bluuebunny/crossref_metadata_2025 huggingface dataset:
Agonist-induced desensitization and phosphorylation of m1-muscarinic receptors
Authors: WAUGH Mark G. , CHALLISS R. A. John , BERSTEIN Gabriel , NAHORSKI Stefan R. , TOBIN Andrew B.
Published: August 2015

Pre-stimulation of Chinese hamster ovary (CHO) cells expressing the human m1-muscarinic receptor (CHO-m1 cells) with a maximally effective concentration of the muscarinic agonist methacholine resulted in desensitization of Ins(1,4,5)P3 accumulation, apparent as a ∼ 4-fold shift in the agonist dose–response curve. Agonist-induced desensitization was rapid (detectable by 10 s) and concentration dependent (EC50 = 8.2±2.2 µM) and resulted in a complete loss of receptor reserve for the agonist-stimulated Ins(1,4,5)P3 response. An investigation of the possible mechanisms involved in m1-muscarinic receptor desensitization indicated that agonist-induced receptor internalization, PtdIns-(4,5)P2 depletion or an increased rate of Ins(1,4,5)P3 metabolism were not involved. m1-Muscarinic receptors did, however, undergo rapid agonist-induced phosphorylation with a time course that was consistent with an involvement in receptor desensitization. Characterization studies indicated that the receptor-specific kinase involved was distinct from protein kinase C and other second-messenger-dependent protein kinases. Since previous studies have suggested that the m3-muscarinic receptor subtype undergoes agonist-dependent phosphorylation via casein kinase 1α (CK1α) [Tobin, Totty, Sterlin and Nahorski (1997) J. Biol. Chem. 272, 20844–20849], we examined the ability of m1-muscarinic receptors to be phosphorylated by this kinase. In reconstitution experiments, CK1α was able to phosphorylate purified, soluble m1-muscarinic receptors in an agonist-dependent manner.
Differential regulation of vascular endothelial growth factor and its receptor fms-like-tyrosine kinase is mediated by nitric oxide in rat renal mesangial cells
Authors: FRANK Stefan , STALLMEYER Birgit , KÄMPFER Heiko , SCHAFFNER Christian , PFEILSCHIFTER Josef
Published: August 2015

Under conditions associated with local and systemic inflammation, mesangial cells and invading immune cells are likely to be responsible for the release of large amounts of nitric oxide (NO) in the glomerulus. To further define the mechanisms of NO action in the glomerulus, we attempted to identify genes which are regulated by NO in rat glomerular mesangial cells. We identified vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase (FLT-1) to be under the regulatory control of exogenously applied NO in these cells. Using S-nitroso-glutathione (GSNO) as an NO-donating agent, VEGF expression was strongly induced, whereas expression of its FLT-1 receptor simultaneously decreased. Expressional regulation of VEGF and FLT-1 mRNA was transient and occurred rapidly within 1–3 h after GSNO treatment. Expression of a second VEGF-specific receptor, fetal liver kinase-1 (FLK-1/KDR), could not be detected. The inflammatory cytokine interleukin-1β mediated a moderate increase in VEGF expression after 24 h and had no influence on FLT-1 expression. In contrast, platelet-derived growth factor–BB and basic fibroblast growth factor had no effect on VEGF expression, but strongly induced FLT-1 mRNA levels. Obviously, there is a differential regulation of VEGF and its receptor FLT-1 by NO, cytokines and growth factors in rat mesangial cells.
Thyroid hormone promotes the phosphorylation of STAT3 and potentiates the action of epidermal growth factor in cultured cells
Authors: LIN Hung-Yun , SHIH Ai , DAVIS Faith B. , DAVIS Paul J.
Published: August 2015

We have examined the effects of l-thyroxine (T4) on the activation of signal transducer and activator of transcription 3 (STAT3) and on the STAT3-dependent induction of c-Fos expression by epidermal growth factor (EGF). T4, at a physiological concentration of 100 nM, caused tyrosine phosphorylation and nuclear translocation (i.e. activation) of STAT3 in HeLa cells in as little as 10–20 min. Activation by T4 of STAT3 was maximal at 30 min (15±4-fold enhancement; mean±S.E.M.) in 18 experiments. This effect was reproduced by T4–agarose (100 nM) and blocked by CGP 41251, genistein, PD 98059 and geldanamycin, inhibitors of protein kinase C (PKC), protein tyrosine kinase (PTK), mitogen-activated protein kinase (MAPK) kinase and Raf-1 respectively. Tyrosine-phosphorylated MAPK also appeared in nuclear fractions within 10 min of treatment with T4. In the nuclear fraction of T4-treated cells, MAPK immunoprecipitate also contained STAT3. The actions of T4 were similar in HeLa and CV-1 cells, which lack thyroid hormone receptor (TR), and in TR-replete skin fibroblasts (BG-9). T4 also potentiated the EGF-induced nuclear translocation of activated STAT1α and STAT3 and enhanced the EGF-stimulated expression of c-Fos. Hormone potentiation of EGF-induced signal transduction and c-Fos expression was inhibited by CGP 41251, geldanamycin and PD 98059. Therefore the non-genomically induced activation by T4 of STAT3, and the potentiation of EGF by T4, require activities of PKC, PTK and an intact MAPK pathway.
Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor
Authors: MACPHEE Colin H. , MOORES Kitty E. , BOYD Helen F. , DHANAK Dash , IFE Robert J. , LEACH Colin A. , LEAKE David S. , MILLINER Kevin J. , PATTERSON Rebecca A. , SUCKLING Keith E. , TEW David G. , HICKEY Deirdre M. B.
Published: August 2015

A novel and potent azetidinone inhibitor of the lipoprotein-associated phospholipase A2 (Lp-PLA2), i.e. platelet-activating factor acetylhydrolase, is described for the first time. This inhibitor, SB-222657 (Ki = 40±3 nM, kobs/[I] = 6.6×105 M-1·s-1), is inactive against paraoxonase, is a poor inhibitor of lecithin:cholesterol acyltransferase and has been used to investigate the role of Lp-PLA2 in the oxidative modification of lipoproteins. Although pretreatment with SB-222657 did not affect the kinetics of low-density lipoprotein (LDL) oxidation by Cu2+ or an azo free-radical generator as determined by assay of lipid hydroperoxides (LOOHs), conjugated dienes and thiobarbituric acid-reacting substances, in both cases it inhibited the elevation in lysophosphatidylcholine content. Moreover, the significantly increased monocyte chemoattractant activity found in a non-esterified fatty acid fraction from LDL oxidized by Cu2+ was also prevented by pretreatment with SB-222657, with an IC50 value of 5.0±0.4 nM. The less potent diastereoisomer of SB-222657, SB-223777 (Ki = 6.3±0.5 µM, kobs/[I] = 1.6×104 M-1·s-1), was found to be significantly less active in both assays. Thus, in addition to generating lysophosphatidylcholine, a known biologically active lipid, these results demonstrate that Lp-PLA2 is capable of generating oxidized non-esterified fatty acid moieties that are also bioactive. These findings are consistent with our proposal that Lp-PLA2 has a predominantly pro-inflammatory role in atherogenesis. Finally, similar studies have demonstrated that a different situation exists during the oxidation of high-density lipoprotein, with enzyme(s) other than Lp-PLA2 apparently being responsible for generating lysophosphatidylcholine.
Physical characterization of a low-charge glycoform of the MUC5B mucin comprising the gel-phase of an asthmatic respiratory mucous plug
Authors: SHEEHAN John K. , HOWARD Marjorie , RICHARDSON Paul S. , LONGWILL Toby , THORNTON David J.
Published: August 2015

We have previously noted that sequential extraction of an asthmatic mucous exudate with 6 M guanidinium chloride yielded a fraction of the mucins that were most resistant to solubilization and of high Mr [Sheehan, Richardson, Fung, Howard and Thornton (1995) Am. J. Respir. Cell Mol. Biol. 13, 748–756]. Here we show that this mucin fraction is dominated (at least 96% of the total) by the low-charge glycoform of the MUC5B gene product. Seen in the electron microscope the mucins appeared mainly as compact ‘island ’ structures composed of linear threads often emanating from globular ‘nodes ’ rather than the discrete linear threads more typical of mucins that we have previously described. The effect of reducing agents was as expected for other gel-forming mucins, i.e. reduced subunits or monomers of Mr 3×106) were produced within 15 min of treatment. Kinetic experiments on the cleavage of the intact mucins with the proteinase trypsin indicated two clear regimes of fragmentation. An initial rapid cleavage generated mucins ranging from Mr = 4×106 to 30×106 that in the electron microscope appeared as polydisperse threads (500–3000 nm in length), similar to normal and other respiratory mucins that we have previously characterized. A subsequent slower fragmentation over many hours yielded a major fragment of Mr 3×106 and length 200–600 nm, very similar in size and Mr to the subunits obtained by reduction. The results suggest that the MUC5B mucin is assembled, first into polydisperse linear threads, which are then linked together via a protein-mediated process. This might involve part of the mucin polypeptide or an as yet unidentified protein(s). The high proteinase susceptibility of the linkage suggests that it might be a point of control for mucin size and thus mucus rheology.
Purinogen is not an endogenous substrate used in endothelial cells during substrate deprivation
Authors: CULIC Ognjen , DECKING Ulrich K. M. , BERGSCHNEIDER Eva , SCHRADER Jürgen
Published: August 2015

Porcine aortic endothelial cells (PAEC) are known to be metabolically robust. They are capable of surviving extended periods of complete lack of exogenous substrate, and purine release has been shown to be significantly up-regulated. The endogenous substrates used during substrate deprivation, as well as the sources responsible for the increased purine release, have not been completely identified. We tested the possibility that a phosphoglyceroyl-ATP-containing polymer, purinogen, might support PAEC hibernation induced by lack of exogenous substrate. This involved isolation of the acid-insoluble fraction of PAEC, which was presumed to contain purinogen, and analysis by HPLC and 31P NMR. No evidence supporting the presence of triphosphate-containing compounds (purinogen) was found. Similar results were obtained in the rat heart. The majority of the products in the acid-insoluble, alkaline-treated fraction were identified as RNA degradation products (2´- and 3´-nucleoside monophosphates). A [14C]adenosine labelling experiment showed that incorporation of adenosine into the acid-insoluble fraction was almost completely prevented after inhibition of RNA synthesis with actinomycin D. Furthermore, RNA isolated from PAEC and subsequently treated with alkali showed a profile that was almost identical with the HPLC profile of the acid-insoluble fraction. Finally, substrate-free incubation of the cells did not quantitatively or qualitatively influence the distribution of acid-insoluble derivatives. We conclude that PAEC survival during the absence of exogenous substrate is not supported by purinogen but rather by some other, yet-to-be-identified, endogenous substrate.
Regulation of UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosamine-1-phosphate transferase by retinoic acid in P19 cells
Authors: MEISSNER Joachim D. , NAUMANN Andreas , MUELLER Walter H. , SCHEIBE Renate J.
Published: August 2015

UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosamine-1-phosphate transferase (GPT) is the first enzyme in the dolichol pathway of protein N-glycosylation, and is implicated in the developmental programmes of a variety of eukaryotes. In the present study we describe the effects of all-trans-retinoic acid (RA) on the levels of GPT protein and enzymic activity, and on the transcription rate of the GPT gene, in mouse P19 teratocarcinoma cells. RA caused a dose-dependent and protein-synthesis-dependent induction of enzyme activity. The maximum induction of GPT activity (about 3-fold) required 2 days of exposure to 1 µM RA. Induced GPT activity also resulted in an increase in the rate of incorporation of [3H]mannose into Glc3Man9GlcNAc2. Enzymic activities paralleled GPT gene expression. The GPT gene was induced (2-fold) after 7 h of RA treatment. An approx. 3-fold increase in a 48 kDa GPT protein and approx. 4-fold increases in the levels of three GPT transcripts (1.8, 2.0 and 2.2 kb) were observed after 2 days of RA treatment. The enhanced levels of GPT protein and mRNAs began to decline 3 days after the initiation of differentiation, and GPT expression was down-regulated during cellular differentiation. GPT activity decreased about 2.8-fold to a constant level in differentiated P19 cells. The results indicate that the RA-induced enzyme activity was mainly determined by increased transcription of the GPT gene. RA-treated P19 cells were about 4-fold more resistant to tunicamycin, a fungal antibiotic which inhibits GPT, than were control cells. In addition, GPT activity in membranes from RA-treated P19 cells exhibited approx. 4-fold increased resistance to tunicamycin compared with activity in membranes from untreated control cells, demonstrating that resistance to tunicamycin is correlated with induced GPT activity. Furthermore, increased GPT activity had regulatory significance with regard to the rate of incorporation of [3H]mannose into Glc3Man9GlcNAc2-P-P-dolichol and into glycoproteins. Together, the data provide additional insights into the hormonal regulation of GPT and present evidence that the RA-mediated induction of GPT has a regulatory impact on the dolichol pathway.
Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: a control enzyme in ketogenesis
Authors: HEGARDT Fausto G.
Published: August 2015

Cytosolic and mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthases were first recognized as different chemical entities in 1975, when they were purified and characterized by Lane's group. Since then, the two enzymes have been studied extensively, one as a control site of the cholesterol biosynthetic pathway and the other as an important control site of ketogenesis. This review describes some key developments over the last 25 years that have led to our current understanding of the physiology of mitochondrial HMG-CoA synthase in the HMG-CoA pathway and in ketogenesis in the liver and small intestine of suckling animals. The enzyme is regulated by two systems: succinylation and desuccinylation in the short term, and transcriptional regulation in the long term. Both control mechanisms are influenced by nutritional and hormonal factors, which explains the incidence of ketogenesis in diabetes and starvation, during intense lipolysis, and in the foetal–neonatal and suckling–weaning transitions. The DNA-binding properties of the peroxisome-proliferator-activated receptor and other transcription factors on the nuclear-receptor-responsive element of the mitochondrial HMG-CoA synthase promoter have revealed how ketogenesis can be regulated by fatty acids. Finally, the expression of mitochondrial HMG-CoA synthase in the gonads and the correction of auxotrophy for mevalonate in cells deficient in cytosolic HMG-CoA synthase suggest that the mitochondrial enzyme may play a role in cholesterogenesis in gonadal and other tissues.
Tumour necrosis factor-α regulates expression of the CCAAT-enhancer-binding proteins (C/EBPs) α and β and determines the occupation of the C/EBP site in the promoter of the insulin-responsive glucose-transporter gene in 3T3-L1 adipocytes
Authors: JAIN Renu , POLICE Shailaja , PHELPS Kelle , PEKALA Phillip H.
Published: August 2015

We have demonstrated previously that treatment of 3T3-L1 adipocytes with tumour necrosis factor-α (TNF) results in a rapid (4 h) and significant (75–80%) reduction in the rate of transcription of the GLUT4 gene. Control of GLUT4 gene transcription has been suggested at least in part to reside with the CCAAT-enhancer-binding protein (C/EBP) family (α, β and δ isoforms) of transcription factors. Using electrophoretic mobility shift assays, we have examined the ability of TNF to alter the occupation of the C/EBP site in the GLUT4 promoter. The data suggest that in fully differentiated adipocytes the C/EBP site is a ligand for predominantly α/α homodimers; however, after exposure to TNF, a shift in occupancy of the site occurs and the ligands become α/β heterodimers and β/β homodimers. Partner selection in dimer formation appears to be controlled by selective translocation of the β-isoform from the cytosol to the nucleus after exposure of the cells to TNF.
The protein disulphide-isomerase family: unravelling a string of folds
Authors: FERRARI David M. , SÖLING Hans-Dieter
Published: August 2015

The mammalian protein disulphide-isomerase (PDI) family encompasses several highly divergent proteins that are involved in the processing and maturation of secretory proteins in the endoplasmic reticulum. These proteins are characterized by the presence of one or more domains of roughly 95–110 amino acids related to the cytoplasmic protein thioredoxin. All but the PDI-D subfamily are composed entirely of repeats of such domains, with at least one domain containing and one domain lacking a redox-active -Cys-Xaa-Xaa-Cys- tetrapeptide. In addition to their known roles as redox catalysts and isomerases, the last few years have revealed additional functions of the PDI proteins, including peptide binding, cell adhesion and perhaps chaperone activities. Attention is now turning to the non-redox-active domains of the PDIs, which may play an important role in all of the known activities of these proteins. Thus the presence of both redox-active and -inactive domains within these proteins portends a complexity of functions differentially accommodated by the various family members.
Conserved polar residues in the transmembrane domain of the human tachykinin NK2 receptor: functional roles and structural implications
Authors: DONNELLY Dan , MAUDSLEY Stuart , GENT J. Paul , MOSER Rachel N. , HURRELL Craig R. , FINDLAY John B. C.
Published: August 2015

We have studied the effects of agonist and antagonist binding, agonist-induced activation and agonist-induced desensitization of the human tachykinin NK2 receptor mutated at polar residues Asn-51 [in transmembrane helix 1 (TM1)], Asp-79 (TM2) and Asn-303 (TM7), which are highly conserved in the transmembrane domain in the rhodopsin family of G-protein-coupled receptors. Wild-type and mutant receptors were expressed in both COS-1 cells and Xenopus oocytes. The results show that the N51D mutation results in a receptor which, in contrast with the wild-type receptor, is desensitized by the application of a concentration of 1 µM of the partial agonist GR64349, indicating that the mutant is more sensitive to agonist activation than is the wild-type receptor. In addition, we show that, whereas the D79E mutant displayed activation properties similar to those of the wild-type receptor, the D79N and D79A mutants displayed a severely impaired ability to activate the calcium-dependent chloride current. This suggests that it is the negative charge at Asn-79, rather than the ability of this residue to hydrogen-bond, that is critical for the activity of the receptor. Interestingly, the placement of a negative charge at position 303 could compensate for the removal of the negative charge at position 79, since the double mutant D79N/N303D displayed activation properties similar to those of the wild-type receptor. This suggests that these two residues are functionally coupled, and may even be in close proximity in the three-dimensional structure of the human tachykinin NK2 receptor. A three-dimensional model of the receptor displaying this putative interaction is presented.
Selective loss of PMA-stimulated expression of matrix metalloproteinase 1 in HaCaT keratinocytes is correlated with the inability to induce mitogen-activated protein family kinases
Authors: SUDBECK Barry D. , BAUMANN Petra , RYAN Gavin J. , BREITKOPF Katja , NISCHT Roswitha , KRIEG Thomas , MAUCH Cornelia
Published: August 2015

Many cell types, including fibroblasts and primary keratinocytes, increase matrix metalloproteinase 1 (MMP-1) production in response to agonists such as growth factors and phorbol esters. However, the spontaneously transformed human keratinocyte cell line HaCaT, although it increases MMP-1 production in response to epidermal growth factor (EGF), does not respond similarly to stimulation with PMA. This phenomenon occurs even though HaCaT cells remain proliferatively responsive to both agonists, suggesting a HaCaT-specific defect in a PMA-mediated signal transduction pathway. Using an inside-out approach to elucidate the source of this defect, we found that EGF, but not PMA, stimulated MMP-1 promoter activity in transiently transfected HaCaT keratinocytes. In addition, an assessment of fibroblast and HaCaT c-fos and c-jun gene expression after exposure to EGF and PMA showed that although both agonists increased the expression of c-fos and c-jun mRNA in fibroblasts, only EGF did so in HaCaT keratinocytes. Finally, we looked at the activation of mitogen-activated protein (MAP) family kinases after stimulation with EGF or PMA and found that both agonists increased the phosphorylation and activation of fibroblast extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but only EGF activated the same kinase activities in HaCaT cells. Further, the EGF-mediated increase in MMP-1 gene expression was inhibited by the MAP kinase/ERK kinase (MEK)-specific inhibitor PD98059 and the p38 kinase-specific inhibitor SB203580. Our evidence indicates that although HaCaT MAP kinases are functional, they are not properly regulated in response to the activation of protein kinase C, and that the defect that bars HaCaT MMP-1 expression in response to stimulation with PMA lies before MAP kinase activation.
Mammalian PIG-L and its yeast homologue Gpi12p are N-acetylglucosaminylphosphatidylinositol de-N-acetylases essential in glycosylphosphatidylinositol biosynthesis
Authors: WATANABE Reika , OHISHI Kazuhito , MAEDA Yusuke , NAKAMURA Nobuo , KINOSHITA Taroh
Published: August 2015

Glycosylphosphatidylinositol (GPI) is used as a membrane anchor by many eukaryotic cell-surface proteins. The second step of GPI biosynthesis is de-N-acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI). We have previously cloned the rat PIG-L gene by expression cloning that complemented a mutant Chinese hamster ovary cell line defective in this step. Here we show that recombinant rat PIG-L protein purified from Escherichia coli as a complex with GroEL has GlcNAc-PI de-N-acetylase activity in vitro. The activity was not enhanced by GTP, which is known to enhance the de-N-acetylase activity of mammalian cell microsomes. As with other de-N-acetylases that act on the GlcNAc moiety, metal ions, in particular Mn2+ and Ni2+, enhanced the enzyme activity of PIG-L. The Saccharomyces cerevisiae YMR281W open reading frame encodes a protein (termed Gpi12p) with 24% amino acid identity with rat PIG-L. On transfection into mammalian PIG-L-deficient cells, this gene, GPI12, restored the cell-surface expression of GPI-anchored proteins and GlcNAc-PI de-N-acetylase activity. The disruption of the gene caused lethality in S. cerevisiae. These results indicate that GlcNAc-PI de-N-acetylase is conserved between mammals and yeasts and that the de-N-acetylation step is also indispensable in yeasts.
Kinetic properties and tissular distribution of mammalian phosphomannomutase isozymes
Authors: PIRARD Michel , ACHOURI Younes , COLLET Jean-François , SCHOLLEN Els , MATTHIJS Gert , SCHAFTINGEN Emile VAN
Published: August 2015

Human tissues contain two types of phosphomannomutase, PMM1 and PMM2. Mutations in the PMM2 gene are responsible for the most common form of carbohydrate-deficient glycoprotein syndrome [Matthijs, Schollen, Pardon, Veiga-da-Cunha, Jaeken, Cassiman and Van Schaftingen (1997) Nat. Genet.19, 88–92]. The protein encoded by this gene has now been produced in Escherichia coli and purified to homogeneity, and its properties have been compared with those of recombinant human PMM1. PMM2 converts mannose 1-phosphate into mannose 6-phosphate about 20 times more rapidly than glucose 1-phosphate to glucose 6-phosphate, whereas PMM1 displays identical Vmax values with both substrates. The Ka values for both mannose 1,6-bisphosphate and glucose 1,6-bisphosphate are significantly lower in the case of PMM2 than in the case of PMM1. Like PMM1, PMM2 forms a phosphoenzyme with the chemical characteristics of an acyl-phosphate. PMM1 and PMM2 hydrolyse different hexose bisphosphates (glucose 1,6-bisphosphate, mannose 1,6-bisphosphate, fructose 1,6-bisphosphate) at maximal rates of ≈ 3.5 and 0.3% of their PMM activity, respectively. Fructose 1,6-bisphosphate does not activate PMM2 but causes a time-dependent stimulation of PMM1 due to the progressive formation of mannose 1,6-bisphosphate from fructose 1,6-bisphosphate and mannose 1-phosphate. Experiments with specific antibodies, kinetic studies and Northern blots indicated that PMM2 is the only detectable isozyme in most rat tissues except brain and lung, where PMM1 accounts for about 66 and 13% of the total activities, respectively.
Identification of the Mg2+-binding site in the P-type ATPase and phosphatase members of the HAD (haloacid dehalogenase) superfamily by structural similarity to the response regulator protein CheY
Authors: RIDDER Ivo S. , DIJKSTRA Bauke W.
Published: August 2015

The large HAD (haloacid dehalogenase) superfamily of hydrolases comprises P-type ATPases, phosphatases, epoxide hydrolases and l-2-haloacid dehalogenases. A comparison of the three-dimensional structure of l-2-haloacid dehalogenase with that of the response regulator protein CheY allowed the assignment of a conserved pair of aspartate residues as the Mg2+-binding site in the P-type ATPase and phosphatase members of the superfamily. From the resulting model of the active site, a conserved serine/threonine residue is suggested to be involved in phosphate binding, and a mechanism comprising a phosphoaspartate intermediate is postulated.
Synthesis and assembly of connexins in vitro into homomeric and heteromeric functional gap junction hemichannels
Authors: AHMAD Shoeb , DIEZ Juan A. , GEORGE Christopher H. , EVANS W. Howard
Published: August 2015

The biogenesis of connexins and their assembly into functional gap junction hemichannels (connexons) was studied with the use of a cell-free transcription/translation system. Velocity sedimentation on sucrose gradients showed that a small proportion of connexin (Cx) 26 and Cx32 that were co-translationally translocated into microsomes were oligomers of Cx26 and Cx32. Chemical cross-linking studies showed that these corresponded to hexameric connexons. Reconstitution of connexons synthesized in vitro into liposomes induced permeability properties consistent with the view that open gap junction hemichannels were produced. By using an immunoprecipitation approach, a simultaneous translation of Cx26 and Cx32 incorporated into microsomes resulted in homomeric connexons. However, supplementation of the translation system in vitro with liver Golgi membranes produced heteromeric connexons constructed of Cx32 and Cx26, and also resulted in an increased oligomerization especially of Cx32. All of the connexins analysed were inserted co-translationally into canine pancreatic microsomal membranes. In addition, Cx26 and Cx43, but not Cx32, were also inserted into microsomal membranes post-translationally. Analysis of various connexin constructs in which the cytoplasmic carboxy tails were transposed, the cytoplasmic tail of Cx43 was truncated or a reporter protein, aequorin, was attached to the C-terminus showed that tail length was not the major determinant of the post-translational membrane insertion of connexins.
Transmembrane folding of the human erythrocyte anion exchanger (AE1, Band 3) determined by scanning and insertional N-glycosylation mutagenesis
Authors: POPOV Milka , LI Jing , REITHMEIER Reinhart A. F.
Published: August 2015

The human erythrocyte anion exchanger (AE1, Band 3) contains up to 14 transmembrane segments, with a single site of N-glycosylation at Asn642 in extracellular (EC) loop 4. Scanning and insertional N-glycosylation mutagenesis were used to determine the folding pattern of AE1 in the membrane. Full-length AE1, when expressed in transfected human embryonic kidney (HEK)-293 or COS-7 cells, retained a high-mannose oligosaccharide structure. Scanning N-glycosylation mutagenesis of EC loop 4 showed that N-glycosylation acceptor sites (Asn-Xaa-Ser/Thr) spaced 12 residues from the ends of adjacent transmembrane segments could be N-glycosylated. An acceptor site introduced at position 743 in intracellular (IC) loop 5 that could be N-glycosylated in a cell-free translation system was not N-glycosylated in transfected cells. Mutations designed to disrupt the folding of this loop enhanced the level of N-glycosylation at Asn743in vitro. The results suggest that this loop might be transiently exposed to the lumen of the endoplasmic reticulum during biosynthesis but normally folds rapidly, precluding N-glycosylation. EC loop 4 insertions into positions 428, 484, 754 and 854 in EC loops 1, 2, 6 and 7 respectively were efficiently N-glycosylated, showing that these regions were extracellular. EC loop 4 insertions into positions 731 or 785 were poorly N-glycosylated, which was inconsistent with an extracellular disposition for these regions of AE1. Insertion of EC loop 4 into positions 599 and 820 in IC loops 3 and 6 respectively were not N-glycosylated in cells, which was consistent with a cytosolic disposition for these loops. Inhibitor-affinity chromatography with 4-acetamido-4´-isothiocyanostilbene-2,2´-disulphonate (SITS)-Affi-Gel was used to assess whether the AE1 mutants were in a native state. Mutants with insertions at positions 428, 484, 599, 731 and 785 showed impaired inhibitor binding, whereas insertions at positions 754, 820 and 854 retained binding. The results indicate that the folding of the C-terminal region of AE1 is more complex than originally proposed and that this region of the transporter might have a dynamic aspect.
N-glycosylation requirements for the AT1a angiotensin II receptor delivery to the plasma membrane
Authors: DESLAURIERS Benoit , PONCE Cecilia , LOMBARD Colette , LARGUIER Renée , BONNAFOUS Jean-Claude , MARIE Jacky
Published: August 2015

The purpose of this work was to investigate the role of N-glycosylation in the expression and pharmacological properties of the the rat AT1a angiotensin II (AII) receptor. Glycosylation-site suppression was carried out by site-directed mutagenesis (Asn → Gln) of Asn176 and Asn188 (located on the second extracellular loop) and by the removal of Asn4 at the N-terminal end combined with the replacement of the first four amino acids by a 10 amino acid peptide epitope (c-Myc). We generated seven possible N-glycosylation-site-defective mutants, all tagged at their C-terminal ends with the c-Myc epitope. This double-tagging strategy, associated with photoaffinity labelling, allowed evaluation of the molecular masses and immunocytochemical cellular localization of the various receptors transiently expressed in COS-7 cells. We showed that: (i) each of the three N-glycosylation sites are utilized in COS-7 cells; (ii) the mutant with three defective N-glycosylation sites was not (or was very inefficiently) expressed at the plasma membrane and accumulated inside the cell at the perinuclear zone; (iii) the preservation of two sites allowed normal receptor delivery to the plasma membrane, the presence of only Asn176 ensuring a behaviour similar to that of the wild-type receptor; and (iv) all expressed receptors displayed unchanged pharmacological properties (Kd for 125I-sarcosine1-AII; sarcosine1-AII-induced inositol phosphate production). These results demonstrate that N-glycosylation is required for the AT1 receptor expression. They are discussed in the light of current knowledge of membrane-protein maturation and future prospects of receptor overexpression for structural studies.
MgATP-independent hydrogen evolution catalysed by nitrogenase: an explanation for the missing electron(s) in the MgADP–AlF4 transition-state complex
Authors: YOUSAFZAI Faridoon K. , EADY Robert R.
Published: August 2015

When the MoFe (Kp1) and Fe (Kp2) component proteins of Klebsiella pneumoniae nitrogenase are incubated with MgADP and AlF4- in the presence of dithionite as a reducing agent, a stable putative transition-state complex is produced [Yousafzai and Eady (1997) Biochem. J. 326, 637-640]. Surprisingly, the EPR signal associated with reduced Kp2 is not detectable, but Kp1 retains the S = 3/2 EPR signal arising from the dithionite reduced state of the MoFe cofactor centre of the protein. This is consistent with the [Fe4S4] centre of the Fe protein in the complex being oxidized, and similar observations have been made with the complex of Azotobacter vinelandii [Spee, Arendsen, Wassink, Marritt, Hagen and Haaker (1998) FEBS Lett. 432, 55-58]. No satisfactory explanation for the fate of the electrons lost by Kp2 has been forthcoming. However, we report here that during the preparation of the MgADP-AlF4K. pneumoniae complex under argon, H2 was evolved in amounts corresponding to one half of the FeMoco content of the Kp1 (FeMoco is the likely catalytic site of nitrogenase with a composition Mo:Fe7:S9:homocitrate). This is surprising, since activity is observed during incubation in the absence of MgATP, normally regarded as being essential for nitrogenase function, and in the presence of MgADP, a strong competitive inhibitor of nitrogenase. The formation of H2 by nitrogenase in the absence of AlF4- was also observed in reaction mixtures containing MgADP but not MgATP. The reaction showed saturation kinetics when Kp1 was titrated with increasing amounts of Kp2 and, at saturation, the amount of H2 formed was stoichiometric with the FeMoco content of Kp1. The dependence of the rate of formation of H2 on [MgADP] was inconsistent with the activity arising from MgATP contamination. We conclude that MgATP is not obligatory for H+ reduction by nitrogenase since MgADP supports a very low rate of hydrogen evolution.
Structural characterization of DNA and RNA sequences recognized by the gene 5 protein of bacteriophage fd
Authors: OLIVER Antony W. , KNEALE G. Geoff
Published: August 2015

The single-stranded DNA sequence d(GT5G4CT4C) occurs close to the origin of replication within the intergenic region of the viral strand of bacteriophage fd. The RNA analogue of this sequence r(GU5G4CU4C) forms part of the untranslated leader sequence of the gene 2 mRNA and is specifically bound by the fd gene 5 protein in its role as a translational repressor. The structure of these sequences is likely to have an important role in the control of both DNA replication and RNA translation in the phage. We show that this 16 nt sequence, in both a DNA and an RNA context, can exist in a structured and unstructured form as determined by high-resolution gel filtration and non-denaturing gel electrophoresis. The CD spectrum of the structured form is characteristic of parallel guanine tetraplexes. The structured form of the DNA sequence melts at approx. 47 °C in the presence of Na+ ions but the structure is stabilized up to 75 °C in the presence of K+ ions. The RNA structure is more stable than the equivalent DNA structure (melting temperature approx. 62 °C), and its stability is further enhanced in the presence of K+ ions. Two of the central guanine residues are fully protected from cleavage as determined by dimethyl sulphate protection experiments, whereas methylation interference studies show that methylation of any of the four central guanine residues inhibits structure formation. Our results demonstrate that the structured form of the nucleic acid is mediated through the formation of a guanine-tetraplex core region, in RNA this might be further stabilized by the presence of weaker uracil quartets.
Arrest of endosome acidification by bafilomycin A1 mimics insulin action on GLUT4 translocation in 3T3-L1 adipocytes
Authors: CHINNI Sreenivasa R. , SHISHEVA Assia
Published: August 2015

In insulin-sensitive fat and muscle cells, the major glucose transporter GLUT4 is constitutively sequestered in endosomal tubulovesicular membranes, and moves to the cell surface in response to insulin. While sequence information within GLUT4 appears to be responsible for its constitutive intracellular sequestration, the regulatory elements and mechanisms that enable this protein to achieve its unique sorting pattern under basal and insulin-stimulated conditions are poorly understood. We show here that arrest of endosome acidification in insulin-sensitive 3T3-L1 adipocytes by bafilomycin A1, a specific inhibitor of the vacuolar proton pump, results in the rapid and dose-dependent translocation of GLUT4 from the cell interior to the membrane surface; the effects of maximally stimulatory concentrations of bafilomycin A1 (400-800 nM) were equivalent to 50-65% of the effects of acute insulin treatment. Like insulin, bafilomycin A1 induced the redistribution of GLUT1 and Rab4, but not that of other proteins whose membrane localization has been shown to be insulin-insensitive. Studies to address the mechanism of this effect demonstrated that neither autophosphorylation nor internalization of the insulin receptor was altered by bafilomycin A1 treatment. Bafilomycin-induced GLUT4 translocation was not blocked by cell pretreatment with wortmannin. Taken together, these data indicate that arrest of endosome acidification mimics insulin action on GLUT4 and GLUT1 translocation by a mechanism distal to insulin receptor and phosphatidylinositol 3-kinase activation, and suggest an important role for endosomal pH in the membrane dynamics of the glucose transporters.
Structural organization and splice variants of the POLE1 gene encoding the catalytic subunit of human DNA polymerase ε
Authors: HUANG Deqi , POSPIECH Helmut , KESTI Tapio , SYVÄOJA Juhani E.
Published: August 2015

The catalytic subunit of human DNA polymerase ϵ, an enzyme involved in nuclear DNA replication and repair, is encoded by the POLE1 gene. This gene is composed of 51 exons spanning at least 97 kb of genomic DNA. It was found to encode three alternative mRNA splice variants that differ in their 5ʹ-terminal sequences and in the N-termini of the predicted proteins. A CpG island covers the promoter region for the major transcript in HeLa cells. This promoter is TATA-less and contains several putative binding sites for transcription factors typical of S-phase-up-regulated and serum-responsive promoters. Potential promoter regions were also identified for the two other alternative transcripts. Interestingly, no nuclear polyadenylation signal sequence was detected in the 3ʹ-untranslated region, although a poly(A) tail was present. These results suggest a complicated regulatory machinery for the expression of the human POLE1 gene, including three alternative transcripts expressed from three promoters.
Analysis of RNA–protein interactions of mouse liver cytochrome P4502A5 mRNA
Authors: TILLOY-ELLUL Anne , RAFFALLI-MATHIEU Francoise , LANG Matti A.
Published: August 2015

In our previous studies we have identified a 37/39 kDa, pyrazole-inducible, cytochrome P4502A5 (CYP2A5) mRNA binding protein and provided evidence that it may play a role in the stabilization and processing of the RNA [Geneste, Rafalli and Lang (1996) Biochem. J. 313, 1029-1037; Thulke-Gross, Hergenhahn, Tilloy-Ellul, Lang and Bartsch (1998) Biochem. J. 331, 473-481]. Details of the RNA-protein interactions are, however, not known. In this report we have performed an analysis of the interaction between the CYP2A5 mRNA and the 37/39 kDa protein. With UV-cross linking experiments, using RNA probes corresponding to various parts of the CYP2A5 mRNA, and with antisense oligonucleotides complementary to certain areas of the 3ʹ-untranslated region (3ʹUTR), we could map the primary binding site to the tip of a 71 nt hair-pin loop at the 3ʹ-UTR. This analysis also showed that the protein may have more than one site of interaction with the RNA and/or that, within the binding region, there could be more than one protein molecule binding to the RNA. Analysis of the probable conformations of the various probes used in the UV cross-linking experiments, in combination with the estimated binding affinities of the protein to the different probes, suggests that important factors in the high-affinity binding are the UAG triplet flanked by GA-rich sequences at the tip of the hair-pin loop, in addition to the conformation of the loop itself. Within the binding region, similarities with known binding sites of heterogeneous nuclear ribonucleoprotein (hnRNP) A1 in other RNA molecules were revealed by sequence alignment analysis. Moreover, competition experiments with an oligoribonucleotide corresponding to a known high-affinity binding site of hnRNP A1, and immunoprecipitation of the UV cross-linked 37/39 kDa complex showed that the protein binding to the CYP2A5 mRNA could be hnRNP A1 or its close analogue. It was also shown that the 37/39 kDa protein binds with less affinity to CYP2A4 mRNA than to CYP2A5 mRNA. This is in accordance with experiments characterizing the binding site, since these two otherwise highly homologous genes are kown to have a three nucleotide difference within the region important for the high binding affinity. Since the response of CYP2A4 to pyrazole is known to be weak, as compared with CYP2A5, this observation provides further evidence for a regulatory role of the 37/39 kDa protein in CYP2A5 mRNA metabolism.
Peroxynitrite induces haem oxygenase-1 in vascular endothelial cells: a link to apoptosis
Authors: FORESTI Roberta , SARATHCHANDRA Padmini , CLARK James E. , GREEN Colin J. , MOTTERLINI Roberto
Published: August 2015

Peroxynitrite (ONOO-) is a potent oxidizing agent generated by the interaction of nitric oxide (NO) and the superoxide anion. In physiological solution, ONOO- rapidly decomposes to a hydroxyl radical, one of the most reactive free radicals, and nitrogen dioxide, another species able to cause oxidative damage. In the present study we investigated the effect of ONOO- on the expression of haem oxygenase-1 (HO-1), an inducible protein that is highly up-regulated by oxidative stress. Exposure of bovine aortic endothelial cells to ONOO- (250-1000 μM) produced a concentration-dependent increase in haem oxygenase activity and HO-1 protein expression. This effect was completely abolished by the ONOO- scavengers uric acid and N-acetylcysteine, and partly attenuated by 1,3-dimethyl-2-thiourea, a scavenger of hydroxyl radicals. ONOO- also produced a concentration-dependent increase in apoptosis and cytotoxicity, which were considerably decreased by uric acid and N-acetylcysteine. A 70% decrease in apoptosis was observed when cells were exposed to ONOO- in the presence of 10 μM tin protoporphyrin IX (SnPPIX), an inhibitor of haem oxygenase activity. When SnPPIX was added 5 min after ONOO-, apoptosis decreased by only 40%, which suggests that an interaction between ONOO- and the protoporphyrin occurs in our system. Increased haem oxygenase activity by pretreatment of cells with haemin resulted in elevated bilirubin production and was associated with a substantial decrease (35%) in ONOO--mediated apoptosis. These results indicate the ability of ONOO- to modulate the expression of the stress protein HO-1 and suggest that the haem oxygenase pathway contributes to protection against the cytotoxic action of ONOO-.
Glucocorticoid inhibition of human SP-A1 promoter activity in NCI-H441 cells
Authors: HOOVER Russell R. , THOMAS Klaus H. , FLOROS Joanna
Published: August 2015

Glucocorticoids have complex effects on human surfactant protein (SP) SP-A1 and SP-A2 gene expression that occur at both transcriptional and post-transcriptional levels. In the lung adenocarcinoma cell line NCI-H441, dexamethasone causes a dose-dependent decrease in total SP-A mRNA levels and inhibits SP-A gene transcription. In this study, a deletional analysis of the SP-A1 promoter was performed in order to identify cis-acting elements that mediate dexamethasone responsiveness in NCI-H441 cells. The region -32/+63 relative to the start of SP-A1 transcription mediated both basal promoter activity and dexamethasone repression of transcription. Removal of the region +18/+63 abolished dexamethasone responsiveness, indicating that sequences within this region are necessary for the inhibitory effect. Furthermore, the region -32/+63 formed a sequence-specific DNA-protein complex with NCI-H441 nuclear extract. This DNA-protein complex was induced by dexamethasone exposure and its formation was mediated partially by sequences within the region +26/+63.
Fish macrophages express a cyclo-oxygenase-2 homologue after activation
Authors: ZOU Jun , NEUMANN Norman F. , HOLLAND Jason W. , BELOSEVIC Miodrag , CUNNINGHAM Charles , SECOMBES Christopher J. , ROWLEY Andrew F.
Published: August 2015

In mammals, the increased generation of prostaglandins (PG) during the onset of inflammatory responses and activation of immune cell types has been attributed to the induction of a novel cyclo-oxygenase (COX) isoform, termed COX-2, which is distinct from the well-characterized constitutive activity (COX-1). Goldfish (Carassius auratus) macrophages exposed to bacterial lipopolysaccharide and leucocyte-derived macrophage-activating factor(s) showed a significant increase in the generation of the major COX product, PGE2, within the first 6 h of stimulation. The selective COX-2 inhibitor, NS398, inhibited this elevated generation of PGE, whereas the basal level of this product synthesized by unstimulated macrophages was unaffected by such exposure. PGE generation by goldfish macrophages was similarly inhibited by the glucocorticoid, dexamethasone, and an inhibitor of protein synthesis, cycloheximide, suggesting that this stimulation may be due to an inducible enzyme equivalent to mammalian COX-2. The complete coding sequence of rainbow trout (Oncorhynchus mykiss) COX-2 was obtained by PCR. The gene contains a 61 bp 5ʹ-untranslated region (UTR), a 1821 bp open reading frame and a 771 bp 3ʹUTR containing multiple copies of an mRNA instability motif (ATTTA). The predicted translation product had high homology to known mammalian and chicken COX-2 (83-84%) and COX-1 (77%) sequences. Reverse-transcriptase PCR with cDNA from control and bacterially challenged fish revealed that trout COX-2 expression was not constitutive but could be induced. Overall, these studies show for the first time that the inducible isoform of COX has a long evolutionary history, probably dating back to the evolution of fish over 500 million years ago.