bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium entry in Trypanosoma brucei is regulated by phospholipase A2 and arachidonic acid
Authors: EINTRACHT Jason , MAATHAI Ronald , MELLORS Alan , RUBEN Larry
Published: August 2015

In contrast with mammalian cells, little is known about the control of Ca2+ entry into primitive protozoans. Here we report that Ca2+ influx in pathogenic Trypanosoma brucei can be regulated by phospholipase A2 (PLA2) and the subsequent release of arachidonic acid (AA). Several PLA2 inhibitors blocked Ca2+ entry; 3-(4-octadecyl)-benzoylacrylic acid (OBAA; IC50 0.4±0.1 µM) was the most potent. We identified in live trypanosomes PLA2 activity that was sensitive to OBAA and could be stimulated by Ca2+, suggesting the presence of positive feedback control. The cell-associated PLA2 activity was able to release [14C]AA from labelled phospholipid substrates. Exogenous AA (5–50 µM) also initiated Ca2+ entry in a manner that was inhibited by the Ca2+ antagonist La3+ (100 µM). Ca2+ entry did not depend on AA metabolism or protein kinase activation. The cell response was specific for AA, and fatty acids with greater saturation than tetraeicosanoic acid (AA) or with chain lengths less than C20 exhibited greatly diminished ability to initiate Ca2+ influx. Myristate and palmitate inhibited PLA2 activity and also inhibited Ca2+ influx. Overall, these results demonstrate that Ca2+ entry into T. bruceican result from phospholipid hydrolysis and the release of eicosanoic acids.
Calsequestrin: more than ‘only’ a luminal Ca2+ buffer inside the sarcoplasmic reticulum
Authors: SZEGEDI Csaba , SÁRKÖZI Sándor , HERZOG Anke , JÓNA István , VARSÁNYI Magdolna
Published: August 2015

In striated muscle, the sarcoplasmic reticulum (SR) Ca2+ release/ryanodine receptor (RyR) channel provides the pathway through which stored Ca2+ is released into the myoplasm during excitation–contraction coupling. Various luminal Ca2+-binding proteins are responsible for maintaining the free [Ca2+] at 10-3–10-4 M in the SR lumen; in skeletal-muscle SR, it is mainly calsequestrin. Here we show that, depending on its phosphorylation state, calsequestrin selectively controls the RyR channel activity at 1 mM free luminal [Ca2+]. Calsequestrin exclusively in the dephosphorylated state enhanced the open probability by approx. 5-fold with a Hill coefficient (h) of 3.3, and increased the mean open time by about 2-fold, i.e. solely dephosphorylated calsequestrin regulates Ca2+ release from the SR. Because calsequestrin has been found to occur mainly in the phosphorylated state in the skeletal-muscle SR for the regulation of RyR channel activity, the dephosphorylation of calsequestrin would appear to be a quintessential physiological event.
N-lobe versus C-lobe complexation of bismuth by human transferrin
Authors: SUN Hongzhe , LI Hongyan , MASON Anne B. , WOODWORTH Robert C. , SADLER Peter J.
Published: August 2015

Interactions of recombinant N-lobe of human serum transferrin (hTF/2N) with Bi3+, a metal ion widely used in medicine, have been investigated by both UV and NMR spectroscopy. The bicarbonate-independent stability constant for Bi3+ binding (K*) to hTF/2N was determined to be log K* 18.9±0.2 in 5 mM bicarbonate/10 mM Hepes buffer at 310 K, pH 7.4. The presence of Fe3+ in the C-lobe of intact hTF perturbed Bi3+ binding to the N-lobe, whereas binding of Bi3+ to the C-lobe was unaffected by the presence of Fe3+ in the N-lobe. Reactions of Bi3+ (as bismuth nitrilotriacetate or ranitidine bismuth citrate) with hTF/2N in solutions containing 10 mM bicarbonate induced specific changes to high-field 1H-NMR peaks. The 1H co-ordination shifts induced by Bi3+ were similar to those induced by Fe3+ and Ga3+, suggesting that Bi3+ binding causes similar structural changes to those induced by hTF/2N. 13C-NMR data showed that carbonate binds to hTF/2N concomitantly with Bi3+.
Disruption of the utrophin–actin interaction by monoclonal antibodies and prediction of an actin-binding surface of utrophin
Authors: MORRIS Glenn E. , MAN Nguyen thi , HUYEN Nguyen thi Ngoc , PEREBOEV Alexander , KENDRICK-JONES John , WINDER Steven J.
Published: August 2015

Monoclonal antibody (mAb) binding sites in the N-terminal actin-binding domain of utrophin have been identified using phage-displayed peptide libraries, and the mAbs have been used to probe functional regions of utrophin involved in actin binding. mAbs were characterized for their ability to interact with the utrophin actin-binding domain and to affect actin binding to utrophin in sedimentation assays. One of these antibodies was able to inhibit utrophin–F-actin binding and was shown to recognize a predicted helical region at residues 13–22 of utrophin, close to a previously predicted actin-binding site. Two other mAbs which did not affect actin binding recognized predicted loops in the second calponin homology domain of the utrophin actin-binding domain. Using the known three-dimensional structure of the homologous actin-binding domain of fimbrin, these results have enabled us to determine the likely orientation of the utrophin actin-binding domain with respect to the actin filament.
Co-operation between the PAI and RED subdomains of Pax-8 in the interaction with the thyroglobulin promoter
Authors: PELLIZZARI Lucia , TELL Gianluca , DAMANTE Giuseppe
Published: August 2015

Pax proteins are transcription factors that play an important role in the differentiation of several cell types. These proteins bind to specific DNA sequences through the paired domain. This evolutionarily conserved element is composed of two subdomains (PAI and RED), located at the N- and C-terminals, respectively. Due to the presence of these two subdomains, Pax proteins may recognize DNA in different modes, a possibility that has not been exhaustively explored yet. The C site of the thyroglobulin promoter is bound by the thyroid-specific transcription factor Pax-8. In this study we have characterized the mode by which the Pax-8 paired domain interacts with the C site. Results allow the identification of the respective positions of the PAI and RED subdomains when the full-length protein is bound to the C site. The binding of the isolated PAI and RED subdomains to the C site and to several related mutants was also evaluated. Both subdomains interact with DNA as a monomer and display a lower binding affinity than the full-length protein. Therefore, the Pax-8 paired domain–C site interaction occurs through a co-operation between the two subdomains. The binding properties of the PAI subdomain suggest that the co-operation between PAI and RED subdomains does not merely consist of the sum of contacts established by the single subdomain: the presence of the RED subdomain is necessary for correct DNA recognition by the PAI subdomain, thus accounting for a sort of chronology of events during DNA binding. Since the RED subdomain is much more variable than the PAI subdomain among Pax proteins, these results could explain how distinct Pax proteins may select different target genes.
Molecular characterization of the Acremonium chrysogenum cefG gene product: the native deacetylcephalosporin C acetyltransferase is not processed into subunits
Authors: VELASCO Javier , GUTIERREZ Santiago , CAMPOY Sonia , MARTIN Juan F.
Published: August 2015

Constructions starting at each of the three in-frame ATG codons of the Acremonium chrysogenum cefG gene (Met1, Met46 and Met60) were expressed in Escherichia coli, obtaining proteins of 49, 44 and 43 kDa, respectively. All three proteins showed deacetylcephalosporin C (DAC) acetyltransferase activity. The native A. chrysogenum DAC acetyltransferase was purified to electrophoretic homogeneity by immunoaffinity chromatography. It showed a molecular mass of 50 kDa by filtration in calibrated Sephadex G-75 SF or Superose 12 (FPLC) columns. The N-terminal end of the pure DAC acetyltransferase was Met-Leu-Pro-Ser-Ala-Gln-Val-Ala-Arg-Leu, which matched perfectly the deduced amino acid sequence starting at Met1. The putative α- and β-subunits of DAC acetyltransferase were also obtained in E. coli but showed no enzymic activity either separately or in combination. Immunoblotting (Western) analysis revealed that the 50 kDa DAC acetyltransferase showed high protein levels in A. chrysogenum cultures at 72 and 96 h and decreased sharply thereafter, but in all cases no detectable processing of the enzyme into subunits was found. Three different A. chrysogenum strains (including the wild-type Brotzu strain and two high-cephalosporin-producing mutants) showed the same unprocessed 50 kDa DAC acetyltransferase. The non-producer mutant ATCC 20371 showed no DAC acetyltransferase protein band but formed a normal transcript of 1.4 kb.
A sensitive immunoassay for rat fatty acid translocase (CD36) using phage antibodies selected on cell transfectants: abundant presence of fatty acid translocase/CD36 in cardiac and red skeletal muscle and up-regulation in diabetes
Authors: PELSERS Maurice M. A. L. , LUTGERINK Jan T. , NIEUWENHOVEN Frans A. van , TANDON Narendra N. , VUSSE Ger J. van der , ARENDS Jan-Willem , HOOGENBOOM Hennie R. , GLATZ Jan F. C.
Published: August 2015

The rat membrane protein fatty acid translocase (FAT), which shows sequence similarity to human CD36 (a membrane protein supposedly involved in a variety of membrane processes), is implicated in the transport of long-chain fatty acids across cellular membranes. To set up an immunoassay for quantification of FAT in different tissues, we isolated a series of anti-FAT antibodies by panning a large naive phage antibody library on FAT-transfected H9c2 cells. All seven different phage antibody fragments isolated reacted specifically with FAT, and most likely recognize the same or closely located immunodominant sites on FAT, as a competitive monoclonal antibody (mAb) (CLB-IV7) completely blocked the binding of all these phage antibodies to cells. A sandwich ELISA was set up using mAb 131.4 (directed against purified CD36 from human platelets) as capture antibody and phage antibodies and anti-phage sera as detector. With this ELISA (sensitivity 0.05 µg/ml), the FAT content in isolated cardiomyocytes was found to be comparable with that of total heart (≈ 3 mg/g of protein), while liver tissue and endothelial cells were below the detection limit (< 0.1 mg of FAT/g of protein). During rat heart development, protein levels of FAT rose from 1.7±0.7 mg/g of protein on the day before birth to 3.6±0.4 mg/g of protein on day 70. Comparing control with streptozotocin-induced diabetic rats, a statistically significant (P< 0.05) 2–4-fold increase of FAT was seen in heart (from 4.2±2.3 to 11.0±5.7 mg/g of protein), soleus (from 0.6±0.1 to 1.4±0.5 mg/g of protein) and extensor digitorum longus (EDL) muscle (from 0.3±0.1 to 1.2±0.8 mg/g of protein). In addition, the FAT contents of each of these muscles were found to be of similar magnitude to the contents of cytoplasmic heart-type fatty-acid-binding protein in both diabetic rats and controls, supporting the suggested roles of these two proteins in cellular fatty acid metabolism. This is the first time phage display technology has been succesfully applied for direct selection, from a large naive antibody library, of antibodies that recognize selected membrane proteins in their natural context.
Rat-2 fibroblasts express specific adrenomedullin receptors, but not calcitonin-gene-related-peptide receptors, which mediate increased intracellular cAMP and inhibit mitogen-activated protein kinase activity
Authors: COPPOCK Hedley A. , OWJI Ali A. , AUSTIN Carol , UPTON Paul D. , JACKSON Mary L. , GARDINER James V. , GHATEI Mohammad A. , BLOOM Stephen R. , SMITH David M.
Published: August 2015

Rat-2 fibroblasts demonstrate specific binding of 125I-labelled rat adrenomedullin (KD = 0.43nM; Bmax = 50fmol/mg of protein) in the absence of 125I-labelled calcitonin-gene-related peptide (CGRP) binding. Therefore Rat-2 cells were used to examine the pharmacology and signal transduction pathways of adrenomedullin receptors. We examined the effects of adrenomedullin, the CGRP receptor antagonist CGRP-(8–37) and the amylin antagonists AC187 and AC253 on receptor binding and cAMP production. AC253, AC187 and CGRP-(8–37) inhibited 125I-adrenomedullin binding, with respective IC50 values of 25±8, 129±39 and 214±56nM. Adrenomedullin dose-dependently increased intracellular cAMP (approximate EC50 = 1.0nM). CGRP-(8–37), AC253 and AC187 antagonized adrenomedullin-stimulated cAMP production at micromolar concentrations. Using kinase-substrate assays, Mono Q FPLC and ‘phospho-specific ’ Western blotting, we found that adrenomedullin alone abolished basal mitogen-activated protein kinase (MAPK) activity and dose-dependently inhibited platelet-derived-growth-factor-stimulated MAPK activity. Radioimmunoassay for adrenomedullin of media from Rat-2 cells showed a linear release of adrenomedullin-like immunoreactivity of 3.1fmol/h per 2×106 cells. Gel-filtration chromatography showed that this adrenomedullin-like immunoreactivity co-eluted with synthetic rat adrenomedullin. Northern blotting with a rat adrenomedullin cDNA probe was used to confirm the presence of adrenomedullin mRNA. However, neither Northern blotting nor reverse transcriptase–PCR showed the presence of the cloned adrenomedullin receptor (L1). We conclude that the Rat-2 cell line expresses a specific adrenomedullin receptor (coupled to cAMP production and regulation of MAPK) and secretes adrenomedullin, which may participate in a regulatory control loop.
Synthesis and characterization of bactericidal oligopeptides designed on the basis of an insect anti-bacterial peptide
Authors: SAIDO-SAKANAKA Hisako , ISHIBASHI Jun , SAGISAKA Aki , MOMOTANI Eiichi , YAMAKAWA Minoru
Published: August 2015

Defensin from a beetle, Allomyrina dichotoma, is known to have anti-bacterial activity against Gram-positive bacteria. This peptide, which comprises 43 amino acid residues, was effective against methicillin-resistant Staphylococcus aureus. We identified the active site of beetle defensin by measuring anti-bacterial activity against S. aureus of 64 overlapping 12-mer peptides with either a free carboxylate or a free amide group at their C-termini. An LCAAHCLAIGRR-NH2 (19L–30R-NH2) fragment showed the greatest activity of the synthetic oligopeptides. The 19L–30R-NH2 fragment was effective against both Gram-positive and Gram-negative bacteria. CD spectra showed that the 19L–30R-NH2 fragment formed an α-helical structure in the lipidic environment. The anti-bacterial effect of the 19L–30R-NH2 fragment was due to its interaction with bacterial membranes, judging from the leakage of liposome-entrapped glucose. Its anti-bacterial activity was increased when certain amino acid residues were replaced. Truncated peptides having had some amino acids removed from the N-terminus of the 19L–30R-NH2 fragment (8–10-mer peptides) still had strong anti-bacterial activity. Deleting some amino acids from the C-terminal region of the fragment dramatically reduced activity, indicating that the C-terminal region of the 19L–30R-NH2 fragment, i.e. RR-NH2, is important for exerting anti-bacterial activity. The AHCLAIGRR-NH2 (22A–30R-NH2) fragment and its analogues exhibited about 3-fold and 9–12-fold higher activity against S. aureus than did the 19L–30R-NH2 fragment, and these analogues were effective against methicillin-resistant S. aureus and Pseudomonas aeruginosa isolated from patients. These oligopeptides showed no haemolytic activity and did not inhibit the growth of murine fibroblast cells.
Inhibition of proprotein convertases-1, -7 and furin by diterpines of Andrographis paniculata and their succinoyl esters
Authors: BASAK Ajoy , COOPER Sam , ROBERGE Andreé G. , BANIK Upen K. , CHRÉTIEN Michel , SEIDAH Nabil G.
Published: August 2015

Studies were performed to investigate the prohormone/proprotein convertase (PC)-inhibitory properties of chemical constituents of the medicinally active plant Andrographis paniculata (AP; from the family Acanthaceae), also known as ‘King of Bitters ’. Among the individual components tested against the clinically important convertases, furin and PC1, neoandrographolide (a C3 O-glucoside derivative of the major constituent andrographolide) exhibited the highest inhibitory action with an IC50 of 53.5 µM against furin. The data further revealed that although andrographolide, the major bitter principle of AP, exhibited a relatively small enzyme inhibition (IC50 = 1.0 mM and Ki = 200 µM against furin), upon succinoylation, its inhibitory action against the above convertases was enhanced significantly with a Ki in the low micromolar range (< 30 µM), suggesting that a specific structural modification of the andrographolide skeleton may be exploited to develop a new class of non-peptide inhibitors of PCs. When tested against PC7, these succinoylated derivatives of andrographolide also displayed strong inhibitory action, with Ki values again in the low micromolar range. This potentially interesting observation may be attributed to the reported anti-HIV property of 14-dehydroandrographolide succinic acid monoester (DASM). It is suggested here that DASM, by virtue of this protease inhibitory property, possibly acts by suppressing the proteolytic cleavage of envelope glycoprotein gp160 of HIV, which is known to be PC-mediated, particularly by furin and PC7.
Upstream region of rat serum albumin gene promoter contributes to promoter activity: presence of functional binding site for hepatocyte nuclear factor-3
Authors: HSIANG Chin-Hui , MARTEN Norman W. , STRAUS Daniel S.
Published: August 2015

Transcription of the serum albumin gene occurs almost exclusively in the liver and is controlled in part by a strong liver-specific promoter. The upstream region of the serum albumin gene promoter is highly conserved among species and is footprinted in vitro by a number of nuclear proteins. However, the role of the upstream promoter region in regulating transcription and the identity of the transcription factors that bind to this region have not been established. In the present study, deletion analysis of the rat serum albumin promoter in transiently transfected HepG2 cells demonstrated that elimination of the region between -207 and -153 bp caused a two-fold decrease in promoter activity (P< 0.05). Additional analysis of the -207 to -124 bp promoter interval led to the identification of two potential binding sites for hepatocyte nuclear factor-3 (HNF-3) located at -168 to -157 bp (site X) and -145 to -134 bp (site Y). Electrophoretic mobility-shift assays performed with the HNF-3 X and Y sites demonstrated that both sites are capable of binding HNF-3α and HNF-3β. Placement of a single copy of the HNF-3 X site upstream from a minimal promoter increased promoter activity by about four-fold in HepG2 cells, and the reporter construct containing this site could be transactivated if co-transfected with an HNF-3 expression construct. Furthermore, inactivation of the HNF-3 X site by site-directed mutagenesis within the context of the -261 bp albumin promoter construct resulted in a 40% decrease in transcription (P< 0.05). These results indicate that the positive effect of the -207 to -153 bp promoter interval is attributable to the presence of the HNF-3 X site within this interval. Additional results obtained with transfected HepG2 cells suggest that the HNF-3 Y site plays a lesser role in activation of transcription than the X site.
Baculovirus expression and biochemical characterization of the human microsomal triglyceride transfer protein
Authors: RITCHIE Penelope J. , DECOUT Anne , AMEY Joanna , MANN Christopher J. , READ Jacqueline , ROSSENEU Maryvonne , SCOTT James , SHOULDERS Carol C.
Published: August 2015

The microsomal triglyceride transfer protein (MTP) complexed to protein disulphide isomerase (PDI) is obligatory for the assembly of chylomicrons and very-low-density lipoproteins. The determination of the atomic structure of the MTP–PDI heterodimer has important implications for the treatment of those forms of hyperlipidaemia associated with the overproduction of very-low-density lipoproteins, which predispose to premature coronary heart disease. To perform structural studies of the human MTP–PDI complex it was necessary to produce milligram quantities of pure protein. We chose the baculovirus expression system for this purpose. Insects cells were co-infected with recombinant viruses encoding FLAG-tagged MTP and His-tagged PDI; the resulting heterodimer was purified by affinity chromatography. From 5 litres of insect cells, 4–6 mg of more than 95% pure recombinant protein was obtained. CD and attenuated total reflection Fourier-transform infrared spectroscopy indicate that the purified protein has around 34% α-helical and 33% β-structure content. The recombinant protein had a comparable triglyceride transfer activity to that of bovine MTP–PDI. The production of polyclonal antibodies raised against the MTP and PDI subunits of the purified protein is described. The present study demonstrates the feasibility of expressing two proteins at high levels in insect cells and describes a transferable methodology for the purification of the resulting protein complex.
The endosome fusion regulator early-endosomal autoantigen 1 (EEA1) is a dimer
Authors: CALLAGHAN Judy , SIMONSEN Anne , GAULLIER Jean-Michel , TOH Ban-Hock , STENMARK Harald
Published: August 2015

EEA1, an early-endosomal protein originally identified as an autoantigen, is essential for endocytic membrane fusion. It interacts with early endosomes via binding to the membrane lipid phosphatidylinositol 3-phosphate (PtdIns3P) and the active form of the small GTPase Rab5. Most of the EEA1 sequence contains heptad repeats characteristic of proteins involved in coiled-coil protein–protein interactions. Here we have investigated the ability of EEA1 to self-interact. Crosslinking of cytosolic and recombinant EEA1 resulted in the disappearance of the 180-kDa monomer in SDS/PAGE and the strong appearance of a ∼ 350-kDa crosslinked product. Glycerol gradient centrifugation experiments indicated that native EEA1 had the same hydrodynamic properties as the ∼ 350-kDa crosslinked complex. Two-hybrid analysis indicated that N- and C-terminal fragments of EEA1 can interact with themselves, but not with each other, suggesting that EEA1 forms parallel coiled-coil dimers. The ability of the C-terminus of EEA1 to dimerize correlates with its ability to bind to Rab5 and early endosomes, whereas its binding to PtdIns3P is independent of dimerization. These data enable us to propose a model for the quaternary structure of EEA1.
Dual role for transcription factor AP-2 in the regulation of the major fetal promoter P3 of the gene for human insulin-like growth factor II
Authors: RIETVELD Luc E. G. , KOONEN-REEMST Annemie M. C. B. , SUSSENBACH John S. , HOLTHUIZEN P. Elly
Published: August 2015

The human insulin-like growth factor II (IGF-II) gene contains four promoters that are differentially active during cell growth and development. Promoter 3 (P3) is the most active promoter in fetal and non-hepatic adult tissues. In addition to its expression during development, P3 is also the major promoter in many tumour tissues and IGF-II-expressing cell lines. Here we show that AP-2 has a dual function in P3 regulation in vivo as well as in vitro. In cells expressing low levels of endogenous AP-2, AP-2 overexpression activates P3, whereas P3 promoter activity is inhibited in cells containing abundant AP-2. Four potential AP-2-binding sites were identified in footprinting studies with recombinant AP-2. One of these AP-2-binding sites is located within the previously identified element P3-4 that contains two adjacent binding sites for IGF-II promoter-binding proteins IPBP3 and IPBP4/5. By applying binding competition assays and mutational analysis it is shown that AP-2 interferes with IPBP3 binding and transactivation in vivo as well as in vitro. Furthermore, AP-2 can bind additional elements in the proximal P3 promoter that also contribute to AP-2-mediated transactivation as shown by transient transfection assays. From these results we conclude that AP-2 is an important regulator in vivo and in vitro of IGF-II P3 activity.
Functional production and reconstitution of the human equilibrative nucleoside transporter (hENT1) in Saccharomyces cerevisiae
Authors: VICKERS Mark F. , MANI Rajam S. , SUNDARAM Manickavasagam , HOGUE Douglas L. , YOUNG James D. , BALDWIN Stephen A. , CASS Carol E.
Published: August 2015

We have produced recombinant human equilibrative nucleoside transporter (hENT1) in the yeast Saccharomyces cerevisiae and have compared the binding of inhibitors of equilibrative nucleoside transport with the wild-type transporter and a N-glycosylation-defective mutant transporter. Equilibrium binding of 3H-labelled nitrobenzylmercaptopurine ribonucleoside {6-[(4-nitrobenzyl)thio]-9-β-d-ribofuranosyl purine; NBMPR} to hENT1-producing yeast revealed a single class of high-affinity sites that were shown to be in membrane fractions by (1) equilibrium binding (means±S.D.) of [3H]NBMPR to intact yeast (Kd 1.2±0.2 nM; Bmax 5.0±0.5 pmol/mg of protein) and membranes (Kd 0.7±0.2 nM; Bmax 6.5±1 pmol/mg of protein), and (2) reconstitution of hENT1-mediated [3H]thymidine transport into proteoliposomes that was potently inhibited by NBMPR. Dilazep and dipyridamole inhibited NBMPR binding to hENT1 with IC50 values of 130±10 and 380±20 nM respectively. The role of N-linked glycosylation in the interaction of NBMPR with hENT1 was examined by the quantification of binding of [3H]NBMPR to yeast producing either wild-type hENT1 or a glycosylation-defective mutant (hENT1/N48Q) in which Asn-48 was converted into Gln. The Kd for binding of NBMPR to hENT1/N48Q was 10.5±1.6 nM, indicating that the replacement of an Asn residue with Gln decreased the affinity of hENT1 for NBMPR. The decreased affinity of hENT1/N48Q for NBMPR was due to an increased rate of dissociation (koff) and a decreased rate of association (kon) of specifically bound [3H]NBMPR because the values for hENT1-producing and hENT1/N48Q-producing yeast were respectively 0.14±0.02 and 0.36±0.05 min-1 for koff, and (1.2±0.1)×108 and (0.40±0.04)×108 M-1·min-1 for kon. These results indicated that the conservative conversion of an Asn residue into Gln at position 48 of hENT1 and/or the loss of N-linked glycosylation capability altered the binding characteristics of the transporter for NBMPR, dilazep and dipyridamole.
Only one of the charged amino acids located in membrane-spanning regions is important for the function of the Saccharomyces cerevisiae uracil permease
Authors: PINSON Benoît , CHEVALLIER Jean , URBAN-GRIMAL Danièle
Published: August 2015

The transport of uracil into the yeast Saccharomyces cerevisiae is mediated by uracil permease, a specific co-transporter encoded by the FUR4 gene. Uracil permease is a multispan membrane protein that is delivered to the plasma membrane via the secretory pathway. Experimental results led to the proposal of a two-dimensional model of the protein's topology. According to this model, the membrane domain of Fur4p contains three charged amino acid residues (Glu-243, Lys-272 and Glu-539) that are conserved in the members of the FUR family of yeast transporters. We have previously shown that a mis-sense mutation leading to the replacement of Lys-272 by Glu severely impairs the function of uracil permease. In the present paper, the role of the three charged residues present in the membrane-spanning regions of Fur4p was further investigated by using site-directed mutagenesis. The variant permeases were correctly targeted to the plasma membrane and their stabilities were similar to that of the wild-type permease. The effect of the mutations was studied by measuring the uptake constants for uracil on whole cells and equilibrium binding parameters on plasma membrane-enriched fractions. We found no evidence for ionic interaction between either of the glutamic residues in transmembrane segments 3 and 9 and the lysine residue in transmembrane segment 4. Of the three charged residues, only Lys-272 was important for the transport activity of the transporter. Its replacement by Ala, Glu or even Arg strongly impaired both the binding and the translocation of uracil.
Electron transfer reactions in the alkene mono-oxygenase complex from Nocardia corallina B-276
Authors: GALLAGHER Stephen C. , CAMMACK Richard , DALTON Howard
Published: August 2015

Nocardia corallina B-276 possesses a multi-component enzyme, alkene mono-oxygenase (AMO), that catalyses the stereoselective epoxygenation of alkenes. The reductase component of this system has been shown by EPR and fluorescence spectroscopy to contain two prosthetic groups, an FAD centre and a [2Fe–2S] cluster. The role of these centres in the epoxygenation reaction was determined by midpoint potential measurements and electron transfer kinetics. The order of potentials of the prosthetic groups of the reductase were FAD/FAD• = -216 mV, [2Fe–2S]/[2Fe–2S]• = -160 mV and FAD•/FAD•• = -134 mV. Combined, these data implied that the reductase component supplied the energy required for the epoxygenation reaction and allowed a prediction of the mechanism of electron transfer within the AMO complex. The FAD moiety was reduced by bound NADH in a two-electron reaction. The electrons were then transported to the [2Fe–2S] centre one at a time, which in turn reduced the di-iron centre of the epoxygenase. Reduction of the di-iron centre is required for oxygen binding and substrate oxidation.
Characterization of β2 (CD18) integrin phosphorylation in phorbol ester-activated T lymphocytes
Authors: VALMU Leena , HILDEN Tiina J. , WILLIGEN Gijsbert van , GAHMBERG Carl G.
Published: August 2015

Integrins are transmembrane proteins involved in cell–cell and cell–extracellular-matrix interactions. The affinity and avidity of integrins for their ligands change in response to cytoplasmic signals. This ‘inside-out ’ activation has been reported to occur also with β2 integrins (CD18). The β2 integrin subunit has previously been shown to become phosphorylated in T lymphocytes on cytoplasmic serine and the functionally important threonine residues after treatment with phorbol esters or on triggering of T-cell receptors. We have now characterized the phosphorylation of β2 integrins in T-cells in more detail. When T-cells were activated by phorbol esters the phosphorylation was mainly on Ser756. After inhibition of serine/threonine phosphatases, phosphorylation was also found in two of the threonine residues in the threonine triplet 758–760 of the β2 cytoplasmic domain. Activation of T-cells by phorbol esters resulted in phosphorylation in only approx. 10% of the integrin molecules. Okadaic acid increased this phosphorylation to approx. 30% of the β2 molecules, assuming three phosphorylation sites. This indicates that a strong dynamic phosphorylation exists in serine and threonine residues of the β2 integrins.
Glucose and thyroid hormone co-regulate the expression of the intestinal fructose transporter GLUT5
Authors: MATOSIN-MATEKALO Miriam , MESONERO José E. , LAROCHE Thibaut J. , LACASA Michel , BROT-LAROCHE Edith
Published: August 2015

Expression of the fructose transporter GLUT5 in Caco-2 cells is controlled by the carbohydrate content of the culture media [Mesonero, Matosin, Cambier, Rodriguez-Yoldi and Brot-Laroche (1995) Biochem. J. 312, 757–762] and by the metabolic status of the cells [Mahraoui, Takeda, Mesonero, Chantret, Dussaulx, Bell, and Brot-Laroche (1994) Biochem. J. 301, 169–175]. In this study we show that, in fully differentiated Caco-2/TC7 cells, thyroid hormone and glucose increase GLUT5 mRNA abundance in a dose-dependent manner. Using Caco-2/TC7 cells stably transformed with various fragments of the GLUT5 promoter inserted upstream of the luciferase reporter gene, we localized the sequences that confer 3,3´,5-l-tri-iodothyronine (T3)- and/or glucose-sensitivity to the gene. Glucose responsiveness is conferred by the -272/+41 fragment of the promoter, but it is only with the -338/+41 region that transcription of the luciferase reporter gene is stimulated by T3. This 70 bp fragment from position -338 to -272 of the GLUT5 gene is able to confer T3/glucose-responsiveness to the heterologous thymidine kinase promoter. Electrophoretic-mobility-shift assays demonstrate that thyroid hormone receptors α and β are expressed in Caco-2/TC7 cells. They further show that the -308/-290 region of the GLUT5 promoter binds thyroid hormone receptor/retinoid X receptor heterodimers, and that glucose and/or T3 exert a deleterious effect on the binding of the nuclear protein complex.
Calcium-binding protein S100A7 and epidermal-type fatty acid-binding protein are associated in the cytosol of human keratinocytes
Authors: HAGENS Gerry , MASOUYÉ Isabelle , AUGSBURGER Eric , HOTZ Raymonde , SAURAT Jean-Hilaire , SIEGENTHALER Georges
Published: August 2015

Expression of epidermal-type fatty acid-binding protein (E-FABP) and S100A7 has previously been shown to be elevated in psoriatic skin, a disease characterized by abnormal keratinocyte differentiation. However, no causal relationship between the up-regulation of these proteins and the disease has been shown. E-FABP is thought to be involved in cytosolic fatty acid (FA) transport, whereas the role of S100A7 is still unknown. In this report, we show by overlay assays that E-FABP, immobilized on nitrocellulose, is able to capture S100A7 from cytosolic psoriatic protein extracts and vice versa, suggesting the formation of a complex between the two proteins. Using purified E-FABP and S100A7, the complex can be reconstituted only in presence of EDTA. Moreover, we show that increased EDTA concentrations in psoriatic cytosolic protein extracts enhance complex formation. Partial complex disruption was obtained by the addition of physiological concentrations of Zn2+ (0.1 mM), whereas Ca2+ at 5 mM and Mg2+ at 30 mM had no effect. On the other hand, high Ca2+ concentrations (30 mM) resulted in partial complex disruption. Oleic acid-binding properties were observed for free E-FABP and the complex E-FABP–S100A7, but not for free S100A7. By using confocal microscopy we show that S100A7 and E-FABP are co-localized in the cytoplasm of differentiating keratinocytes from lesional psoriatic skin. These data indicate that formation of the E-FABP–S100A7 complex and its FA-binding function might be regulated at least by bivalent cations.
Specificity of human tissue kallikrein towards substrates containing Phe–Phe pair of amino acids
Authors: PIMENTA Daniel C. , CHAO Julie , CHAO Lee , JULIANO Maria A. , JULIANO Luiz
Published: August 2015

We have explored in detail the determinants of specificity for the hydrolysis by human tissue kallikrein (hK1) of substrates containing the Phe–Phe amino acid pair, after which hK1 cleaves kallistatin (human kallikrein-binding protein), a specific serpin for this protease, as well as somatostatin 1–14. Internally quenched fluorogenic peptides were synthesized with the general structure Abz-peptidyl-EDDnp [Abz, o-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)ethylenediamine], based on the natural reactive-centre loop sequence of kallistatin from P9 to P´13, and the kinetic parameters of their hydrolysis by hK1 were determined. All these peptides were cleaved after the Phe–Phe pair. For comparison, we have also examined peptides containing the reactive-centre loop sequences of human protein-C inhibitor (PCI) and rat kallikrein-binding protein, which were hydrolysed after Phe–Arg and Leu–Lys bonds, respectively. Hybrid peptides containing kallistatin–PCI sequences showed that the efficiency of hK1 activity on the peptides containing kallistatin and PCI sequences depended on both the nature of the P1 amino acid as well as on residues at the P- and P´-sides. Moreover, we have made systematic modifications on the hydrophobic pair Phe–Phe, and on Lys and Ile at the P3 and P4 positions according to the peptide substrate, Abz-AIKFFSRQ-EDDnp. All together, we concluded that tissue kallikrein was very effective on short substrates that are cleaved after the Phe–Arg pair; however, hydrolysis after Phe–Phe or other hydrophobic pairs of amino acids was more restrictive, requiring additional enzyme–substrate interaction and/or particular substrate conformations.
Pancreatic lipase/colipase-mediated triacylglycerol hydrolysis is required for cholesterol transport from lipid emulsions to intestinal cells
Authors: YOUNG Simon C. , HUI David Y.
Published: August 2015

This study tested the hypothesis that dietary cholesterol uptake by intestinal cells is dependent on the structure and composition of the lipid carriers in the extracellular milieu. In in vivo experiments with female C57BL/6 mice, cholesterol absorption from phospholipid/triacylglycerol emulsions was significantly reduced by administration of tetrahydrolipstatin, an inhibitor of pancreatic lipase. This inhibitor had no effect on the absorption of cholesterol from phospholipid vesicles. The importance of pancreatic-lipase-mediated triacylglycerol hydrolysis for cholesterol transport from emulsions to intestinal cells was confirmed by in vitro experiments with rat IEC-6 intestinal cells. Cellular uptake of cholesterol from emulsions with a phospholipid/triacylglycerol molar ratio of < 0.3 could be stimulated by pancreatic lipase/colipase hydrolysis of the core neutral lipids. However, pancreatic lipase/colipase was ineffective in hydrolysing triacylglycerols in emulsions with a phospholipid/triacylglycerol molar ratio of > 0.3. Phospholipase A2-mediated hydrolysis of the surface phospholipids was necessary prior to triacylglycerol hydrolysis in these phospholipid-rich emulsions and to the stimulation of cholesterol transport from these particles to IEC-6 cells. The data also revealed that minimal triacylglycerol hydrolysis was sufficient to significantly increase cholesterol transport from lipid emulsions to the intestinal cells. Thus the products of triacylglycerol hydrolysis, namely monoacylglycerol and non-esterified fatty acids, are key determinants in mediating cholesterol transport from lipid emulsions to intestinal cells. Taken together, these results support the hypothesis that remodelling of the surface and core components of lipid carriers is necessary prior to absorption of dietary cholesterol from the gastrointestinal tract.
Identification of protein components of the microsomal glucose 6-phosphate transporter by photoaffinity labelling
Authors: KRAMER Werner , BURGER Hans-Joerg , ARION William J. , CORSIERO Daniel , GIRBIG Frank , WEYLAND Claudia , HEMMERLE Horst , PETRY Stefan , HABERMANN Paul , HERLING Andreas
Published: August 2015

The glucose-6-phosphatase system catalyses the terminal step of hepatic glucose production from both gluconeogenesis and glycogenolysis and is thus a key regulatory factor of blood glucose homoeostasis. To identify the glucose 6-phosphate transporter T1, we have performed photoaffinity labelling of human and rat liver microsomes by using the specific photoreactive glucose-6-phosphate translocase inhibitors S 0957 and S 1743. Membrane proteins of molecular mass 70, 55, 33 and 31 kDa were labelled in human microsomes by [3H]S 0957, whereas in rat liver microsomes bands at 95, 70, 57, 54, 50, 41, 33 and 31 kDa were detectable. The photoprobe [3H]S 1743 led to the predominant labelling of a 57 kDa and a 50 kDa protein in the rat. Stripping of microsomes with 0.3% CHAPS retains the specific binding of T1 inhibitors; photoaffinity labelling of such CHAPS-treated microsomes resulted in the labelling of membrane proteins of molecular mass 55, 33 and 31 kDa in human liver and 50, 33 and 31 kDa in rat liver. Photoaffinity labelling of human liver tissue samples from a healthy individual and from liver samples of patients with a diagnosed glycogen-storage disease type 1b (GSD type 1b; von Gierke's disease) revealed the absence of the 55 kDa protein from one of the patients with GSD type 1. These findings support the identity of the glucose 6-phosphate transporter T1, with endoplasmic reticulum protein of molecular mass 50 kDa in rat liver and 55 kDa in human liver.
Extrahepatic synthesis of plasminogen in the human cornea is up-regulated by interleukins-1α and -1β
Authors: TWINING Sally S. , WILSON Patricia M. , NGAMKITIDECHAKUL Chatri
Published: August 2015

The avascular cornea has limited access to plasma proteins, including plasminogen, a protein that is synthesized by the liver and supplied to most tissues via the blood. Recent experiments by others using plasminogen-deficient mice revealed the importance of plasmin, the active form of plasminogen, for the maintenance of the normal cornea and for corneal wound healing [Kao, Kao, Bugge, Kaufman, Kombrinck, Converse, Good and Degan (1998) Invest. Ophthalmol. Vis. Sci. 39, 502-508; Drew, Kaufman, Kombrinck, Danton, Daugherty, Degen and Bugge (1998) Blood 91, 1616-1624]. In the present experiments, plasmin was identified as a major serine proteinase in the human cornea. The major plasminogen and plasmin forms on non-reducing zymograms and Western blots had Mr values of 76×103 and 85×103, with minor forms of Mr 200×103, 135×103, 68×103 and 45×103. Angiostatin-like peptides with Mrs of 48×103, 45×103 and 38×103 were observed which bound to lysine-Sepharose and reacted with anti-plasminogen monoclonal antibodies directed towards kringle domains 1-3 of plasminogen. The cornea contained 1.1±0.15 μg of plasminogen+plasmin/cornea, or 0.54±0.05 μg of plasminogen+plasmin/mg of protein. Cornea conditioned medium contained nine times the amount of plasminogen+plasmin that could be extracted from the cornea. These data suggested that corneal cells, unlike most extrahepatic cells, synthesize plasminogen. The synthesis of plasminogen by the cornea was confirmed by immunoprecipitation of metabolically labelled plasminogen, sequencing of its cDNA obtained by reverse transcriptase-PCR and inhibition of protein synthesis. Interleukins-1α and -1β stimulated corneal plasminogen synthesis 2-3-fold; however, interleukin-6 decreased corneal plasminogen synthesis by approx. 40% at early times after addition of the cytokine. By 24 h of culture, no differences were noted in the presence and absence of interleukin-6. Thus the cornea can synthesize plasminogen and regulate its synthesis in response to its environment, including cytokines induced in the cornea by injury and inflammation. Therefore the cornea can control the amount of plasminogen, the precursor of both plasmin and angiostatin.
Purification, characterization and cDNA cloning of an endo-exonuclease from the basidiomycete fungus Armillaria mellea
Authors: HEALY Vincent , DOONAN Shawn , McCARTHY Tommie V.
Published: August 2015

We have purified an endo-exonuclease from the fruiting body of the basidiomycete fungus Armillaria mellea by using an ethanol fractionation step, followed by two rounds of column chromatography. The enzyme had an apparent molecular mass of 17500 Da and was shown to exist as a monomer by gel-filtration analysis. The nuclease was active on both double-stranded and single-stranded DNA but not on RNA. It was optimally active at pH 8.5 and also exhibited a significant degree of thermostability. Three bivalent metal ions, Mg2+, Co2+ and Mn2+, acted as cofactors in the catalysis. It was also inhibited by high salt concentrations: activity was completely abolished at 150 mM NaCl. The nuclease possessed both endonuclease activity on supercoiled DNA and a 3ʹ-5ʹ (but not a 5ʹ-3ʹ) exonuclease activity. It generated 5ʹ-phosphomonoesters on its products that, after a prolonged incubation, were hydrolysed to a mixture of free mononucleotides and small oligonucleotides ranging in size from two to eight bases. Elucidation of its N-terminal amino acid sequence permitted the cDNA cloning of the A. mellea nuclease via a PCR-based approach. Peptide mapping of the purified enzyme generated patterns consistent with the amino acid sequence coded for by the cloned cDNA. A BLAST search of the SwissProt database revealed that A. mellea nuclease shared significant amino acid similarity with two nucleases from Bacillussubtilis, suggesting that the three might constitute a distinct class of nucleolytic enzymes.
Purification of xyloglucan endotransglycosylases (XETs): a generally applicable and simple method based on reversible formation of an enzyme–substrate complex
Authors: STEELE Nancy M. , FRY Stephen C.
Published: August 2015

We describe a novel and general, mechanism-based, method for purification of xyloglucan endotransglycosylases (XETs) from crude plant extracts. Putative isoforms, obtained by step-wise precipitation with (NH4)2SO4, were incubated with tamarind xyloglucan (≈ 1 MDa) to form stable xyloglucan-XET complexes with apparent molecular masses > 500 kDa on gel-permeation chromatography (GPC). Subsequent addition of xyloglucan-derived oligosaccharides (a mixture of XET acceptor substrates) caused a shift in the GPC elution volume of the activity back to that expected of a ≈ 32 kDa protein, presumably by completing the transglycosylation reaction and so freeing the enzyme from the xyloglucan (donor substrate). This simple two-step method enabled the isolation of each XET activity attempted [various (NH4)2SO4 cuts from extracts of cauliflower florets and mung bean seedlings], in pure form as judged by SDS/PAGE.
The N-terminal segment of endothelin-converting enzyme (ECE)-1b contains a di-leucine motif that can redirect neprilysin to an intracellular compartment in Madin–Darby canine kidney (MDCK) cells
Authors: CAILLER Françoise , ZAPPULLA Jacques P. , BOILEAU Guy , CRINE Philippe
Published: August 2015

Endothelin-converting enzyme (ECE)-1 is a membrane-bound metallopeptidase of the neprilysin (NEP) family. ECE-1 is responsible for the conversion of inactive big-endothelins into active endothelins. Three different isoforms of human ECE-1 (ECE-1a, ECE-1b and ECE-1c) have been identified. They differ in their N-terminal cytosolic regions, have distinct tissue distribution and intracellular localization. ECE-1a and ECE-1c are both located at the cell surface whereas ECE-1b is targeted to an intracellular compartment. To better understand the nature of the signal responsible for the targeting of ECE-1b to the intracellular compartment, we have constructed several ECE/NEP chimaeric proteins and expressed them by transfection into Madin-Darby canine kidney (MDCK) cells. This allowed us to identify a nine amino acid segment in the cytosolic tail of ECE-1b that is sufficient to relocate NEP from the cell surface to an intracellular compartment. Site-directed mutagenesis on these chimaeras led to the identification of two leucine residues as part of the intracellular retention signal.
Angiotensin II stimulates serine phosphorylation of the adaptor protein Nck: physical association with the serine/threonine kinases Pak1 and casein kinase I
Authors: VOISIN Laure , LAROSE Louise , MELOCHE Sylvain
Published: August 2015

Nck is a small adaptor protein consisting exclusively of three SH3 domains and one SH2 domain. Nck is thought to have an important role in cell signalling by coupling receptor tyrosine kinases, via its SH2 domain, to downstream SH3-binding effectors. We report here that angiotensin II, working through the AT1 receptor subtype, stimulates the phosphorylation of Nck in rat aortic smooth muscle cells. Phosphopeptide mapping analysis revealed that Nck is phosphorylated on four peptides containing exclusively phosphoserine in quiescent cells. Treatment with angiotensin II resulted in increased phosphorylation of these four peptides, without the appearance of new phosphopeptides. We show that Nck, via its SH3 domains, specifically binds three major phosphoproteins of 95, 82 and 66 kDa both in vitro and in intact cells. Notably, the phosphorylation of these Nck-binding proteins was found to increase in parallel with that of Nck on stimulation by angiotensin II. One candidate for the 66 kDa phosphoprotein is the serine/threonine kinase p21-activated kinase 1 (Pak1), which was found to form a stable complex with Nck in aortic smooth muscle cells. We have also identified the γ2 isoform of casein kinase I as another protein kinase that associates with Nck in these cells. These findings indicate that Nck is a target of G-protein-coupled receptors and suggest a role for Pak1 and casein kinase I-γ2 in downstream signalling or regulation of the AT1 receptor.