bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancement of the thermostability and hydrolytic activity of xylanase by random gene shuffling
Authors: SHIBUYA Hajime , KANEKO Satoshi , HAYASHI Kiyoshi
Published: August 2015

The thermostability of Streptomyces lividans xylanase B (SlxB-cat) was significantly increased by the replacement of its N-terminal region with the corresponding region from Thermomonospora fusca xylanase A (TfxA-cat) without observing a decrease in enzyme activity. In spite of the significant similarity between the amino acid sequences of the two xylanases, their thermostabilities are quite different. To facilitate an understanding of the contribution of structure to the thermostability observed, chimaeric enzymes were constructed by random gene shuffling and the thermostable chimaeric enzymes were selected for further study. A comparative study of the chimaeric and parental enzymes indicated that the N-terminus of TfxA-cat contributed to the observed thermostability. However, too many substitutions decreased both the thermostability and the activity of the enzyme. The mutants with the most desirable characteristics, Stx15 and Stx18, exhibited significant thermostabilities at 70 °C with optimum temperatures which were 20 °C higher than that of SlxB-cat and equal to that of TfxA-cat. The ability of these two chimaeric enzymes to produce reducing sugar from xylan was enhanced in comparison with the parental enzymes. These results suggest that these chimaeric enzymes inherit both their thermostability from TfxA-cat and their increased reactivity from SlxB-cat. Our study also demonstrates that random shuffling between a mesophilic enzyme and its thermophilic counterpart represents a facile approach for the improvement of the thermostability of a mesophilic enzyme.
RGS14 is a novel Rap effector that preferentially regulates the GTPase activity of Gαo
Authors: TRAVER Sabine , BIDOT Carole , SPASSKY Nathalie , BALTAUSS Tania , DE TAND Marie-France , THOMAS Jean-Léon , ZALC Bernard , JANOUEIX-LEROSEY Isabelle , DE GUNZBURG Jean
Published: August 2015

In an attempt to elucidate the physiological function(s) of the Ras-related Rap proteins, we used the yeast two-hybrid system and isolated a cDNA encoding a protein that interacts with both Rap1 and Rap2, but not with Ras; the use of Rap2 mutants showed that this interaction is characteristic of a potential Rap effector. This protein was identified as RGS14, a member of the recently discovered family of RGS (‘regulators of G-protein signalling’) proteins that stimulate the GTPase activity of the GTP-binding α subunit of heterotrimeric G-proteins (Gα). Deletion analysis, as well as in vitro binding experiments, revealed that RGS14 binds Rap proteins through a domain distinct from that carrying the RGS identity, and that this domain shares sequence identity with the Ras/Rap binding domain of B-Raf and Raf-1 kinases. RGS14 is distinguished from other RGS proteins by its marked preference for Gαo over other Gα subunits: RGS14 binds preferentially to Gαo in isolated brain membranes, and also interacts preferentially with Gαo (as compared with Gαi1) to stimulate its GTPase activity. In adult mice, RGS14 expression is restricted to spleen and brain. In situ hybridization studies showed that it is highly expressed only in certain areas of mouse brain (such as the CA1 and CA2 regions of the hippocampus), and that this pattern closely resembles that of Rap2, but not Rap1, expression. Double in situ hybridization experiments revealed that certain cells in the hippocampus express both RGS14 and Gαo, as well as both RGS14 and Rap2, showing that the interaction of RGS14 with Gαo and Rap2 is physiologically possible. Taken together, these results suggest that RGS14 could constitute a bridging molecule that allows cross-regulation of signalling pathways downstream from G-protein-coupled receptors involving heterotrimeric proteins of the Gi/o family and those involving the Ras-related GTPase Rap2.
Red-cell glycophorin A–band 3 interactions associated with the movement of band 3 to the cell surface
Authors: YOUNG Mark T. , BECKMANN Roland , TOYE Ashley M. , TANNER Michael J. A.
Published: August 2015

We have examined the mechanism by which glycophorin A (GPA) facilitates the movement of the human red-cell anion exchanger (band 3, AE1) to the cell surface. GPA itself forms stable dimers in membranes and detergent solution. Four mutants of human GPA with impaired dimerization were prepared (L75I, I76A, G79L and G83L). All four GPA mutants enhanced band 3 translocation to the Xenopus oocyte plasma membrane in the same way as wild-type GPA, showing that the GPA monomer is sufficient to mediate this process. Cell-surface expression of the natural band 3 mutant G701D has an absolute requirement for GPA. GPA monomers also rescued the cell-surface expression of this mutant band 3. Taking into account other evidence, we infer that the site of GPA interaction with band 3 is located outside the GPA dimerization interface but within the GPA transmembrane span. The results of examination of the cell-surface expression of GPA and band 3 in different K562 erythroleukaemia cell clones stably transfected with band 3 are consistent with the movement of GPA and band 3 to the cell surface together. We discuss the pathways by which band 3 moves to the cell surface in the presence and the absence of GPA, concluding that GPA has a role in enhancing the folding and maturation of band 3. We propose that GPA functions in erythroid cells to assist with the incorporation of large amounts of properly folded band 3 into the membrane within a limited time span during erythroid maturation.
Characterization of derivatives of the high-molecular-mass penicillin-binding protein (PBP) 1 of Mycobacterium leprae
Authors: MAHAPATRA Sebabrata , BHAKTA Sanjib , AHAMED Jasimuddin , BASU Joyoti
Published: August 2015

Mycobacterium leprae has two high-molecular-mass multimodular penicillin-binding proteins (PBPs) of class A, termed PBP1 and PBP1* [Lepage, Dubois, Ghosh, Joris, Mahapatra, Kundu, Basu, Chakrabarti, Cole, Nguyen-Disteche and Ghuysen (1997) J. Bacteriol. 179, 4627–4630]. PBP1-Xaa–β-lactamase fusions generated periplasmic β-lactamase activity when Xaa (the amino acid of PBP1 at the fusion junction) was residue 314, 363, 407, 450 or 480. Truncation of the N-terminal part of the protein up to residue Leu-147 generated a penicillin-binding polypeptide which could still associate with the plasma membrane, whereas [∆M1–R314]PBP1 (PBP1 lacking residues Met-1 to Arg-314) failed to associate with the membrane, suggesting that the region between residues Leu-147 and Arg-314 harbours an additional plasma membrane association site for PBP1. Truncation of the C-terminus up to 42 residues downstream of the KTG (Lys-Thr-Gly) motif also generated a polypeptide that retained penicillin-binding activity. [∆M1–R314]PBP1 could be extracted from inclusion bodies and refolded under appropriate conditions to give a form capable of binding penicillin with the same efficiency as full-length PBP1. This is, to the best of our knowledge, the first report of a soluble derivative of a penicillin-resistant high-molecular-mass PBP of class A that is capable of binding penicillin. A chimaeric PBP in which the penicillin-binding (PB) module of PBP1 was fused at its N-terminal end with the non-penicillin-binding (n-PB) module of PBP1* retained pencillin-binding activity similar to that of PBP1, corroborating the finding that the n-PB module of PBP1 is dispensable for its penicillin-binding activity.
Regulation of GLUT5, GLUT2 and intestinal brush-border fructose absorption by the extracellular signal-regulated kinase, p38 mitogen-activated kinase and phosphatidylinositol 3-kinase intracellular signalling pathways: implications for adaptation to diabetes
Authors: HELLIWELL Philip A. , RICHARDSON Michael , AFFLECK Julie , KELLETT George L.
Published: August 2015

We have investigated the role of the extracellular signal-regulated kinase (ERK), p38 and phosphatidylinositol 3-kinase (PI 3-kinase) pathways in the regulation of intestinal fructose transport. Different combinations of anisomycin, PD98059 and wortmannin had very different effects on fructose transport in perfused isolated loops of rat jejunum. Transport was stimulated maximally by anisomycin (2µM) and blocked by SB203580 (20µM), confirming involvement of the p38 pathway. PD98059 (50µM) alone had little effect on fructose transport. However, it had a dramatic effect on stimulation by anisomycin, diminishing the Ka 50-fold from 1µM to 20nM to show that the ERK pathway restrains the p38 pathway. The Ka for diabetic jejunum was 30nM and PD98059 had no effect. Transport in the presence of anisomycin was 3.4-fold that for anisomycin plus PD98059 plus wortmannin. Transport was mediated by both GLUT5 and GLUT2. In general, GLUT2 levels increased up to 4-fold within minutes and with only minimal changes in GLUT5 or SGLT1 levels, demonstrating that GLUT2 trafficks by a rapid trafficking pathway distinct from that of GLUT5 and SGLT1. GLUT2 intrinsic activity was regulated over a 9-fold range. It is concluded that there is extensive cross-talk between the ERK, p38 and PI 3-kinase pathways in their control of brush-border fructose transport by modulation of both the levels and intrinsic activities of GLUT5 and GLUT2. The potential of the intracellular signalling pathways to regulate short-term nutrient transport during the assimilation of a meal and longer-term adaptation to diabetes and hyperglycaemia is discussed.
Substrates of semicarbazide-sensitive amine oxidase co-operate with vanadate to stimulate tyrosine phosphorylation of insulin-receptor-substrate proteins, phosphoinositide 3-kinase activity and GLUT4 translocation in adipose cells
Authors: ENRIQUE-TARANCÓN Gemma , CASTAN Isabelle , MORIN Nathalie , MARTI Luc , ABELLA Anna , CAMPS Marta , CASAMITJANA Roser , PALACÍN Manuel , TESTAR Xavier , DEGERMAN Eva , CARPÉNÉ Christian , ZORZANO Antonio
Published: August 2015

It has been shown that the combination of benzylamine or tyramine and low concentrations of vanadate markedly stimulates glucose transport in rat adipocytes by a mechanism that requires semicarbazide-sensitive amine oxidase (SSAO) activity and H2O2 formation. Here we have further analysed the insulin-like effects of the combination of SSAO substrates and vanadate and we have studied the signal-transduction pathway activated in rat adipocytes. We found that several SSAO substrates (benzylamine, tyramine, methylamine, n-decylamine, histamine, tryptamine or β-phenylethylamine), in combination with low concentrations of vanadate, stimulate glucose transport in isolated rat adipocytes. Furthermore, SSAO substrates together with vanadate stimulated the recruitment of GLUT4 to the cell surface in isolated rat adipocytes. Benzylamine plus vanadate also stimulated glucose transport and GLUT4 translocation in 3T3-L1 adipocytes. Benzylamine or tyramine in combination with vanadate potently stimulated the tyrosine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-3. In contrast, benzylamine and vanadate caused only a weak stimulation of insulin receptor kinase. Benzylamine or tyramine in combination with vanadate also stimulated phosphoinositide 3-kinase activity; wortmannin abolished the stimulatory effect of benzylamine and vanadate on glucose transport in adipose cells. Furthermore, the administration of benzylamine and vanadate in vivo caused a rapid lowering of plasma glucose levels, which took place in the absence of alterations in plasma insulin. On the basis of these results we propose that SSAO activity regulates glucose transport in adipocytes. SSAO oxidative activity stimulates glucose transport via the translocation of GLUT4 carriers to the cell surface, resulting from a potent tyrosine phosphorylation of IRS-1 and IRS-3 and phosphoinositide 3-kinase activation. Our results also indicate that substrates of SSAO might regulate glucose disposal in vivo.
Catabolism of aggrecan, decorin and biglycan in tendon
Authors: REES Sarah G. , FLANNERY Carl R. , LITTLE Chris B. , HUGHES Clare E. , CATERSON Bruce , DENT Colin M.
Published: August 2015

We have examined the catabolism of the proteoglycans aggrecan, decorin and biglycan in fresh tendon samples and in explant cultures of tissue from the tensional and compressed regions of young and mature bovine tendons. A panel of well-characterized antibodies that recognize glycosaminoglycan or protein (linear or neoepitope) sequences was used to detect proteoglycans and proteoglycan degradation products that were both retained within the tissue and released into the culture medium. In addition, a reverse-transcriptase-mediated PCR analysis was used to examine the mRNA expression patterns of tendon proteoglycans and aggrecanases. The results of this study indicate a major role for aggrecanase(s) in the catabolism of aggrecan in bovine tendon. The study also provides a characterization of glycosaminoglycan epitopes associated with the proteoglycans of tendon, illustrating age-related changes in the isomers of chondroitin sulphate disaccharides that remain attached to the core protein glycosaminoglycan linkage region after digestion with chondroitinase ABC. Evidence for a rapid turnover of the small proteoglycans decorin and biglycan was also observed, indicating additional molecular pathways that might compromise the integrity of the collagen matrix and potentially contribute to tendon dysfunction after injury and during disease.
Cloning and characterization of a novel human histone deacetylase, HDAC8
Authors: BUGGY Joseph J. , SIDERIS Mindy L. , MAK Polly , LORIMER Donald D. , MCINTOSH Brian , CLARK James M.
Published: August 2015

Histone deacetylases (HDACs) are a growing family of enzymes implicated in transcriptional regulation by affecting the acetylation state of core histones in the nucleus of cells. HDACs are known to have key roles in the regulation of cell proliferation [Brehm, Miska, McCance, Reid, Bannister and Kouzarides (1998) Nature (London) 391, 597–600], and aberrant recruitment of an HDAC complex has been shown to be a key step in the mechanism of cell transformation in acute promyelocytic leukaemia [Grignani, De Matteis, Nervi, Tomassoni, Gelmetti, Cioce, Fanelli, Ruthardt, Ferrara, Zamir et al. (1998) Nature (London) 391, 815–818; Lin, Nagy, Inoue, Shao, Miller and Evans (1998), Nature (London) 391, 811–814]. Here we present the complete nucleotide sequence of a cDNA clone, termed HDAC8, that encodes a protein product with similarity to the RPD3 class (I) of HDACs. The predicted 377-residue HDAC8 product contains a shorter C-terminal extension relative to other members of its class. After expression in two cell systems, immunopurified HDAC8 is shown to possess trichostatin A- and sodium butyrate-inhibitable HDAC activity on histone H4 peptide substrates as well as on core histones. Expression profiling reveals the expression of HDAC8 to various degrees in every tissue tested and also in several tumour lines. Mutation of two adjacent histidine residues within the predicted active site severely decreases activity, confirming these residues as important for HDAC8 enzyme activity. Finally, linkage analysis after radiation hybrid mapping has localized HDAC8 to chromosomal position Xq21.2–Xq21.3. These results confirm HDAC8 as a new member of the HDAC family.
Expression of Escherichia coli otsA in a Saccharomyces cerevisiae tps1 mutant restores trehalose 6-phosphate levels and partly restores growth and fermentation with glucose and control of glucose influx into glycolysis
Authors: BONINI Beatriz M. , VAN VAECK Christophe , LARSSON Christer , GUSTAFSSON Lena , MA Pingsheng , WINDERICKX Joris , VAN DIJCK Patrick , THEVELEIN Johan M.
Published: August 2015

The TPS1 gene, encoding trehalose-6-phosphate synthase (TPS), exerts an essential control on the influx of glucose into glycolysis in the yeast Saccharomyces cerevisiae. The deletion of TPS1 causes an inability to grow on glucose because of a hyperaccumulation of sugar phosphates and depletion of ATP and phosphate. We show that expression of the Escherichia coli homologue, otsA, in a yeast tps1 mutant results in high TPS activity. Although the trehalose 6-phosphate (Tre6P) level during exponential growth on glucose was at least as high as in a wild-type yeast strain, growth on glucose was only partly restored and the lag phase was much longer. Measurement of the glycolytic metabolites immediately after the addition of glucose showed that in spite of a normal Tre6P accumulation there was still a partial hyperaccumulation of sugar phosphates. Strong elevation of the Tre6P level by the additional deletion of the TPS2 gene, which encodes Tre6P phosphatase, was not able to cause a strong decrease in the sugar phosphate levels in comparison with the wild-type strain. In addition, in chemostat experiments the short-term response to a glucose pulse was delayed, but normal metabolism was regained over a longer period. These results show that Tre6P synthesis from a heterologous TPS enzyme can to some extent restore the control of glucose influx into glycolysis and growth on glucose in yeast. However, they also indicate that the yeast TPS enzyme, as opposed to the E. coli otsA gene product, is able to increase the efficiency of the Tre6P control on glucose influx into yeast glycolysis.
Interaction of the collagen-like tail of asymmetric acetylcholinesterase with heparin depends on triple-helical conformation, sequence and stability
Authors: DEPREZ Paola , DOSS-PEPE Ellen , BRODSKY Barbara , INESTROSA Nibaldo C.
Published: August 2015

The collagen-like tail of asymmetric acetylcholinesterase (AChE) contains two heparin-binding domains (HBDs) that interact with heparan sulphate proteoglycans, determining the anchoring of the enzyme at the basal lamina and its specific localization at the neuromuscular junction. Both HBDs are characterized by a cluster of basic residues containing a core with the BBXB consensus sequence (where B represents a basic residue and X a non-basic residue). To study the interaction of such HBDs with heparin we have used synthetic peptides to model the N-terminal and C-terminal sites. CD spectroscopy showed that all peptides are triple-helical at low temperatures, and undergo trimer-to-monomer transitions. Displacement assays of asymmetric AChE bound to heparin were performed using the peptides in both monomeric and triple-helical states. In the monomeric conformation, all the peptides were able to displace low levels of AChE depending on the basic charge content. In the triple-helical conformation, peptides containing the consensus sequence showed a large increase in the ability to displace bound AChE. Results suggest that the specific binding of the collagen-like-tail peptides to heparin depends both on the presence of the core sequence and on the triple-helical conformation. Moreover, BBXB-containing peptides that are less stable are more effective in displacing AChE, suggesting that the interaction region needs a significant amount of structural flexibility to better accommodate the ligand.
Oxygen and haem regulate the synthesis of peroxisomal proteins: catalase A, acyl-CoA oxidase and Pex1p in the yeast Saccharomyces cerevisiae; the regulation of these proteins by oxygen is not mediated by haem
Authors: SKONECZNY Marek , RYTKA Joanna
Published: August 2015

Saccharomyces cerevisiae genes related to respiration are typically controlled by oxygen and haem. Usually the regulation by these factors is co-ordinated; haem is indicated as the oxygen sensor. However, the responsiveness of peroxisome functions to these regulatory factors is poorly understood. The expression of CTA1, POX1 and PEX1 genes encoding the peroxisomal proteins catalase A, acyl-CoA oxidase and Pex1p peroxin respectively was studied under various conditions: in anaerobiosis, in the absence of haem and in respiratory incompetence caused by the lack of a mitochondrial genome (ρ0). The influence of haem deficiency or ρ0 on peroxisomal morphology was also investigated. Respiratory incompetence has no effect on the expression of CTA1 and POX1, whereas in the absence of haem their expression is markedly decreased. The synthesis of Pex1p is decreased in ρ0 cells and is decreased even more in haem-deficient cells. Nevertheless, peroxisomal morphology in both these types of cell does not differ significantly from the morphology of peroxisomes in wild-type cells. The down-regulating effect of anoxia on the expression of CTA1 and POX1 is even stronger than the effect of haem deficiency and is not reversed by the addition of exogenous haem or the presence of endogenous haem. Moreover, neither of these genes responds to the known haem-controlled transcriptional factor Hap1p. In contrast with the other two genes studied, PEX1 is up-regulated in anaerobiosis. The existence of one or more novel mechanisms of regulation of peroxisomal genes by haem and oxygen, different from those already known in S. cerevisiae, is postulated.
Purification and characterization of Ak.1 protease, a thermostable subtilisin with a disulphide bond in the substrate-binding cleft
Authors: TOOGOOD Helen S. , SMITH Clyde A. , BAKER Edward N. , DANIEL Roy M.
Published: August 2015

Ak.1 protease, a thermostable subtilisin isolated originally from Bacillus st. Ak.1, was purified to homogeneity from the Escherichia coli clone PB5517. It is active against substrates containing neutral or hydrophobic branched-chain amino acids at the P1 site, such as valine, alanine or phenylalanine. The Km and kcat of the enzyme decrease with decreasing temperature, though not to the same degree with all substrates, suggesting that specificity changes with temperature. The protease is markedly stabilized by Ca2+ ions. At 70°C, a 10-fold increase in Ca2+ concentration increases the half-life by three orders of magnitude. Ak.1 protease is stabilized by Ca2+ to a greater extent than is thermitase. This may be due, in part, to the presence of an extra Ca2+-binding site in Ak.1 protease. Other metal ions, such as Sr2+, increase the thermostability of the enzyme, but to a significantly lower degree than does Ca2+. The structure of the protease showed the presence of a disulphide bond located within the active-site cleft. This bond influences both enzyme activity and thermostability. The disulphide bond appears to have a dual role: maintaining the integrity of the substrate-binding cleft and increasing the thermostability of the protease. The protease was originally investigated to determine its usefulness in the clean-up of DNA at high temperatures. However, it was found that this protease has a limited substrate specificity, so this application was not explored further.
A role for serine-175 in modulating the molecular conformation of calponin
Authors: JIN Jian-Ping , WALSH Michael P. , SUTHERLAND Cindy , CHEN Wenhua
Published: August 2015

Calponin is an actin filament-associated protein found in smooth muscle and non-muscle cells. Calponin inhibits actin–myosin interaction in a manner that is prevented by protein kinase C (PKC)-catalysed phosphorylation of serine-175. To investigate the molecular basis of serine-175-mediated regulation, we examined the effect of phosphorylation on the conformation of calponin using monoclonal antibody (mAb) epitope analysis. Eight mAbs against different epitopes on chicken gizzard calponin were developed to monitor the conformational changes in calponin induced by PKC-mediated phosphorylation or serine-175 → alanine (S175A) substitution. The relative affinities of the mAbs for calponins immobilized on microtitre plates or bound to actin–tropomyosin thin filaments were determined, and epitope competitions between free and immobilized calponins were carried out. The changes in binding affinity between mAb paratopes and calponin epitopes demonstrate several serine-175 modification-induced conformational effects: (a) structures of calponin are reconfigured by serine-175 modification, supporting the regulatory function of serine-175; (b) there are submolecular structures unaffected by modification of serine-175 in both free and thin filament-associated calponins, suggesting that the serine-175-based conformational modulation is a targeted allosteric effect; (c) significant conformational changes are detected between free and thin filament-associated calponins, indicating two functional states of the molecular conformation; and (d) the different epitope characteristics between thin filament-bound and free calponins suggest that calponin is a flexible molecule, and the modifications of serine-175 may also determine the structural flexibility to increase the epitope accessibility. These results provide novel information concerning the structure–function relationships of calponin and its regulation by phosphorylation.
The skin of atopic dermatitis patients contains a novel enzyme, glucosylceramide sphingomyelin deacylase, which cleaves the N-acyl linkage of sphingomyelin and glucosylceramide
Authors: HIGUCHI Kazuhiko , HARA Junko , OKAMOTO Reiko , KAWASHIMA Makoto , IMOKAWA Genji
Published: August 2015

We have demonstrated previously that there is an abnormal expression of sphingomyelin (SM) deacylase in the epidermis of patients with atopic dermatitis (ADe). In the present study, we have prepared N-[palmitic acid-1-14C]SM and N-[palmitic acid-1-14C]glucosylceramide (GCer) to use as substrates and have quantified SM deacylase activity by detecting the release of [14C]palmitic acid in extracts of the stratum corneum or the epidermis of ADe patients. In studies using [palmitic acid-1-14C]SM as a substrate, a pH dependency of catalytic activity with a peak at pH 5.0 was found. Preparative SDS/PAGE using an extract of ADe epidermis revealed that the molecular mass of SM deacylase is 40000Da, which is consistent with its apparent molecular mass of 42000Da estimated by gel-filtration analysis of stratum corneum extracts. Analytical isoelectric focusing (IEF) chromatography demonstrated that the pI values of SM deacylase, β-glucocerebrosidase (GlcCDase), sphingomyelinase (SMase) and acid ceramidase were 4.2, 7.4, 7.0 and 5.7, respectively. In enzymic analysis using pI-4.2 SM deacylase partially purified by IEF, which had no detectable contamination with acid ceramidase, GlcCDase or SMase, radio-TLC analysis revealed that radiolabelled sphingosylphosphocholine or [1-14C]palmitic acid was enzymically liberated from [choline-methyl-14C]SM or N-[palmitoyl-1-14C]GCer, respectively, used as substrates. Further the pI-4.2 protein purified from extracts of the stratum corneum of ADe patients was able to hydrolyse N-[palmitoyl-1-14C]SM and GCer, but not N-[palmitoyl-1-14C]ceramide. These results indicate that a hitherto undiscovered epidermal enzyme, termed here glucosylceramide sphingomyelin deacylase, is expressed in the skin of ADe patients, which plays an important role in ceramide deficiency (including acylceramides) in the stratum corneum.
The chloride effect is related to anion binding in determining the rate of iron release from the human transferrin N-lobe
Authors: HE Qing-Yu , MASON Anne B. , NGUYEN Vinh , MacGILLIVRAY Ross T. A. , WOODWORTH Robert C.
Published: August 2015

The major function of human transferrin is to deliver iron from the bloodstream to actively dividing cells. Upon iron release, the protein changes its conformation from ‘closed’ to ‘open’. Extensive studies in vitro indicate that iron release from transferrin is very complex and involves many factors, including pH, the chelator used, an anion effect, temperature, receptor binding and intra-lobe interactions. Our earlier work [He, Mason and Woodworth (1997) Biochem. J. 328, 439–445] using the isolated transferrin N-lobe (recombinant N-lobe of human transferrin comprising residues 1–337; hTF/2N) has shown that anions and pH modulate iron release from hTF/2N in an interdependent manner: chloride retards iron release at neutral pH, but accelerates the reaction at acidic pH. The present study supports this idea and further details the nature of the dual effect of chloride: the anion effect on iron release is closely related to the strength of anion binding to the apoprotein. The negative effect seems to originate from competition between chloride and the chelator for an anion-binding site(s) near the metal centre. With decreasing pH, the strength of anion binding to hTF/2N increases linearly, decreasing the contribution of competition with the chelator. In the meantime, the ‘open’ or ‘loose’ conformation of hTF/2N, induced by the protonation of critical residues such as the Lys-206/Lys-296 pair at low pH, enables chloride to enter the cleft and bind to exposed side chains, thereby promoting cleft opening and synergistically allowing removal of iron by the chelator, leading to a positive anion effect. Disabling one or more of the primary anion-binding residues, namely Arg-124, Lys-206 and Lys-296, substantially decreases the anion-binding ability of the resulting mutant proteins. In these cases, the competition for the remaining binding residue(s) is increased, leading to a negative chloride effect or, at most, a very small positive effect, even at low pH.
Association of several small heat-shock proteins with reproductive tissues in the nematode Caenorhabditis elegans
Authors: DING Lily , CANDIDO E. Peter M.
Published: August 2015

Immunohistochemical data on 10 of the 14 small heat-shock (smHSPs) proteins in fourth larval stage and adult Caenorhabditis elegans show that the tissues expressing the greatest number of smHSPs are vulva (HSP12s, HSP43 and, under stress, HSP16s) and spermatheca (HSP12s, HSP25, HSP43 and, under stress, HSP16s). HSP43 is also expressed in male tail structures, and following heat-shock HSP16s are expressed in spermatids and spermatozoa.
Effects of insulin-like growth factor I on basal and stimulated glucose fluxes in rat liver
Authors: ENGLISCH Rainer , WURZINGER Reingard , FÜRNSINN Clemens , SCHNEIDER Barbara , FRISCH Herwig , WALDHÄUSL Werner , GRAF Jürg , RODEN Michael
Published: August 2015

Effects of insulin-like growth factor I (IGF-I) and insulin on glucose and potassium fluxes were examined by measuring transhepatic glucose and potassium balance in isolated perfused rat livers. At 1nM, both IGF-I and insulin decreased basal glucose release by ≈ 64% (P < 0.05). Adrenaline (epinephrine)-stimulated glucose release (42.6±4.5µmol/g of liver within 30 min) was inhibited (P < 0.05) by ≈ 32 and ≈ 52% during IGF-I and insulin exposure, which was accompanied by reduced cAMP release (-71 and -80%, P < 0.05). IGF-I- and insulin-induced reduction of glucose release only decreased during calcium-free perfusion, but not during inhibition of phosphoinositide 3-kinase by wortmannin. Both IGF-I and insulin induced net potassium uptake, while insulin also attenuated the response to adrenaline. In conclusion, IGF-I causes (i) insulin-like inhibition of hepatic glycogenolysis, even at low, nanomolar concentrations, which is associated with decreased cAMP release, reduced in the absence of Ca2+, but not mediated by phosphoinositide 3-kinase, (ii) reduction of adrenaline-induced glycogenolysis and (iii) net potassium uptake under basal conditions.
Stimulation of hormone-sensitive lipase activity by contractions in rat skeletal muscle
Authors: LANGFORT Jozef , PLOUG Thorkil , IHLEMANN Jacob , HOLM Cecilia , GALBO Henrik
Published: August 2015

Because the enzymic regulation of muscle triglyceride breakdown is poorly understood we studied whether neutral lipase in skeletal muscle is activated by contractions. Incubated soleus muscles from 70 g rats were electrically stimulated for 60min. Neutral lipase activity against triacylglycerol increased after 1 and 5min of contractions [0.36±0.02 (basal) versus 0.49±0.05 (1min) and 0.54±0.05 (5min) m-unit·mg of protein-1, means±S.E.M., P < 0.05]. After 10min the neutral lipase activity (0.40±0.05m-unit·mg of protein-1) had decreased to basal values (P > 0.05). The contraction-mediated increase in lipase activity was increased by ≈ 110% when muscle was stimulated in the presence of okadaic acid. Conversely, treatment of muscle homogenate with alkaline phosphatase completely reversed the contraction-mediated lipase activation. Lipase activity did not change during contractions when analysed in the presence of anti-hormone-sensitive-lipase (HSL) antibody [0.17±0.02 (basal) versus 0.21±0.02 (5min) m-unit·mg of protein-1, P > 0.05]. Furthermore, immunoprecipitation with affinity-purified anti-HSL antibody reduced muscle-HSL protein concentration by 81±4% and caused similar reductions in lipase activity against triacylglycerol and in the contraction-induced increase in this activity. Neither prior sympathectomy [0.33±0.02 (basal) versus 0.53±0.06 (5min) m-unit·mg of protein-1, P < 0.05] nor propranolol impaired the lipase response to contractions. Glycogen phosphorylase activity in the absence of AMP increased after 1min [27.3±3.1 versus 8.9±1.8% (activity without AMP/total activity with AMP), P < 0.05] and returned to basal levels after 5min. In conclusion, skeletal-muscle-immunoreactive HSL is transiently stimulated by contractions and the mechanism probably involves phosphorylation. The time course of HSL activation is similar to that of glycogen phosphorylase. Apparently, the two enzymes are regulated in parallel by contraction-induced as well as hormonal mechanisms, allowing simultaneous recruitment of all major extra- and intra-muscular energy stores.
Ischaemia induces changes in the association of the binding protein 4E-BP1 and eukaryotic initiation factor (eIF) 4G to eIF4E in differentiated PC12 cells
Authors: MARTÍN M. Elena , MUÑOZ Francisco M. , SALINAS Matilde , FANDO Juan L.
Published: August 2015

Ischaemia was obtained in vitro by subjecting nerve-growth-factor-differentiated PC12 cells to glucose deprivation plus anoxia. During ischaemia the rate of protein synthesis was significantly inhibited, and eIF4E-binding protein (4E-BP1) and eukaryotic initiation factor 4E (eIF4E) were significantly dephosphorylated in parallel. In addition, ischaemia induced an enhancement of the association of 4E-BP1 to eIF4E, which in turn decreased eIF4F formation, whereas no degradation of initiation factor 4G was observed. The treatment of PC12 cells with the specific p38 mitogen-activated protein kinase inhibitor SB203580 induced eIF4E dephosphorylation but did not cause any effect on protein synthesis rate. Rapamycin, the inhibitor of mammalian target of rapamycin (‘mTOR’), but not PD98059, the inhibitor of extracellular signal-regulated protein kinases (‘ERK1/2’), induced similar effects on 4E-BP1 phosphorylation to ischaemia; nevertheless, 4E-BP1–eIF4E complex levels were higher in ischaemia than in rapamycin-treated cells. In addition, both protein synthesis rate and eIF4F formation were lower in ischaemic cells than in rapamycin-treated cells.
The tissue-specific regulation of the carboxyl ester lipase gene in exocrine pancreas differs significantly between mouse and human
Authors: KANNIUS-JANSON Marie , LIDBERG Ulf , BJURSELL Gunnar , NILSSON Jeanette
Published: August 2015

The carboxyl ester lipase (CEL) gene is highly expressed in exocrine pancreas and expression of the human CEL gene is mediated by a strong tissue-specific enhancer, which is absolutely necessary for high-level expression. The mouse promoter, on the other hand, does not contain a corresponding enhancer element, but instead is totally dependent on another pancreas-specific element. This element is identified as a pancreatic transcription factor 1 (PTF 1)-binding site. The human CEL promoter also contains a putative PTF 1 element located at a position corresponding to the essential PTF 1 site in the mouse promoter. However, nucleotide changes in the human promoter 5´ flanking this PTF 1 site have created an overlapping CCAAT/enhancer-binding protein (C/EBP)-like binding motif, interfering with the binding of PTF 1. Hence, our findings provide an example of genetic divergence between species not accompanied by difference in function.
DNA repair protein O6-alkylguanine-DNA alkyltransferase is phosphorylated by two distinct and novel protein kinases in human brain tumour cells
Authors: MULLAPUDI Srinivas R. S. , ALI-OSMAN Francis , SHOU Jiang , SRIVENUGOPAL Kalkunte S.
Published: August 2015

We showed recently that human O6-alkylguanine-DNA alkyltransferase (AGT), an important target for improving cancer chemotherapy, is a phosphoprotein and that phosphorylation inhibits its activity [Srivenugopal, Mullapudi, Shou, Hazra and Ali-Osman (2000) Cancer Res. 60, 282–287]. In the present study we characterized the cellular kinases that phosphorylate AGT in the human medulloblastoma cell line HBT228. Crude cell extracts used Mg2+ more efficiently than Mn2+ for phosphorylating human recombinant AGT (rAGT) protein. Both [γ-32P]ATP and [γ-32P]GTP served as phosphate donors, with the former being twice as efficient. Specific components known to activate protein kinase A, protein kinase C and calmodulin-dependent kinases did not stimulate the phosphorylation of rAGT. Phosphoaminoacid analysis after reaction in vitro with ATP or GTP showed that AGT was modified at the same amino acids (serine, threonine and tyrosine) as in intact HBT228 cells. Although some of these properties pointed to casein kinase II as a candidate enzyme, known inhibitors and activators of casein kinase II did not affect rAGT phosphorylation. Fractionation of the cell extracts on poly(Glu/Tyr)-Sepharose resulted in the adsorption of an AGT kinase that modified the tyrosine residues and the exclusion of a fraction that phosphorylated AGT on serine and threonine residues. In-gel kinase assays after SDS/PAGE and non-denaturing PAGE revealed the presence of two AGT kinases of 75 and 130kDa in HBT228 cells. The partly purified tyrosine kinase, identified as the 130kDa enzyme by the same assays, was strongly inhibited by tyrphostin 25 but not by genestein. The tyrosine kinase used ATP or GTP to phosphorylate the AGT protein; this reaction inhibited the DNA repair activity of AGT. Evidence that the kinases might physically associate with AGT in cells was also provided. These results demonstrate that two novel cellular protein kinases, a tyrosine kinase and a serine/threonine kinase, both capable of using GTP as a donor, phosphorylate the AGT protein and affect its function. The new kinases might serve as potential targets for strengthening the biochemical modulation of AGT in human tumours.
Multitasking in signal transduction by a promiscuous human Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4)P3 5/6-kinase
Authors: YANG Xiaonian , SHEARS Stephen B.
Published: August 2015

We describe a human cDNA encoding 1-kinase activity that inactivates Ins(3,4,5,6)P4, an inhibitor of chloride-channel conductance that regulates epithelial salt and fluid secretion, as well as membrane excitability. Unexpectedly, we further discovered that this enzyme has alternative positional specificity (5/6-kinase activity) towards a different substrate, namely Ins(1,3,4)P3. Kinetic data from a recombinant enzyme indicate that Ins(1,3,4)P3 (Km = 0.3µM; Vmax = 320 pmol/min per µg) and Ins(3,4,5,6)P4 (Km = 0.1µM; Vmax = 780pmol/min per µg) actively compete for phosphorylation in vivo. This competition empowers the kinase with multitasking capability in several key aspects of inositol phosphate signalling.
Stichopus japonicus arginine kinase: gene structure and unique substrate recognition system
Authors: SUZUKI Tomohiko , YAMAMOTO Yasufumi , UMEKAWA Masahiro
Published: August 2015

Arginine kinase from the sea cucumber Stichopus japonicus underwent a unique molecular evolution. Unlike the monomeric 40kDa arginine kinases from molluscs and arthropods, Stichopus arginine kinase is dimeric, the same as cytoplasmic isoenzymes of the vertebrate creatine kinases. Its entire amino acid sequence is more similar to creatine kinases than to other arginine kinases, but the guanidino specificity region (GS region) is of the arginine kinase type. To elucidate its unusual evolution, the structure of the Stichopus arginine kinase gene was determined. It consisted of seven exons and six introns, and a part of the exon 2 of the Stichopus gene corresponds to the GS region. Compared with the structure of the human muscle creatine kinase gene (seven exons, six introns), the splice junctions of five introns were conserved exactly between the two genes, suggesting that these introns had been conserved for at least 500 million years. The entire sequence of Stichopus arginine kinase is distinctly included in the creatine kinase cluster in all tree construction methods examined. On the other hand, if the tree is constructed only from sequences corresponding to Stichopus exon 2, it is placed in the arginine kinase cluster. Thus we conclude that Stichopus arginine kinase evolved not from the arginine kinase gene but from the creatine kinase gene, and suggest that its GS region, determining substrate specificity, has been replaced by an arginine kinase type via exon shuffling. In typical arginine kinases four residues, Ser63, Gly64, Val65 and Tyr68 (numbering from the Limulus polyphemus sequence), in the GS region are highly conserved and are associated with substrate binding. Among them, Tyr68 appears to play a crucial role by forming a hydrogen bond with the substrate, and is conserved exactly in all arginine kinases. However, in Stichopus arginine kinase, none of these four conserved residues were present. Nevertheless, the enzyme displays an affinity for the substrate arginine (Km = 0.8mM) comparable with other arginine kinases. This implies that a completely different substrate-binding system has been developed in Stichopus arginine kinase. We propose that the His64 in Stichopus arginine kinase acts as a substitute for the Tyr68 in other arginine kinases, and that the imidazole ring of His64 is hydrogen bonded with the substrate arginine, thus stabilizing it.
Transmembrane and cytoplasmic domains of syndecan mediate a multi-step endocytic pathway involving detergent-insoluble membrane rafts
Authors: FUKI Ilia V. , MEYER Marie E. , WILLIAMS Kevin Jon
Published: August 2015

Syndecan heparan sulphate proteoglycans directly mediate a novel endocytic pathway. Using Chinese hamster ovary cells expressing the human syndecan 1 core protein or a chimaeric receptor, FcR-Synd, consisting of the ectodomain of the IgG Fc receptor Ia linked to the transmembrane and cytoplasmic domains of syndecan 1, we previously reported that efficient internalization is triggered by ligand clustering, requires intact actin microfilaments and tyrosine kinases, proceeds with a t1/2 of approx. 1h and is distinct from coated-pit pathways. We have now examined the involvement of cholesterol-rich, detergent-insoluble membrane rafts. On clustering, 125I-labelled IgG bound to FcR-Synd rapidly became insoluble in cold Triton X-100, well before endocytosis. Insolubility of clustered FcR-Synd ligand did not require the syndecan ectodomain, linkage of the cytoplasmic tail to the cytoskeleton, or energy-dependent cellular metabolism. Pretreatment of cells with cyclodextrin to deplete cholesterol from rafts abolished insolubility of the clustered ligand and inhibited endocytosis in a dose-responsive fashion. Similar results were obtained with 125I-labelled lipoprotein lipase bound to authentic cell-surface syndecan. In contrast, the 39kDa receptor-associated protein (RAP), a coated-pit ligand, was more than 80% soluble in cold Triton even after internalization; cellular cholesterol depletion failed to substantially affect the internalization of 125I-RAP. Overall, our results indicate a multi-step endocytic process consisting of ligand binding, clustering, energy-independent lateral movement into detergent-insoluble membrane rafts and finally recruitment of actin and tyrosine kinases to bring the ligands into the cell.
Insulin-induced phospholipase D1 and phospholipase D2 activity in human embryonic kidney-293 cells mediated by the phospholipase Cγ and protein kinase Cα signalling cascade
Authors: SLAABY Rita , DU Guangwei , ALTSHULLER Yelena M. , FROHMAN Michael A. , SEEDORF Klaus
Published: August 2015

Phospholipase D (PLD)1 is quiescent in vitro and in vivo until stimulated by classical protein kinase C (PKC) isoforms, ADP-ribosylation factor or Rho family members. By contrast, PLD2 has high basal activity, and the mechanisms involved in agonist-induced activation of PLD2 are poorly understood. Using transiently transfected human embryonic kidney (HEK)-293 cells as a model system, we report in the present study that PLD2 overexpressed in HEK-293 cells exhibits regulatory properties similar to PLD1 when stimulated in response to insulin and phorbol ester. Co-expression of PLD1 or PLD2 with PKCα results in constitutive activation of both PLD isoforms, which cannot be further stimulated by insulin. Co-expression of PLD1 with phospholipase C (PLC)γ has the same effect, while co-expression of PLD2 with PLCγ allows PLD2 activity to be stimulated in an insulin-dependent manner. The PKC-specific inhibitors bisindolylmaleimide and Gö 6976 abolish insulin-induced PLD2 activation in HEK-293 cells co-expressing the insulin receptor, PLCγ and PLD2, confirming that not only PLD1, but PLD2 as well, is regulated in a PKC-dependent manner. Finally, we provide evidence that PKCα is constitutively associated with PLD2. In summary, we demonstrate that insulin treatment results in activation of both PLD1 and PLD2 in appropriate cell types when the appropriate upstream intermediate signalling components, i.e. PKCα and PLCγ, are expressed at sufficient levels.
Platelet αIIb-β3 integrin engagement induces the tyrosine phosphorylation of Cbl and its association with phosphoinositide 3-kinase and Syk
Authors: SACI Abdelhafid , RENDU Francine , BACHELOT-LOZA Christilla
Published: August 2015

Agonist-induced platelet activation triggers ‘inside-out’signalling which activates αIIb-β3, the most abundant integrin in platelet membranes. The engagement of activated αIIb-β3 integrin by linking fibrinogen is necessary for platelet aggregation, and this induces subsequent outside-in signalling, which enhances platelet activation. Here we studied the involvement of Cbl during αIIb-β3-integrin-mediated signal transduction. During thrombin-induced platelet activation, Cbl was tyrosine phosphorylated, and phosphoinositide 3-kinase (PI 3-kinase) activity measured in Cbl immunoprecipitates was increased. Both Cbl phosphorylation and its association with PI 3-kinase were dependent on αIIb-β3 engagement by linking fibrinogen. The P256 and anti-LIBS6 (ligand-induced binding site 6) antibodies, which activate platelets directly through αIIb-β3, induced Cbl phosphorylation and increased the PI 3-kinase activity associated with Cbl. Both thrombin and antibodies to αIIb-β3 induced association of Cbl with the tyrosine kinase, Syk. Experiments performed with inhibitors of tyrosine kinases indicated that both Src-family kinases and Syk contribute to phosphorylation of Cbl and its consequent association with PI 3-kinase. The results show that, following integrin αIIb-β3 engagement, Cbl is tyrosine phosphorylated, recruits PI 3-kinase to this integrin signalling pathway and possibly enhances PI 3-kinase activity, downstream of Src-family tyrosine kinases and Syk activation.