bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross-talk between interleukin 1β (IL-1β) and IL-6 signalling pathways: IL-1β selectively inhibits IL-6-activated signal transducer and activator of transcription factor 1 (STAT1) by a proteasome-dependent mechanism
Authors: SHEN Xuening , TIAN Zhigang , HOLTZMAN Michael J. , GAO Bin
Published: August 2015

Interleukin 1β (IL-1β) suppresses the IL-6-dependent induction of type II acute-phase response genes, but the underlying mechanism for this suppression remains uncertain. Here we report that treatment of human hepatocullular carcinoma HepG2 cells with IL-1β inhibited the IL-6-dependent binding of signal transducer and activator of transcription factor (STAT)1, but not that of STAT3, to the high-affinity serum-inducible element (‘SIE’). Furthermore, IL-1β selectively down-regulated the IL-6-induced tyrosine phosphorylation of STAT1 without affecting the level of STAT1 or tyrosine phosphorylation of STAT3. Kinase assays in vitro indicated that the inhibition of STAT1 phosphorylation by IL-1β was not due to an upstream blockade of Janus kinase (JAK1 or JAK2) activation. However, pretreatment with the proteasome inhibitor MG132 under conditions that prevented the IL-1β-dependent activation of the nuclear factor NF-κB also blocked the inhibitory effect of IL-1β on IL-6-activated STAT1. In related experiments, the protein tyrosine phosphatase inhibitor Na3VO4 also antagonized the inhibitory effect of IL-1β on the activation of STAT1 by IL-6. Taken together, these findings indicate that, by using a proteasome-dependent mechanism, IL-1β concomitantly induces NF-κB activation and dephosphorylates IL-6-activated STAT1; the latter might partly account for the inhibition by IL-1β of the IL-6-dependent induction of type II acute-phase genes.
Apoptosis of haematopoietic cells upon thymidylate synthase inhibition is independent of p53 accumulation and CD95–CD95 ligand interaction
Authors: MUÑOZ-PINEDO Cristina , OLIVER F. Javier , LÓPEZ-RIVAS Abelardo
Published: August 2015

Treatment of haematopoietic BA/F3 cells with the thymidylate synthase inhibitor 5-fluoro-2′-deoxyuridine (FUdR) activated apoptosis through a mechanism that required continuous protein synthesis and was inhibited by Bcl-2 over-expression. Analysis of p53 levels in cells treated with FUdR indicated a marked accumulation of this protein. Accumulation of p53 was also observed in cells over-expressing Bcl-2. In BA/F3 cells transfected with a cDNA coding for the human papilloma virus protein E6, p53 accumulation after FUdR treatment was inhibited markedly. However, apoptosis was induced in both control and E6 cells to a similar extent. The role of the CD95/CD95 ligand (CD95L) system in FUdR-induced apoptosis was also assessed. As determined by reverse transcriptase PCR, BA/F3 expressed a low constitutive level of CD95L mRNA, which decreased following FUdR treatment. Moreover, blocking CD95–CD95L interactions with antagonistic CD95 monoclonal antibody did not prevent drug-induced apoptosis. Furthermore, analysis of caspase involvement showed important differences in apoptosis induced by CD95-triggering or FUdR treatment. In summary, these results suggest that apoptosis induced by thymineless stress in haematopoietic BA/F3 cells occurs by a mechanism that does not require accumulation of p53 and which is independent of CD95–CD95L interactions.
Haem-linked interactions in horseradish peroxidase revealed by spectroscopic analysis of the Phe-221→Met mutant
Authors: HOWES Barry D. , VEITCH Nigel C. , SMITH Andrew T. , WHITE Christopher G. , SMULEVICH Giulietta
Published: August 2015

A gene encoding a Phe-221-to-Met substitution in the haem enzyme horseradish peroxidase has been constructed and expressed in Escherichia coli. In the wild-type enzyme the side chain of Phe-221 is tightly stacked against the imidazole ring of His-170, which provides the only axial ligand to the haem iron atom. The Phe-221 → Met enzyme is active, and forms characteristic complexes with typical peroxidase ligands (CO, cyanide, fluoride), and with benzhydroxamic acid. Significant differences between the mutant and wild-type enzymes can be detected spectroscopically. These include a change in the Fe(III) resting state of the enzyme to an unusual quantum mechanically mixed-spin haem species, a marked decrease in the pKa of the alkaline transition and a reduction in enzyme stability at alkaline pH for both Fe(III) and Fe(II) forms. The perturbation of the haem pocket in the mutant can be attributed to several factors, including the increased steric freedom and solvent accessibility of the His-170 ligand, as indicated by 1H-NMR data, and the loss of the πŐπ interaction between His-170 and Phe-221.
Escherichia coli flavohaemoglobin (Hmp) with equistoichiometric FAD and haem contents has a low affinity for dioxygen in the absence or presence of nitric oxide
Authors: MILLS Catherine E. , SEDELNIKOVA Svetlana , SØBALLE Britta , HUGHES Martin N. , POOLE Robert K.
Published: August 2015

A purification procedure for flavohaemoglobin Hmp (NO oxygenase) is described that gives high yields of protein with equistoichiometric haem and FAD contents. H2O2 accumulated on NADH oxidation by the purified protein and in cell extracts with elevated Hmp contents. H2O2 probably arose by dismutation from superoxide, which was also detectable during oxygen reduction; water was not a product. In the absence of agents that scavenge superoxide and peroxide, the mean Km for oxygen was 80µM; the addition of 15µM FAD decreased the Km for oxygen to 15µM without a change in Vmax but catalysed cyanide-insensitive oxygen consumption, attributed to electron transfer from flavins to O2. Purified Hmp consumed NO in the absence of added FAD (approx. 1 O2 per NO), which is consistent with NO oxygenation. However, half-maximal rates of NO-stimulated O2 consumption required approx. 47µM O2; NO removal was ineffective at physiologically relevant O2 concentrations (below approx. 30µM O2). On exhaustion of O2, NO was removed by a cyanide-sensitive process attributed to NO reduction, with a turnover number approx. 1% of that for oxygenase activity. These results suggest that the ability of Hmp to detoxify NO might be compromised in hypoxic environments.
Peroxisomal bifunctional enzyme binds and activates the activationfunction-1 region of the peroxisome proliferator-activated receptor α
Authors: JUGE-AUBRY Cristiana E. , KUENZLI Stéphane , SANCHEZ Jean-Charles , HOCHSTRASSER Denis , MEIER Christoph A.
Published: August 2015

The transcriptional activity of peroxisome proliferator-activated receptors (PPARs), and of nuclear hormone receptors in general, is subject to modulation by cofactors. However, most currently known co-activating proteins interact in a ligand-dependent manner with the C-terminal ligand-regulated activation function (AF)-2 domain of nuclear receptors. Since PPARα exhibits a strong constitutive transactivating function contained within an N-terminal AF-1 region, it can be speculated that a different set of cofactors might interact with this region of PPARs. An affinity purification approach was used to identify the peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (bifunctional enzyme, BFE) as a protein which strongly and specifically interacted with the N-terminal 92 amino acids of PPARα. ProteinŐprotein interaction assays with the cloned BFE confirmed this interaction, which could be mapped to amino acids 307Ő514 of the BFE and the N-terminal 70 amino acids of PPARα. Moreover, transient transfection experiments in hepatoma cells revealed a 2.2-fold increase in the basal and ligand-stimulated transcriptional activity of PPARα in the presence of BFE. This stimulatory effect is preferentially observed for the PPARα isoform and it is significantly stronger (4.8-fold) in non-hepatic cells, which presumably express lower levels of endogenous BFE. Hence, the BFE represents the first known cofactor capable of activating the AF-1 domain of PPAR without requiring additional regions of this receptor. These data are compatible with a model whereby the PPAR-regulated BFE is able to modulate its own expression through an enhancement of the activity of PPARα, representing a novel peroxisomalŐnuclear feed-forward regulatory loop.
Catalytic and spectroscopic analysis of blue copper-containing nitrite reductase mutants altered in the environment of the type 2 copper centre: implications for substrate interaction
Authors: PRUDÊNCIO Miguel , EADY Robert R. , SAWERS Gary
Published: August 2015

The blue dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans is a trimer containing two types of Cu centre, three type 1 electron transfer centres and three type 2 centres. The latter have been implicated in the binding and reduction of nitrite. The Cu ion of the type 2 centre of the oxidized enzyme is ligated by three His residues, and additionally has a co-ordinated water molecule that is also hydrogen-bonded to the carboxyl of Asp92 [Dodd, Van Beeumen, Eady and Hasnain (1998), J. Mol. Biol. 282, 369Ő382]. Two mutations of this residue have been made, one to a glutamic acid residue and a second to an asparagine residue; the effects of both mutations on the spectroscopic and catalytic properties of the enzyme have been analysed. EPR spectroscopy revealed that both mutants retained intact type 1 Cu centres with g‖ = 2.12 (A‖ = 0mT) and g⊥ = 2.30 (A⊥ = 6.4mT), which was consistent with their blue colour, but differed in their activities and in the spectroscopic properties of the type 2 centres. The D92E mutant had an altered geometry of its type 2 centre such that nitrite was no longer capable of binding to elicit changes in the EPR parameters of this centre. Accordingly, this mutation resulted in a form of NiR that had very low enzyme activity with the artificial electron donors reduced Methyl Viologen and sodium dithionite. As isolated, the EPR spectrum of the Asp92 → Asn (D92N) mutant showed no characteristic type 2hyperfine lines. However, oxidation with iridium hexachloride partly restored a type 2 EPR signal, suggesting that type 2 copper is present in the enzyme but in a reduced, EPR-silent form. Like the Asp92 → Glu mutant, D92N had very low enzyme activities with either Methyl Viologen or dithionite. Remarkably, when the physiological electron donor reduced azurin I was used, both mutant proteins exhibited restoration of enzyme activity. The degree of restoration differed for the two mutants, with the D92N derivative exhibiting approx. 60% of the activity seen for the wild-type NiR. These findings suggest that on formation of an electron transfer complex with azurin, a conformational change in NiR occurs that returns the catalytic Cu centre to a functionally active state capable of binding and reducing nitrite.
Studies on a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus: the role of tyrosine-53 in the reaction with human IgG
Authors: BECKINGHAM Jennifer A. , HOUSDEN Nicholas G. , MUIR Nicola M. , BOTTOMLEY Stephen P. , GORE Michael G.
Published: August 2015

Chemical modification experiments with tetranitromethane (TNM) have been used to investigate the role of tyrosine residues in the formation of the complex between PpL (the single Ig-binding domain of protein L, isolated from P. magnus strain 3316) and the kappa light chain (κ-chain). Reaction of PpL with TNM causes the modification of 1.9 equiv. of tyrosine (Tyr51 and Tyr53) and results in an approx. 140-fold decrease in affinity for human IgG. Similar experiments with mutated PpL proteins suggest that nitration predominantly inactivates the protein by modification of Tyr53. Reduction of the nitrotyrosine groups to aminotyrosine by incubation with sodium hydrosulphite does not restore high affinity for IgG. Modification of κ-chain by TNM resulted in the nitration of 3.1±0.09 tyrosine residues. When the PpLŐκ-chain complex was incubated with TNM, 4.1±0.04 tyrosine residues were nitrated, indicating that one tyrosine residue previously modified by the reagent was protected from TNM when the proteins are in complex with each other. The Kd for the equilibrium between PpL, human IgG and their complex has been shown by ELISA to be 112±20nM. A similar value (153±33nM) was obtained for the complex formed between IgG and the Tyr64 → Trp mutant (Y64W). However, the Kd values for the equilibria involving the PpL mutants Y53F and Y53F,Y64W were found to be 3.2±0.2 and 4.6±1µM respectively. These suggest that the phenol group of Tyr53 in PpL is important to the stability of the PpLŐκ-chain complex.
Vascular endothelial growth factor-induced prostacyclin production is mediated by a protein kinase C (PKC)-dependent activation of extracellular signal-regulated protein kinases 1 and 2 involving PKC-δ and by mobilization of intracellular Ca2+
Authors: GLIKI Georgia , ABU-GHAZALEH Robin , JEZEQUEL Sylvie , WHEELER-JONES Caroline , ZACHARY Ian
Published: August 2015

We reported previously that vascular endothelial growth factor (VEGF) stimulates prostacyclin (PGI2) production via activation of the extracellular signal-regulated kinase (ERK) cascade. In this paper, we examined the role of protein kinase C (PKC) in this pathway. VEGF-induced PGI2 generation and arachidonic acid release in human umbilical vein endothelial cells were inhibited by the PKC inhibitors GF109203X and calphostin C. VEGF increased PKC activity and immunoreactivity of the PKCδ, α and ε isoforms in particulate fractions of cells. PKC inhibitors blocked VEGF-induced activation of ERK, MEK (mitogen-activated protein kinase kinase) and the cytosolic phospholipase A2, but had little effect on ERK activation induced by basic fibroblast growth factor. GF109203X, calphostin C and the PKCδ-selective inhibitor, rottlerin, did not inhibit activation of the KDR receptor for VEGF. Inhibition of Ca2+ fluxes using BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)] blocked VEGF-induced PGI2 production but did not inhibit ERK activation. Neither activation nor inhibition of the NO/cGMP pathway had any effect on VEGF induction of ERK activity and PGI2 synthesis. Wortmannin partially inhibited VEGF stimulation of PGI2 production, but did not inhibit VEGF-induced ERK activity. VEGF-induced ERK activation and PGI2 production were blocked by rottlerin, and VEGF increased association of PKCδ with Raf-1, the upstream activator of MEK. The PKC-selective inhibitor Go6976 did not inhibit ERK activation and had only a partial effect on PGI2 production. These findings indicate that activation of PKC plays a crucial role in VEGF signalling via the ERK cascade leading to PGI2 synthesis and suggest that the PKCδ isoform may be a key mediator of VEGF-induced activation of the ERK pathway via increased association with Raf-1.
Localization of membrane-type 1 matrix metalloproteinase in caveolae membrane domains
Authors: ANNABI Borhane , LACHAMBRE Marie-Paule , BOUSQUET-GAGNON Nathalie , PAGÉ Martine , GINGRAS Denis , BÉLIVEAU Richard
Published: August 2015

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-associated MMP that has been recently reported to have a central role in tumour cell invasion. Here we report that both the native and overexpressed recombinant forms of MT1-MMP are highly enriched in low-density Triton X-100-insoluble membrane domains that contain the caveolar marker protein caveolin 1. Moreover, the MT1-MMP-dependent activation of proMMP-2 induced by concanavalin A and cytochalasin D was correlated with the processing of MT1-MMP to its proteolytically inactive 43kDa fragment in U-87 glioblastoma and HT-1080 fibrosarcoma tumour cell lines; this processing was also preferentially observed within the caveolar fraction. Interestingly, whereas the expression of caveolin 1had no effect on the MT1-MMP-dependent activation of proMMP-2, its co-expression with MT1-MMP antagonized the MT1-MMP-increased migratory potential of COS-7 cells. Taken together, our results provide evidence that MT1-MMP is preferentially compartmentalized and proteolytically processed in caveolae of cancer cells. The inhibition of MT1-MMP-dependent cell migration by caveolin 1 also suggests that the localization of MT1-MMP to caveolin-enriched domains might have an important function in the control of its enzymic activity.
Role of Ins(1,4,5)P3, cADP-ribose and nicotinic acid–adenine dinucleotide phosphate in Ca2+ signalling in mouse submandibular acinar cells
Authors: HARMER Alexander R. , GALLACHER David V. , SMITH Peter M.
Published: August 2015

cADP-ribose (cADPr) and nicotinic acid–adenine dinucleotide phosphate (NAADP) are two putative second messengers; they were first shown to stimulate Ca2+ mobilization in sea urchin eggs. We have used the patch-clamp whole-cell technique to determine the role of cADPr and NAADP in relation to that of Ins(1,4,5)P3 in mouse submandibular acinar cells by measuring agonist-evoked and second-messenger-evoked changes in Ca2+-dependent K+ and Cl- currents. Both Ins(1,4,5)P3 and cADPr were capable of reproducing the full range of responses normally seen in response to stimulation with acetylcholine (ACh). Low concentrations of agonist (10–20nM ACh) or second messenger [1–10µM Ins(1,4,5)P3 or cADPr] elicited a sporadic transient activation of the Ca2+-dependent currents; mid-range concentrations [50–500nM ACh, 50µM Ins(1,4,5)P3 or 50–100µM cADPr] elicited high-frequency (approx. 2Hz) trains of current spikes; and high concentrations [more than 500nM ACh, more than 50µM Ins(1,4,5)P3 or more than 100µM cADPr] gave rise to a sustained current response. The response to ACh was inhibited by antagonists of both the Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] and the ryanodine receptor (RyR) but could be completely blocked only by an Ins(1,4,5)P3R antagonist (heparin). NAADP (50nM to 100µM) did not itself activate the Ca2+-dependent ion currents, nor did it inhibit the activation of these currents by ACh. These results show that, in these cells, both Ins(1,4,5)P3R and RyR are involved in the propagation of the Ca2+ signal stimulated by ACh and that cADPr can function as an endogenous regulator of RyR. Furthermore, although NAADP might have a role in hormone-stimulated secretion in pancreatic acinar cells, it does not contribute to ACh-evoked secretion in submandibular acinar cells.
Signalling properties of an HIV-encoded angiogenic peptide mimicking vascular endothelial growth factor activity
Authors: SCHEIDEGGER Patrick , WEIGLHOFER Wolfgang , SUAREZ Stéphanie , CONSOLE Sandra , WALTENBERGER Johannes , PEPPER Michael S. , JAUSSI Rolf , BALLMER-HOFER Kurt
Published: August 2015

HIV-1 expresses a multifunctional protein called TAT (trans-acting transcriptional activator), the function of which in vivo is tightly correlated with the incidence of Kaposi's sarcoma in AIDS patients. TAT is angiogenic and apparently binds to receptors specific for vascular endothelial growth factor (VEGF). Amino acids 46–60 of HIV-TAT, known as the basic peptide, have been shown to be responsible for its functional interaction with VEGF receptors. To characterize further the binding properties of this peptide, its coding sequence was fused to the reading frame of bacterial thioredoxin, allowing the production of large amounts of chimaeric polypeptides in bacteria in a biologically active form. Binding of chimaeric proteins to VEGF receptors was studied in vitro in endothelial cell cultures expressing either of the two receptors. Chimaeric thioredoxin proteins carrying the basic domain of TAT bound to both VEGF receptors with affinities similar to those of HIV-TAT or VEGF. Interestingly, these polypeptides competed only partially with VEGF for receptor binding, implying different binding sites for the TAT peptide and VEGF. This suggests that TAT binds VEGF receptors at new sites that might be useful targets for pharmacological intervention during pathological angiogenesis. The thioredoxin/basic-peptide chimaeras are functional agonists that mediate VEGF receptor signalling: (1) they stimulate the growth of endothelial cells; (2) together with basic fibroblast growth factor they cause tube formation of endothelial cells in collagen gels; (3) they induce blood vessel formation on the chicken chorioallantoic membrane; and (4) they activate VEGF receptor kinase and mitogen-activated protein kinase activity.
Androgen-receptor-specific DNA binding to an element in the first exon of the human secretory component gene
Authors: HAELENS Annemie , VERRIJDT Guy , CALLEWAERT Leen , PEETERS Ben , ROMBAUTS Wilfried , CLAESSENS Frank
Published: August 2015

Androgens and glucocorticoids are steroid hormones, which exert their effects in vivo by binding and activating their cognate receptors. These intracellular receptors are transcription factors that can bind specific DNA sequences, called hormone response elements, located near the target genes. Although the androgen receptor (AR) and the glucocorticoid receptor (GR) bind the same consensus DNA sequence, androgen-specific responses can be achieved by non-conventional androgen response elements (AREs). Here we determine the specificity mechanism of such a selective element recently identified in the first exon of the human gene for secretory component (sc ARE). This sc ARE consists of two receptor-binding hexamers separated by three nucleotides. The DNA-binding domains of the AR and GR both bind the sc ARE, but, although the AR fragment dimerizes on the element, the GR fragment does not. Comparing the affinities of the DNA-binding domains for mutant forms of the sc ARE revealed that dimeric GR binding is actively excluded by the left hexamer and more precisely by the presence of a G residue at position -3, relative to the central spacer nucleotide. Inserting a G at this position changed a non-selective element into an androgen-selective one. We postulate that the AR recognizes the sc ARE as a direct repeat of two 5′-TGTTCT-3′-like core sequences instead of the classical inverted repeat. Direct repeat binding is not possible for the GR, thus explaining the selectivity of the sc ARE. This alternative dimerization by the AR on the sc ARE is also indicated by the DNA-binding characteristics of receptor fragments in which the dimerization interfaces were swapped. In addition, the flanking and spacer sequences seem to affect the functionality of the sc ARE.
Molecular dynamics study of active-site interactions with tetracoordinate transients in acetylcholinesterase and its mutants
Authors: ENYEDY Istvan J. , KOVACH Ildiko M. , BENCSURA Akos
Published: August 2015

The role of active-site residues in the dealkylation reaction in the PSCS diastereomer of 2-(3,3-dimethylbutyl)methylphosphonofluoridate (soman)-inhibited Torpedo californicaacetylcholinesterase (AChE) was investigated by full-scale molecular dynamics simulations using CHARMM: > 400ps equilibration was followed by 150–200ps production runs with the fully solvated tetracoordinate phosphonate adduct of the wild-type, Trp84Ala and Gly199Gln mutants of AChE. Parallel simulations were carried out with the tetrahedral intermediate formed between serine-200 Oγ of AChE and acetylcholine. We found that the NεH in histidine H+-440 is positioned to protonate the oxygen in choline and thus promote its departure. In contrast, NεH in histidine H+-440 is not aligned for a favourable proton transfer to the pinacolyl O to promote dealkylation, but electrostatic stabilization by histidine H+-440 of the developing anion on the phosphonate monoester occurs. Destabilizing interactions between residues and the alkyl fragment of the inhibitor enforce methyl migration from Cβ to Cα concerted with C—O bond breaking in soman-inhibited AChE. Tryptophan-84, phenyalanine-331 and glutamic acid-199 are within 3.7–3.9 Å (1 Å=10-10 m) from a methyl group in Cβ, 4.5–5.1 Å from Cβ and 4.8–5.8 Å from Cα, and can better stabilize the developing carbenium ion on Cβ than on Cα. The Trp84Ala mutation eliminates interactions between the incipient carbenium ion and the indole ring, but also reduces its interactions with phenylalanine-331 and aspartic acid-72. Tyrosine-130 promotes dealkylation by interacting with the indole ring of tryptophan-84. Glutamic acid-443 can influence the orientation of active-site residues through tyrosine-421, tyrosine-442 and histidine-440 in soman-inhibited AChE, and thus facilitate dealkylation.
Oxidized low-density lipoprotein and peroxisome-proliferator-activated receptor α down-regulate platelet-activating-factor receptor expression in human macrophages
Authors: HOURTON Delphine , DELERIVE Philippe , STANKOVA Jana , STAELS Bart , CHAPMAN M. John , NINIO Ewa
Published: August 2015

Regulation of the expression of platelet-activating factor (PAF) receptor by atherogenic lipoproteins might contribute to atherogenesis. We show that progressive oxidation of low-density lipoprotein (LDL) gradually inhibits PAF receptor expression on the macrophage cell surface. We tested the effect of oxidized LDL (oxLDL) on PAF receptor expression in human monocytes that do not contain peroxisome-proliferator-activated receptor γ (PPARγ), a nuclear receptor activated by oxLDL. OxLDL decreased by 50% (P ⩽0.001) and by 29% (P⩽0.05) the binding of PAF and the expression of PAF receptor mRNA respectively. Next we demonstrated that progressive oxidation of LDLs significantly activated PPARα-dependent transcription in transfected mouse aortic endothelial cells. Finally we demonstrated, in mature macrophages, that fenofibrate (20µM), a specific PPARα agonist, but not the specific PPARγ agonist BRL49653 (20nM), significantly decreased both PAF binding and PAF receptor mRNA expression, by 65% and 40% (P⩽0.001) respectively. Additionally, another PPARα agonist, Wy14,643, decreased PAF receptor promoter activity by 70% (P⩽0.05) in transfected THP-1 cells, suggesting the involvement of the proximal promoter region (-980 to -500) containing a series of four nuclear factor (NF)-κB motifs. Thus PPARα might be involved in the down-regulation of PAF receptor gene expression by oxLDLs in human monocytes/macrophages. The oxidation of one or more lipid components of LDLs might result in the formation of natural activators of PPARα. It is hypothesized that such activators might modulate inflammation and apoptosis upon atherogenesis by decreasing the expression of PAF receptor.
Post-translational modifications of the β-1 subunit of AMP-activated protein kinase affect enzyme activity and cellular localization
Authors: WARDEN Scott M. , RICHARDSON Christine , O'DONNELL John , STAPLETON David , KEMP Bruce E. , WITTERS Lee A.
Published: August 2015

The AMP-activated protein kinase (AMPK) is a ubiquitous mammalian protein kinase important in the adaptation of cells to metabolic stress. The enzyme is a heterotrimer, consisting of a catalytic α subunit and regulatory β and γ subunits, each of which is a member of a larger isoform family. The enzyme is allosterically regulated by AMP and by phosphorylation of the α subunit. The β subunit is post-translationally modified by myristoylation and multi-site phosphorylation. In the present study, we have examined the impact of post-translational modification of the β-1 subunit on enzyme activity, heterotrimer assembly and subcellular localization, using site-directed mutagenesis and expression of subunits in mammalian cells. Removal of the myristoylation site (G2A mutant) results in a 4-fold activation of the enzyme and relocalization of the β subunit from a particulate extranuclear distribution to a more homogenous cell distribution. Mutation of the serine-108 phosphorylation site to alanine is associated with enzyme inhibition, but no change in cell localization. In contrast, the phosphorylation site mutations, SS24,25AA and S182A, while having no effects on enzyme activity, are associated with nuclear redistribution of the subunit. Taken together, these results indicate that both myristoylation and phosphorylation of the β subunit of AMPK modulate enzyme activity and subunit cellular localization, increasing the complexity of AMPK regulation.
Evidence that 2-aminoethyl diphenylborate is a novel inhibitor of store-operated Ca2+ channels in liver cells, and acts through a mechanism which does not involve inositol trisphosphate receptors
Authors: GREGORY Roland B. , RYCHKOV Grigori , BARRITT Greg J.
Published: August 2015

The compound 2-aminoethyl diphenylborate (2-APB), an inhibitor of Ins(1,4,5)P3 receptor action in some cell types, has been used to assess the role of Ins(1,4,5)P3 receptors in the activation of store-operated Ca2+ channels (SOCs) [Ma, Patterson, van Rossum, Birnbaumer, Mikoshiba and Gill (2000) Science 287, 1647–1651]. In freshly-isolated rat hepatocytes, 2-APB inhibited thapsigargin- and vasopressin-stimulated Ca2+ inflow (measured using fura-2) with no detectable effect on the release of Ca2+ from intracellular stores. The concentration of 2-APB which gave half-maximal inhibition of Ca2+ inflow was approx. 10µM. 2-APB also inhibited Ca2+ inflow initiated by a low concentration of adenophostin A but had no effect on maitotoxin-stimulated Ca2+ inflow through non-selective cation channels. The onset of the inhibitory effect of 2-APB on thapsigargin-stimulated Ca2+ inflow was rapid. When 2-APB was added to rat hepatocytes in the presence of extracellular Ca2+ after a vasopressin-induced plateau in the cytoplasmic free Ca2+ concentration ([Ca2+]cyt) had been established, the kinetics of the decrease in [Ca2+]cyt were identical with those induced by the addition of 50µM Gd3+ (gadolinium). 2-APB did not inhibit the release of Ca2+ from intracellular stores induced by the addition of Ins(1,4,5)P3 to permeabilized hepatocytes. In the H4-IIE rat hepatoma cell line, 2-APB inhibited thapsigargin-stimulated Ca2+ inflow (measured using fura-2) and, in whole-cell patch-clamp experiments, the Ins(1,4,5)P3-induced inward current carried by Ca2+. It was concluded that, in liver cells, 2-APB inhibited SOCs through a mechanism which involved the binding of 2-APB to either the channel protein or an associated regulatory protein. 2-APB appeared to be a novel inhibitor of SOCs in liver cells with a mechanism of action which, in this cell type, is unlikely to involve an interaction of 2-APB with Ins(1,4,5)P3 receptors. The need for caution in the use of 2-APB as a probe for the involvement of Ins(1,4,5)P3 receptors in the activation of SOCs in other cell types is briefly discussed.
GLUT4 vesicle trafficking in rat adipocytes after ethanol feeding: regulation by heterotrimeric G-proteins
Authors: POIRIER Laurie A. , RACHDAOUI Nadia , NAGY Laura E.
Published: August 2015

Long-term ethanol consumption decreases insulin-stimulated glucose uptake in isolated rat adipocytes. Here we investigate the mechanisms for this decrease. Male Wistar rats were fed for 4 weeks with a liquid diet containing 35% of the calories from ethanol and compared with pair-fed controls. Stimulation of 3-O-methylglucose transport in isolated adipocytes by insulin was decreased by 70% after ethanol feeding. However, stimulation by insulin of the tyrosine phosphorylation of the p85 subunit of phosphoinositide 3-kinase and the phosphorylation of Akt were not affected by ethanol feeding. GLUT4 was mobilized from intracellular light microsomes in response to insulin in both pair-fed and ethanol-fed rats, resulting in 4.3-fold and 3.3-fold increases in GLUT4 associated with plasma membrane in pair-fed and ethanol-fed rats respectively. Surface-accessible GLUT4, assessed by a trypsin cleavage assay or cell-surface labelling with bis-mannose photolabel, was increased 2.3-fold and 1.6-fold respectively, in pair-fed rats after treatment with insulin. In contrast, insulin did not increase surface-accessible GLUT4 in ethanol-fed rats. Treatment of adipocytes with R-phenylisopropyladenosine, an adenosine A1 receptor agonist, increased the transport of 3-O-methylglucose and trypsin-accessible GLUT4, in adipocytes from both pair-fed and ethanol-fed rats. These results demonstrate that whereas the insulin-mediated signalling and translocation of GLUT4 to the plasma membrane is maintained after ethanol feeding, the final fusion of GLUT4 vesicles to the plasma membrane is disrupted, preventing the stimulation of glucose uptake by insulin. Fusion of GLUT4 with the plasma membrane can be stimulated by the activation of adenosine A1 receptors.
Role of complex asparagine-linked oligosaccharides in the expression of a functional thyrotropin receptor
Authors: SIFFROI-FERNANDEZ Sandrine , COSTAGLIOLA Sabine , PAUMEL Sophie , GIRAUD Annie , BANGA J. Paul , FRANC Jean-Louis
Published: August 2015

To evaluate the functional role of complex asparagine-linked oligosaccharides of the human thyrotropin receptor (TSHR), a Chinese hamster ovary cell line (JP09) and a K562 cell line (K562-TSHR) expressing this receptor were treated with deoxymannojirimycin (dMM), a mannosidase I inhibitor. dMM blocks the formation of complex-type structures and leads to the formation of high-mannose-type structures. Treatment of cells with dMM led to a decrease in the number of thyrotropin (TSH)-binding sites at the cell surface. Detection of the TSHR at the cell surface using a monoclonal antibody directed against the A subunit showed that this decrease was not due to a decrease in the number of TSHRs expressed at the cell surface. However the recognition of TSHR by a monoclonal antibody directed against the C peptide was greatly decreased. On immunoblotting, after deglycosylation using peptide N-glycanase F, the A subunit was visualized as a doublet (36 and 41kDa). In control cells the species of higher molecular mass was more abundant whereas after dMM treatment the species of lower molecular mass became more abundant. This difference in molecular mass between the two peptides is compatible with the removal of the C peptide. In conclusion, the results show that inhibition of complex-type structure formation leads to (i) an incapacity for TSHR to bind TSH, without affecting its intracellular transport and (ii) an increase of TSHR susceptibility to proteases that remove the C peptide. We then hypothesized that removal of the C peptide could contribute to the formation of a non-functional TSHR.
Selective inactivation of guanine-nucleotide-binding regulatory protein (G-protein) α and βγ subunits by urea
Authors: LIM William K. , NEUBIG Richard R.
Published: August 2015

G-protein-coupled receptors activate signal-transducing G-proteins, which consist of an α subunit and a βγ dimer. Membrane extraction with 5–7M urea has been used to uncouple receptors from endogenous G-proteins to permit reconstitution with purified G-proteins. We show that αi subunits are inactivated with 5M urea whereas the βγ dimer requires at least 7M urea for its inactivation. There is no significant loss of receptors. Surprisingly, Western-blot analysis indicates that the urea-denatured αi subunit remains mostly membrane-bound and that β is only partially removed. After 7M urea treatment, both αi1 and βγ subunits are required to restore high-affinity agonist binding and receptor-catalysed guanosine 5′-[γ-thio]triphosphate binding. We demonstrate the generality of this approach for four Gi-coupled receptors (α2A-adrenergic, adenosine A1, 5-hydroxytryptamine1A and µ-opioid) expressed in insect cells and two mammalian cell lines. Thus a selectivity of urea for G-protein α versus βγ subunits is established in both concentration and mechanism.
Characterization of a cDNA encoding a novel avian hypothalamic neuropeptide exerting an inhibitory effect on gonadotropin release
Authors: SATAKE Honoo , HISADA Miki , KAWADA Tsuyoshi , MINAKATA Hiroyuki , UKENA Kazuyoshi , TSUTSUI Kazuyoshi
Published: August 2015

We previously isolated a novel dodecapeptide containing a C-terminal -Arg-Phe-NH2 sequence, SIKPSAYLPLRF-NH2 (RFamide peptide), from the quail brain. This quail RFamide peptide was shown to decrease gonadotropin release from the cultured anterior pituitary and to be located at least in the quail hypothalamo-hypophysial system. We therefore designated this RFamide peptide gonadotropin inhibitory hormone (GnIH). In the present study we characterized the GnIH cDNA from the quail brain by a combination of 3′ and 5′ rapid amplification of cDNA ends (‘RACE’). The deduced GnIH precursor consisted of 173 amino acid residues, encoding one GnIH and two putative gene-related peptide (GnIH-RP-1 and GnIH-RP-2) sequences that included -LPXRF (X = L or Q) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Southern blotting analysis of reverse-transcriptase-mediated PCR products demonstrated a specific expression of the gene encoding GnIH in the diencephalon including the hypothalamus. Furthermore, mass spectrometric analyses detected the mass numbers for matured GnIH and GnIH-RP-2, revealing that both peptides are produced from the precursor in the diencephalon as an endogenous ligand. Taken together, these results lead to the conclusion that GnIH is a hypothalamic factor responsible for the negative regulation of gonadotropin secretion. Furthermore, the presence of a novel RFamide peptide family containing a C-terminal -LPXRF-NH2 sequence has been revealed.
Tyr199 in transmembrane domain 5 of the β2-adrenergic receptor interacts directly with the pharmacophore of a unique fluorenone-based antagonist
Authors: WU Zhongren , THIRIOT David S. , RUOHO Arnold E.
Published: August 2015

Mutagenesis of the β2-adrenergic receptor (β2AR) has suggested that amino acids in transmembrane domain 5 (TMD 5) play an important role in the interaction of the receptor with the catechol end of adrenergic agonists. However, little direct biochemical evidence for the interaction of any β2AR agonist or antagonist with TMD 5 has been reported. To identify receptor amino acids that contribute to the β2AR antagonist binding site, we identified the precise amino acid photoinsertion site of a novel carazolol-like fluorenone antagonist photoaffinity label, [125I]iodoaminoflisopolol ([125I]IAmF). A unique property of this photolabel is that the photoreactive centre is also the binding pharmacophore, which corresponds to the catechol end of related β2AR agonists. [125I]IAmF specifically photolabels membrane-bound and purified β2AR from a baculovirus/Spodoptera frugiperda (fall armyworm) (‘Sf9’) expression system. When the photolabelled β2AR was cleaved by trypsin or Factor Xa, 30kDa labelled peptides were generated. On the basis of concanavalin A binding and amino acid sequencing, these contain the N-terminus of the β2AR, including TMDs 1–5. Further cleavage of the 30kDa peptides with endoproteinase Lys-C generated a 4kDa labelled peptide with an N-terminal amino acid sequence between TMDs 4 and 5. Radiosequencing of this peptide demonstrated that the precise [125I]IAmF photoinsertion site was Tyr199 in TMD 5. Since the photoreactive centre and the binding pharmacophore of IAmF are the same, these data demonstrate that Tyr199 interacts with the planar fluorenone moiety of a carazolol-like β2AR antagonist, and contributes significant new information regarding the binding site for β2AR antagonists.
Human ornithine transcarbamylase: crystallographic insights into substrate recognition and conformational changes
Authors: SHI Dashuang , MORIZONO Hiroki , YU Xiaolin , TONG Liang , ALLEWELL Norma M. , TUCHMAN Mendel
Published: August 2015

Two crystal structures of human ornithine transcarbamylase (OTCase) complexed with the substrate carbamoyl phosphate (CP) have been solved. One structure, whose crystals were prepared by substituting N-phosphonacetyl-L-ornithine (PALO) liganded crystals with CP, has been refined at 2.4 Å (1 Å = 0.1nm) resolution to a crystallographic R factor of 18.4%. The second structure, whose crystals were prepared by co-crystallization with CP, has been refined at 2.6 Å resolution to a crystallographic R factor of 20.2%. These structures provide important new insights into substrate recognition and ligand-induced conformational changes. Comparison of these structures with the structures of OTCase complexed with the bisubstrate analogue PALO or CP and L-norvaline reveals that binding of the first substrate, CP, induces a global conformational change involving relative domain movement, whereas the binding of the second substrate brings the flexible SMG loop, which is equivalent to the 240s loop in aspartate transcarbamylase, into the active site. The model reveals structural features that define the substrate specificity of the enzyme and that regulate the order of binding and release of products.

Atomic coordinates and structural factors (codes 1EP9 and 1FVO respectively) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ, U.S.A.
Natural-resistance-associated macrophage protein 1 is an H+/bivalent cation antiporter
Authors: GOSWAMI Tapasree , BHATTACHARJEE Arin , BABAL Paul , SEARLE Susan , MOORE Elizabeth , LI Ming , BLACKWELL Jenefer M.
Published: August 2015

In mammals, natural-resistance-associated macrophage protein 1 (Nramp1) regulates macrophage activation and is associated with infectious and autoimmune diseases. Nramp2 is associated with anaemia. Both belong to a highly conserved eukaryote/prokaryote protein family. We used Xenopus oocytes to demonstrate that, like Nramp2, Nramp1 is a bivalent cation (Fe2+, Zn2+ and Mn2+) transporter. Strikingly, however, where Nramp2 is a symporter of H+ and metal ions, Nramp1 is a highly pH-dependent antiporter that fluxes metal ions in either direction against a proton gradient. At pH9.0, oocytes injected with cRNA from wild-type murine Nramp1 with a glycine residue at position 169 (Nramp1G169; P = 3.22×10-6) and human NRAMP1 (P = 3.87×10-5) showed significantly enhanced uptake of radiolabelled Zn2+ compared with water-injected controls. At pH5.5, Nramp1G169 (P = 1.34×10-13) and NRAMP1 (P = 1.09×10-6) oocytes showed significant efflux of Zn2+. Zn2+ transport was abolished when the proton gradient was dissipated using carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Using pre-acidified oocytes, currents of 130±57 nA were evoked by 100µM Zn2+ at pH7.5, and 139±47 nA by 100µM Fe2+ at pH7.0, in Nramp1G169 oocytes; currents of 254±49 nA and 242±26 nA were evoked, respectively, in NRAMP1 oocytes. Steady-state currents evoked by increasing concentrations of Zn2+ were saturable, with apparent affinity constants of approx. 614nM for Nramp1G169 and approx. 562nM for NRAMP1 oocytes, and a curvilinear voltage dependence of transporter activity (i.e. the data points approximate to a curve that approaches a linear asymptote). In the present study we propose a new model for metal ion homoeostasis in macrophages. Under normal physiological conditions, Nramp2, localized to early endosomal membranes, delivers extracellularly acquired bivalent cations into the cytosol. Nramp1, localized to late endosomal/lysosomal membranes, delivers bivalent cations from the cytosol into this acidic compartment where they may directly affect antimicrobial activity.
Effect of human acetylcholinesterase subunit assembly on its circulatory residence
Authors: CHITLARU Theodor , KRONMAN Chanoch , VELAN Baruch , SHAFFERMAN Avigdor
Published: August 2015

Sialylated recombinant human acetylcholinesterase (rHuAChE), produced by stably transfected cells, is composed of a mixed population of monomers, dimers and tetramers and manifests a time-dependent circulatory enrichment of the higher-order oligomeric forms. To investigate this phenomenon further, homogeneous preparations of rHuAChE differing in their oligomerization statuses were generated: (1) monomers, represented by the oligomerization-impaired C580A-rHuAChE mutant, (2) wild-type (WT) dimers and (3) tetramers of WT-rHuAChE generated in vitro by complexation with a synthetic ColQ-derived proline-rich attachment domain (‘PRAD’) peptide. Three different series of each of these three oligoform preparations were produced: (1) partly sialylated, derived from HEK-293 cells; (2) fully sialylated, derived from engineered HEK-293 cells expressing high levels of sialyltransferase; and (3) desialylated, after treatment with sialidase to remove sialic acid termini quantitatively. The oligosaccharides associated with each of the various preparations were extensively analysed by matrix-assisted laser desorption ionization–time-of-flight MS. With the enzyme preparations comprising the fully sialylated series, a clear linear relationship between oligomerization and circulatory mean residence time (MRT) was observed. Thus monomers, dimers and tetramers exhibited MRTs of 110, 195 and 740min respectively. As the level of sialylation decreased, this differential behaviour became less pronounced; eventually, after desialylation all oligoforms had the same MRT (5min). These observations suggest that multiple removal systems contribute to the elimination of AChE from the circulation. Here we also demonstrate that by the combined modulation of sialylation and tetramerization it is possible to generate a rHuAChE displaying a circulatory residence exceeding that of all other known forms of native or recombinant human AChE.
Differential expression of chicken dimerization cofactor of hepatocyte nuclear factor-1 (DcoH) and its novel counterpart, DcoHα
Authors: KIM Hyunggee , YOU Seungkwon , FOSTER Linda K. , FARRIS James , CHOI Yun-Jaie , FOSTER Douglas N.
Published: August 2015

We have used differential display PCR to study altered gene expression in immortalized chicken embryo fibroblasts (CEFs) that have been established in our laboratory. This technique resulted in the cloning of a novel counterpart of the previously cloned chicken dimerization cofactor of hepatocyte nuclear factor (HNF)-1 (cDcoH), which was identified as cDcoHα. The steady-state mRNA levels of cDcoHα were up-regulated in all immortal CEFs tested compared with primary CEF cells. cDcoH and cDcoHα showed opposite patterns of mRNA expression due to differential regulation of transcription rates, but not mRNA half-lives, in primary and immortal CEFs. Expression of cDcoHα increased in the late G1 and early S phases of the cell cycle, while cDcoH mRNA increased in the late S and G2/M phases. In contrast with consistent expression of both genes in primary quiescent cells, cDcoH mRNA, but not cDcoHα mRNA, was dramatically decreased in primary senescent cells. The highest levels of cDcoHα mRNA were found in the kidney, liver, heart and ovarian follicles, while the major tissues expressing cDcoH were hypothalamus, kidney and liver. cDcoH and cDcoHα probes did not cross-hybridize to human hepatocyte mRNA. When transfected into human HepG2 cells, both cDcoH and cDcoHα showed similar functional activity as measured by increased expression of a reporter gene, as well as α-fetoprotein and albumin genes that both contain HNF-1 binding elements in their promoters. Our results suggest that the novel chicken DcoHα might function as a transcriptional cofactor for HNF-1 in specific cellular-environmental states.
Guanylate cyclase in Dictyostelium discoideum with the topology of mammalian adenylate cyclase
Authors: ROELOFS Jeroen , SNIPPE Helena , KLEINEIDAM Reinhard G. , HAASTERT Peter J.M. Van
Published: August 2015

The core of adenylate and guanylate cyclases is formed by an intramolecular or intermolecular dimer of two cyclase domains arranged in an antiparallel fashion. Metazoan membrane-bound adenylate cyclases are composed of 12 transmembrane spanning regions, and two cyclase domains which function as a heterodimer and are activated by G-proteins. In contrast, membrane-bound guanylate cyclases have only one transmembrane spanning region and one cyclase domain, and are activated by extracellular ligands to form a homodimer. In the cellular slime mould, Dictyosteliumdiscoideum, membrane-bound guanylate cyclase activity is induced after cAMP stimulation; a G-protein-coupled cAMP receptor and G-proteins are essential for this activation. We have cloned a Dictyostelium gene, DdGCA, encoding a protein with 12 transmembrane spanning regions and two cyclase domains. Sequence alignment demonstrates that the two cyclase domains are transposed, relative to these domains in adenylate cyclases. DdGCA expressed in Dictyostelium exhibits high guanylate cyclase activity and no detectable adenylate cyclase activity. Deletion of the gene indicates that DdGCA is not essential for chemotaxis or osmo-regulation. The knock-out strain still exhibits substantial guanylate cyclase activity, demonstrating that Dictyostelium contains at least one other guanylate cyclase.