bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of neuropeptide Y gene expression by brain-derived neurotrophic factor requires both the phospholipase Cγ and Shc binding sites on its receptor, TrkB
Authors: WILLIAMS Alan G. , HARGREAVES Andrew C. , GUNN-MOORE Frank J. , TAVARÉ Jeremy M.
Published: August 2015

In PC12 cells, it has been previously reported that nerve growth factor stimulates neuropeptide Y (NPY) gene expression. In the current study we examined the signalling pathways involved in this effect by transiently expressing in PC12 cells the receptor (TrkB) for the related neurotrophin, brain-derived neurotrophic factor (BDNF). BDNF caused a 3-fold induction of luciferase expression from a transiently co-transfected plasmid possessing the firefly luciferase gene under the control of the NPY promoter. This effect of BDNF was completely blocked by either a Y484F mutation in TrkB (which blocks high-affinity Shc binding to TrkB) or by a Y785F substitution [which blocks the binding, phosphorylation and activation of phospholipase Cγ (PLCγ)]. Activation of the NPY promoter by neurotrophin-3 in PC12 cells overexpressing TrkC was also completely blocked by a naturally occurring kinase insert which prevents the high-affinity binding of Shc and PLCγ. NPY promoter activation by BDNF was blocked by PD98059, suggesting a role for mitogen-activated protein kinase (MAP kinase). Stimulation of NPY gene expression by PMA, but not by BDNF, was blocked by Ro-31-8220, a protein kinase C inhibitor, excluding a role for this serine/threonine protein kinase in the effect of BDNF. In addition, BDNF did not cause an elevation in cytosolic Ca2+ concentration. Taken together, our results suggest that stimulation of the NPY promoter by BDNF requires the simultaneous activation of two distinct pathways; one involves Shc and MAP kinase, and the other appears to be PLCγ-independent but requires an intact tyrosine-785 on TrkB and so may involve an effector of TrkB signalling that remains to be identified.
4-Aminobutyrate (GABA) transporters from the amine-polyamine-choline superfamily: substrate specificity and ligand recognition profile of the 4-aminobutyrate permease from Bacillus subtilis
Authors: BRECHTEL Casey E. , KING Steven C.
Published: August 2015

A previous study [Ferson, Wray and Fisher (1996) Mol. Microbiol. 22, 693–701] has shown that transposon-mediated disruption of a protein 47% identical to the Escherichia coli GABA (4-aminobutyrate) transporter abolishes the ability of nitrogen-limited culture conditions to induce expression of a GABA transport activity in Bacillus subtilis. Here it is demonstrated directly that the B. subtilis GABA permease (gabP) gene can complement the transport defect in the gabP-negative E. colistrain. Unexpectedly, the ligand-recognition profile of the B. subtilis GabP was found to differ substantially from that of the highly homologous E. coli GabP. Unlike the E. coli GabP, the B. subtilis GabP: (i) exhibits approx. equal preference for the 3-carbon (β-alanine, Km = 9.6 µM) and the 4-carbon (GABA, Km = 37 µM) amino acids, and (ii) resists inhibition by bulky, conformationally constrained compounds (e.g. nipecotic acid, guvacine), which are active against GABA transporters from brain. The present study shows additionally that the B. subtilis GabP can translocate several open-chain GABA analogues (3-aminobutyrate, 3-aminopropanoate, cis-4-aminobutenoate) across the membrane via counterflow against [3H]GABA. Thus, consistent with the idea that the ligand-recognition domain of the B. subtilis GabP is less spacious than that of the close homologue from E. coli, the former exhibits more stringent requirements than the latter for substrate recognition and translocation. These distinct functional characteristics of the E. coli and B. subtilis GABA transporters provide a basis by which to identify ligand-recognition domains within the amine-polyamine-choline transporter superfamily.
Linear and cyclic peptides as substrates and modulators of P-glycoprotein: peptide binding and effects on drug transport and accumulation
Authors: SHAROM Frances J. , LU Peihua , LIU Ronghua , YU Xiaohong
Published: August 2015

One cause of multidrug resistance (MDR) in human cancers is the overexpression of the P-glycoprotein multidrug transporter, a member of the ABC superfamily of membrane proteins. Natural products and chemotherapeutic drugs are pumped out of the cell by P-glycoprotein in an ATP-dependent fashion. There is growing evidence that many hydrophobic peptides are also P-glycoprotein substrates. With the use of a fluorescence-quenching assay, we have shown that some linear and cyclic hydrophobic peptides interact with P-glycoprotein, whereas others do not. The measured values of the quenching constant, Kq, for interaction of peptides with P-glycoprotein ranged from 200 nM for cyclosporine A to 138 µM for the tripeptide N-acetyl-leucyl-leucyl-norleucinal. Peptides that interacted with P-glycoprotein in the fluorescence assay also blocked colchicine transport into plasma membrane vesicles from MDR cells. The values of Dm, the peptide concentration causing 50% inhibition of drug uptake, were highly correlated with the values of Kq, over three orders of magnitude. The P-glycoprotein ATPase stimulation/inhibition profile of the peptides was not helpful in making a quantitative assessment of the ability of a peptide to interact with P-glycoprotein or to block drug transport. Some hydrophobic peptides were able to restore accumulation in MDR cells of the chemotherapeutic drug daunorubicin and the fluorescent dye rhodamine 123 to the levels observed in the drug-sensitive parent. Peptides that interacted with P-glycoprotein also displayed a relatively low overall toxicity to intact MDR cells, and inhibited drug transport at concentrations below the toxic range. Hydrophobic peptides should be given serious consideration for development as clinical chemosensitizing agents.
Herbicide-resistant forms of Arabidopsis thaliana acetohydroxyacid synthase: characterization of the catalytic properties and sensitivity to inhibitors of four defined mutants
Authors: CHANG Alan K. , DUGGLEBY Ronald G.
Published: August 2015

Acetohydroxyacid synthase (AHAS) catalyses the first step in the synthesis of the branched-chain amino acids and is the target of several classes of herbicides. Four mutants (A122V, W574S, W574L and S653N) of the AHAS gene from Arabidopsis thaliana were constructed, expressed in Escherichia coli, and the enzymes were purified. Each mutant form and wild-type was characterized with respect to its catalytic properties and sensitivity to nine herbicides. Each enzyme had a pH optimum near 7.5. The specific activity varied from 13% (A122V) to 131% (W574L) of the wild-type and the Km for pyruvate of the mutants was similar to the wild-type, except for W574L where it was five-fold higher. The activation by cofactors (FAD, Mg2+ and thiamine diphosphate) was examined. A122V showed reduced affinity for all three cofactors, whereas S653N bound FAD more strongly than wild-type AHAS. Six sulphonylurea herbicides inhibited A122V to a similar degree as the wild-type but S653N showed a somewhat greater reduction in sensitivity to these compounds. In contrast, the W574 mutants were insensitive to these sulphonylureas, with increases in the Kiapp (apparent inhibition constant) of several hundred fold. All four mutants were resistant to three imidazolinone herbicides with decreases in sensitivity ranging from 100-fold to more than 1000-fold.
Molecular cloning and characterization of Ca2+-dependent inducible nitric oxide synthase from guinea-pig lung
Authors: SHIRATO Manabu , SAKAMOTO Tohru , UCHIDA Yoshiyuki , NOMURA Akihiro , ISHII Yukio , IIJIMA Hiroaki , GOTO Yukio , HASEGAWA Shizuo
Published: August 2015

We have isolated a full-length cDNA for an inducible nitric oxide synthase (iNOS) from guinea-pig lung. The cDNA has a 3447 bp open reading frame encoding 1149 amino acid residues. The deduced amino acid sequence is approx. 80% identical with iNOS of human epithelial cells and murine macrophages. Consensus recognition sites for cofactors are highly conserved. COS cell lysate transfected with the guinea-pig iNOS shows significant levels of nitric oxide synthase (NOS) activity, and this is inhibited by 79% by chelation of Ca2+ ions. The NOS activity is restored in a concentration-dependent manner by increasing the free Ca2+ level. The NOS activity is also inhibited by trifluoperazine, a calmodulin antagonist, which suggests that the Ca2+ dependence is due to Ca2+-dependent calmodulin binding to the enzyme. Northern blot analysis reveals that the cloned iNOS mRNA is expressed in the lung and the colon in normal guinea pigs. Stimulation in vivo by lipopolysaccharide induces the expression of iNOS in the kidney, the spleen and the colon, but in the lung the same stimulation decreases its expression. These results suggest that the cloned guinea-pig iNOS is distinct in characteristics and expression from previously described iNOS forms.
Thioltransferase activity of bovine lens glutathione S-transferase
Authors: MONTE Massimo DAL , CECCONI Ilaria , BUONO Francesca , VILARDO Pier Giuseppe , CORSO Antonella DEL , MURA Umberto
Published: August 2015

A Mu-class glutathione S-transferase purified to electrophoretic homogeneity from bovine lens displayed thioltransferase activity, catalysing the transthiolation reaction between GSH and hydroxyethyldisulphide. The thiol-transfer reaction is composed of two steps, the formation of GSSG occurring through the generation of an intermediate mixed disulphide between GSH and the target disulphide. Unlike glutaredoxin, which is only able to catalyse the second step of the transthiolation process, glutathioneS-transferase catalyses both steps of the reaction. Data are presented showing that bovine lens glutathione S-transferase and rat liver glutaredoxin, which was used as a thioltransferase enzyme model, can operate in synergy to catalyse the GSH-dependent reduction of hydroxyethyldisulphide.
Transcriptional activation of the haem oxygenase-1 gene by cGMP via a cAMP response element/activator protein-1 element in primary cultures of rat hepatocytes
Authors: IMMENSCHUH Stephan , HINKE Vera , OHLMANN Andreas , GIFHORN-KATZ Susanne , KATZ Norbert , JUNGERMANN Kurt , KIETZMANN Thomas
Published: August 2015

The expression of the rate-limiting enzyme of haem degradation, haem oxygenase-1 (HO-1), can be induced by various stimuli, including lipopolysaccharide, tumour necrosis factor α and NO. The NO signal can be transmitted by cGMP, therefore this study was aimed at testing the activation of the HO-1 gene by cGMP. In primary cultures of rat hepatocytes, both HO-1 mRNA and protein were induced by the NO donor sodium nitroprusside and 8-bromo-cGMP. The HO-1 mRNA induction by cGMP was prevented by the specific protein kinase G inhibitor KT5823. The cGMP-dependent HO-1 mRNA induction was dose-dependent and transcriptionally regulated, as determined by studies with actinomycin D and a nuclear run-on assay. Cycloheximide lowered the cGMP-dependent induction of HO-1 mRNA to about one half. Luciferase reporter constructs driven by about 800 bp of the 5´-flanking region of the rat HO-1 gene were transiently transfected into primary rat hepatocytes; 8-bromo-cGMP caused a 6-fold induction, which was obliterated by deletion and mutation of the cAMP response element/activator protein-1 (CRE/AP-1) (-665/-654) site. Thus HO-1 induction by cGMP appears to be stimulated by the protein kinase G pathway and may be mediated mainly via a CRE/AP-1 element in the rat HO-1 promoter.
Temperature-induced conformational changes in prosomatostatin-II: implications for processing
Authors: MITRA Joydeep , TANG Xue-jun , ALMO Steven C. , SHIELDS Dennis
Published: August 2015

Somatostatin (SRIF) is a 14-residue peptide hormone synthesized in the hypothalamus and pancreatic islets. SRIF-14 and an N-terminally extended form, SRIF-28, are generated by the proteolytic processing of an approx. 102-residue precursor prosomatostatin (proSRIF) at a single set of paired basic residues (Arg-Lys) and at a monobasic (Arg) site respectively. Previous work in our laboratory demonstrated that the propeptide of SRIF mediates intracellular sorting; we suggested that this information resides in the prohormone structure. To identify putative sorting domains we have investigated structural features of recombinant anglerfish proSRIF-II purified from Escherichia coli. Two species of proSRIF-II were obtained: a monomeric form and a disulphide-linked dimer. CD analyses revealed that monomeric proSRIF-II lacks appreciable periodic secondary structure; however, on slow heating (2 °C/min) and cooling, it assumed a predominantly α-helical conformation. When subjected to a second heating-and-cooling cycle, the α-helical conformation was maintained. In contrast, the dimeric form of proSRIF-II was predominantly α-helical and its helicity did not increase in response to heating and recooling. Our results suggest that proSRIF-II might exist in several different folding intermediate states.
5-Octadecenoic acid: evidence for a novel type of fatty acid modification in schistosomes
Authors: BROUWERS Jos F. H. M. , VERSLUIS Cornelis , GOLDE Lambert M. G. van , TIELENS Aloysius G. M.
Published: August 2015

The lipid metabolism of schistosomes is characterized by several intriguing adaptations to a parasitic way of living. The surface of the parasite consists of two closely apposed phospholipid bilayers, a structure unique to blood flukes. Schistosomes do not synthesize fatty acids de novo, but are able to modify fatty acids, which they obtain from the host, by chain elongation. Here we present evidence that schistosomes are capable of another type of fatty acid modification, resulting in the formation of 5-octadecenoic acid [C18:1(5)]. This highly unusual fatty acid, which is absent in the blood of the host, was shown to be almost exclusively located in the outer membrane complex of the schistosome. Within these membranes, it was almost exclusively present in one molecular phospholipid species, 1-palmitoyl-2,5-octadecenoyl phosphatidylcholine [C16:0–18:1(5)PtdCho]. Apart from dipalmitoyl phosphatidylcholine, this was the most abundant phosphatidylcholine species in the outer membrane complex. The specific synthesis by the schistosome of C18:1(5) and the highly specific localization of this fatty acid to the tegumental membranes suggest an important tegument-mediated role for this lipid.
Muscle-specific mRNA isoform encodes a protein composed mainly of the N-terminal 175 residues of type 2 Ins(1,4,5)P3 receptor
Authors: FUTATSUGI Akira , KUWAJIMA Goro , MIKOSHIBA Katsuhiko
Published: August 2015

We have found a novel isoform of the mouse type 2 Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] mRNA by reverse transcriptase-mediated PCR analysis. The novel isoform, which was expressed specifically in skeletal muscle and heart, was generated by the inclusion of a novel exon. As this exon contains a stop codon, the isoform encodes a putative protein (designated TIPR) consisting of 175 acid residues of the type 2 Ins(1,4,5)P3R and the following six residues derived from this exon. We transfected the cDNA of this isoform into COS-7 cells; these cells expressed a 24 kDa protein that was recognized by an antibody against TIPR produced in Escherichia coli. The isoform encoding TIPR was also found in human skeletal muscle and heart. The N-terminal region of Ins(1,4,5)P3R is suggested to have a role in ligand binding and to interact with the C-terminal channel domain of Ins(1,4,5)P3R itself. TIPR might regulate the Ins(1,4,5)P3 signal pathway in both muscles.
Plant mitochondrial pyruvate dehydrogenase complex: purification and identification of catalytic components in potato
Authors: MILLAR A. Harvey , KNORPP Carina , LEAVER Christopher J. , HILL Steven A.
Published: August 2015

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) tuber mitochondria was purified 40-fold to a specific activity of 5.60 µmol/min per mg of protein. The activity of the complex depended on pyruvate, divalent cations, NAD+ and CoA and was competitively inhibited by both NADH and acetyl-CoA. SDS/PAGE revealed the complex consisted of seven polypeptide bands with apparent molecular masses of 78, 60, 58, 55, 43, 41 and 37 kDa. N-terminal sequencing revealed that the 78 kDa protein was dihydrolipoamide transacetylase (E2), the 58 kDa protein was dihydrolipoamide dehydrogenase (E3), the 43 and 41 kDa proteins were α subunits of pyruvate dehydrogenase, and the 37 kDa protein was the β subunit of pyruvate dehydrogenase. N-terminal sequencing of the 55 kDa protein band yielded two protein sequences: one was another E3; the other was similar to the sequence of E2 from plant and yeast sources but was distinctly different from the sequence of the 78 kDa protein. Incubation of the mPDC with [2-14C]pyruvate resulted in the acetylation of both the 78 and 55 kDa proteins.
Mixed-lineage kinase 2-SH3 domain binds dynamin and greatly enhances activation of GTPase by phospholipid
Authors: RASMUSSEN Richele K. , RUSAK Jenny , PRICE Gareth , ROBINSON Phillip J. , SIMPSON Richard J. , DOROW Donna S.
Published: August 2015

Mixed-lineage kinase 2 (MLK2) is a cytoplasmic protein kinase expressed at high levels in mammalian brain. The MLK2 structure is composed of a Src homology 3 (SH3) domain, two leucine zippers, a basic motif, a Cdc42/Rac interactive binding motif and a large C-terminal domain rich in proline, serine and threonine residues. To begin to define the role of MLK2 in mammalian brain, we used an MLK2-SH3 domain–glutathione S-transferase fusion protein (GST–MLK2-SH3) to isolate MLK2-binding proteins from rat brain extract. This analysis revealed that the major MLK2-SH3-domain-binding protein in rat brain is the GTPase dynamin. By using two different forms of the dynamin proline-rich domain as affinity ligands, the binding site for MLK2-SH3 was mapped to the C-terminal region of dynamin between residues 832 and 864. In GTPase assays, the addition of MLK2-SH3 stimulated the activity of purified dynamin I by 3-fold over the basal level, whereas the addition of a known dynamin activator, phosphatidylserine (PtdSer), stimulated a 6-fold increase. When MLK2-SH3 was added to the assay together with PtdSer, however, dynamin GTPase activity accelerated by more than 23-fold over basal level. An MLK2 mutant (MLK2-W59A-SH3), with alanine replacing a conserved tryptophan residue in the SH3 domain consensus motif, had no effect on dynamin activity, either alone or in the presence of PtdSer. In the same assay the SH3 domain from the regulatory subunit of phosphatidylinositol 3´-kinase stimulated a similar synergistic acceleration of dynamin GTPase activity in the presence of PtdSer. These results suggest that synergy between phospholipid and SH3 domain binding might be a general mechanism for the regulation of GTP hydrolysis by dynamin.
Characterization of the hypertonically induced tyrosine phosphorylation of erythrocyte band 3
Authors: MINETTI Giampaolo , SEPPI Claudio , CIANA Annarita , BALDUINI Cesare , LOW Philip S. , BROVELLI Augusta
Published: August 2015

Human erythrocyte band 3 becomes rapidly phosphorylated on tyrosine residues after exposure of erythrocytes to hypertonic conditions. The driving force for this phosphorylation reaction seems to be a decrease in cell volume, because (1) changes in band 3 phosphotyrosine content accurately track repeated changes in erythrocyte volume through several cycles of swelling and shrinking; (2) the level of band 3 phosphorylation is independent of the osmolyte employed but strongly sensitive to the magnitude of cell shrinkage; and (3) exposure of erythrocytes to hypertonic buffers under conditions in which intracellular osmolarity increases but volume does not change (nystatin-treated cells) does not promote an increase in tyrosine phosphorylation. We hypothesize that shrinkage-induced tyrosine phosphorylation results either from an excluded-volume effect, stemming from an increase in intracellular crowding, or from changes in membrane curvature that accompany the decrease in cell volume. Although the net phosphorylation state of band 3 is shown to be due to a delicate balance between a constitutively active tyrosine phosphatase and constitutively active tyrosine kinase, the increase in phosphorylation during cell shrinkage was demonstrated to derive specifically from an activation of the latter. Further, a peculiar inhibition pattern of the volume-sensitive erythrocyte tyrosine kinase that matched that of p72syk, a tyrosine kinase already known to associate with band 3 in vivo, suggested the involvement of this kinase in the volume-dependent response.
Glucose induces expression of stearoyl-CoA desaturase in 3T3-L1 adipocytes
Authors: JONES Brynn H. , STANDRIDGE Melissa K. , CLAYCOMBE Kate J. , SMITH Pamela J. , MOUSTAÏD-MOUSSA Naïma
Published: August 2015

Stearoyl-CoA desaturase (SCD; EC 1.14.99.5) is a key enzyme in the synthesis of polyunsaturated fatty acids. Liver and adipose tissue are the predominant sites of SCD expression. Regulation of hepatic SCD by various nutritional and hormonal factors, such as insulin, dietary carbohydrates and polyunsaturated fatty acids, has been well documented. Little is known, however, about adipocyte SCD regulation despite high levels of SCD activity in adipose tissue. The present study was designed to investigate SCD regulation in adipocytes by examining the effects of glucose and insulin on SCD expression. We report here that glucose availability directly increased SCD gene transcription in 3T3-L1 adipocytes. This response was independent of insulin, and insulin alone in the absence of glucose had no effect on SCD mRNA levels. SCD thus represents a novel model in which to investigate the mechanisms of direct regulation of gene expression by glucose in adipose cells.
Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane
Authors: ILANGUMARAN Subburaj , HOESSLI Daniel C.
Published: August 2015

Sphingolipid microdomains are thought to result from the organization of plasma membrane sphingolipids and cholesterol into a liquid ordered phase, wherein the glycosylphosphatidylinositol (GPI)-anchored proteins are enriched. These domains, resistant to extraction by cold Triton X-100, can be isolated as buoyant membrane complexes (detergent-resistant membranes) in isopycnic density gradients. Here the effects of methyl-β-cyclodextrin (MBCD), a specific cholesterol-binding agent that neither binds nor inserts into the plasma membrane, were investigated on the sphingolipid microdomains of lymphocytes. MBCD released substantial quantities of GPI-anchored Thy-1 and glycosphingolipid GM1, and also other surface proteins including CD45, and intracellular Lck and Fyn kinases. From endothelial cells, MBCD released GPI-anchored CD59, and CD44, but only a negligible amount of caveolin. Most MBCD-released Thy-1 and CD59 were not sedimentable and thus differed from Thy-1 released by membrane-active cholesterol-binding agents such as saponin and streptolysin O, or Triton X-100. Unlike that released by Triton X-100, only part of the Thy-1 molecules released by MBCD was buoyant in density gradients and co-isolated with GM1. Finally, treatment of Triton X-100-isolated detergent-resistant membranes with MBCD extracted most of the cholesterol without affecting the buoyant properties of Thy-1 or GM1. We suggest that (1) MBCD preferentially extracts cholesterol from outside, rather than within the sphingolipid microdomains and (2) this partly solubilizes GPI-anchored and transmembrane proteins from the glycerophospholipid-rich membrane and releases sphingolipid microdomains in both vesicular and non-vesicular form.
Mechanism of activation of the gastric aspartic proteinases: pepsinogen, progastricsin and prochymosin
Authors: RICHTER Catherine , TANAKA Takuji , YADA Rickey Y.
Published: August 2015

The gastric aspartic proteinases (pepsin A, pepsin B, gastricsin and chymosin) are synthesized in the gastric mucosa as inactive precursors, known as zymogens. The gastric zymogens each contain a prosegment (i.e. additional residues at the N-terminus of the active enzyme) that serves to stabilize the inactive form and prevent entry of the substrate to the active site. Upon ingestion of food, each of the zymogens is released into the gastric lumen and undergoes conversion into active enzyme in the acidic gastric juice. This activation reaction is initiated by the disruption of electrostatic interactions between the prosegment and the active enzyme moiety at acidic pH values. The conversion of the zymogen into its active form is a complex process, involving a series of conformational changes and bond cleavage steps that lead to the unveiling of the active site and ultimately the removal and dissociation of the prosegment from the active centre of the enzyme. During this activation reaction, both the prosegment and the active enzyme undergo changes in conformation, and the proteolytic cleavage of the prosegment can occur in one or more steps by either an intra- or inter-molecular reaction. This variability in the mechanism of proteolysis appears to be attributable in part to the structure of the prosegment. Because of the differences in the activation mechanisms among the four types of gastric zymogens and between species of the same zymogen type, no single model of activation can be proposed. The mechanism of activation of the gastric aspartic proteinases and the contribution of the prosegment to this mechanism are discussed, along with future directions for research.
Mutant DNA polymerase δ from thermosensitive Schizosaccharomyces pombe strains display reduced stimulation by proliferating cell nuclear antigen
Authors: PERDERISET Mylène , MAGA Giovanni , PIARD Karine , FRANCESCONI Stefania , TRATNER Isabelle , HÜBSCHER Ulrich , BALDACCI Giuseppe
Published: August 2015

We have isolated and characterized DNA polymerase δ (pol δ) from two thermosensitive Schizosaccharomyces pombe strains, polδts1 and polδts3, mutated in two different evolutionarily conserved domains of the catalytic subunit. At the restrictive temperature of 37 °C polδts1 and polδts3 mutant strains arrest growth in the S phase of the cell cycle. We show that at low levels of primer ends, in vitro stimulation by proliferating cell nuclear antigen (PCNA) of mutant enzymes is lower than stimulation of wild-type pol δ. Affinity for primer (3´-OH) ends and processivity of mutant enzymes do not appear different from wild-type pol δ. In contrast, Vmax values are lower than the wild-type value. The major in vitro defect appears to be decreased stimulation of mutant enzymes by PCNA, resulting in reduced velocity of DNA synthesis. In addition, ts1 pol δ is not stimulated by low PCNA concentration at 37 °C, although low concentrations stimulate activity at 25 °C, suggesting that this thermolability at low levels of primer ends could be its critical defect in vivo. Thus, both ts1 and ts3 pol δ mutations are located in regions of the catalytic subunit that seem necessary, directly or indirectly, for its efficient interaction with PCNA.
Reversible penetration of α-glutathione S-transferase into biological membranes revealed by photosensitized labelling in situ
Authors: MEREZHINSKAYA Natasha , KUIJPERS Gemma A. J. , RAVIV Yossef
Published: August 2015

Fluorescent lipid analogue 3,3´-dioctadecyloxacarbocyanine incorporated into biological membranes was used to induce photoactivation of a hydrophobic probe 5-[125I]iodonaphthyl-1-azide (125INA) by energy transfer and to thereby confine subsequent radiolabelling of proteins to the lipid bilayer. This approach was applied in bovine chromaffin cells to discover cytosolic proteins that reversibly penetrate into membrane domains. α-Glutathione S-transferase (α-GST) was identified as the only labelled protein in bovine chromaffin-cell cytosol, indicating that it inserts reversibly into the membrane lipid bilayer. The selectivity of the labelling towards the lipid bilayer is demonstrated by showing that influenza virus haemagglutinin becomes labelled by 125INA only after the insertion of this protein into the target membrane. The molar 125INA:protein ratio was used as a quantitative criterion for evaluation of the penetration of proteins into the membrane lipid bilayer. This ratio was calculated for four integral membrane proteins and four soluble proteins that interact with biological membranes. The values for four integral membrane proteins (erythrocyte anion transporter, multidrug transporter gp-170, dopamine transporter and fusion-competent influenza virus haemagglutinin) were 1, 8, 2 and 2, respectively, whereas for soluble proteins (annexin VII, protein kinase C, BSA and influenza virus haemagglutinin) the values were 0.002, 0, 0.002 and 0.02, respectively. The molar ratio for α-GST was found to be 1, compatible with the values obtained for integral membrane proteins.
Inhibition of the intrinsic NAD+ glycohydrolase activity of CD38 by carbocyclic NAD analogues
Authors: WALL Katherine A. , KLIS Mariola , KORNET John , COYLE Donna , AMÉ Jean-Christophe , JACOBSON Myron K. , SLAMA James T.
Published: August 2015

Carba-NAD and pseudocarba-NAD are carbocyclic analogues of NAD+ in which a 2,3-dihydroxycyclopentane methanol replaces the β-d-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ [Slama and Simmons (1988) Biochemistry 27, 183–193]. These carbocyclic NAD+ analogues, related to each other as diastereomers, have been tested as inhibitors of the intrinsic NAD+ glycohydrolase activity of human CD38, dog spleen NAD+ glycohydrolase, mouse CD38 and Aplysia californicacADP-ribose synthetase. Pseudocarba-NAD, the carbocyclic dinucleotide in which l-2,3-dihydroxycyclopentane methanol replaces the d-ribose of the nicotinamide riboside moiety of NAD+, was found to be the more potent inhibitor. Pseudocarba-NAD was shown to inhibit the intrinsic NAD+ glycohydrolase activity of human CD38 competitively, with Ki = 148 µM determined for the recombinant extracellular protein domain and Ki = 180 µM determined for the native protein expressed as a cell-surface enzyme on cultured Jurkat cells. Pseudocarba-NAD was shown to be a non-competitive inhibitor of the purified dog spleen NAD+ glycohydrolase, with Kis = 47 µM and Kii = 198 µM. Neither pseudocarba-NAD nor carba-NAD inhibited mouse CD38 or Aplysia californicacADP-ribose synthetase significantly at concentrations up to 1 mM. The results underscore significant species differences in the sensitivity of these enzymes to inhibition, and indicate that pseudocarba-NAD will be useful as an inhibitor of the enzymic activity of human but not mouse CD38 in studies using cultured cells.
Characterization of the human proline/arginine-rich end leucine-rich repeat protein (PRELP) gene promoter and identification of a repressor element
Authors: GROVER Judy , ROUGHLEY Peter J.
Published: August 2015

The 5´-flanking region of the human proline/arginine-rich end leucine-rich repeat protein (PRELP) gene has been characterized for both promoter and repressor activity by using a variety of reporter gene constructs and transient transfection into chondrocytes or fibroblasts. The human PRELP gene lacks a TATA box, and in its absence a Sp1-binding site residing 29 bp upstream of the transcription start site is essential for initiating gene expression. In contrast, an Ets-binding site residing 497 bp upstream of the transcription start site can lead to the repression of gene expression. The analysis of nuclear proteins by gel retardation studies with the repressor element identified a common protein, presumably an Ets family member, present in neonatal chondrocytes and skin fibroblasts that do not express the PRELP gene. The factor was not detected in nuclear protein preparations from adult chondrocytes in which the PRELP gene is expressed.
The linear C-terminal regions of epidermal growth factor (EGF) and transforming growth factor-α bind to different epitopes on the human EGF receptor
Authors: LENFERINK Anne E. G. , De ROOS Albert D. G. , VUGT Marianne J. H. Van , De POLL Monique L. M. Van , ZOELEN Everardus J. J. Van
Published: August 2015

Epidermal growth factor (EGF) and transforming growth factor-α (TGFα) bind with similar affinities in a competitive fashion to the human EGF receptor, and basically induce similar mitogenic responses. In spite of the fact that EGF and TGFα are structurally alike, it is still not clear if the two growth factors bind the receptor in an identical manner. The observation that the 13A9 antibody blocks binding of TGFα, but not that of EGF, to the human EGF receptor [Winkler, O'Connor, Winget and Fendly (1989) Biochemistry 28, 6373–6378] suggests that their binding characteristics are not identical. In the present study we have made use of a set of EGF/TGFα chimaeric molecules to show that the 13A9 antibody blocks receptor binding of ligands with TGFα sequences, but not of ligands with EGF sequences, in their C-terminal linear regions. Using HaCaT human keratinocyte cells in culture, it was determined that ligands that are able to bind the EGF receptor in the presence of 13A9 are also able to induce calcium release from intracellular stores in these cells, indicating that these ligands have the ability to activate the EGF receptor in the presence of the antibody. From these data it is concluded that the flexible C-terminal linear domains of EGF and TGFα bind to separate sequences on the EGF receptor, such that the binding domain of TGFα, but not that of EGF, overlaps with the binding epitope of the 13A9 antibody.
Human mitogen-activated protein kinase kinase kinase mediates the stress-induced activation of mitogen-activated protein kinase cascades
Authors: CHAN-HUI Po-Ying , WEAVER Robert
Published: August 2015

The mitogen-activated protein kinase (MAPK) cascades represent one of the important signalling mechanisms in response to environmental stimuli. We report the identification of a human MAPK kinase kinase, MAPKKK4, via sequence similarity with other MAPKKKs. When truncated MAPKKK4 (ΔMAPKKK4) was overexpressed in HEK293 cells, it was constitutively active and induced the activation of endogenous p38α, c-Jun N-terminal kinase (JNK)1/2 and extracellular signal-regulated kinase (ERK)2 in vivo. Kinase-inactive ΔMAPKKK4 partly inhibited the activation of p38α, JNK1/2 and ERK2 induced by stress, tumour necrosis factor α or epidermal growth factor, suggesting that MAPKKK4 might be physiologically involved in all three MAPK cascades. Co-expressed MAP kinase kinase (MKK)-1, MKK-4, MKK-3 and MKK-6 were activated in vivo by ΔMAPKKK4. All of the above MKKs purified from Escherichia coli were phosphorylated and activated by ΔMAPKKK4 immunoprecipitates in vitro. When expressed by lower plasmid doses, ΔMAPKKK4 preferentially activated MKK-3 and p38α in vivo. Overexpression of ΔMAPKKK4 did not activate the NF-κB pathway. Immunoprecipitation of endogenous MAPKKK4 by specific antibodies showed that MAPKKK4 was activated after the treatment of K562 cells with various stress conditions. As a broadly distributed kinase, MAPKKK4 might serve as a stress responder. MAPKKK4 is 91% identical with the recently described murine MEKK-4β and might be its human homologue. It is also identical with the recently cloned human MAP three kinase 1 except for the lack of an internal sequence homologous to the murine MEKK-4α isoform. Differences in the reported functional activities of the three kinases are discussed.
A dimeric form of prothrombin on membrane surfaces
Authors: ANDERSON Peter J.
Published: August 2015

Blood coagulation requires the conversion of zymogens to active enzymes. These reactions are facilitated by Ca2+-dependent protein binding to membrane surfaces containing anionic phospholipids. Here it is shown that only in the presence of both Ca2+ and phospholipid vesicles composed of phosphatidylcholine and phosphatidylserine can a prothrombin dimer be chemically cross-linked. A cross-linker containing evenly spaced reactive groups was prepared by activating the carboxy groups of a ten-residue glutamic acid peptide and allowed to react with physiological concentrations of prothrombin. When Ca2+ and anionic phospholipids were both present during exposure to the cross-linker, it was found that more than 50% of the prothrombin was trapped as a chemically defined dimer with reaction times of the order of 1 min. The dimer yield remained high even when reactions were performed at high phospholipid-to-protein ratios at protein concentrations an order of magnitude less than physiological. Amino acid sequencing of a CNBr peptide produced from the purified dimer localized the cross-link to residues Lys341 and Lys427 of prothrombin. The specificity and high yield under mild conditions of the cross-linking suggest that dimeric membrane bound prothrombin might be a physiologically relevant substrate for the formation of thrombin. Prothrombinase converts this modified protein to an enzyme that catalyses the hydrolysis of a thrombin chromogenic substrate as efficiently as thrombin and is inhibited by a thrombin active-site directed inhibitor, but is a thrombin dimer. The thrombin dimer has impaired activity compared with thrombin with respect to physiological functions requiring binding to exosite I. A model based on the known structure of thrombin is presented that can account for the prothrombin dimer and the properties of the dimeric thrombin formed from it.
Sugars and light/dark exposure trigger differential regulation of ADP-glucose pyrophosphorylase genes in Arabidopsis thaliana (thale cress)
Authors: SOKOLOV Lubomir N. , DÉJARDIN Annabelle , KLECZKOWSKI Leszek A.
Published: August 2015

Expression of four Arabidopsis (thale cress) genes corresponding to the small (ApS) and large subunits (ApL1, ApL2, ApL3) of ADP-glucose pyrophosphorylase (AGPase), a key enzyme of starch biosynthesis, was found to be profoundly and differentially regulated by sugar and light/dark exposures. Transcript levels of both ApL2 and ApL3, and to a lesser extent ApS, increased severalfold upon feeding sucrose or glucose to the detached leaves in the dark, whereas the mRNA content for ApL1 decreased under the same conditions. Glucose was, in general, less effective than sucrose in inducing regulation of AGPase genes, possibly due to observed limitations in its uptake when compared with sucrose uptake by detached leaves. Osmotic agents [sorbitol, poly(ethylene glycol)] had no effect on ApS, ApL2 and ApL3 transcript level, but they did mimic the effect of sucrose on ApL1 gene, suggesting that the latter is regulated by osmotic pressure rather than any particular sugar. For all the genes the sugar effect was closely mimicked by an exposure of the dark-pre-adapted leaves to the light. Under both dark and light conditions, sucrose fed to the detached leaves was found to be rapidly metabolized to hexoses and, to some extent, starch. Starch production reflected most probably an increase in substrate availability for AGPase reaction rather than being due to changes in AGPase protein content, since both the sugar feeding and light exposure had little or no effect on the activity of AGPase or on the levels of its small and large subunit proteins in leaf extracts. The data suggest tight translational or post-translational control, but they may also reflect spatial control of AGPase gene expression within a leaf. The sugar/light-dependent regulation of AGPase gene expression may represent a part of a general cellular response to the availability/allocation of carbohydrates during photosynthesis.
Distinct sites of action of Bcl-2 and Bcl-xL in the ceramide pathway of apoptosis
Authors: EL-ASSAAD Wissal , EL-SABBAN Marwan , AWARAJI Christian , ABBOUSHI Nour , DBAIBO Ghassan S.
Published: August 2015

We studied the inhibition of tumour necrosis factor α (TNFα)- and camptothecin-induced apoptosis by Bcl-2 and Bcl-xL as they relate to the ceramide pathway. Expression of either Bcl-2 or Bcl-xL provided significant protection from the apoptotic effects of TNFα or camptothecin. In contrast to Bcl-2, Bcl-xL overexpression did not protect cells from ceramide-induced apoptosis. On the other hand, Bcl-xL prevented the accumulation of endogenous ceramide in response to TNFα or camptothecin, whereas Bcl-2 showed little effect on ceramide formation. Moreover, Bcl-xL, but not Bcl-2, totally inhibited a caspase-8-like activity in cell lysates stimulated with TNFα. These results identify a different mechanism of action for Bcl-xL compared with Bcl-2 and they demonstrate that Bcl-xL targets a point upstream of ceramide generation, whereas Bcl-2 functions downstream of ceramide in the TNFα- and camptothecin-activated pathways of apoptosis.
Association of 4F2hc with light chains LAT1, LAT2 or y+LAT2 requires different domains
Authors: BRÖER Angelika , FRIEDRICH Björn , WAGNER Carsten A. , FILLON Sophie , GANAPATHY Vadivel , LANG Florian , BRÖER Stefan
Published: August 2015

Heterodimeric amino acid transporters are comprised of a type-II membrane protein named the heavy chain (4F2hc or rBAT) that may associate with a number of different polytopic membrane proteins, called light chains. It is thought that the heavy chain is mainly involved in the trafficking of the complex to the plasma membrane, whereas the transport process itself is catalysed by the light chain. The 4F2heavy chain (4F2hc) associates with at least six different light chains to induce distinct amino acid-transport activites. To test if the light chains are specifically recognized and to identify domains involved in the recognition of light chains, C-terminally truncated mutants of 4F2hc were constructed and co-expressed with the light chains LAT1, LAT2 and y+LAT2. The truncated isoform T1, comprised of only 133 amino acids that form the cytosolic N-terminus and the transmembrane helix, displayed only a slight reduction in its ability to promote LAT1 expression at the membrane surface compared with the 529 amino acid wild-type 4F2hc protein. Co-expression of increasingly larger 4F2hc mutants caused a delayed translocation of LAT1. In contrast to the weak effects of 4F2hc truncations on LAT1 expression, surface expression of LAT2 and y+LAT2 was almost completely lost with all truncated heavy chains. Co-expression of LAT1 together with the other light chains did not result in displacement of LAT2 and y+LAT2. The results suggest that extracellular domains of the heavy chain are responsible mainly for recognition of light chains other than LAT1 and that the extracellular domain ensures proper translocation to the plasma membrane.
Determination of interaction sites of phospholipase D1 for RhoA
Authors: CAI Songmin , EXTON John H.
Published: August 2015

Phospholipase D (PLD) is regulated by many factors, including protein kinase C (PKC) and small G-proteins of the Rho and ADP-ribosylation factor families. Previous studies revealed that the interaction site of human PLD1 for RhoA is located in its C-terminus, but the exact locus has not been determined. The purpose of the present study was to determine the interaction site of rat PLD1 (rPLD1) with RhoA. Selection with phage display of different peptides of rPLD1 confirmed that GTP-bound RhoA interacted with a site in the amino acid sequence 873–1024 at the C-terminus of rPLD1. RhoA also associated with this peptide in a GTP-dependent manner in COS-7 cell lysates and the peptide inhibited RhoA stimulation of PLD activity in membranes from COS-7 cells expressing rPLD1. A series of alanine mutations of non-conserved residues were made in this sequence, and the enzymes were expressed in COS-7 cells and checked for responses to activation of PKC, which interacts with the N-terminus of PLD1, and also to the constitutively active V14RhoA. Mutations in the C-terminus of rPLD1 (K946A, V950A, R955A and K962A) caused partial loss of V14RhoA stimulation, and double mutations (K946A/K962A, K946A/V950A and K962A/V950A) caused an almost total loss. Co-immunoprecipitation studies also showed that the mutated forms of rPLD1 described above failed to bind V14RhoA compared with wild-type rPLD1, whereas rPLD1 with mutations outside the region K946–K962 bound V14RhoA normally. It is concluded that basic amino acids in a restricted C-terminal region of rPLD1 are important for binding of RhoA and its activation of PLD activity.
Isoaspartyl dipeptidase activity of plant-type asparaginases
Authors: HEJAZI Mahdi , PIOTUKH Kirill , MATTOW Jens , DEUTZMANN Rainer , VOLKMER-ENGERT Rudolf , LOCKAU Wolfgang
Published: August 2015

Recombinant plant-type asparaginases from the cyanobacteria Synechocystis sp. PCC (Pasteur culture collection) 6803 and Anabaena sp. PCC 7120, from Escherichia coli and from the plant Arabidopsis thaliana were expressed in E. coli with either an N-terminal or a C-terminal His tag, and purified. Although each of the four enzymes is encoded by a single gene, their mature forms consist of two protein subunits that are generated by autoproteolytic cleavage of the primary translation products at the Gly—Thr bond within the sequence GTI/VG. The enzymes not only deamidated asparagine but also hydrolysed a range of isoaspartyl dipeptides. As various isoaspartyl peptides are known to arise from proteolytic degradation of post-translationally altered proteins containing isoaspartyl residues, and from depolymerization of the cyanobacterial reserve polymer multi-l-arginyl-poly-l-aspartic acid (cyanophycin), plant-type asparaginases may not only function in asparagine catabolism but also in the final steps of protein and cyanophycin degradation. The properties of these enzymes are compared with those of the sequence-related glycosylasparaginases.
Crystal structure of human dehydroepiandrosterone sulphotransferase in complex with substrate
Authors: REHSE Peter H. , ZHOU Ming , LIN Sheng-Xiang
Published: August 2015

Dehydroepiandrosterone sulphotransferase (DHEA-ST) is an enzyme that converts dehydroepiandrosterone (DHEA), and some other steroids, into their sulphonated forms. The enzyme catalyses the sulphonation of DHEA on the 3α-oxygen, with 3′-phosphoadenosine-5′-phosphosulphate contributing the sulphate. The structure of human DHEA-ST in complex with its preferred substrate DHEA has been solved here to 1.99Å using molecular replacement with oestradiol sulphotransferase (37% sequence identity) as a model. Two alternative substrate-binding orientations have been identified. The primary, catalytic, orientation has the DHEA 3α-oxygen and the highly conserved catalytic histidine in nearly identical positions as are seen for the related oestradiol sulphotransferase. The substrate, however, shows rotations of up to 30°, and there is a corresponding rearrangement of the protein loops contributing to the active site. This may also reflect the low identity between the two enzymes. The second orientation penetrates further into the active site and can form a potential hydrogen bond with the desulphonated cofactor 3′,5′-phosphoadenosine (PAP). This second site contains more van der Waal interactions with hydrophobic residues than the catalytic site and may also reflect the substrate-inhibition site. The PAP position was obtained from the previously solved structure of DHEA-ST co-crystallized with PAP. This latter structure, due to the arrangement of loops within the active site and monomer interactions, cannot bind substrate. The results presented here describe details of substrate binding to DHEA-ST and the potential relationship to substrate inhibition.