bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterodimerization of the epidermal-growth-factor (EGF) receptor and ErbB2 and the affinity of EGF binding are regulated by different mechanisms
Authors: JOHANNESSEN Lene E. , HAUGEN Karianne E. , ØSTVOLD Anne Carine , STANG Espen , MADSHUS Inger Helene
Published: August 2015

When clathrin-dependent endocytosis is inhibited in HeLa cells by overexpression of a K44A (Lys44 → Ala) mutant of the GTPase dynamin, high-affinity binding of epidermal growth factor (EGF) to the EGF receptor (EGFR) is disrupted [Ringerike, Stang, Johannessen, Sandnes, Levy and Madshus (1998) J. Biol. Chem. 273, 16639–16642]. We now report that the effect of [K44A]dynamin on EGF binding was counteracted by incubation with the non-specific kinase inhibitor staurosporine (SSP), implying that a protein kinase is responsible for disrupted high-affinity binding of EGF upon overexpression of [K44A]dynamin. The effect of [K44A]dynamin on EGF binding was not due to altered phosphorylation of the EGFR, suggesting that the activated kinase is responsible for phosphorylation of a substrate other than EGFR. The number of EGFR molecules was increased in cells overexpressing [K44A]dynamin, while the number of proto-oncoprotein ErbB2 molecules was unaltered. EGF-induced receptor dimerization was not influenced by overexpression of [K44A]dynamin. ErbB2–EGFR heterodimer formation was found to be ligand-independent, and the number of heterodimers was not altered by overexpression of [K44A]dynamin. Neither SSP nor the phorbol ester PMA, which disrupts high-affinity EGF–EGFR interaction, had any effect on the EGFR homo- or hetero-dimerization. Furthermore, the EGF-induced tyrosine phosphorylation of ErbB2 was not affected by overexpression of [K44A]dynamin, implying that EGFR–ErbB2 dimers were fully functional. Our results strongly suggest that high-affinity binding of EGF and EGFR–ErbB2 heterodimerization are regulated by different mechanisms.
HC fragment (C-terminal portion of the heavy chain) of tetanus toxin activates protein kinase C isoforms and phosphoproteins involved in signal transduction
Authors: GIL Carles , CHAIB-OUKADOUR Imane , BLASI Juan , AGUILERA José
Published: August 2015

A recent report [Gil, Chaib-Oukadour, Pelliccioni and Aguilera (2000) FEBS Lett. 481, 177–182] describes activation of signal transduction pathways by tetanus toxin (TeTx), a Zn2+-dependent endopeptidase synthesized by the Clostridium tetani bacillus, which is responsible for tetanus disease. In the present work, specific activation of protein kinase C (PKC) isoforms and of intracellular signal-transduction pathways, which include nerve-growth-factor (NGF) receptor trkA, phospholipase C(PLC)γ-1 and extracellular regulated kinases (ERKs) 1 and 2, by the recombinant C-terminal portion of the TeTx heavy chain (HC-TeTx) is reported. The activation of PKC isoforms was assessed through their translocation from the soluble (cytosolic) compartment to the membranous compartment, showing that clear translocation of PKC-α, −β, −γ and −δ isoforms exists, whereas PKC-∊ showed a slight decrease in its soluble fraction immunoreactivity. The PKC-∊ isoform showed no consistent response. Using immunoprecipitation assays against phosphotyrosine residues, time- and dose-dependent increases in tyrosine phosphorylation were observed in the trkA receptor, PLCγ-1 and ERK-1/2. The effects shown by the HC-TeTx fragment on tyrosine phosphorylation were compared with the effects produced by NGF. The trkA and ERK-1/2 activation were corroborated using phospho-specific antibodies against trkA phosphorylated on Tyr490, and antibodies against Thr/Tyr phosphorylated ERK-1/2. Moreover, PLCγ-1 phosphorylation was supported by its HC-TeTx-induced translocation to the membranous compartment, an event related to PLCγ-1 activation. Since HC-TeTx is the domain responsible for membrane binding and lacks catalytic activity, the activations described here must be exclusively triggered by the interaction of TeTx with a membrane component.
Simple, sensitive and accurate method for the quantification of prothrombin mRNA by using competitive PCR
Authors: GROVER Phulwinder K. , STAPLETON Alan M. F. , MIYAZAWA Katsuhito , RYALL Rosemary Lyons
Published: August 2015

A method for the quantification of prothrombin (PT) mRNA species in hepatic tissues of rats was developed with the use of competitive PCR. To validate the quantification approach, sequential dilutions of total RNA from one of the samples were reverse transcribed. Their equivalent volumes were amplified together with a known amount of non-homologous competitor cDNA with identical nucleotide primers. The disparate sizes of target and competitor permitted the easy identification and quantification of bands in samples after densitometric analysis of ethidium bromide-stained agarose gels. Ratios of intensities of target and competitor bands were plotted against the initial amounts of total RNA species used, giving a linear relationship. The slope of this line was virtually identical with that obtained when the sample RNA was replaced with recombinant target cDNA, indicating that recombinant cDNA behaved in PCR identically with that made by reverse transcription and permitting the estimation of transcripts in reverse transcription reactions by using the recombinant counterpart of each as a standard. To avoid variation in the final results, the amount of competitor used in the assay was calculated separately from the equivalence point of the reverse-transcribed total RNA of one of the tissue samples; PCR was performed only for the minimum number of cycles required to detect products. A standard curve was made in each PCR run by amplifying differing amounts of recombinant cDNA species of PT or β-actin together with a constant amount of its competitor. The numbers of transcripts in the tissues were then determined directly by PCR incorporating the same amount of respective competitor (as used in the standard curve) and comparing the ratios of products with the standard curve. Application of this method revealed that the median ratio of PT message to β-actin message in hepatic tissues of 10 normal rats was 0.37, with a mean±S.D. of 0.37±0.07 (range 0.27–0.47). Although the method was developed for the quantification of PT transcripts in liver, it can easily be used for non-hepatic tissues as well. The technique is simple, quick and sensitive and requires only a very small amount of substrate.
Crustacean hyperglycaemic hormone (CHH)-like peptides and CHH-precursor-related peptides from pericardial organ neurosecretory cells in the shore crab, Carcinus maenas, are putatively spliced and modified products of multiple genes
Authors: DIRCKSEN Heinrich , BÖCKING Detlef , HEYN Uwe , MANDEL Christa , CHUNG J. Sook , BAGGERMAN Geert , VERHAERT Peter , DAUFELDT Sabine , PLÖSCH Torsten , JAROS Peter P. , WAELKENS Etienne , KELLER Rainer , WEBSTER Simon G.
Published: August 2015

About 24 intrinsic neurosecretory neurons within the pericardial organs (POs) of the crab Carcinus maenas produce a novel crustacean hyperglycaemic hormone (CHH)-like peptide (PO-CHH) and two CHH-precursor-related peptides (PO-CPRP I and II) as identified immunochemically and by peptide chemistry. Edman sequencing and MS revealed PO-CHH as a 73 amino acid peptide (8630Da) with a free C-terminus. PO-CHH and sinus gland CHH (SG-CHH) share an identical N-terminal sequence, positions 1–40, but the remaining sequence, positions 41–73 or 41–72, differs considerably. PO-CHH may have different precursors, as cDNA cloning of PO-derived mRNAs has revealed several similar forms, one exactly encoding the peptide. All PO-CHH cDNAs contain a nucleotide stretch coding for the SG-CHH41–76 sequence in the 3′-untranslated region (UTR). Cloning of crab testis genomic DNA revealed at least four CHH genes, the structure of which suggest that PO-CHH and SG-CHH arise by alternative splicing of precursors and possibly post-transcriptional modification of PO-CHH. The genes encode four exons, separated by three variable introns, encoding part of a signal peptide (exon I), the remaining signal peptide residues, a CPRP, the PO-CHH1–40/SG-CHH1–40 sequences (exon II), the remaining PO-CHH residues (exon III) and the remaining SG-CHH residues and a 3′-UTR (exon IV). Precursor and gene structures are more closely related to those encoding related insect ion-transport peptides than to penaeid shrimp CHH genes. PO-CHH neither exhibits hyperglycaemic activity in vivo, nor does it inhibit Y-organ ecdysteroid synthesis in vitro. From the morphology of the neurons it seems likely that novel functions remain to be discovered.
Activation of store-mediated calcium entry by secretion-like coupling between the inositol 1,4,5-trisphosphate receptor type II and human transient receptor potential (hTrp1) channels in human platelets
Authors: ROSADO Juan A. , SAGE Stewart O.
Published: August 2015

Physical coupling between inositol 1,4,5-trisphosphate (IP3) receptors and transient receptor potential (Trp) channels has been demonstrated in both transfected and normal cells as a candidate mechanism for the activation of store-mediated Ca2+ entry (SMCE). We have investigated the properties of the coupling between the type II IP3 receptor and naturally expressed human Trp1 (hTrp1) in human platelets. Treatment with xestospongin C, an inhibitor of IP3 receptor function, abolished SMCE and coupling between the IP3 receptor and hTrp1. The coupling was activated by depletion of the intracellular Ca2+ stores, and was reversed by refilling of the stores. We have also examined the role of actin filaments in the activation and maintenance of the coupling. Stabilization of the cortical actin network with jasplakinolide prevented the coupling, indicating that, as with secretion, the actin filaments at the cell periphery act as a negative clamp which prevents constitutive coupling. In addition, the actin cytoskeleton plays a positive role, since disruption of the actin network inhibited the coupling when the Ca2+ stores were depleted. These results provide strong evidence for the activation of SMCE by a secretion-like coupling mechanism involving a reversible association between IP3 receptors and hTrp1 in normal human cells.
Isolation of ubiquitin–E2 (ubiquitin-conjugating enzyme) complexes from erythroleukaemia cells using immunoaffinity techniques
Authors: TAKADA Koji , HIRAKAWA Tae , YOKOSAWA Hideyoshi , OKAWA Yutaka , TAGUCHI Hideki , OHKAWA Kiyoshi
Published: August 2015

A variety of ubiquitin-associated (or conjugated) proteins, including substrates and enzymes for the ubiquitin system, are present in eukaryotic cells. In the present study we developed a simple method for their isolation, consisting of immunoaffinity chromatography using the monoclonal antibody FK2, which recognizes the conjugated ubiquitin molecule. Using this method followed by gel filtration, we isolated multi-ubiquitinated proteins with high molecular masses (> 30kDa) and also ubiquitinthioester-linked and mono-ubiquitinated forms of ubiquitin-conjugating (E2) enzymes, UbcH7 and UBE2N, together with mono-, di- and tri-ubiquitin molecules, from the cytoplasmic extract of heat-shock-treated K562 erythroleukaemia cells. We also demonstrated that the FK2 antibody was capable of precipitating a ubiquitin–UbcH7 thioester, but not free UbcH7, which enabled the measurement of the respective cellular levels separately. The immunoprecipitable ubiquitin–UbcH7 thioester was found only when the cells were treated with heat-shock. These results suggest the usefulness of the immunoaffinity techniques for identifying and analysing the cellular enzyme/protein–ubiquitin complexes.
Self-association and precursor protein binding of Saccharomyces cerevisiae Tom40p, the core component of the protein translocation channel of the mitochondrial outer membrane
Authors: GORDON Donna M. , WANG Jing , AMUTHA Boominathan , PAIN Debkumar
Published: August 2015

The precursor protein translocase of the mitochondrial outer membrane (Tom) is a multi-subunit complex containing receptors and a general import channel, of which the core component is Tom40p. Nuclear-encoded mitochondrial precursor proteins are first recognized by surface receptors and then pass through the import channel. The Tom complex has been purified; however, the protein–protein interactions that drive its assembly and maintain its stability have been difficult to study. Here we show that Saccharomyces cerevisiae Tom40p expressed in bacteria and purified to homogeneity associates efficiently with itself. The self-association is very strong and can withstand up to 4M urea or 1M salt. The tight self-association does not require the N-terminal segment of Tom40p. Furthermore, purified Tom40p preferentially recognizes the targeting sequence of mitochondrial precursor proteins. Although the binding of the targeting sequence to Tom40p is inhibited by urea concentrations in excess of 1M, it is moderately resistant to 1M salt. Simultaneous self-assembly and precursor protein binding suggest that Tom40p contains at least two different domains mediating these processes. The experimental approach described here should be useful for analysing protein–protein interactions involving individual or groups of components of the mitochondrial import machinery.
Inhibition of Escherichia coli CTP synthase by glutamate γ-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis
Authors: BEARNE Stephen L. , HEKMAT Omid , MacDONNELL Jennifer E.
Published: August 2015

Cytidine 5′-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specific activity with the use of metal-ion-affinity chromatography. Unfused CTP synthase, generated by the enzymic removal of the hexahistidine tag, displayed an activity identical with that of the purified native enzyme and was used to study the effect of GTP on the inhibition of enzymic activity by glutamate γ-semialdehyde. Glutamate γ-semialdehyde is expected to inhibit CTP synthase by reacting reversibly with the active-site Cys-379 to form an analogue of a tetrahedral intermediate in glutamine hydrolysis. Indeed, glutamate γ-semialdehyde is a potent linear mixed-type inhibitor of CTP synthase with respect to glutamine (Kis 0.16±0.03mM; Kii 0.4±0.1mM) and a competitive inhibitor with respect to ammonia (Ki 0.39±0.06mM) in the presence of GTP at pH8.0. The mutant enzyme (C379A), which is fully active with ammonia but has no glutamine-dependent activity, is not inhibited by glutamate γ-semialdehyde. Although glutamate γ-semialdehyde exists in solution primarily in its cyclic form, Δ1-pyrroline-5-carboxylate, the variation of inhibition with pH, and the weak inhibition by cyclic analogues of Δ1-pyrroline-5-carboxylate (l-proline, l-2-pyrrolidone and pyrrole-2-carboxylate) confirm that the rare open-chain aldehyde species causes the inhibition. When ammonia is employed as the substrate in the absence of GTP, the enzyme's affinity for glutamate γ-semialdehyde is decreased approx. 10-fold, indicating that the allosteric effector, GTP, functions by stabilizing the protein conformation that binds the tetrahedral intermediate(s) formed during glutamine hydrolysis.
Human oestrogenic 17β-hydroxysteroid dehydrogenase specificity: enzyme regulation through an NADPH-dependent substrate inhibition towards the highly specific oestrone reduction
Authors: GANGLOFF Anne , GARNEAU Amélie , HUANG Yi-Wei , YANG Fu , LIN Sheng-Xiang
Published: August 2015

Human oestrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1) catalyses the final step in the biosynthesis of all active oestrogens. Here we report the steady-state kinetics for 17β-HSD1 at 37°C and pH7.5, using a homogeneous enzyme preparation with oestrone, dehydroepiandrosterone (DHEA) or dihydrotestosterone (DHT) as substrate and NADP(H) as the cofactor. Kinetic studies made over a wide range of oestrone concentrations (10nM–10μM) revealed a typical substrate-inhibition phenomenon. Data analysis using the substrate-inhibition equation v = V·[s]/{Km+[s](1+[s]/Ki)} gave a Km of 0.07±0.01μM, a kcat (for the dimer) of 1.5±0.1s−1, a specificity of 21μM−1·s−1 and a Ki of 1.3μM. When NADH was used instead of NADPH, substrate inhibition was no longer observed and the kinetic constants were significantly modified to 0.42±0.07μM for the Km, 0.8±0.04s−1 for the kcat and 1.9μM−1·s−1 for the specificity. The modification of an amino acid in the cofactor-binding site (Leu36Asp) eliminated the substrate inhibition observed in the presence of NADPH, confirming the NADPH-dependence of the phenomenon. The possible formation of an enzyme–NADP+–oestrone dead-end complex during the substrate-inhibition process is supported by the competitive inhibition of oestradiol oxidation by oestrone. Kinetic studies performed with either DHEA (Km = 24±4μM; kcat = 0.47±0.06s−1; specificity = 0.002μM−1·s−1) or DHT (Km = 26±6μM; kcat = 0.2±0.02s−1; specificity = 0.0008μM−1·s−1) in the presence of NADP(H) resulted in low specificities and no substrate inhibition. Taken together, our results demonstrate that the high specificity of 17β-HSD1 towards oestrone is coupled with an NADPH-dependent substrate inhibition, suggesting that both the specificity and the enzyme control are provided for the cognate substrate.
Homology modelling and structural analysis of human arylamine N-acetyltransferase NAT1: evidence for the conservation of a cysteine protease catalytic domain and an active-site loop
Authors: RODRIGUES-LIMA Fernando , DELOMÉNIE Claudine , GOODFELLOW Geoffrey H. , GRANT Denis M. , DUPRET Jean-Marie
Published: August 2015

Arylamine N-acetyltransferases (EC 2.3.1.5) (NATs) catalyse the biotransformation of many primary arylamines, hydrazines and their N-hydroxylated metabolites, thereby playing an important role in both the detoxification and metabolic activation of numerous xenobiotics. The recently published crystal structure of the Salmonella typhimurium NAT (StNAT) revealed the existence of a cysteine protease-like (Cys-His-Asp) catalytic triad. In the present study, a three-dimensional homology model of human NAT1, based upon the crystal structure of StNAT [Sinclair, Sandy, Delgoda, Sim and Noble (2000) Nat. Struct. Biol. 7, 560–564], is demonstrated. Alignment of StNAT and NAT1, together with secondary structure predictions, have defined a consensus region (residues 29–131) in which 37% of the residues are conserved. Homology modelling provided a good quality model of the corresponding region in human NAT1. The location of the catalytic triad was found to be identical in StNAT and NAT1. Comparison of active-site structural elements revealed that a similar length loop is conserved in both species (residues 122–131 in NAT1 model and residues 122–133 in StNAT). This observation may explain the involvement of residues 125, 127 and 129 in human NAT substrate selectivity. Our model, and the fact that cysteine protease inhibitors do not affect the activity of NAT1, suggests that human NATs may have adapted a common catalytic mechanism from cysteine proteases to accommodate it for acetyl-transfer reactions.
Pressure- and heat-induced inactivation of butyrylcholinesterase: evidence for multiple intermediates and the remnant inactivation process
Authors: WEINGAND-ZIADE Alexandra , RIBES Fabien , RENAULT Frédérique , MASSON Patrick
Published: August 2015

The inactivation process of native (N) human butyrylcholinesterase (BuChE) by pressure and/or heat was found to be multi-step. It led to irreversible formation of an active intermediate (I) state and a denatured state. This series-inactivation process was described by expanding the Lumry–Eyring [Lumry, R. and Eyring, H. (1954) J. Phys. Chem. 58, 110–120] model. The intermediate state (I) was found to have a Km identical with that of the native state and a turnover rate (kcat) twofold higher than that of the native state with butyrylthiocholine as the substrate. The increased catalytic efficiency (kcat/Km) of I can be explained by a conformational change in the active-site gorge and/or restructuring of the water-molecule network in the active-site pocket, making the catalytic steps faster. However, a pressure/heat-induced covalent modification of native BuChE, affecting the catalytic machinery, cannot be ruled out. The inactivation process of BuChE induced by the combined action of pressure and heat was found to continue after interruption of pressure/temperature treatment. This secondary inactivation process was termed ‘remnant inactivation’. We hypothesized that N and I were in equilibrium with populated metastable N′ and I′ states. The N′ and I′ states can either return to the active forms, N and I, or develop into inactive forms, N′in and I′in. Both active N′ and I′ intermediate states displayed different rates of remnant inactivation depending on the pressure and temperature pretreatments and on the storage temperature. A first-order deactivation model describing the kinetics of the remnant inactivation of BuChE is proposed.
Functional analysis of the human annexin A5 gene promoter: a downstream DNA element and an upstream long terminal repeat regulate transcription
Authors: CARCEDO Maria-Teresa , IGLESIAS Juan-Manuel , BANCES Patricia , MORGAN Reginald O. , FERNANDEZ Maria-Pilar
Published: August 2015

Human annexin A5 is a ubiquitous protein implicated in diverse signal transduction processes associated with cell growth and differentiation, and its gene regulation is an important component of this function. Promoter transcriptional activity was determined for a wide 5′ portion of the human annexin A5 gene, from bp −1275 to +79 relative to the most 5′ of several discrete transcription start points. Transfection experiments carried out in HeLa cells identified the segment from bp −202 to +79 as the minimal promoter conferring optimal transcriptional activity. Two canonical Sp1 sites in the immediate 5′ flanking region of a CpG island were required for significant transcription. Strong repressive activity in the distal promoter region between bp −717 to −1153 was attributed to the presence of an endogenous retroviral long terminal repeat, homologous with long terminal repeat 47B. The downstream sequence from bp position +31 to +79 in untranslated exon 1 was also essential for transcription, as its deletion from any of the plasmid constructs abolished activity in transfection assays. Electrophoretic mobility-shift assays, Southwestern-blot analysis and affinity chromatography were used to identify a protein doublet of relative molecular mass 35kDa that bound an octanucleotide palindromic sequence in exon 1. The DNA cis-element resembled an E-box, but did not bind higher molecular mass transcription factors, such as upstream stimulatory factor or activator protein 4. The discovery of a downstream element crucial for annexin A5 gene transcription, and its interaction with a potentially novel transcription factor or complex, may provide a clue to understanding the initiation of transcription by TATA-less, multiple start site promoters.
Characterization of the gene family encoding alternative oxidase from Candida albicans
Authors: HUH Won-Ki , KANG Sa-Ouk
Published: August 2015

Candida albicans possesses a cyanide-resistant respiratory pathway mediated by alternative oxidase (AOX), which seems to be encoded by a gene family with two members. Cloning and expression of AOX1a, one of the genes encoding alternative oxidase from C. albicans, has previously been reported [Huh and Kang (1999) J. Bacteriol. 181, 4098–4102]. In the present study we report the isolation of another gene coding for alternative oxidase, designated AOX1b. AOX1b contains a continuous open reading frame that encodes a polypeptide consisting of 365 amino acids. Interestingly, AOX1a and AOX1b were found to be located in tandem on one of the chromosomes of C. albicans. The presence of cyanide in the culture medium remarkably retarded the growth of the aox1a/aox1a mutants. The growth of the aox1b/aox1b mutants and the aox1a/aox1a aox1b/aox1b double mutants was almost completely inhibited in the same medium. β-Galactosidase reporter assays indicated that, whereas AOX1a was expressed constitutively, the expression of AOX1b was dependent on growth phase and was induced by treatment with cyanide, antimycin A, H2O2, menadione and paraquat. Growth of the cells in media with non-fermentable carbon sources also enhanced the expression of AOX1b. CaSLN1, which encodes a histidine kinase, seems to be involved in the regulation of AOX expression in C. albicans on the basis of the observation that the activity of cyanide-resistant respiration and the expression level of AOX in the casln1/casln1 mutants were found to be significantly low under normal conditions and slightly increased in the presence of respiratory inhibitors compared with the wild-type strain. Like AOX1a, AOX1b could also be functionally expressed in AOX-deficient Saccharomyces cerevisiae and confer cyanide-resistant respiration on the organism.
Signal transducer gp130: biochemical characterization of the three membrane-proximal extracellular domains and evaluation of their oligomerization potential
Authors: PFLANZ Stefan , KERNEBECK Thomas , GIESE Bernd , HERRMANN Andreas , PACHTA-NICK Michael , STAHL Jürgen , WOLLMER Axel , HEINRICH Peter C. , MÜLLER-NEWEN Gerhard , GRÖTZINGER Joachim
Published: August 2015

Glycoprotein 130 (gp130) is a type I transmembrane protein and serves as the common signal-transducing receptor subunit of the interleukin-6-type cytokines. Whereas the membrane-distal half of the gp130 extracellular part confers ligand binding and has been subject to intense investigation, the structural and functional features of its membrane-proximal half are poorly understood. On the basis of predictions of tertiary structure, the membrane-proximal part consists of three fibronectin-type-III-like domains D4, D5 and D6. Here we describe the bacterial expression of the polypeptides predicted to comprise each of these three domains. The recombinant proteins were refolded from solubilized inclusion bodies in vitro, purified to homogeneity and characterized by means of size-exclusion chromatography and CD spectroscopy. For the first time the prediction of three individual membrane-proximal protein domains for gp130has been verified experimentally. The three domains do not show intermediate-affinity or high-affinity interactions between each other. Mapping of a neutralizing gp130 monoclonal antibody against D4 suggested a particular functional role of this domain for gp130 activation, because above that an intrinsic tendency for low-affinity oligomerization was demonstrated for D4.
Role of the System L permease LAT1 in amino acid and iodothyronine transport in placenta
Authors: RITCHIE James W. A. , TAYLOR Peter M.
Published: August 2015

The feto-placental unit relies on a maternal supply of indispensable amino acids and iodothyronines for early development and normal growth. We examined the role of the System L transporter in placental uptake of these substances, using the human placental choriocarcinoma cell line BeWo as a model experimental system. BeWo cells express both heavy (4F2hc) and light (LAT1, LAT2) chains of the System L holotransporter. Saturable transport of both l-[3H]tryptophan and [125I]tri-iodo-l-thyronine in BeWo cells includes components sensitive to inhibition by the System-L-specific substrate 2-endoamino-bicycloheptane-2-carboxylic acid; kinetic properties of these components indicate that the 4F2hc-LAT1 transporter isoform is likely to predominate for the carriage of both substances at physiologically relevant concentrations. Both 4F2hc and LAT1 proteins are also expressed in human placental membranes and LAT1 at least is localized largely to the syncytiotrophoblast layer of the term human placenta. The 4F2hc-LAT1 transporter might therefore serve a vital role in supplying the developing fetus and the placenta with both thyroid hormones and indispensable amino acids from the maternal circulation.
Fungal trehalose phosphorylase: kinetic mechanism, pH-dependence of the reaction and some structural properties of the enzyme from Schizophyllum commune
Authors: EIS Christian , WATKINS Mark , PROHASKA Thomas , NIDETZKY Bernd
Published: August 2015

Initial-velocity measurements for the phospholysis and synthesis of α,α-trehalose catalysed by trehalose phosphorylase from Schizophyllum commune and product and dead-end inhibitor studies show that this enzyme has an ordered Bi Bi kinetic mechanism, in which phosphate binds before α,α-trehalose, and α-d-glucose is released before α-d-glucose 1-phosphate. The free-energy profile for the enzymic reaction at physiological reactant concentrations displays its largest barriers for steps involved in reverse glucosyl transfer to d-glucose, and reveals the direction of phospholysis to be favoured thermodynamically. The pH dependence of kinetic parameters for all substrates and the dissociation constant of d-glucal, a competitive dead-end inhibitor against d-glucose (Ki = 0.3mM at pH6.6 and 30°C), were determined. Maximum velocities and catalytic efficiencies for the forward and reverse reactions decrease at high and low pH, giving apparent pK values of 7.2–7.8 and 5.5–6.0 for two groups whose correct protonation state is required for catalysis. The pH dependences of kcat/K are interpreted in terms of monoanionic phosphate and α-d-glucose 1-phosphate being the substrates, and of the pK value seen at high pH corresponding to the phosphate group in solution or bound to the enzyme. The Ki value for the inhibitor decreases outside the optimum pH range for catalysis, indicating that binding of d-glucal is tighter with incorrectly ionized forms of the complex between the enzyme and α-d-glucose 1-phosphate. Each molecule of trehalose phosphorylase contains one Mg2+ that is non-dissociable in the presence of metal chelators. Measurements of the 26Mg2+/24Mg2+ ratio in the solvent and on the enzyme by using inductively coupled plasma MS show that exchange of metal ion between protein and solution does not occur at measurable rates. Tryptic peptide mass mapping reveals close structural similarity between trehalose phosphorylases from basidiomycete fungi.
Amine oxidase substrates mimic several of the insulin effects on adipocyte differentiation in 3T3 F442A cells
Authors: FONTANA Emi , BOUCHER Jérémie , MARTI Luc , LIZCANO José Miguel , TESTAR Xavier , ZORZANO Antonio , CARPÉNÉ Christian
Published: August 2015

We have previously reported that substrates of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) exert short-term insulin-like effects in rat adipocytes, such as stimulation of glucose transport. In the present work, we studied whether these substrates could also mimic long-term actions of insulin. Adipose differentiation of 3T3 F442A cells, which is highly insulin-dependent, served as a model to test the effects of sustained administration of amine oxidase substrates. Daily treatment of confluent cells with 0.75mM tyramine (a substrate of MAO and SSAO) or benzylamine (a substrate of SSAO) over 1 week caused the acquisition of typical adipocyte morphology. The stimulation of protein synthesis and triacylglycerol accumulation caused by tyramine or benzylamine reached one half of that promoted by insulin. This effect was insensitive to pargyline (an MAO inhibitor), but was inhibited by semicarbazide (an SSAO inhibitor) and by N-acetylcysteine (an antioxidant agent), suggesting the involvement of the H2O2 generated during SSAO-dependent amine oxidation. Chronic administration of amine oxidase substrates also induced the emergence of adipose conversion markers, such as aP2, glycerol-3-phosphate dehydrogenase, the glucose transporter GLUT4, and SSAO itself. Moreover, cells treated with amines acquired the same insulin sensitivity regarding glucose transport as adipocytes classically differentiated with insulin. In all, most of the adipogenic effects of amines were additive to insulin. Our data reveal that amine oxidase substrates partially mimic the adipogenic effect of insulin in cultured preadipocytes. Furthermore, they suggest that SSAO not only represents a novel late marker of adipogenesis, but could also be directly involved in the triggering of terminal adipocyte differentiation.
Overproduction, purification and novel redox properties of the dihaem cytochrome c, NapB, from Haemophilus influenzae
Authors: BRIGÉ Ann , COLE Jeffrey A. , HAGEN Wilfred R. , GUISEZ Yves , Van BEEUMEN Jozef J.
Published: August 2015

The napB gene of the pathogenic bacterium Haemophilus influenzae encodes a dihaem cytochrome c, the small subunit of a heterodimeric periplasmic nitrate reductase similar to those found in other bacteria. In order to obtain sufficient protein for biophysical studies, we aimed to overproduce the recombinant dihaem protein in Escherichia coli. Initial expression experiments indicated that the NapB signal peptide was not cleaved by the leader peptidase of the host organism. Apocytochrome was formed under aerobic, semi-aerobic and anaerobic growth conditions in either Luria–Bertani or minimal salts medium. The highest amounts of apo-NapB were produced in the latter medium, and the bulk was inserted into the cytoplasmic membrane. The two haem groups were covalently attached to the pre-apocytochrome only under anaerobic growth conditions, and with 2.5mM nitrite or at least 10mM nitrate supplemented to the minimal salts growth medium. In order to obtain holocytochrome, the gene sequence encoding mature NapB was cloned in-frame with the E. coli ompA (outer membrane protein A) signal sequence. Under anaerobic conditions, NapB was secreted into the periplasmic space, with the OmpA signal peptide being correctly processed and with both haem c groups attached covalently. Unless expressed in the DegP-protease-deficient strain HM125, some of the recombinant NapB polypeptides were N-terminally truncated as a result of proteolytic activity. Under aerobic growth conditions, co-expression with the E. coli ccm (cytochrome c maturation) genes resulted in a higher yield of holocytochrome c. The pure recombinant NapB protein showed absorption maxima at 419, 522 and 550nm in the reduced form. The midpoint reduction potentials of the two haem groups were determined to be −25mV and −175mV. These results support our hypothesis that the Nap system fulfils a nitrate-scavenging role in H. influenzae.
Thrombopoietin stimulates cortactin translocation to the cytoskeleton independently of tyrosine phosphorylation
Authors: LOPEZ Isabelle , DUPREZ Véronique , MELLE Josiane , DREYFUS François , LÉVY-TOLÉDANO Sylviane , FONTENAY-ROUPIE Michaëla
Published: August 2015

Cortactin is an F-actin-binding protein expressed in platelets. During aggregation by thrombin, cortactin associates with Src, is tyrosine phosphorylated, and then translocates to the cytoskeleton. It is also found to associate with Syk during platelet shape change. Since cortactin undergoes tyrosine phosphorylation in platelets activated by thrombopoietin (TPO) that exhibit neither shape change nor aggregation, we investigated whether it could also relocalize to the detergent-insoluble fraction. We demonstrate that cortactin was present as a tyrosine-phosphorylated protein and co-localized with Syk in the Triton X-100-insoluble fraction of TPO-activated platelets. TPO stimulated Syk activation and association with cortactin. Conversely, cortactin associated with the kinases, Syk and Src. Cortactin tyrosine phosphorylation was blocked by Syk kinase inhibitor, piceatannol or Src family kinase inhibitor, PP2, suggesting that it depends on these two kinases. However, piceatannol or PP2 did not prevent cortactin translocation to the detergent-insoluble fraction. These data suggest that tyrosine phosphorylation is not required for cortactin translocation to the detergent-insoluble compartment. Furthermore, TPO activates, through its receptor c-Mpl, a signalling pathway to the cytoskeleton.
Inhibition of peroxisome-proliferator-activated receptor (PPAR)α by MK886
Authors: KEHRER James P. , BISWAL Shyam S. , LA Eunhye , THUILLIER Philippe , DATTA Kaushik , FISCHER Susan M. , VANDEN HEUVEL John P.
Published: August 2015

Although MK886 was originally identified as an inhibitor of 5-lipoxygenase activating protein (FLAP), recent data demonstrate that this activity does not underlie its ability to induce apoptosis [Datta, Biswal and Kehrer (1999) Biochem. J. 340, 371–375]. Since FLAP is a fatty-acid binding protein, it is conceivable that MK886 may affect other such proteins. A family of nuclear receptors that are activated by fatty acids and their metabolites, the peroxisome-proliferator-activated receptors (PPARs), have been implicated in apoptosis and may represent a target for MK886. The ability of MK886 to inhibit PPAR-α, −β and −γ activity was assessed using reporter assay systems (peroxisome-proliferator response element–luciferase). Using a transient transfection system in monkey kidney fibroblast CV-1 cells, mouse keratinocyte 308 cells and human lung adenocarcinoma A549 cells, 10–20μM MK886 inhibited Wy14,643 activation of PPARα by approximately 80%. Similar inhibition of PPARα by MK886 was observed with a stable transfection reporter system in CV-1 cells. Only minimal inhibitory effects were seen on PPARβ and PPARγ. MK886 inhibited PPARα by a non-competitive mechanism as shown by its effects on the binding of arachidonic acid to PPARα protein, and a dose–response study using a transient transfection reporter assay in COS-1 cells. An assay assessing PPAR ligand–receptor interactions showed that MK886 prevents the conformational change necessary for active-complex formation. The expression of keratin-1, a protein encoded by a PPARα-responsive gene, was reduced by MK886 in a culture of mouse primary keratinocytes, suggesting that PPAR inhibition has functional consequences in normal cells. Although Jurkat cells express all PPAR isoforms, various PPARα and PPARγ agonists were unable to prevent MK886-induced apoptosis. This is consistent with MK886 functioning as a non-competitive inhibitor of PPARα, but may also indicate that PPARα is not directly involved in MK886-induced apoptosis. Although numerous PPAR activators have been identified, the results show that MK886 can inhibit PPARα, making it the first compound identified to have such an effect.
Cloning and characterization of human liver cytosolic β-glycosidase
Authors: de GRAAF Michelle , van VEEN Irene C. , van der MEULEN-MUILEMAN Ida H. , GERRITSEN Winald R. , PINEDO Herbert M. , HAISMA Hidde J.
Published: August 2015

Cytosolic β-glucosidase (EC 3.2.1.21) from mammalian liver is a member of the family 1 glycoside hydrolases and is known for its ability to hydrolyse a range of β-d-glycosides, including β-d-glucoside and β-d-galactoside. We therefore refer to this enzyme as cytosolic β-glycosidase. We cloned the cDNA encoding the human cytosolic β-glycosidase by performing PCR on cDNA prepared from total human liver RNA. Specific primers were based on human expressed sequence tags found in the expressed sequence tag database. The cloned cDNA contained 1407nt with an open reading frame encoding 469 amino acid residues. Amino acid sequence analysis indicates that human cytosolic β-glycosidase is most closely related to lactase phlorizin hydrolase and klotho protein. The enzyme was characterized by using cell lysates of COS-7 cells transfected with a eukaryotic expression vector containing the cDNA. The biochemical, kinetic and inhibition properties of the cloned enzyme were found to be identical with those reported for the enzyme purified from human liver.
Functional role of polar amino acid residues in Na+/H+ exchangers
Authors: WIEBE Christine A. , DiBATTISTA Emily R. , FLIEGEL Larry
Published: August 2015

Na+/H+ exchangers are a family of ubiquitous membrane proteins. In higher eukaryotes they regulate cytosolic pH by removing an intracellular H+ in exchange for an extracellular Na+. In yeast and Escherichia coli, Na+/H+ exchangers function in the opposite direction to remove intracellular Na+ in exchange for extracellular H+. Na+/H+ exchangers display an internal pH-sensitivity that varies with the different antiporter types. Only recently have investigations examined the amino acids involved in pH-sensitivity and in cation binding and transport. Histidine residues are good candidates for H+-sensing amino acids, since they can ionize within the physiological pH range. Histidine residues have been shown to be important in the function of the E. coli Na+/H+ exchanger NhaA and in the yeast Na+/H+ exchanger sod2. In E. coli, His225 of NhaA may function to interact with, or regulate, the pH-sensory region of NhaA. In sod2, His367 is also critical to transport and may be a functional analogue of His225 of NhaA. Histidine residues are not critical for the function of the mammalian Na+/H+ exchanger, although an unusual histidine-rich sequence of the C-terminal tail has some influence on activity. Other amino acids involved in cation binding and transport by Na+/H+ exchangers are only beginning to be studied. Amino acids with polar side chains such as aspartate and glutamate have been implicated in transport activity of NhaA and sod2, but have not been studied in the mammalian Na+/H+ exchanger. Further studies are needed to elucidate the mechanisms involved in pH-sensitivity and cation binding and transport by Na+/H+ exchangers.
Cholesterol depletion reduces apical transport capacity in epithelial Madin–Darby canine kidney cells
Authors: PRYDZ Kristian , SIMONS Kai
Published: August 2015

Reduction of the cholesterol level in membranes of epithelial Madin–Darby canine kidney (MDCK) cells reverses the apical-to-basolateral transport ratio of the apical membrane marker protein influenza virus haemagglutinin and the secreted glycoprotein gp80. At the same time, basolateral transport of the vesicular stomatitis virus G protein is unaffected [Keller and Simons (1998) J. Cell Biol. 140, 1357–1367]. To investigate whether cholesterol depletion influences apical sorting mechanisms specifically, or apical transport capacity more generally, we studied the effect of cholesterol depletion on the secretion of three different classes of molecules from the apical and basolateral surfaces of MDCK cell layers: glycoprotein gp80, sulphated proteoglycans and proteins, and non-glycosylated rat growth hormone. In each case, cholesterol depletion reduced the fraction secreted to the apical medium and increased the fraction secreted basolaterally. The fact that this was observed for all sulphated proteins and proteoglycans and for the non-glycosylated rat growth hormone, which is randomly secreted in untreated cells, indicates that cholesterol depletion reduces the apical transport capacity, rather than interfering with specific recognition and sorting processes.
The mouse Nudt7 gene encodes a peroxisomal nudix hydrolase specific for coenzyme A and its derivatives
Authors: GASMI Lakhdar , McLENNAN Alexander G.
Published: August 2015

A mouse homologue of the Saccharomyces cerevisiae Pcd1p coenzyme A diphosphatase, NUDT7α, has been expressed as a thioredoxin fusion protein in Escherichia coli. NUDT7α is also a CoA diphosphatase of the nudix hydrolase family, and hydrolyses CoA, CoA esters and oxidized CoA with similar efficiences, yielding 3′,5′-ADP and the corresponding 4′-phosphopantetheine derivative as products. Km and kcat values with CoA were 240μM and 3.8s−1. Activity was optimal at pH8.0 with 5mM Mg2+ or Mn2+ ions, while fluoride was inhibitory with an IC50 value of 20μM. Expression of the Nudt7 gene was highest in liver, intermediate in lung and kidney, and lowest in brain and heart, producing a 1.5kb transcript. A similar pattern of expression was found for the human orthologue, NUDT7. An enzymically inactive splice variant, NUDT7β, which lacks 20 amino acids downstream of the nudix motif, was also found to be expressed in mouse tissues. Transfection of HeLa cells with a vector expressing the Nudt7α gene fused to the C-terminus of red fluorescent protein showed that NUDT7α, like Pcd1p, was a peroxisomal enzyme. The function of the NUDT7 enzyme may be the elimination of oxidized CoA from peroxisomes, or the regulation of CoA and acyl-CoA levels in this organelle in response to metabolic demand.
Protein kinase C-δ C2-like domain is a binding site for actin and enables actin redistribution in neutrophils
Authors: LÓPEZ-LLUCH Guillermo , BIRD Margaret M. , CANAS Benito , GODOVAC-ZIMMERMAN Jasminka , RIDLEY Anne , SEGAL Anthony W. , DEKKER Lodewijk V.
Published: August 2015

Neutrophils play a key role in host-defence mechanisms against invading pathogens, using their capacity to migrate, engulf micro-organisms and produce toxic radicals. Protein kinase C (PKC) isotypes are important intracellular regulators of these processes in neutrophils. PKC isotypes themselves are controlled by interactions with lipids, Ca2+ and proteins. The C2-like domain of PKC-δ (δC2) has been identified as a protein-interaction domain in this PKC isotype. In the present paper we have investigated the contribution of protein interactions at this domain to the regulation/function of PKC-δ in neutrophils. Using affinity chromatography we identified actin as a δC2 binding partner in these cells. Fluorescein-labelled δC2, microinjected into immobilized neutrophils, interacts with filamentous actin (F-actin) inside the cell. PKC-δ co-localizes with F-actin in neutrophils, in lamellipodia at the leading edge of the cell. Stimulation with phorbol ester or IgG-opsonized Staphylococcus aureus results in co-ordinated redistribution of PKC-δ and F-actin, and a PKC-δ inhibitor inhibits these changes. Microinjection of δC2 also inhibits F-actin redistribution. Thus PKC-δ binds to F-actin through its C2 domain, and these interactions are important in regulating actin redistribution in neutrophils.
Production and characterization of recombinant human CLN2 protein for enzyme-replacement therapy in late infantile neuronal ceroid lipofuscinosis
Authors: LIN Li , LOBEL Peter
Published: August 2015

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal recessive childhood disease caused by mutations in the CLN2 gene, which encodes the lysosomal enzyme tripeptidyl peptidase I. As a step towards understanding the protein and developing therapeutics for the disease, we have produced and characterized recombinant human CLN2 (ceroid lipofuscinosis, neuronal 2) protein from Chinese-hamster ovary cells engineered to secrete high levels of the enzyme. The protein was secreted as an inactive soluble proenzyme of ≈ 65kDa that appears as a monomer by gel filtration. Upon acidification, the protein is processed to mature form and acquires activity. The enzyme is efficiently delivered to the lysosomes of LINCL fibroblasts by mannose 6-phosphate-receptor-mediated endocytosis (EC50≈ 2nM), where it remains active for long periods of time (t1/2≈ 12 days). In addition, the enzyme is taken up by rat cerebellar granule neurons by mannose 6-phosphate-dependent and -independent mechanisms. Treatment of LINCL fibroblasts with recombinant CLN2 protein restores normal enzyme activity and ameliorates accumulation of the major storage protein, mitochondrial ATP synthase subunit c.