bluuebunny/crossref_metadata_2025 huggingface dataset:
Polycitone A, a novel and potent general inhibitor of retroviral reverse transcriptases and cellular DNA polymerases
Authors: LOYA Shoshana , RUDI Amira , KASHMAN Yoel , HIZI Amnon
Published: August 2015

Polycitone A, an aromatic alkaloid isolated from the ascidian Polycitorsp. exhibits potent inhibitory capacity of both RNA- and DNA-directed DNA polymerases. The drug inhibits retroviral reverse transcriptase (RT) [i.e. of human immunodeficiency virus type 1 (HIV), murine leukaemia virus (MLV) and mouse mammary tumour virus (MMTV)] as efficiently as cellular DNA polymerases (i.e. of both DNA polymerases α and β and Escherichia coliDNA polymerase I). The mode and mechanism of inhibition of the DNA-polymerase activity associated with HIV-1 RT by polycitone A have been studied. The results suggest that the inhibitory capacity of the DNA polymerase activity is independent of the template-primer used. The RNase H function, on the other hand, is hardly affected by this inhibitor. Polycitone A has been shown to interfere with DNA primer extension as well as with the formation of the RT-DNA complex. Steady-state kinetic studies demonstrate that this inhibitor can be considered as an allosteric inhibitor of HIV-1 RT. The target site on the enzyme may be also spatially related to the substrate binding site, since this inhibitor behaves competitively with respect to dTTP with poly(rA)˙oligo(dT) as template primer. Chemical transformations of the five phenol groups of polycitone A by methoxy groups have a determinant effect on the inhibitory potency. Thus, the pentamethoxy derivative which is devoid of all hydroxy moieties, loses significantly, by 40-fold, the ability to inhibit the DNA polymerase function. Furthermore, this analogue lacks the ability to inhibit DNA primer extension as well as the formation of the RT-DNA complex. Indeed, inhibition of the first step in DNA polymerization, the formation of the RT-DNA complex, and hence, of the overall process, could serve as a model for a universal inhibitor of the superfamily of DNA polymerases.
Extracellular regulated kinase (ERK) interaction with actin and the calponin homology (CH) domain of actin-binding proteins
Authors: LEINWEBER Barbara D. , LEAVIS Paul C. , GRABAREK Zenon , WANG C.-L. Albert , MORGAN Kathleen G.
Published: August 2015

An interaction between extracellular regulated kinase 1 (ERK1) and calponin has previously been reported (Menice, Hulvershorn, Adam, Wang and Morgan (1997) J. Biol. Chem.272(40), 25157-25161) and has been suggested to reflect a function of calponin as a signalling molecule. We report in this study that calponin binds to both ERK1 and ERK2 under native conditions as well as in an overlay assay. Using chymotryptic fragments of calponin, the binding site of ERK on calponin was identified as the calponin homology (CH) domain, an N-terminal region of calponin found in other actin-binding proteins. ERK also bound, in a gel overlay assay, α-actinin, a protein with two tandem CH domains, as well as a 27 kDa thermolysin product of α-actinin containing the CH domains of α-actinin. The CH domain of calponin could compete with intact calponin or α-actinin for ERK binding. Titration of acrylodan-labelled calponin with ERK gave a Ka of 6×106 M-1 and titration of acrylodan-labelled calponin with a peptide from the αL16 helix of ERK gave a Ka of 1×106 M-1. Recombinant ERK was found to co-sediment with purified actin and induced a fluorescence change in pyrene-labelled F-actin (Ka = 5×106 M-1). The interaction of ERK with CH domains points to a new potential function for CH domains. The interaction of ERK with actin raises the possibility that actin may provide a scaffold for ERK signalling complexes in both muscle and non-muscle cells.
Central modules of the vaccinia virus complement control protein are not in extensive contact
Authors: KIRKITADZE Marina D. , HENDERSON Colin , PRICE Nicholas C. , KELLY Sharon M. , MULLIN Nicholas P. , PARKINSON John , DRYDEN David T. F. , BARLOW Paul N.
Published: August 2015

The 28.6 kDa vaccinia virus complement control protein (VCP) is an inhibitor of the complement system and has therapeutic potential. It is composed of four domains or modules and is a homologue of complement receptor 1 (CR1) and other mammalian regulators of complement activation. A key aspect to structure-function relationships in these proteins is the extent of intramolecular module-module interactions, since these dictate the overall shape and flexibility of the molecules. A protein fragment (VCP∼ 2,3) encompassing modules 2 and 3 of VCP was over-expressed in Pichia pastoris. Ultracentrifugation showed that VCP∼ 2,3 is highly asymmetric with an axial ratio of 5.3:1, which is consistent with an end-to-end arrangement of the two modules. NMR spectroscopy, differential scanning calorimetry, CD and intrinsic tryptophan fluorescence were used to monitor unfolding of VCP∼ 2,3. Experiments performed over a range of temperatures and concentrations of guanidinium chloride revealed that module 2 unfolds under milder conditions than, and independently of, module 3. Unfolding of module 2 is not associated with extensive changes in amide 15N and 1H chemical shifts of module 3, implying that the modules do not form an extensive intermodular interface. Results obtained in this work for VCP∼ 2,3 are compared with those obtained in a study of CR1 modules 15-17 [Kirkitadze, Krych, Uhrin, Dryden, Smith, Cooper, Wang, Hauhart, Atkinson and Barlow (1999) Biochemistry 38, 7019-7031].
Predominant expression of the mitochondrial dicarboxylate carrier in white adipose tissue
Authors: DAS Kallol , LEWIS Renée Y. , COMBATSIARIS Terry P. , LIN Ying , SHAPIRO Lawrence , CHARRON Maureen J. , SCHERER Philipp E.
Published: August 2015

We report the identification of a novel mouse protein closely related to the family of mitochondrial uncoupling proteins and the oxoglutarate carrier. The cDNA encodes a protein of 287 amino acids that shares all the hallmark features of the mitochondrial transporter superfamily, including six predicted transmembrane domains. It is nearly identical to the sequence recently reported for the rat mitochondrial dicarboxylate carrier (DIC). We find that murine DIC (mDIC) is expressed at very high levels in mitochondria of white adipocytes and is strongly induced in the course of 3T3-L1 adipogenesis. To determine the consequences of the presence of mDIC on the mitochondrial membrane potential, we transiently expressed mDIC in 293-T cells. Overexpression of mDIC leads to significant mitochondrial hyperpolarization. In addition, exposure to cold down-regulates mDIC levels in vivo. In contrast, free fatty acids lead to an up-regulation of mDIC protein in 3T3-L1 adipocytes. This is the first report demonstrating preferential expression in white adipose tissue of any mitochondrial transporter. However, it remains to be determined which metabolic pathways most critically depend on high level expression of mDIC in the adipocyte.
Identification of MUC5B, MUC5AC and small amounts of MUC2 mucins in cystic fibrosis airway secretions
Authors: DAVIES Julia R. , SVITACHEVA Naila , LANNEFORS Louise , KORNFÄLT Ragnhild , CARLSTEDT Ingemar
Published: August 2015

To investigate the genetic identities of the mucins secreted in cystic fibrosis (CF) airways, sputum was collected from five individuals. Samples were separated into gel and sol phases by high-speed centrifugation and the gel phase was extracted in 6 M guanidinium chloride. The ‘insoluble’ residue remaining after extraction of the gel phase was brought into solution by reduction/alkylation. Density-gradient centrifugation in CsCl revealed polydisperse distributions of sialic acid-containing mucins in the gel phase, insoluble residue and sol phase fractions and the degree of variation between the different individuals was low. Antibodies recognizing MUC5AC and MUC5B identified these mucins in each of the fractions. MUC2, however, was present only as a component of the insoluble residue from the gel which accounted for less than 4% by mass of the total mucins. MUC5B and MUC5AC from the gel phase were large oligomeric species composed of disulphide-bond linked subunits and MUC5B was present as two populations with different charge densities which are likely to correspond to MUC5B ‘glycoforms’. The sol phase contained, in addition to MUC5AC and MUC5B, mainly smaller mucins which did not react with the antisera and which were probably degraded. MUC5AC appeared to be enriched in the sol, suggesting that this mucin may be more susceptible to proteolytic degradation than MUC5B. The mucins present in sputum remained broadly similar during acute exacerbation and following antibiotic treatment, although the relative amount of an acidic MUC5B glycoform was decreased during infection.
Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation
Authors: NAVÉ Barbara T. , OUWENS D. Margriet , WITHERS Dominic J. , ALESSI Dario R. , SHEPHERD Peter R.
Published: August 2015

Growth factor induced activation of phosphoinositide 3-kinase and protein kinase B (PKB) leads to increased activity of the mammalian target of rapamycin (mTOR). This subsequently leads to increased phosphorylation of eIF4E binding protein-1 (4EBP1) and activation of p70 ribosomal S6 protein kinase (p70S6K), both of which are important steps in the stimulation of protein translation. The stimulation of translation is attenuated in cells deprived of amino acids and this is associated with the attenuation of 4EBP1 phosphorylation and p70S6K activation. It has been suggested that PKB regulates mTOR function by phosphorylation although direct phosphorylation of mTOR by PKB has not been demonstrated previously. In the present work, we have found that PKB directly phosphorylates mTOR and, using phosphospecific antibodies, we have shown this phosphorylation occurs at Ser2448. Insulin also induces phosphorylation on Ser2448 and this effect is blocked by wortmannin but not rapamycin, consistent with the effect being mediated by PKB. Amino-acid starvation rapidly attenuated the reactivity of the Ser2448 phosphospecific antibody with mTOR and this could not be restored by either insulin stimulation of cells or incubation with PKB in vitro. Our findings demonstrate that mTOR is a direct target for PKB and support the conclusion that regulation of phosphorylation of Ser2448 is a point of convergence for the counteracting regulatory effects of growth factors and amino acid levels.
Copper inhibits β-amyloid production and stimulates the non-amyloidogenic pathway of amyloid-precursor-protein secretion
Authors: BORCHARDT Thilo , CAMAKARIS James , CAPPAI Roberto , MASTERS Colin L. , BEYREUTHER Konrad , MULTHAUP Gerd
Published: August 2015

Previous studies have demonstrated that amyloid precursor protein (APP) can bind and reduce Cu(II) to Cu(I), leading to oxidative modification of APP. In the present study we show that adding copper to Chinese-hamster ovary (CHO) cells greatly reduced the levels of amyloid Aβ peptide (Aβ) both in parental CHO-K1 and in copper-resistant CHO-CUR3 cells, which have lower intracellular copper levels. Copper also caused an increase in the secretion of the APP ectodomain, indicating that the large decrease in Aβ release was not due to a general inhibition in protein secretion. There was an increase in intracellular full-length APP levels which paralleled the decrease in Aβ generation, suggesting the existence of two distinct regulating mechanisms, one acting on Aβ production and the other on APP synthesis. Maximal inhibition of Aβ production and stimulation of APP secretion was achieved in CHO-K1 cells at about 10 μM copper and in CHO-CUR3 cells at about 50 μM copper. This dose ‘window of opportunity’ at which copper promoted the non-amyloidogenic pathway of APP was confirmed by an increase in the non-amyloidogenic p3 fragment produced by α-secretase cleavage. Our findings suggest that copper or copper agonists might be useful tools to discover novel targets for anti-Alzheimer drugs and may prove beneficial for the prevention of Alzheimer's disease.
Melatonin: an endogenous negative modulator of 12-lipoxygenation in the rat pineal gland
Authors: ZHANG Hongjian , AKBAR Mohammed , KIM Hee-Yong
Published: August 2015

Major biochemical activities of the pineal gland include melatonin biosynthesis and 12-lipoxygenation. In this paper, we provide evidence in vivo that melatonin regulates 12-lipoxygenation via 12-lipoxygenase (LOX) expression. The relationship between these two biochemical activities was established by monitoring levels of endogenous melatonin and a 12-LOX metabolite, 12-hydroxyeicosatetraenoic acid (12-HETE), in the rat pineal gland both during the light-dark cycle and after isoproterenol injection using GC/MS with negative ion chemical ionization. As pineal melatonin production reflected a typical diurnal variation, 12-HETE levels showed an off-phase diurnal pattern in relation to melatonin levels. Intravenous administration of isoproterenol, which has been shown to elevate melatonin production, decreased the 12-HETE level significantly. The reduction of 12-HETE levels during the dark phase and after isoproterenol injection was accompanied by decreases in 12-LOX mRNA and protein levels. Direct administration of melatonin to rats by intravenous injection decreased pineal 12-LOX protein levels significantly, indicating that melatonin plays a role in down-regulating 12-LOX expression. When pineal glands were incubated with exogenous melatonin in culture, time-dependent reduction of 12-LOX protein levels was observed. The melatonin-induced reduction in 12-LOX protein was abolished in the presence of the melatonin receptor antagonist luzindole, establishing further the role of melatonin in this process. Incubation of pineal homogenates with exogenous melatonin partially inhibited 12-LOX activity. Taken together, an inverse relationship exists in the endogenous production of 12-HETE, 12-LOX mRNA and protein with respect to melatonin production in the rat pineal gland. Melatonin decreased both 12-LOX mRNA and protein levels in addition to 12-LOX enzyme activity, indicating that melatonin is an endogenous modulator of pineal 12-lipoxygenation.
Confocal imaging of the subcellular distribution of phosphatidylinositol 3,4,5-trisphosphate in insulin- and PDGF-stimulated 3T3-L1 adipocytes
Authors: OATEY Paru B. , VENKATESWARLU Kanamarlapudi , WILLIAMS Alan G. , FLETCHER Laura M. , FOULSTONE Emily J. , CULLEN Peter J. , TAVARÉ Jeremy M.
Published: August 2015

The activation of phosphatidylinositol 3-kinase (PI 3-kinase) and production of PtdIns(3,4,5)P3 is crucial in the actions of numerous extracellular stimuli, including insulin-stimulated glucose uptake. Platelet-derived growth factor (PDGF) also stimulates PI 3-kinase, but only weakly promotes glucose uptake when compared with insulin. Insulin and PDGF have thus been proposed to have differential effects on the subcellular targeting of PI 3-kinase. However, owing to a lack of suitable methodologies, the subcellular localization of the PtdIns(3,4,5)P3 generated has not been examined. The pleckstrin-homology (PH) domains of the nucleotide exchange factors, ADP-ribosylation factor nucleotide-binding-site opener (ARNO) and general receptor for 3-phosphoinositides (GRP1), which have a high affinity and specificity for PtdIns(3,4,5)P3, were fused to green fluorescent protein and used to examine the subcellular localization of PtdIns(3,4,5)P3 generation in living 3T3-L1 adipocytes. PtdIns(3,4,5)P3 was produced almost exclusively in the plasma membrane in response to both agonists, although the response to insulin was greater in magnitude and occurred in considerably more cells. The results suggest that the greater ability of insulin to stimulate glucose uptake may be the result of its ability to generate significantly more plasma-membrane PtdIns(3,4,5)P3 than PDGF. ARNO and GRP1 are nucleotide exchange factors for the small GTP-binding protein ADP-ribosylation factor 6 (ARF6). The inability of a constitutively active GTPase-deficient mutant of ARF6 (ARF6-Q67L; Gln67 → Leu) to cause glucose transporter GLUT4 translocation suggests that activation of this pathway is not sufficient to cause GLUT4 translocation.
Platelet-derived growth factor-dependent association of the GTPase-activating protein of Ras and Src
Authors: SCHLESINGER Thomas K. , DEMALI Kris A. , JOHNSON Gary L. , KAZLAUSKAS Andrius
Published: August 2015

Here we report that the platelet-derived growth factor β receptor (βPDGFR) is not the only tyrosine kinase able to associate with the GTPase-activating protein of Ras (RasGAP). The interaction of non-βPDGFR kinase(s) with RasGAP was dependent on stimulation with platelet-derived growth factor (PDGF) and seemed to require tyrosine phosphorylation of RasGAP. Because the tyrosine phosphorylation site of RasGAP is in a sequence context that is favoured by the Src homology 2 (‘SH2’) domain of Src family members, we tested the possibility that Src was the kinase that associated with RasGAP. Indeed, Src interacted with phosphorylated RasGAP fusion proteins; immunodepletion of Src markedly decreased the recovery of the RasGAP-associated kinase activity. Thus PDGF-dependent tyrosine phosphorylation of RasGAP results in the formation of a complex between RasGAP and Src. To begin to address the relevance of these observations, we focused on the consequences of the interaction of Src and RasGAP. We found that a receptor mutant that did not activate Src was unable to efficiently mediate the tyrosine phosphorylation of phospholipase Cγ (PLCγ). Taken together, these observations support the following hypothesis. When RasGAP is recruited to the βPDGFR, it is phosphorylated and associates with Src. Once bound to RasGAP, Src is no longer able to promote the phosphorylation of PLCγ. This hypothesis offers a mechanistic explanation for our previously published findings that the recruitment of RasGAP to the βPDGFR attenuates the tyrosine phosphorylation of PLCγ. Finally, these findings suggest a novel way in which RasGAP negatively regulates signal relay by the βPDGFR.
Trafficking of Glut4–Green Fluorescent Protein chimaeras in 3T3-L1 adipocytes suggests distinct internalization mechanisms regulating cell surface Glut4 levels
Authors: POWELL Kate A. , CAMPBELL Lachlan C. , TAVARÉ Jeremy M. , LEADER David P. , WAKEFIELD Jill A. , GOULD Gwyn W.
Published: August 2015

Insulin stimulates glucose transport in adipose and muscle tissue by stimulating the movement (‘translocation’) of an intracellular pool of glucose transporters (the Glut4 isoform) to the plasma membrane. We have engineered a series of chimaeras between Glut4 and green fluorescent protein (GFP) from Aequoria victoria and expressed these proteins in 3T3-L1 adipocytes by microinjection of plasmid cDNA. In the absence of insulin, GFP-Glut4 is localized intracellularly within a perinuclear compartment and multiple intracellular punctate structures. In response to insulin, chimaeric GFP-Glut4 species exhibit a profound redistribution to the cell surface with kinetics comparable with the endogenous protein. The intracellular localization of GFP-Glut4 overlaps partially with compartments labelled with Texas Red transferrin, but is largely distinct from intracellular structures identified using Lysotracker-Red®. K+-depletion resulted in the accumulation of GFP-Glut4 at the cell surface, but to an lesser extent than that observed in response to insulin. In contrast with native Glut4, removal of the insulin stimulus or treatment of insulin-stimulated cells with phosphatidylinositol 3′-kinase inhibitors did not result in re-internalization of the chimaeric GFP-Glut4 from the plasma membrane, suggesting that the recycling properties of this species differ from the native Glut4 molecule. We suggest that the recycling pathway utilized by GFP-Glut4 in the absence of insulin is distinct from that used to internalize GFP-Glut4 from the plasma membrane after withdrawal of the insulin stimulus, which may reflect distinct pathways for internalization of endogenous Glut4 in the presence or absence of insulin.
Ca2+-sensitive phosphoinositide hydrolysis is activated in synovial cells but not in articular chondrocytes
Authors: CAPOZZI Ilaria , TONON Rossana , d'ANDREA Paola
Published: August 2015

Cell-to-cell diffusion of second messengers across intercellular channels allows tissues to co-ordinate responses to extracellular stimuli. Intercellular diffusion of inositol 1,4,5-trisphosphate, locally produced by focal stimulations, sustains the propagation of intercellular Ca2+ waves, by stimulating the release of intracellular Ca2+ in neighbouring cells. We previously demonstrated that in cultured articular chondrocytes and HIG-82 synovial cells, studied with digitial fluorescence video imaging, mechanical stimulation of a single cell induced intercellular Ca2+ waves dependent on the presence of gap junctions. In the absence of extracellular Ca2+ the propagating distance of the wave decreased significantly in HIG-82 cells, but appeared unaffected in chondrocytes. We now show that both cells types express connexin 43 and a similar functional coupling, thus suggesting that the different Ca2+ sensitivity of intercellular waves is not due to major differences in gap junction constituent proteins. In HIG-82 synoviocytes, but not in chondrocytes, the Ca2+ ionophore ionomycin stimulated phosphoinositide hydrolysis in a concentration-dependent manner, an effect strictly dependent on the presence of extracellular Ca2+, suggesting the expression, in these cells, of a Ca2+-sensitive phospholipase C activity. Such an activity could be stimulated also by Ca2+ influx induced by P2Y receptor activation and considerably amplifies ATP-induced inositol phosphate (InsP) production. In contrast, Ca2+ influx did not affect considerably the response of chondrocytes to ATP stimulation. In HIG-82 cells, the combined application of ionomycin and ATP maximally stimulated InsP synthesis, suggesting the involvement of two independent mechanisms in inositol phosphate generation. These results suggest that in HIG-82 synovial cells the recruitment of a Ca2+-sensitive phospholipase C activity could amplify the cell response to a focally applied extracellular stimulus, thus providing a positive feedback mechanism for intercellular wave propagation.
Adenomatous polyposis coli protein (APC)-independent regulation of β-catenin/Tcf-4 mediated transcription in intestinal cells
Authors: BAULIDA Josep , BATLLE Eduard , HERREROS Antonio GARCÍA DE
Published: August 2015

Alterations in the transcriptional activity of the β-catenin-Tcf complex have been associated with the earlier stages of colonic transformation. We show here that the activation of protein kinase C by the phorbol ester PMA in several intestinal cell lines increases the levels of β-catenin detected in the nucleus and augments the transcriptional activity mediated by β-catenin. The response to PMA was not related to modifications in the cytosolic levels of β-catenin and was observed not only in cells with wild-type adenomatous polyposis coli protein (APC) but also in APC-deficient cells. Binding assays in vitro revealed that PMA facilitates the interaction of the β-catenin with the nuclear structure. Our results therefore show that β-catenin-mediated transcription can be regulated independently of the presence of APC.
Structural model for the organization of the transmembrane spans of the human red-cell anion exchanger (band 3; AE1)
Authors: GROVES Jonathan D. , TANNER Michael J. A.
Published: August 2015

We have examined the functional co-assembly of non-complementary pairs of N- and C-terminal polypeptide fragments of the anion transport domain (b3mem) of human red-cell band 3. cDNA clones encoding non-contiguous pairs of fragments with one transmembrane (TM) region omitted, or overlapping pairs of fragments with between one and ten TM regions duplicated, were co-expressed in Xenopus oocytes and a cell-free translation system. Stilbene disulphonate-sensitive chloride uptake assays in oocytes revealed that the omission of any single TM region of b3mem except spans 6 and 7 caused a complete loss of functional expression. In contrast, co-expressed pairs of fragments overlapping a single TM region 5, 6, 7, 8, 9-10 or 11-12 retained a high level of functionality, whereas fragments overlapping the clusters of TM regions 2-5, 4-5, 5-8 and 8-10 also mediated some stilbene disulphonate-sensitive uptake. The co-assembly of N- or C-terminal fragments with intact band 3, b3mem or other fragments was examined by co-immunoprecipitation in non-denaturing detergent solutions by using monoclonal antibodies against the termini of b3mem. All the fragments, except for TM spans 13-14, co-immunoprecipitated with b3mem. The medium-sized N-terminal fragments comprising spans 1-6, 1-7 or 1-8 co-immunoprecipitated particularly strongly with the C-terminal fragments containing spans 8-14 or 9-14. The fragments comprising spans 1-4 or 1-12 co-immunoprecipitated less extensively than the other N-terminal fragments with either b3mem or C-terminal fragments. There is sufficient flexibility in the structure of b3mem to allow the inclusion of at least one duplicated TM span without a loss of function. We propose a working model for the organization of TM spans of dimeric band 3 based on current evidence.
Biosynthesis of glycosylphosphatidylinositols of Plasmodium falciparum in a cell-free incubation system: inositol acylation is needed for mannosylation of glycosylphosphatidylinositols
Authors: GEROLD Peter , JUNG Nicole , AZZOUZ Nahid , FREIBERG Nicole , KOBE Sabine , SCHWARZ Ralph T.
Published: August 2015

The structures of glycosylphosphatidylinositols (GPIs) in Plasmodium have been described [Gerold, Schuppert and Schwarz (1994) J. Biol. Chem. 269, 2597-2606]. A detailed understanding of GPI synthesis in Plasmodium is a prerequisite for identifying differences present in biosynthetic pathways of parasites and host cells. A comparison of the biosynthetic pathway of GPIs has revealed differences between mammalian cells and parasitic protozoans. A cell-free incubation system prepared from asexual erythrocytic stages of Plasmodium falciparum, the causative agent of malaria in humans, is capable of synthesizing the same spectrum of GPIs as that found in metabolically labelled parasites. The formation of mannosylated GPIs in the cell-free system is shown to be inhibited by GTP and, unexpectedly, micromolar concentrations of GDP-Man. Lower concentrations of GDP-Man affect the spectrum of GPIs synthesized. The inositol ring of GPIs of P. falciparum is modified by an acyl group. The preferred donor of this fatty acid at the inositol ring is myristoyl-CoA. Inositol acylation has to precede the mannosylation of GPIs because, in the absence of acyl-CoA or CoA, mannosylated GPIs were not detected. Inositol myristoylation is a unique feature of plasmodial GPIs and thus might provide a potential target for drug therapy.
Biochemical and spectroscopic characterization of Escherichia coli aconitases (AcnA and AcnB)
Authors: JORDAN Peter A. , TANG Yue , BRADBURY Alan J. , THOMSON Andrew J. , GUEST John R.
Published: August 2015

Escherichia coli contains two major aconitases (Acns), AcnA and AcnB. They are distantly related monomeric Fe-S proteins that contain different arrangements of four structural domains. On the basis of the differential expression of the acnA and acnB genes, AcnA has been designated as an aerobic-stationary-phase enzyme that is specifically induced by iron and oxidative stress, whereas AcnB functions as the major citric-acid-cycle enzyme during exponential growth. The biochemical and kinetic properties of the purified enzymes have now shown that AcnA is more stable than AcnB, has a higher affinity for citrate, and operates optimally over a wider pH range, consistent with its role as a maintenance or survival enzyme during nutritional or oxidative stress. In contrast, the better performance at high substrate concentrations and greater instability of AcnB indicate that AcnB is specifically adapted to function as the main catabolic enzyme and, by inactivation, to rapidly modulate energy metabolism in response to oxidative or pH stress, either directly or indirectly by regulating post-transcriptional gene expression. EPR and magnetic-CD spectroscopy showed that the iron-sulphur clusters of the bacterial Acns (and their binding sites) strongly resemble those of the mammalian enzymes. The EPR and MCD spectra of the oxidized inactive form of AcnB confirmed the presence of a [3Fe-4S]1+ (S = 1/2) cluster. Comparisons showed that the EPR spectrum of AcnB more closely resembled that of mammalian mitochondrial Acn (m-Acn), whereas the spectrum of AcnA more closely resembled that of the cytoplasmic enzyme (c-Acn). The MCD spectra revealed spectroscopic signatures similar to that of m-Acn. Reconstitution of the active [4Fe-4S]2+ forms followed by one-electron reduction gave rise to EPR spectra that are almost identical with those reported for the mammalian enzymes.
Insulin stimulates pancreatic–duodenal homoeobox factor-1 (PDX1) DNA-binding activity and insulin promoter activity in pancreatic β cells
Authors: WU Hong , MACFARLANE Wendy M. , TADAYYON Mohammad , ARCH Jonathan R. S. , JAMES Roger F. L. , DOCHERTY Kevin
Published: August 2015

Pancreatic-duodenal homoeobox factor-1 (PDX1) is a homoeodomain transcription factor that plays an important role in linking glucose metabolism in pancreatic β cells to the regulation of insulin gene transcription. Our previous results indicated that glucose activates PDX1 DNA-binding activity and insulin promoter activity via a stress-activated signalling pathway involving phosphatidylinositol 3-kinase (PtdIns 3-kinase) and stress-activated protein kinase 2 (SAPK2/p38). The present study was undertaken to determine the effects of other metabolizable and non-metabolizable nutrients. The results indicate that non-metabolizable nutrients, with the exception of 2-deoxyglucose, had no effect. Metabolizable nutrients that could stimulate calcium uptake and insulin release were shown to activate both PDX1 and the insulin promoter. The possible role of insulin acting via an autoregulatory loop was therefore examined. Insulin was shown to potently activate PDX1 DNA-binding activity and insulin promoter activity. The effects of insulin were inhibited by the PtdIns 3-kinase inhibitors wortmannin and LY294002 and by the SAPK2 inhibitor SB203580, suggesting that its effects were mediated via activation of PtdIns 3-kinase and SAPK2. Further support for the insulin-mediated activation of SAPK2 came from the observation that both glucose and insulin stimulated the phosphorylation of SAPK2. These results suggest that both glucose and insulin stimulate PDX1 DNA-binding activity and insulin promoter activity via a pathway involving PtdIns 3-kinase and SAPK2.
Glyoxalase I is a novel nitric-oxide-responsive protein
Authors: MITSUMOTO Atsushi , KIM Kwi-Ryeon , OSHIMA Genichiro , KUNIMOTO Manabu , OKAWA Katsuya , IWAMATSU Akihiro , NAKAGAWA Yasuhito
Published: August 2015

To clarify the molecular mechanisms of nitric oxide (NO) signalling, we examined the NO-responsive proteins in cultured human endothelial cells by two-dimensional (2D) PAGE. Levels of two proteins [NO-responsive proteins (NORPs)] with different pI values responded to NO donors. One NORP (pI 5.2) appeared in response to NO, whereas another (pI 5.0) disappeared. These proteins were identified as a native form and a modified form of human glyoxalase I (Glox I; EC 4.4.1.5) by peptide mapping, microsequencing and correlation between the activity and the isoelectric shift. Glox I lost activity in response to NO, and all NO donors tested inhibited its activity in a dose-dependent manner. Activity and normal electrophoretic mobility were restored by dithiothreitol and by the removal of sources of NO from the culture medium. Glox I was selectively inactivated by NO; compounds that induce oxidative stress (H2O2, paraquat and arsenite) failed to inhibit this enzyme. Our results suggest that NO oxidatively modifies Glox I and reversibly inhibits the enzyme's activity. The inactivation of Glox I by NO was more effective than that of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), another NO-sensitive enzyme. Thus Glox I seems to be a novel NO-responsive protein that is more sensitive to NO than G3PDH.
Direct interaction between p47phox and protein kinase C: evidence for targeting of protein kinase C by p47phox in neutrophils
Authors: REEVES Emer P. , DEKKER Lodewijk V. , FORBES Louisa V. , WIENTJES Frans B. , GROGAN Ann , PAPPIN Darryl J. C. , SEGAL Anthony W.
Published: August 2015

p47phox is an essential component of the NADPH oxidase, and phosphorylation of p47phox is associated with activation of the enzyme. Here we have used p47phox affinity chromatography to extract a p47phox kinase from neutrophil cytosol. The kinase activity was purified by gel filtration and Mini Q chromatography and shown to be indistinguishable from the catalytic fragments of protein kinase C (PKC)-βI, -βII and -∆. The C-terminus of p47phox represented the site of interaction with PKC. Co-immunoprecipitation experiments revealed that the interaction between PKC isotypes and p47phox takes place in intact cells. However PKC-β and -∆ showed different time courses of co-immunoprecipitation, suggesting that the interactions may serve different functions for the various PKC isotypes. Using cells lacking p47phox, we investigated the functional relevance of the interaction between PKC and p47phox. Subcellular fractionation revealed an abnormal recruitment of PKC-βI and -βII, but not PKC-∆, to particulate fractions in p47phox-deficient cells. Phosphorylation of cytosolic proteins was generally increased in stimulated p47phox-deficient neutrophils as compared with normal neutrophils. Furthermore, the cytoskeletal protein coronin was not phosphorylated upon stimulation of p47phox-deficient neutrophils. These findings were confirmed in an in vitro-reconstituted system using rat brain cytosol in which addition of p47phox affected phosphorylation by PKC/PKM (PKM is the catalytic fragment of PKC). These results indicate that p47phox can act as a regulator of PKC in neutrophils.
Transcription from the P2 promoter of the growth hormone receptor gene involves members of the Sp transcription factor family
Authors: ADAMS Timothy E.
Published: August 2015

The P2 promoter of the gene for growth hormone receptor is developmentally regulated and is differentially active in a number of tissues. Little is known about the identity of the transcription factors that participate to effect this pattern of transcription. Deletion analysis and transient transfection were used to localize a previously identified cis-acting element within the sheep P2 promoter to between positions -99 and -87. Gel mobility-shift assays with nuclear extracts from Chinese hamster ovary (CHO-K1) fibroblasts revealed that this sequence encompasses an atypical binding site for both Sp1 and two isoforms of Sp3. A gel mobility-shift scan of promoter sequences between -88 and +21 indicated the existence of three other binding sites for Sp1 and Sp3. One of these, designated site II and found by using a probe spanning -74 to -54, corresponds to a classical GC box consensus sequence. Site III (-63 to -41) and site IV (-27 to -5) harbour atypical Sp1/Sp3-binding sequences. Site-directed mutagenesis of site II or site IV decreased promoter activity by approx. 40%, whereas a promoter construct incorporating both mutations exhibited negligible (approx. 1%) activity. Co-transfection of expression plasmids encoding either Sp1 or Sp3 significantly transactivated reporter gene activity from a P2 promoter construct carrying all four Sp1/Sp3-binding sites (8-fold compared with 7.1-fold induction respectively). Sp1 is known to interact with a variety of other transcription factors to regulate the transcription of a number of differentially expressed genes. The identification of four binding sites for Sp1 and Sp3 within the P2 promoter of the gene for growth hormone receptor might point to other factors that interact to regulate the activity of this promoter in different tissues during foetal and post-natal development.
Activation of tyrosine kinases by α1A-adrenergic and growth factor receptors in transfected PC12 cells
Authors: ZHONG Hongying , MINNEMAN Kenneth P.
Published: August 2015

We compared the role of tyrosine kinases in α1A-adrenergic receptor (AR) and growth factor receptor stimulation of mitogen-activated protein kinase pathways in PC12 cells. Norepinephrine (NE) (noradrenaline), epidermal growth factor (EGF) and nerve growth factor (NGF) caused different patterns of tyrosine phosphorylation in PC12 cells stably expressing α1A-ARs. NE increased tyrosine phosphorylation of focal adhesion-related kinase Pyk2 and a 70 kDa protein, probably paxillin, whereas EGF strongly stimulated tyrosine phosphorylation of the EGF receptor and cytokine-activated kinase Jak2. The EGF receptor inhibitor AG1478 inhibited activation of extracellular signal-regulated kinases (ERKs) by EGF but not by NE. EGF and NGF strongly activated tyrosine phosphorylation of Shc and caused association of Src-homology collagen (Shc) with growth-factor-receptor-bound protein 2 (Grb2); however, neither NE nor UTP caused substantial activation of the Shc/Grb2 pathway. NE, UTP, EGF and NGF all increased tyrosine phosphorylation of Src, and this was inhibited by the Src inhibitor PP2. However, PP2 inhibited ERK activation in response to NE and UTP, but not in response to EGF or NGF. PP2 also completely blocked NE-induced PC12 cell differentiation, but had no measurable effect on NGF-induced differentiation. These studies show that activation of mitogen-activated protein kinase pathways by G-protein-coupled receptors and tyrosine kinase receptors proceed through distinct molecular pathways in PC12 cells, and support an obligatory role for Src activation in mitogenic responses to α1A-ARs in these cells.
Different roles of protein kinase C α and δ isoforms in the regulation of neutral sphingomyelinase activity in HL-60 cells
Authors: VIŠNJIĆ Dora , BATINIŠ Drago , BANFIŠ Hrvoje
Published: August 2015

The signalling mechanisms responsible for the hydrolysis of sphingomyelin mediated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and interferon γ (IFN-γ) in HL-60 cells were investigated. IFN-γ was found to increase selectively the activity of cytosolic, Mg2+-independent, neutral sphingomyelinase. The treatment of HL-60 cells with the combination of 1,25(OH)2D3 and IFN-γ had an additive effect on sphingomyelin hydrolysis, ceramide release and the activity of cytosolic, Mg2+-independent, neutral sphingomyelinase. The pretreatment of HL-60 cells with staurosporine, chelerythrine chloride and bisindolylmaleimide abolished the activity of sphingomyelinase in response to 1,25(OH)2D3 and IFN-γ. Calphostin C, which acts on the regulatory site of protein kinase C (PKC), and Gö 6976, a selective inhibitor of Ca2+-dependent PKC isoforms, inhibited the effect of 1,25(OH)2D3 but had no effect on the IFN-γ-mediated increase in activity of sphingomyelinase. Isoform-specific antibodies were used to deplete different PKC isoforms from cytosol before the treatment of the cytosolic fraction with 1,25(OH)2D3, arachidonic acid (AA) and PMA. The depletion of PKC isoforms β1, β2, ϵ, η, μ, ζ and λ had no effect on the activation of sphingomyelinase induced by 1,25(OH)2D3 or by AA. The depletion of PKC α from the cytosol completely abolished the effect of 1,25(OH)2D3 on sphingomyelinase activity but had no effect on the AA-induced activity of sphingomyelinase. PMA had no effect on the activity of sphingomyelinase in either untreated or α-depleted cytosol but significantly increased the activity of sphingomyelinase when added to cytosol depleted of PKC ∆. Moreover, PMA inhibited the effect of 1,25(OH)2D3 on sphingomyelinase activation but the inhibitory effect was abolished by prior depletion of PKC ∆ from the cytosol. These studies demonstrate that 1,25(OH)2D3-induced activation of sphingomyelinase is mediated by PKC α. Furthermore, PKC ∆ had an inhibitory effect on sphingomyelinase, suggesting that the difference between the 1,25(OH)2D3- and PMA-mediated effects on sphingomyelin turnover depends on the specific regulation of the PKC α and PKC ∆ isoforms.
Acetylcholinesterase from Schistosoma mansoni: interaction of globular species with heparin
Authors: TARRAB-HAZDAI Rebeca , TOKER Lilly , SILMAN Israel , ARNON Ruth
Published: August 2015

In the cercarial and schistosomal stages of the life cycle of the trematode Schistosoma mansoni, acetylcholinesterase occurs as two principal molecular forms (both globular), present in approximately equal amounts, with sedimentation coefficients of 6.5 S and 8 S. The 6.5 S form is solubilized by bacterial phosphatidylinositol-specific phospholipase C from intact schistosomula. It is thus located on the outer surface of the schistosomal tegument and is most probably analogous to the glycosylphosphatidylinositol-anchored G2 form of acetylcholinesterase found in the electric organ of Torpedo, on the surface of mammalian erythrocytes, and elsewhere. Both forms are fully solubilized by the non-ionic detergent Triton X-100. Upon passing such a detergent extract over a heparin-Sepharose column, only the 8 S form was retained on the column. The bound acetylcholinesterase could be progressively eluted by increasing the salt concentration, with approx. 0.5-0.6 M NaCl being needed for complete elution. Selective inhibition experiments carried out on live parasites using the covalent acetylcholinesterase inhibitor echothiophate (phospholine), which does not penetrate the tegument, selectively inhibited the 6.5 S form, but not the 8 S form, suggesting an internal location for the latter. Monoclonal antibodies raised against S. mansoni acetylcholinesterase also distinguished between the two forms. Thus monoclonal antibody SA7 bound the 6.5 S form selectively, whereas SA57 recognized the 8 S form. The selective binding of the 8 S form to heparin suggests that, within the parasite, this form may be associated with the extracellular matrix of the musculature.
Phosphorylation of P20 is associated with the actions of insulin in rat skeletal and smooth muscle
Authors: WANG Yu , XU Aimin , COOPER Garth J. S.
Published: August 2015

Although a large number of protein kinases/phosphatases involved in insulin's actions have been characterized recently, relatively few of the downstream phosphoproteins have been identified. We have employed two-dimensional gel electrophoresis-based proteome analysis to investigate the insulin-evoked phosphorylation cascade in rat soleus muscle. Insulin reproducibly increased phosphorylation of a 20-kDa protein with a pI value of 6.0, which was identified subsequently as a phospho-isoform of P20, a small heat-shock-related protein. The adenylyl cyclase activator, forskolin, decreased phosphorylation of this P20 isoform and increased phosphorylation of another two P20 isoforms, with pI values of 5.9 and 5.6. Two-dimensional peptide mapping revealed that the phospho-peptides of these three P20 isoforms are different. In contrast to its action in soleus muscle, insulin decreased phosphorylation of the P20 isoform with pI 6.0 and increased phosphorylation of the two isoforms with pI values of 5.9 and 5.6 in vascular smooth muscle. This effect is similar to that induced by vasodilatory stimuli, suggesting that insulin could exert its vasodilatory action by affecting phosphorylation of P20. In summary, these results demonstrate that insulin differently modulates phosphorylation of P20 in skeletal and smooth muscle, and suggest that P20 could be a potential modulator of insulin's functions in these tissues.
Molecular cloning and functional expression of bovine spleen ecto-NAD+ glycohydrolase: structural identity with human CD38
Authors: AUGUSTIN Angélique , MULLER-STEFFNER Hélène , SCHUBER Francis
Published: August 2015

Bovine spleen ecto-NAD+ glycohydrolase, an archetypal member of the mammalian membrane-associated NAD(P)+ glycohydrolase enzyme family (EC 3.2.2.6), displays catalytic features similar to those of CD38, i.e. a protein originally described as a lymphocyte differentiation marker involved in the metabolism of cyclic ADP-ribose and signal transduction. Using amino acid sequence information obtained from NAD+ glycohydrolase and from a truncated and hydrosoluble form of the enzyme (hNADase) purified to homogeneity, a full-length cDNA clone was obtained. The deduced sequence indicates a protein of 278 residues with a molecular mass of 31.5 kDa. It predicts that bovine ecto-NAD+ glycohydrolase is a type II transmembrane protein, with a very short intracellular tail. The bulk of the enzyme, which is extracellular and contains two potential N-glycosylation sites, yields the fully catalytically active hNADase which is truncated by 71 residues. Transfection of HeLa cells with the full-length cDNA resulted in the expression of the expected NAD+ glycohydrolase, ADP-ribosyl cyclase and GDP-ribosyl cyclase activities at the surface of the cells. The bovine enzyme, which is the first ‘classical’ NAD(P)+ glycohydrolase whose structure has been established, presents a particularly high sequence identity with CD38, including the presence of 10 strictly conserved cysteine residues in the ectodomain and putative catalytic residues. However, it lacks two otherwise conserved cysteine residues near its C-terminus. Thus hNADase, the truncated protein of 207 amino acids, represents the smallest functional domain endowed with all the catalytic activities of CD38/NAD+ glycohydrolases so far identified. Altogether, our data strongly suggest that the cloned bovine spleen ecto-NAD+ glycohydrolase is the bovine equivalent of CD38.
Oligomerization reduces heparin affinity but enhances receptor binding of fibroblast growth factor 2
Authors: SAFRAN Michal , EISENSTEIN Miriam , AVIEZER David , YAYON Avner
Published: August 2015

The biological response of cells to fibroblast growth factors (FGFs) depends on heparan sulphate glycosaminoglycans sharing particular structural motifs. Heparin induced FGF dimerization has been suggested to mediate receptor dimerization and activation. Here we demonstrate that heparin-derived oligosaccharides that promote receptor binding and activation specifically induce the dimerization of basic FGF (FGF2). These heparin-induced dimers of FGF2 acquire high affinity for receptor binding and are biologically active. Using biotinylated FGF2 bound to immobilized streptavidin gradually saturated with biotin, enabled a quantitative analysis of heparin-dependent and heparin-independent FGF2 monomers and oligomers. Streptavidin induced FGF2 dimers bind and activate FGF receptors only in the presence of heparin. An excess of streptavidin, forcing biotin-FGF2 into monomers, reduces receptor binding and blocks FGF-dependent cell proliferation. All these suggest predominant receptor binding and activation by heparin associated FGF2 oligomers. Unexpectedly, heparin induced dimers and higher order oligomers lose most of their affinity towards heparin. Direct binding of soluble FGF receptors (FGFRs) to either monomers or dimers of FGF2, immobilized on heparin, confirm the preferred association of FGFRs with dimers of FGF2. Computerized molecular docking predicts a cis-oriented FGF2 dimer, stabilized by heparin, which complies with all the experimental data.