH Robin , KABIR Jahangir , JIA Haiyan , LOBO Mel , ZACHARY Ian
Published: August 2015

Vascular endothelial growth factor (VEGF) stimulates the tyrosine phosphorylation of focal adhesion kinase (FAK), increases focal adhesion formation and is chemotactic for human umbilical-vein endothelial cells (HUVECs). In the present study we identified the major sites of VEGF-induced FAK tyrosine phosphorylation and investigated the mechanism mediating this pathway in the action of VEGF. VEGF increased the focal adhesion localization of FAK phosphorylated at Tyr-397 (Y397) and Y861 but stimulated a marked increase in phosphorylation at Y861 without significantly affecting the total level of phospho-Y397 FAK. Inhibition of Src with the specific inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) completely blocked VEGF-induced Y861 phosphorylation without decreasing the level of phospho-Y397 FAK. We also examined the role of Src in mediating endothelial functions of VEGF in which FAK has been implicated as having a role. PP2 markedly inhibited VEGF-induced chemotaxis and wound-healing cell migration. The Src inhibitor also decreased the anti-apoptotic effect of VEGF determined by surface staining of annexin V but did not increase FAK proteolysis or prevent the VEGF-dependent inhibition of FAK proteolysis. In contrast, the specific PtdIns 3-kinase inhibitor LY294002 induced apoptosis and markedly decreased p125FAK expression and increased FAK proteolysis but had little effect on Y861 phosphorylation. These findings identify Src-dependent FAK phosphorylation at Y861 as a novel VEGF-induced signalling pathway in endothelial cells and suggest that this pathway might be involved in the mechanisms mediating VEGF-induced endothelial cell migration and anti-apoptosis.
Tight-junction protein zonula occludens 2 is a target of phosphorylation by protein kinase C
Authors: AVILA-FLORES Antonia , RENDÓN-HUERTA Erika , MORENO Jacqueline , ISLAS Socorro , BETANZOS Abigail , ROBLES-FLORES Martha , GONZÁLEZ-MARISCAL Lorenza
Published: August 2015

Zonula occludens 2 (ZO-2) protein is a tight-junction phos phorylated protein that belongs to the membrane-associated guanylate kinase (‘MAGUK’) family. Here we study the interaction between ZO-2 and protein kinase C (PKC). We have constructed two ZO-2 fusion proteins of the middle (3PSG) and C-terminal (AP) regions of the molecule and demonstrate that they are phosphorylated by PKC isoenzymes β, ∊, λ and ∊. To understand the physiological significance of the interaction between ZO-2 and PKC, we analysed the phosphorylation state of ZO-2 immunoprecipitated from monolayers with mature tight junctions or from cells that either lack them or have them disassembled through Ca2+ chelation. We found that in the latter condition the phosphorylation level of ZO-2 is significantly higher and is due to the action of both PKC and cAMP-dependent protein kinase. These results therefore suggest that the phosphorylated state of ZO-2 restrains its capacity to operate at the junctional complex.
Evaluation of phospholipid transfer protein and cholesteryl ester transfer protein as contributors to the generation of preβ-high-density lipoproteins
Authors: LIE Jessica , de CROM Rini , JAUHIAINEN Matti , van GENT Teus , van HAPEREN Rien , SCHEEK Leo , JANSEN Hans , EHNHOLM Christian , van TOL Arie
Published: August 2015

High-density lipoproteins (HDLs) are considered anti-atherogenic because they mediate peripheral cell cholesterol transport to the liver for excretion and degradation. An important step in this reverse cholesterol-transport pathway is the uptake of cellular cholesterol by a specific subclass of small, lipid-poor apolipoprotein A-I particles designated preβ-HDL. The two lipid-transfer proteins present in human plasma, cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP), have both been implicated in the formation of preβ-HDL. In order to investigate the relative contribution of each of these proteins, we used transgenic mouse models. Comparisons were made between human CETP transgenic mice (huCETPtg), human PLTP transgenic mice (huPLTPtg) and mice transgenic for both lipid-transfer proteins (huCETPtg/huPLTPtg). These animals showed elevated plasma levels of CETP activity, PLTP activity or both activities, respectively. We evaluated the generation of preβ-HDL in mouse plasma by immunoblotting and crossed immuno-electrophoresis. Generation of preβ-HDL was equal in huCETPtg and wild-type mice. In contrast, in huPLTPtg and huCETPtg/huPLTPtg mice, preβ-HDL generation was 3-fold higher than in plasma from either wild-type or huCETPtg mice. Our findings demonstrate that, of the two plasma lipid-transfer proteins, PLTP rather than CETP is responsible for the generation of preβ-HDL. These data support the hypothesis of a role for PLTP in the initial stage of reverse cholesterol transport.
Ecto-protein kinase substrate p120 revealed as the cell-surface-expressed nucleolar phosphoprotein Nopp140: a candidate protein for extracellular Ca2+-sensing
Authors: KÜBLER Dieter
Published: August 2015

A variety of cell membrane proteins become phosphorylated in their ecto-domains by cell-surface protein kinase (ecto-PK) activities, as detected in a broad spectrum of cell types. This study reports the isolation and identification of a frequent ecto-PK substrate, ecto-p120, using HeLa cells as a model. Data from MS and further biochemical and immunochemical means identified ecto-p120 as a cell-surface homologue of human nucleolar phosphoprotein p140 (hNopp140), which belongs to the family of argyrophilic (AgNOR-stainable) proteins. The superposition of 32P-labelled ecto-nucleolar phosphoprotein p140 (ecto-Nopp140) with anti-Nopp140 immunostaining could be demonstrated in a wide range of cell lines without any exceptions, suggesting a nearly universal occurrence of cell-surface Nopp140. A previous, tentative association of ecto-p120 with the nucleoplasmic pre-mRNA-binding protein hnRNP U has thus been supplanted, since improved purification techniques have allowed unambiguous identification of this ecto-PK cell-surface substrate. Furthermore, we have shown that rapid suppression of ecto-hNopp140 phosphorylation resulted upon a rise in the free extracellular calcium, while lowering the calcium concentrations returned ecto-Nopp140 phosphorylation to the original level. It is important to note that these Ca2+-dependent effects on ecto-Nopp140 phosphorylation are not accompanied by alterations in the phosphorylation of other ecto-PK substrates. Our results indicate that, in addition to nucleolin, a further nucleolar protein, which was considered initially to be strictly intracellular, is identified as a cell-surface phosphoprotein.
Regulation and composition of activator protein 1 (AP-1) transcription factors controlling collagenase and c-Jun promoter activities
Authors: STEINMÜLLER Lars , CIBELLI Giuseppe , MOLL Jonathan R. , VINSON Charles , THIEL Gerald
Published: August 2015

The activator protein 1 (AP-1) transcription factor is composed of heterodimers of the Fos/activating transcription factor (ATF) and Jun subfamilies of basic-region leucine-zipper (B-ZIP) proteins. In order to determine the identities of individual B-ZIP proteins in various AP-1 complexes we tested the effect of dominant-negative mutants to the B-ZIP proteins c-Fos, ATF2, ATF4 and CCAAT-enhancer-binding protein (C/EBP) on the activities of the collagenase and c-Jun promoters. These dominant-negative mutants inhibit DNA binding of wild-type B-ZIP proteins in a leucine-zipper-dependent fashion. Transcription of a collagenase promoter/reporter gene was induced in HepG2 hepatoma cells by expression of c-Fos and c-Jun, administration of PMA (‘TPA’) or by expression of a truncated form of MEK (mitogen-activated/extracellular-signal-regulated kinase kinase) kinase-1, MEKK1Δ. In all cases, the dominant-negative mutants A-Fos and A-ATF2 decreased collagenase promoter activity. However, A-ATF4 and A-C/EBP had no effect. A-Fos and A-ATF2 also reduced MEKK1Δ-induced stimulation of the c-Jun promoter. In contrast, constitutive c-Jun promoter activity was blocked solely by A-ATF2, strongly suggesting that ATF2 and/or an ATF2-dimerizing protein are of major importance for c-Jun transcription in unstimulated cells. These results demonstrate that AP-1 transcription factors of different compositions control c-jun gene transcription in resting or stimulated cells.
Kynurenine aminotransferase and glutamine transaminase K of Escherichia coli: identity with aspartate aminotransferase
Authors: HAN Qian , FANG Jianmin , LI Jianyong
Published: August 2015

The present study describes the isolation of a protein from Escherichia coli possessing kynurenine aminotransferase (KAT) activity and its identification as aspartate aminotransferase (AspAT). KAT catalyses the transamination of kynurenine and 3-hydroxykynurenine to kynurenic acid and xanthurenic acid respectively, and the enzyme activity can be easily detected in E. coli cells. Separation of the E. coli protein possessing KAT activity through various chromatographic steps led to the isolation of the enzyme. N-terminal sequencing of the purified protein determined its first 10 N-terminal amino acid residues, which were identical with those of the E. coli AspAT. Recombinant AspAT (R-AspAT), homologously expressed in an E. coli/pET22b expression system, was capable of catalysing the transamination of both l-kynurenine (Km = 3mM; Vmax = 7.9μmol·min−1·mg−1) and 3-hydroxy-dl-kynurenine (Km = 3.7mM; Vmax = 1.25μmol·min−1·mg−1) in the presence of pyruvate as an amino acceptor, and exhibited its maximum activity at temperatures between 50–60°C and at a pH of approx. 7.0. Like mammalian KATs, R-AspAT also displayed high glutamine transaminase K activity when l-phenylalanine was used as an amino donor (Km = 8mM; Vmax = 20.6μmol·min−1·mg−1). The exact match of the first ten N-terminal amino acid residues of the KAT-active protein with that of AspAT, in conjunction with the high KAT activity of R-AspAT, provides convincing evidence that the identity of the E. coli protein is AspAT.