bluuebunny/crossref_metadata_2025 huggingface dataset:
The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP
Authors: RICQUIER Daniel , BOUILLAUD Frédéric
Published: August 2015

Animal and plant uncoupling protein (UCP) homologues form a subfamily of mitochondrial carriers that are evolutionarily related and possibly derived from a proton/anion transporter ancestor. The brown adipose tissue (BAT) UCP1 has a marked and strongly regulated uncoupling activity, essential to the maintenance of body temperature in small mammals. UCP homologues identified in plants are induced in a cold environment and may be involved in resistance to chilling. The biochemical activities and biological functions of the recently identified mammalian UCP2 and UCP3 are not well known. However, recent data support a role for these UCPs in State 4 respiration, respiration uncoupling and proton leaks in mitochondria. Moreover, genetic studies suggest that UCP2 and UCP3 play a part in energy expenditure in humans. The UCPs may also be involved in adaptation of cellular metabolism to an excessive supply of substrates in order to regulate the ATP level, the NAD+/NADH ratio and various metabolic pathways, and to contain superoxide production. A major goal will be the analysis of mice that either lack the UCP2 or UCP3 gene or overexpress these genes. Other aims will be to investigate the possible roles of UCP2 and UCP3 in response to oxidative stress, lipid peroxidation, inflammatory processes, fever and regulation of temperature in certain specific parts of the body.
Biphasic modulation of ryanodine binding to sarcoplasmic reticulum vesicles of skeletal muscle by Zn2+ ions
Authors: XIA Ruo-Hong , CHENG Xiao-Yang , WANG Hui , CHEN Ke-Ying , WEI Qin-Qin , ZHANG Xiao-Hui , ZHU Pei-Hong
Published: August 2015

With the use of a [3H]ryanodine binding assay, the modulation of skeletal muscle ryanodine receptor (RyR1) by Zn2+ was investigated. In the presence of 100 μM free Ca2+ concentration ([Ca2+]f) as activator, the equilibrium [3H]ryanodine binding to heavy sarcoplasmic reticulum vesicles was biphasically modulated by Zn2+. The binding was increased by a free Zn2+ concentration ([Zn2+]f) of less than 1 μM; a peak binding, approx. 140% of the control (without added Zn2+) was obtained at 0.3 μM [Zn2+]f. An inhibitory effect of Zn2+ became obvious with a [Zn2+]f of more than 1 μM; the [Zn2+]f for producing half inhibition was 2.7±0.5 μM (mean±S.D.). Scatchard analysis indicated that the increase in the binding induced by low [Zn2+]f was due to a decrease in Kd, whereas both an increase in Kd and a possible decrease in Bmax were responsible for the decrease in binding induced by high [Zn2+]f. The binding in the presence of micromolar [Zn2+]f showed a biphasic time course. In the presence of 3 μM [Zn2+]f, after reaching a peak with an increased rate of initial binding, the binding gradually declined. The decline phase could be prevented by decreasing [Zn2+]f to 0.5 μM or by adding 2 mM dithiothreitol, a thiol-reducing agent. The [Ca2+]f dependence of binding was changed significantly by Zn2+, whereas Ca2+ had no clear effect on the [Zn2+]f dependence of binding. Moreover, some interactions were found in the effects between Zn2+ and other RyR1 modulators. It is indicated that Zn2+ can modulate the activation sites and inactivation sites for Ca2+ on RyR1. The physiological significance of the effects of Zn2+ on ryanodine binding is discussed.
The winged-helix/forkhead protein myocyte nuclear factor β (MNF-β) forms a co-repressor complex with mammalian Sin3B
Authors: YANG Quan , KONG Yanfeng , ROTHERMEL Beverly , GARRY Daniel J. , BASSEL-DUBY Rhonda , WILLIAMS R. Sanders
Published: August 2015

Winged-helix/forkhead proteins regulate developmental events in both invertebrate and vertebrate organisms, but biochemical functions that establish a mechanism of action have been defined for only a few members of this extensive gene family. Here we demonstrate that MNF (myocyte nuclear factor)-β, a winged-helix protein expressed selectively and transiently in myogenic precursor cells of the heart and skeletal muscles, collaborates with proteins of the mammalian Sin3 (mSin3) family to repress transcription. Mutated forms of MNF-β that fail to bind mSin3 are defective in transcriptional repression and in negative growth regulation, an overexpression phenotype revealed in oncogenic transformation assays. These data extend the known repertoire of transcription factors with which mSin3 proteins can function as co-repressors to include members of the winged-helix gene family. Transcriptional repression by MNF-β-mSin3 complexes may contribute to the co-ordination of cellular proliferation and terminal differentiation of myogenic precursor cells.
Ca2+-calmodulin inhibits Ca2+ release mediated by type-1, -2 and -3 inositol trisphosphate receptors
Authors: ADKINS Charles E. , MORRIS Stephen A. , DE SMEDT Humbert , SIENAERT Ilse , TÖRÖK Katalin , TAYLOR Colin W.
Published: August 2015

InsP3 binding to type-1, but not type-3, InsP3 receptors is inhibited by calmodulin in a Ca2+-independent fashion [Cardy and Taylor (1998) Biochem. J. 334, 447-455], and Ca2+ mobilization by type-1 InsP3 receptors of cerebellum is inhibited by calmodulin [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 11627-11632]. Using cell types expressing predominantly type-1, -2 or -3 InsP3 receptors, we show that InsP3-evoked Ca2+ mobilization from each is similarly inhibited by calmodulin. In SH-SY5Y cells, which express largely type-1 receptors, calmodulin (IC50 ≈ 15 μM) inhibited InsP3-evoked Ca2+ release only in the presence of Ca2+. The inhibition was unaffected by calcineurin inhibitors. The effect of calmodulin did not result from enhanced metabolism of InsP3 because calmodulin also decreased the sensitivity of the Ca2+ stores to adenophostin A, a non-metabolizable InsP3-receptor agonist. Protein kinase A-catalysed phosphorylation of type-1 InsP3 receptors was unaffected by Ca2+-calmodulin. Using a scintillation proximity assay to measure 125I-calmodulin binding to glutathione S-transferase-fusion proteins, we identified two regions of the type-1 InsP3 receptor (cyt1, residues -6 to 159; and cyt11, residues 1499-1649) that bound 125I-calmodulin. The higher-affinity site (cyt11) was also photoaffinity labelled with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB)-calmodulin. We speculate that Ca2+-independent binding of calmodulin to a site within the first 159 residues of the type-1 InsP3 receptor inhibits InsP3 binding and may thereby regulate the kinetics of Ca2+ release. Ca2+-dependent inhibition of Ca2+ release by calmodulin is mediated by a different site: it may reside on an accessory protein that associates with all three receptor subtypes, or Ca2+-calmodulin binding to a site lying between residues 1499 and 1649 of the type-1 receptor may inhibit Ca2+ release from any tetrameric receptor that includes a type-1 subunit.
Initial-rate kinetics of the flavin reductase reaction catalysed by human biliverdin-IXβ reductase (BVR-B)
Authors: CUNNINGHAM Orla , GORE Michael G. , MANTLE Timothy J.
Published: August 2015

The initial-rate kinetics of the flavin reductase reaction catalysed by biliverdin-IXβ reductase at pH 7.5 are consistent with a rapid-equilibrium ordered mechanism, with the pyridine nucleotide binding first. NADPH binding to the free enzyme was characterized using stopped-flow fluorescence quenching, and a Kd of 15.8 μM was calculated. Equilibrium fluorescence quenching experiments indicated a Kd of 0.55 μM, suggesting that an enzyme-NADPH encounter complex (Kd 15.8 μM) isomerizes to a more stable ‘nucleotide-induced’ conformation. The enzyme was shown to catalyse the reduction of FMN, FAD and riboflavin, with Km values of 52 μM, 125 μM and 53 μM, respectively. Lumichrome was shown to be a competitive inhibitor against FMN, with a Ki of 76 μM, indicating that interactions with the isoalloxazine ring are probably sufficient for binding. During initial experiments it was observed that both the flavin reductase and biliverdin reductase activities of the enzyme exhibit a sharp optimum at pH 5 in citrate buffer. An initial-rate study indicated that the enzyme obeys a steady-state ordered mechanism in this buffer. The initial-rate kinetics in sodium acetate at pH 5 are consistent with a rapid-equilibrium ordered mechanism, indicating that citrate may directly affect the enzyme's behaviour at pH 5. Mesobiliverdin XIIIα, a synthetic biliverdin which binds to flavin reductase but does not act as a substrate for the enzyme, exhibits competitive kinetics with FMN (Ki 0.59 μM) and mixed-inhibition kinetics with NADPH. This is consistent with a single pyridine nucleotide site and competition by FMN and biliverdin for a second site. Interestingly, flavin reductase/biliverdin-IXβ reductase has also been shown to exhibit ferric reductase activity, with an apparent Km of 2.5 μM for the ferric iron. The ferric reductase reaction requires NAD(P)H and FMN. This activity is intriguing, as haem cleavage in the foetus produces non-α isomers of biliverdin and ferric iron, both of which are substrates for flavin reductase/biliverdin-IXβ reductase.
Mouse BTEB3, a new member of the basic transcription element binding protein (BTEB) family, activates expression from GC-rich minimal promoter regions
Authors: MARTIN Karen M. , COOPER Wendy N. , METCALFE James C. , KEMP Paul R.
Published: August 2015

Members of the three-zinc-finger family of transcription factors play an important role in determining basal transcription. We have cloned mouse BTEB3 (mBTEB3), a new member of the basic transcription element binding protein (BTEB) family, which is expressed in a wide variety of tissues. mBTEB3 activates transcription of the simian virus 40 early promoter (4-fold) and of the tissue-specific SM22α promoter (100-fold), suggesting that, like BTEB1 and Sp1, mBTEB3 is a basal transcription factor.
Disulphide-bond pattern and molecular modelling of the dimeric disintegrin EMF-10, a potent and selective integrin α5β1 antagonist from Eristocophis macmahoni venom
Authors: CALVETE Juan J. , JÜRGENS Michael , MARCINKIEWICZ Cezary , ROMERO Antonio , SCHRADER Michael , NIEWIAROWSKI Stefan
Published: August 2015

The disulphide-bond pattern of the heterodimeric disintegrin EMF-10, a potent and selective integrin α5β1 antagonist from Eristocophis macmahoni venom, was established by combination of amino-acid analysis, N-terminal sequencing and collision-induced dissociation by nanoelectrospray ionization quadrupole ion-trap MS of fragments isolated by reversed-phase HPLC after degradation of EMF-10 with oxalic acid. Each EMF-10 subunit contains four intrachain disulphide bonds. Two interchain cystine residues join the EMF-10 polypeptides. The intrachain linkages are conserved in monomeric disintegrins. A molecular model of EMF-10 was built using averaged NMR co-ordinates of flavoridin as a template. The active hairpin loops of the EMF-10 subunits occupy opposite locations at the ends of an elongated disulphide-bond ladder. In the EMF-10 model the N-terminal polypeptide of EMF-10B is close to the RGD-loop of the EMF-10A subunit, suggesting that the N-terminal region of the B-subunit could potentially influence the biological activity of the A-subunit.
LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo
Authors: COLLINS Sean P. , REOMA Junewai L. , GAMM David M. , UHLER Michael D.
Published: August 2015

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by melanocytic macules, hamartomatous polyps and an increased risk for numerous cancers. The human LKB1 (hLKB1) gene encodes a serine/threonine protein kinase that is deficient in the majority of patients with PJS. The murine LKB1 (mLKB1) cDNA was isolated, sequenced and shown to produce a 2.4-kb transcript encoding a 436 amino acid protein with 90% identity with hLKB1. RNA blot and RNase-protection analysis revealed that mLKB1 mRNA is expressed in all tissues and cell lines examined. The widespread expression of LKB1 transcripts is consistent with the elevated risk of multiple cancer types in PJS patients. The predicted LKB1 protein sequence terminates with a conserved prenylation motif (Cys433-Lys-Gln-Gln436) directly downstream from a consensus cAMP-dependent protein kinase (PKA) phosphorylation site (Arg428-Arg-Leu-Ser431). The expression of enhanced green fluorescent protein (EGFP)-mLKB1 chimaeras demonstrated that LKB1 possesses a functional prenylation motif that is capable of targeting EGFP to cellular membranes. Mutation of Cys433 to an alanine residue, but not phosphorylation by PKA, blocked membrane localization. These findings suggest that PKA does phosphorylate LKB1, although this phosphorylation does not alter the cellular localization of LKB1.
Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation
Authors: WALKER Edward H. , FRENCH Christopher E. , RATHBONE Deborah A. , BRUCE Neil C.
Published: August 2015

Morphine dehydrogenase (MDH) of Pseudomonas putida M10 catalyses the NADP+-dependent oxidation of morphine and codeine to morphinone and codeinone. This enzyme forms the basis of a sensitive detection and assay method for heroin metabolites and a biotransformation process for production of hydromorphone and hydrocodone. To improve these processes we have undertaken a thorough examination of the kinetic mechanism of MDH. Sequence comparisons indicated that MDH belongs within the aldose reductase enzyme family. MDH was shown to be specific for the pro-R hydrogen of NADPH. In steady-state kinetic studies, product inhibition patterns suggested that MDH follows a Theorell-Chance mechanism for codeinone reduction at pH 7, and a non-Theorell-Chance sequential ordered mechanism for codeine oxidation at pH 9.5. Residues corresponding to the catalytically important Tyr-48, Lys-77 and Asp-43 of aldose reductase were modified by site-directed mutagenesis, resulting in substantial loss of activity consistent with a catalytic role for these residues. Loss of activity of MDH in the presence of the reaction product morphinone was found to be due to the formation of a covalent adduct with Cys-80; alteration of Cys-80 to serine resulted in an enzyme with greatly enhanced stability.
Characterization and gene structure of a novel retinoblastoma-protein-associated protein similar to the transcription regulator TFII-I
Authors: YAN Xiumin , ZHAO Xiangshan , QIAN Min , GUO Ning , GONG Xiaohua , ZHU Xueliang
Published: August 2015

Retinoblastoma protein (Rb) is an important regulator of vertebrate cell cycle and development. It functions through a direct interaction with protein factors involved in cell cycle progression and differentiation. In the present study we characterized a novel Rb-associated protein, Cream1, which bound to Rb specifically through a C-terminal region. Cream1 contained 959 amino acid residues and migrated as a protein of approx. 120 kDa on SDS/PAGE. It was a widely expressed nuclear protein with a nuclear localization signal resembling that of the large T antigen of simian virus 40. Its primary sequence was characteristic of five direct repeats that were similar to, but distinct from, those of TFII-I, a multifunctional transcription regulator. Three additional regions were also highly conserved in both proteins. Cream1 exhibited an activation activity that was attributed to its N-terminal portion when assayed in yeast. Its relationship with the muscle-enhancer-binding protein MusTRD1 further suggests a role in regulating gene expression. The structural gene, CREAM1, contained 27 exons and spanned more than 150 kb. It was located at human chromosome 7q11.23 in a region deleted for Williams' syndrome, a neurodevelopmental disease with multisystem abnormalities, implying its involvement in certain disorders. Taken together, our results suggest that Cream1 might serve as a positive transcription regulator under the control of Rb.
Rat liver nucleotide pyrophosphatase/phosphodiesterase is an efficient adenylyl transferase
Authors: RIBEIRO João Meireles , LÓPEZ-GÓMEZ Juan , VERGELES José María , COSTAS María Jesús , GARCÍA-DÍAZ Miguel , FERNÁNDEZ Ascensión , FLORES Ana , CAMESELLE José Carlos
Published: August 2015

Rat liver nucleotide pyrophosphatase/phosphodiesterase I (NPP/PDE) catalysed efficiently the transfer of adenylate from ATP to alcohols (methanol, ethanol, propanol, ethylene glycol, glycerol, 2,2-dichloroethanol and glycerol 2-phosphate), which acted as adenylate acceptors competing with water with different efficiencies. NPP/PDE kinetics in alcohol/water mixtures were accounted for by rate equations for competitive substrates, modified to include alcohol negative co-operativity and, depending on the nature of the alcohol, enzyme denaturation by high alcohol concentrations or activation by low alcohol concentrations. The correlation of alcohol efficiencies with alcohol acidities, the comparison of rat liver with snake venom NPP/PDE, and the different effects of ionic additives on the efficiencies of glycerol 2-phosphate and glycerol provided evidence for interaction of the alcohols with a base catalyst, a non-polar and a cationic subsite in the active centre of rat liver NPP/PDE. The enzyme thus appears to be well suited to act as transferase, and we propose that NPP/PDE could be an adenylylating agent in the membrane.
A novel principle for conferring selectivity to poly(A)-binding proteins: interdependence of two ATP synthase β-subunit mRNA-binding proteins
Authors: ANDERSSON Ulf , ANTONICKA Hana , HOUSTEK Josef , CANNON Barbara
Published: August 2015

Based on electrophoretic mobility-shift assays and UV cross-linking experiments, we present evidence in the present work for the existence of two mammalian cytosolic proteins that selectively interact with the 3ʹ-untranslated region of the mRNA coding for the catalytic β-subunit of mitochondrial ATP synthase (β-mtATPase). One of the proteins, β-mtATPase mRNA-binding protein (BARB)1, is a novel poly(A)-binding protein that specifically binds the poly(A) tail of the β-mtATPase transcript. BARB1 achieves this mRNA selectivity through its interaction with a second protein, BARB2, that binds the β-mtATPase mRNA through a 22-bp element with a uridylate core, located 75 bp upstream of the poly(A) tail. Conversely, in the absence of BARB1, BARB2 is still able to bind the β-mtATPase mRNA, but does so with lower affinity. Thus the interaction between BARB1 and BARB2 and β-mtATPase mRNA involves the formation of a complex between the two BARB proteins. We conclude that BARB1 and BARB2 selectively bind the 3ʹ-untranslated region of β-mtATPase mRNA in a novel and interdependent manner. The complex between these two proteins may be involved in post-transcriptional regulation of gene expression.
Human N-benzoyl-l-tyrosyl-p-aminobenzoic acid hydrolase (human meprin): genomic structure of the α and β subunits
Authors: HAHN Dagmar , ILLISSON Rainer , METSPALU Andres , STERCHI Erwin E.
Published: August 2015

N-Benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH, human meprin), a zinc-metalloendopeptidase of the astacin family, consists of two similar subunits. As well as in small-intestinal epithelial cells, the enzyme is found in lamina propria leucocytes, human cancer cells and colorectal cancer tissue, making it a potential candidate for a role in tumour formation and cancer progression. To elucidate the mechanisms that control PPH gene expression and to gain more insights into the evolutionary relationship of the two subunits, we analysed the complete exon-intron organization and searched for putative regulatory elements in 3 kb of the upstream region of both genes. The human gene for the α subunit is approx. 35 kb in size and contains 14 exons. The gene for the β subunit is organized in 15 exons and spans approx. 27 kb. A comparison of both genes indicates strong structural similarities. The exons are almost identical in size, except exon 13 in PPHα, which codes for an additional I domain not present in PPHβ. The locations of the respective exon-intron junctions and the intron phases are almost identical; five of them contain conserved split codons. The main variation is in the intron lengths. It can be concluded that PPHα and PPHβ are derived from a common ancestor. Sequence analysis of the 5ʹ flanking DNA with a computer search for promoter elements and different promoter constructs transfected into Caco-2 cells revealed a number of potential regulatory motifs and suggests that each of the two genes is regulated independently.
Export and transport of tRNA are coupled to a multi-protein complex
Authors: KRUSE Charli , WILLKOMM Dagmar K. , GRÜNWELLER Arnold , VOLLBRANDT Tillmann , SOMMER Stefanie , BUSCH Silke , PFEIFFER Thomas , BRINKMANN Jürgen , HARTMANN Roland K. , MÜLLER Peter K.
Published: August 2015

Vigilin is a ubiquitous multi heterogeneous nuclear ribonucleoprotein (hnRNP) K homologous (KH)-domain protein. Here we demonstrate that purified recombinant human vigilin binds tRNA molecules with high affinity, although with limited specificity. Nuclear microinjection experiments revealed for the first time that the immuno-affinity-purified nuclear vigilin core complex (VCCN) as well as recombinant vigilin accelerate tRNA export from the nucleus in human cells. The nuclear tRNA receptor exportin-t is part of the VCCN. Elongation factor (EF)-1α is enriched in VCCN and its cytoplasmic counterpart VCCC, whereas EF-1β, EF-1γ and EF-1∆ are basically confined to the VCCC. Our results suggest further that vigilin and exportin-t might interact during tRNA export, provide evidence that the channeled tRNA cycle is already initiated in the nucleus, and illustrate that intracellular tRNA trafficking is associated with discrete changes in the composition of cellular cytoplasmic multi-protein complexes containing tRNA.
A general kinetic approach to investigation of active-site availability in macromolecular catalysts
Authors: RESMINI Marina , GUL Sheraz , CARTER Steve , SONKARIA Sanjiv , TOPHAM Christopher M. , GALLACHER Gerrard , BROCKLEHURST Keith
Published: August 2015

A potentially general kinetic method for the investigation of active-site availability in preparations of macromolecular catalysts was developed. Three kinetic models were considered: (a) the conventional two-step model of enzyme catalysis, where the preparation contains only active catalyst (Ea) and inert (i.e. non-binding, non-catalytic) material (Ei); (b) an extension of the conventional model (a) involving only Ea and Ei, but with non-productive binding to Ea (in addition to productive binding); (c) a model in which the preparation contains also binding but non-catalytic material (Eb), predicted to be present in polyclonal catalytic antibody preparations. The method involves comparing the parameters Vmax and Km obtained under catalytic conditions where substrate concentrations greatly exceed catalyst concentration with those (k, the limiting value of the first-order rate constant, kobs, at saturating concentrations of catalyst; and K) for single-turnover kinetics, in which the reverse situation obtains. The active-site contents of systems that adhere to model (a) or extensions that also lack Eb, such as the non-productive binding model (b), may be calculated using [Ea]T = Vmax/k. This was validated by showing that, for α-chymotrypsin, identical values of [Ea]T were obtained by the kinetic method using Suc-Ala-Ala-Pro-Phe-4-nitroanilide as substrate and the well-known ‘all-or-none’ spectroscopic assay using N-trans-cinnamoylimidazole as titrant. For systems that contain Eb, such as polyclonal catalytic antibody preparations, Vmax/k is more complex, but provides an upper limit to [Ea]T. Use of the kinetic method to investigate PCA 271-22, a polyclonal catalytic antibody preparation obtained from the antiserum of sheep 271 in week 22 of the immunization protocol, established that [Ea]T is less than approx. 8% of [IgG], and probably less than approx. 1% of [IgG].
Expression and characterization of novel thrombospondin 1 type I repeat fusion proteins
Authors: QABAR Aziz N. , BULLOCK Jeff , MATEJ Louis , POLVERINI Peter
Published: August 2015

Thrombospondin (TSP)1 is a trimeric extracellular matrix protein that is held together by two cysteine residues. It is one of five TSP proteins that have been described to date with almost a universal heparin binding capability (TSP5 being the exception). The existence of two conformationally distinct structures in the TSP family (trimers and pentamers) prompted us to investigate the contribution of TSP1 trimeric structure to its inhibitory role in angiogenesis. We expressed full-length recombinant human TSP1, its type I repeats, and murine TSP3 in a human embryonic kidney cell line and evaluated their effect on human dermal microvascular endothelial cell (HMVEC) proliferation and sprouting into tube-like structures in vitro. Additionally, two chimaeric molecules were constructed so that the type I repeats of TSP1 were expressed as either dimers (TSP1-Ig chimaera) or pentamers (TSP1-TSP3 chimaera). Dimeric and pentameric type I constructs are novel structures. We found that, similarly to full-length TSP1, intact trimeric type I repeats were inhibitory to HMVEC angiogenesis in vitro. However, dimeric and pentameric type I repeats of TSP1 only partially inhibited HMVEC proliferation and sprouting in vitro. TSP3, which is lacking type I repeats, had no inhibitory activity, confirming that type I repeats elicit the anti-angiogenic activity of TSP1.
Properties of Leishmania major dUTP nucleotidohydrolase, a distinct nucleotide-hydrolysing enzyme in kinetoplastids
Authors: CAMACHO Ana , HIDALGO-ZARCO Fernando , BERNIER-VILLAMOR Victor , RUIZ-PÉREZ Luis M. , GONZÁLEZ-PACANOWSKA Dolores
Published: August 2015

We have previously reported the presence, in the parasitic protozoan Leishmania major, of an enzyme involved in controlling intracellular dUTP levels. The gene encoding this enzyme has now been overexpressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical and enzymic analyses of the Leishmania enzyme show that it is a novel nucleotidohydrolase highly specific for deoxyuridine 5ʹ-triphosphate. The enzyme has proved to be a dimer by gel filtration and is able to hydrolyse both dUTP and dUDP quite efficiently, acting as a dUTP nucleotidohydrolase (dUTPase)-dUDP nucleotidohydrolase but has a limited capacity to act upon other nucleoside di- or triphosphates. The reaction products are dUMP and PPi when dUTP is the substrate and dUMP and Pi in the case of dUDP. The enzyme is sensitive to inhibition by the reaction product dUMP but not by PPi. dUTPase activity is highly dependent on Mg2+ concentrations and markedly sensitive to the phosphatase inhibitor, NaF. In summary, Leishmania dUTPase appears to be markedly different to other proteins characterized previously that accomplish the same function.
Vascular endothelial growth factor stimulates dephosphorylation of the catenins p120 and p100 in endothelial cells
Authors: WONG Elaine Y. M. , MORGAN Louise , SMALES Caroline , LANG Paul , GUBBY Sharon E. , STADDON James M.
Published: August 2015

Vascular endothelial growth factor (VEGF) is an endothelium-specific mitogen that induces angiogenesis and increases vascular permeability. These processes involve regulation of cell-cell adhesion, but molecular mechanisms have yet to be fully established. p120, also termed p120ctn, and its variant p100 are catenins which associate with cadherins and localize to adherens junctions. VEGF was reported to stimulate tyrosine phosphorylation of catenins in endothelial cells. In contrast, we have found that VEGF potently stimulated a rapid and dose-dependent decrease in serine/threonine phosphorylation of p120 and p100. VEGF acted via VEGF receptor 2 to achieve this effect which was independent of activation of the extracellular-signal-regulated kinase pathway. Histamine and activators of protein kinase C had a very similar effect to that of VEGF on phosphorylation of p120 and p100, suggesting that these diverse stimuli may converge on a common signalling element regulating p120/p100 serine/threonine phosphorylation. These data raise the possibility that the dephosphorylation of p120 and p100 triggered by VEGF may contribute to mechanisms regulating permeability and/or motility through modulation of cadherin adhesiveness.
Ca2+ and calmodulin differentially modulate myo-inositol 1,4,5-trisphosphate (IP3)-binding to the recombinant ligand-binding domains of the various IP3 receptor isoforms
Authors: VANLINGEN Sara , SIPMA Henk , DE SMET Patrick , CALLEWAERT Geert , MISSIAEN Ludwig , DE SMEDT Humbert , PARYS Jan B.
Published: August 2015

We have expressed the N-terminal 581 amino acids of type 1 myo-inositol 1,4,5-trisphosphate receptor (IP3R1), IP3R2 and IP3R3 as recombinant proteins [ligand-binding site 1 (lbs-1), lbs-2, lbs-3] in the soluble fraction of Escherichia coli. These recombinant proteins contain the complete IP3-binding domain and bound IP3 and adenophostin A with high affinity. Ca2+ and calmodulin were previously found to maximally inhibit IP3 binding to lbs-1 by 42±6 and 43±6% respectively, and with an IC50 of approx. 200 nM and 3 μM respectively [Sipma, De Smet, Sienaert, Vanlingen, Missiaen, Parys and De Smedt (1999) J. Biol. Chem. 274, 12157-12562]. We now report that Ca2+ inhibited IP3 binding to lbs-3 with an IC50 of approx. 700 nM (37±4% inhibition at 5 μM Ca2+), while IP3 binding to lbs-2 was not affected by increasing [Ca2+] from 100 nM to 25 μM. Calmodulin (10 μM) inhibited IP3 binding to lbs-3 by 37±4%, while IP3 binding to lbs-2 was inhibited by only 11±2%. The inhibition of IP3 binding to lbs-3 by calmodulin was dose-dependent (IC50≈ 2 μM). We conclude that the IP3-binding domains of the various IP3R isoforms differ in binding characteristics for IP3 and adenophostin A, and are differentially modulated by Ca2+ and calmodulin, suggesting that the various IP3R isoforms can have different intracellular functions.
Reagent or myeloperoxidase-generated hypochlorite affects discrete regions in lipid-free and lipid-associated human apolipoprotein A-I
Authors: BERGT Constanze , OETTL Karl , KELLER Walter , ANDREAE Fritz , Jörg LEIS Hans , MALLE Ernst , SATTLER Wolfgang
Published: August 2015

We have previously shown that the modification of high-density lipoprotein subclass 3 (HDL3) by HOCl transformed an anti-atherogenic lipoprotein into a high-uptake form for macrophages and caused a significant impairment of cholesterol efflux capacity [Panzenboeck, Raitmayer, Reicher, Lindner, Glatter, Malle and Sattler (1997) J. Biol. Chem. 272, 29711-29720]. To elucidate the consequences of treatment with OCl- on distinct regions in apolipoprotein A-I (apo A-I), lipid-free and lipid-associated apo A-I were modified with increasing molar ratios of NaOCl or HOCl generated by the myeloperoxidase/H2O2/Cl- system. CD analysis revealed a pronounced decrease in α-helicity for lipid-free apo A-I modified by NaOCl, whereas lipid-associated apo A-I was less affected. The modification of apo A-I by NaOCl (molar oxidant-to-lipoprotein ratio 6:1) resulted in the formation of two distinct oxidized forms of apo A-I with molecular masses 32 or 48 atomic mass units (a.m.u.) higher than that of native apo A-I, indicating the addition of two or three oxygen atoms to the native protein. HPLC analysis of tryptic digests obtained from lipid-free and lipid-associated apo A-I modified with increasing oxidant-to-apolipoprotein molar ratios revealed a concentration-dependent modification of apo A-I: at a low molar oxidant-to-lipoprotein ratio (5:1) the peaks corresponding to the methionine-containing tryptic peptides T11 (residues 84-88), T16 (residues 108-116) and T22 (residues 141-149), located in the central region of apo A-I, disappeared. Their loss was accompanied by the formation of three oxidation products with a molecular mass 16 a.m.u. higher than that of the native peptides. This indicates the addition of oxygen, most probably caused by the oxidation of Met86, Met112 and Met148 to the corresponding methionine sulphoxides. At a molar NaOCl-to-apo A-I ratio of 10:1 the disappearance of peptides T1 (residues 1-10), T7 (residues 46-59) and T9 (residues 62-77) was accompanied by the occurrence of new peaks 33.5 and 33.1 a.m.u. higher than those of the native peptides. Amino acid analyses of peptides T7 and T9 after modification with NaOCl confirmed that Phe57 and Phe71 were primary targets for oxidation by HOCl. GLC-MS analysis of hydrolysates obtained from OCl--modified T7, T9, apo A-I and HDL3 confirmed that Phe residues are an early target for OCl- modification. At molar NaOCl-to-apo A-I ratios of 25:1, the peak areas of peptides T31 (residues 189-195) and T32 (residues 196-206) decreased markedly. Most importantly, incubation of apo A-I with the myeloperoxidase/H2O2/Cl- system (the source of HOCl in vivo) resulted in almost identical modification patterns to those observed with reagent NaOCl.
Enzyme activity and dynamics: xylanase activity in the absence of fast anharmonic dynamics
Authors: DUNN Rachel V. , RÉAT Valerie , FINNEY John , FERRAND Michel , SMITH Jeremy C. , DANIEL Roy M.
Published: August 2015

The activity and dynamics of a simple, single subunit enzyme, the xylanase from Thermotoga maritima strain Fj SS3B.1 have been measured under similar conditions, from -70 to +10 °C. The internal motions of the enzyme, as evidenced by neutron scattering, undergo a sharp transition within this temperature range; they show no evidence for picosecond-timescale anharmonic behaviour (e.g. local diffusive motions or jumps between alternative conformations) at temperatures below -50 °C, whereas these motions are strongly activated at higher temperatures. The activity follows Arrhenius behaviour over the whole of the temperature range investigated, -70 to +10 °C. The results indicate that a temperature range exists over which the enzyme rate-limiting step is independent of fast anharmonic dynamics.
Estimation of systolic and diastolic free intracellular Ca2+ by titration of Ca2+ buffering in the ferret heart
Authors: KIRSCHENLOHR Heide L. , GRACE Andrew A. , VANDENBERG Jamie I. , METCALFE James C. , SMITH Gerry A.
Published: August 2015

Spectroscopic Ca2+-indicators are thought to report values of free intracellular Ca2+ concentration ([Ca2+]i) that may differ from unperturbed values because they add to the buffering capacity of the tissue. To check this for the heart we have synthesized a new 19F-labelled NMR Ca2+ indicator, 1,2-bis-[2-bis(carboxymethyl)amino-4,5-difluorophenoxy]ethane (‘4,5FBAPTA’), with a low affinity (Kd 2950 nM). The new indicator and four previously described 19F-NMR Ca2+ indicators 1,2-bis-[2-(1 - carboxyethyl)(carboxymethyl)amino - 5 - fluorophenoxy]ethane (‘DiMe-5FBAPTA’), 1,2-bis-[2-(1-carboxyethyl)(carboxymethyl)amino-4-fluorophenoxy]ethane (‘DiMe-4FBAPTA’), 1,2-bis-[2-bis(carboxymethyl)amino-5-fluorophenoxy]ethane (‘5FBAPTA’) and 1,2-bis-[2-bis(carboxymethyl)amino-5-fluoro-4-methylphenoxy]ethane (‘MFBAPTA’), with dissociation constants for Ca2+ ranging from 46 to 537 nM, have been used to measure [Ca2+]i, over the range from less than 100 nM to more than 3 μM, in Langendorff-perfused ferret hearts (30 °C, pH 7.4, paced at 1.0 Hz) by 19F-NMR spectroscopy. Loading hearts with indicators resulted in buffering of the Ca2+ transient. The measured end-diastolic and peak-systolic [Ca2+]i were both positively correlated with indicator Kd. The positive correlations between indicator Kd and the measured end-diastolic and peak-systolic [Ca2+]i were used to estimate the unperturbed end-diastolic and peak-systolic [Ca2+]i by extrapolation to Kd = 0 (diastolic) and to Kd = ∞ (systolic) respectively. The extrapolated values in the intact beating heart were 161 nM for end-diastolic [Ca2+]i and 2650 nM for peak-systolic [Ca2+]i, which agree well with values determined from single cells and muscle strips.
Identification of a DNA-binding domain and an active-site residue of pseudorabies virus DNase
Authors: HO Tin-Yun , WU Shih-Lu , HSIANG Chin-Hui , CHANG Tien-Jye , HSIANGǁ Chien-Yun
Published: August 2015

The pseudorabies virus (PRV) DNase gene has an open reading frame of 1476 nt, capable of coding a 492-residue protein. A previous study showed that PRV DNase is an alkaline exonuclease and endonuclease, exhibiting an Escherichia coli RecBCD-like catalytic function. To analyse its catalytic mechanism further, we constructed a set of clones truncated at the N-terminus or C-terminus of PRV DNase. The deleted mutants were expressed in E. coli with the use of pET expression vectors, then purified to homogeneity. Our results indicate that (1) the region spanning residues 274-492 exhibits a DNA-binding ability 7-fold that of the intact DNase; (2) the N-terminal 62 residues and the C-terminal 39 residues have important roles in 3ʹ-exonuclease activity, and (3) residues 63-453 are responsible for 5ʹ- and 3ʹ-exonuclease activities. Further chemical modification of PRV DNase revealed that the inactivation of DNase by diethyl pyrocarbonate, which was reversible on treatment with hydroxylamine, seemed to be attributable solely to the modification of histidyl residues. Because the herpesviral DNases contained only one well-conserved histidine residue, site-directed mutagenesis was performed to replace His371 with Ala. The mutant lost most of its nuclease activity; however, it still exhibited a wild-type level of DNA-binding ability. In summary, these results indicate that PRV DNase contains an independent DNA-binding domain and that His371 is the active-site residue that has an essential role in PRV DNase activity.
Rapid replenishment of sphingomyelin in the plasma membrane upon degradation by sphingomyelinase in NIH3T3 cells overexpressing the phosphatidylinositol transfer protein β
Authors: VAN TIEL Claudia M. , LUBERTO Chiara , SNOEK Gerry T. , HANNUN Yusuf A. , WIRTZ Karel W. A.
Published: August 2015

In order to study the in vivo function of the phosphatidylinositol transfer protein β (PI-TPβ), mouse NIH3T3 fibroblasts were transfected with cDNA encoding mouse PI-TPβ. Two stable cell lines were isolated (SPIβ2 and SPIβ8) in which the levels of PI-TPβ were increased 16- and 11-fold respectively. The doubling time of the SPIβ cells was about 1.7 times that of the wild-type (wt) cells. Because PI-TPβ expresses transfer activity towards sphingomyelin (SM) in vitro, the SM metabolism of the overexpressors was investigated. By measuring the incorporation of [methyl-3H]choline chloride in SM and phosphatidylcholine (PtdCho), it was shown that the rate of de novo SM and PtdCho synthesis was similar in transfected and wt cells. We also determined the ability of the cells to resynthesize SM from ceramide produced in the plasma membrane by the action of bacterial sphingomyelinase (bSMase). In these experiments the cells were labelled to equilibrium (60 h) with [3H]choline. At relatively low bSMase concentrations (50 munits/ml), 50% of [3H]SM in wt NIH3T3 cells was degraded, whereas the levels of [3H]SM in SPIβ cells appeared to be unaffected. Since the release of [3H]choline phosphate into the medium was comparable for both wt NIH3T3 and SPIβ cells, these results strongly suggest that breakdown of SM in SPIβ cells was masked by rapid resynthesis of SM from the ceramide formed. By increasing the bSMase concentrations to 200 munits/ml, a 50% decrease in the level of [3H]SM in SPIβ cells was attained. During a recovery period of 6 h (in the absence of bSMase) the resynthesis of SM was found to be much more pronounced in these SPIβ cells than in 50% [3H]SM-depleted wt NIH3T3 cells. After 6 h of recovery about 50% of the resynthesized SM in the SPIβ cells was available for a second hydrolysis by bSMase. When monensin was present during the recovery period, the resynthesis of SM in bSMase-treated SPIβ cells was not affected. However, under these conditions 100% of the resynthesized SM was available for hydrolysis. On the basis of these results we propose that, under conditions where ceramide is formed in the plasma membrane, PI-TPβ plays an important role in restoring the steady-state levels of SM.
Plasmodium falciparum-infected red blood cells depend on a functional glutathione de novo synthesis attributable to an enhanced loss of glutathione
Authors: LÜERSEN Kai , WALTER Rolf D. , MÜLLER Sylke
Published: August 2015

During the erythrocytic cycle, Plasmodium falciparum is highly dependent on an adequate thiol status for its survival. Glutathione reductase as well as de novo synthesis of GSH are responsible for the maintenance of the intracellular GSH level. The first and rate-limiting step of the synthetic pathway is catalysed by γ-glutamylcysteine synthetase (γ-GCS). Using L-buthionine-(S,R)-sulphoximine (BSO), a specific inhibitor of the γ-GCS, we show that the infection with P. falciparum causes drastic changes in the GSH metabolism of red blood cells (RBCs). Infected RBCs lose GSH at a rate 40-fold higher than non-infected RBCs. The de novo synthesis of the tripeptide was found to be essential for parasite survival. GSH depletion by BSO inhibits the development of P. falciparum with an IC50 of 73 μM. The effect of the drug is abolished by supplementation with GSH or GSH monoethyl ester. Our studies demonstrate that the plasmodicidal effect of the inhibitor BSO does not depend on its specificity towards its target enzyme in the parasite, but on the changed physiological needs for the metabolite GSH in the P. falciparum-infected RBCs. Therefore the depletion of GSH is proposed as a chemotherapeutic strategy for malaria, and γ-GCS is proposed as a potential drug target.
Nuclear targeting of the β isoform of Type II phosphatidylinositol phosphate kinase (phosphatidylinositol 5-phosphate 4-kinase) by its α-helix 7
Authors: CIRUELA Antonio , HINCHLIFFE Katherine A. , DIVECHA Nullin , IRVINE Robin F.
Published: August 2015

Type II phosphatidylinositol phosphate kinases (PIPkins) have recently been found to be primarily phosphatidylinositol 5-phosphate 4-kinases, and their physiological role remains unclear. We have previously shown that a Type II PIPkin [isoform(s) unknown], is localized partly in the nucleus [Divecha, Rhee, Letcher and Irvine (1993) Biochem. J. 289, 617-620], and here we show, by transfection of HeLa cells with green-fluorescent-protein-tagged Type II PIPkins, that this is likely to be the Type IIβ isoform. Type IIβ PIPkin has no obvious nuclear localization sequence, and a detailed analysis of the localization of chimaeras and mutants of the α (cytosolic) and β PIPkins shows that the nuclear localization requires the presence of a 17-amino-acid length of α-helix (α-helix 7) that is specific to the β isoform, and that this helix must be present in its entirety, with a precise orientation. This resembles the nuclear targeting of the HIV protein Vpr, and Type IIβ PIPkin is apparently therefore the first example of a eukaryotic protein that uses the same mechanism.
Neutral sphingomyelinase activity dependent on Mg2+ and anionic phospholipids in the intraerythrocytic malaria parasite Plasmodium falciparum
Authors: HANADA Kentaro , MITAMURA Toshihide , FUKASAWA Masayoshi , MAGISTRADO Pamela A. , HORII Toshihiro , NISHIJIMA Masahiro
Published: August 2015

Sphingolipid metabolism and metabolites are important in various cellular events in eukaryotes. However, little is known about their function in plasmodial parasites. Here we demonstrate that neutral sphingomyelinase (SMase) involved in the sphingomyelin (SM) catabolism is retained by the intraerythrocytic parasite Plasmodium falciparum. When assayed in a neutral pH buffer supplemented with Mg2+ and phosphatidylserine, an activity for the release of the phosphocholine group from SM was detected in parasite-infected, but not in uninfected, erythrocyte ghosts. The SMase activity in the parasite-infected erythrocyte ghosts was enhanced markedly by anionic phospholipids including unsaturated but not saturated phosphatidylserine. Mn2+ could not substitute for Mg2+ to activate SMase in parasite-infected erythrocyte ghosts, whereas both Mn2+ and Mg2+ activated mammalian neutral SMase. The specific activity level of SMase was higher in isolated parasites than in infected erythrocyte ghosts; further fractionation of lysates of the isolated parasites showed that the activity was bound largely to the membrane fraction of the parasites. The plasmodial SMase seemed not to hydrolyse phosphatidylcholine or phosphatidylinositol. The plasmodial SMase, but not SM synthase, was sensitive to scyphostatin, an inhibitor of mammalian neutral SMase, indicating that the plasmodial activities for SM hydrolysis and SM synthesis are mediated by different catalysts. Our finding that the malaria parasites possess SMase activity might explain why the parasites seem to have an SM synthase activity but no activity to synthesize ceramide de novo.
Mapping the catalytic pocket of phospholipases A2 and C using a novel set of phosphatidylcholines
Authors: CARAMELO Julio J. , FLORÍN-CHRISTENSEN Jorge , FLORÍN-CHRISTENSEN Mónica , DELFINO José M.
Published: August 2015

A set of radioiodinatable phosphatidylcholines (PCs) derivatized with the Bolton-Hunter reagent (BHPCs) was synthesized to probe the substrate recognition and activity of phospholipases. A common feature of this series is the presence of a bulky 4-hydroxyphenyl group at the end of the fatty acyl chain attached to position sn-2. The distance between the end group and the glycerol backbone was varied by changing the length of the intervening fatty acyl chain (3-25 atoms). Except for the shortest, this chain includes at least one amide linkage. The usefulness of this series of substrates as a molecular ruler was tested by measuring the hydrolytic activities of Naja naja naja phospholipase A2 (PLA2) and Bacillus cereus phospholipase C (PLC) in Triton X-100 micelles. The activity of PLA2 proved to be highly dependent on the length of the fatty acyl chain linker, the shorter compounds (3-10 atoms) being very poor substrates. In contrast, the PLC activity profile exhibited much less discrimination. In both cases, PCs with 16-21 atom chains at position sn-2 yielded optimal activity. We interpret these findings in terms of fatty acyl chain length-related steric hindrance caused by the terminal aromatic group, affecting the activity of PLA2 and, to a smaller extent, that of PLC. This notion agrees with the more extended recognition of aliphatic chains inside the narrow channel leading to the catalytic site in the former case. Molecular models of these substrates bound to PLA2 were built on the basis of the crystallographic structure of Naja naja atra PLA2 complexed with a phospholipid analogue. Docking of these substrates necessarily requires the intrusion of the bulky 4-hydroxyphenyl group inside the binding pocket and also the failure of the amide group to form hydrogen bonds inside the hydrophobic substrate channel.