bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutational analysis of trans-membrane helices M3, M4, M5 and M7 of the fast-twitch Ca2+-ATPase
Authors: ADAMS Phillip , EAST J. Malcolm , LEE Anthony G. , O'CONNOR C. David
Published: August 2015

Mutational analysis of trans-membrane helices M3, M4, M5 and M7 of the Ca2+-ATPase revealed a novel phenotypic variant, M4 [Y295A (the one-letter symbols are used for amino acid residues throughout)], displaying an increased affinity for Pi and decreased affinity for MgATP, while retaining the ability to translocate Ca2+ ions across the endoplasmic reticulum membrane. The properties of this mutant suggest that the E1–E2 equilibrium is shifted towards E2, and indicate a key role for this aromatic residue (Y295) at the end of trans-membrane helix M4. A mutant containing three amino acid residue substitutions at the end of the seventh trans-membrane helix, M7 (F834A, F835A, T837F), showed a complete loss of ATPase activity and a reduced ability to phosphorylate with Pi, although MgATP-initiated phosphorylation was unaffected. The observation that single mutations in this cluster of residues had no effect on Ca2+ transport suggests that correct anchoring of the helix at the lipid–water interface by these aromatic residues is important in the functioning of the ATPase. Mutation of polar residues in helix M3 did not affect inhibition of the ATPase by thapsigargin, thapsivillosin A or t-butyl hydroquinone, suggesting that hydrogen-bonding partners for the essential -OH groups on these inhibitors lie elsewhere in the ATPase.
Genetic engineering in mice: impact on insulin signalling and action
Authors: LAMOTHE Betty , BAUDRY Anne , DESBOIS Pierrette , LAMOTTE Lucianne , BUCCHINI Danielle , MEYTS Pierre DE , JOSHI Rajiv L.
Published: August 2015

The expression of a number of genes encoding key players in insulin signalling and action, including insulin, insulin receptor (IR), downstream signalling molecules such as insulin receptor substrate-1 (IRS-1) and IRS-2, glucose transporters (GLUT4, GLUT2) and important metabolic enzymes such as glucokinase, has now been altered in transgenic or knockout mice. Such mice presented with phenotypes ranging from mild defects, revealing complementarity between key molecules or pathways, to severe diabetes with ketoacidosis and early postnatal death. Insulin action could also be improved by overproduction of proteins acting at regulatory steps. The development of diabetes by combining mutations, which alone do not lead to major metabolic alterations, validated the ‘diabetogenes ’ concept of non-insulin-dependent diabetes mellitus. Genes encoding insulin-like growth factors (IGF-I and IGF-II) and their type I receptor (IGF-IR) have also been disrupted. It appears that although IR and IGF-IR are both capable of metabolic and mitogenic signalling, they are not fully redundant. However, IR could replace IGF-IR if efficiently activated by IGF-II. Studies with cell lines lacking IR or IGF-IR lend support to such conclusions. Concerning the issues of specificity and redundancy, studies with cell lines derived from IRS-1-deficient mice showed that IRS-1 and IRS-2 are also not completely interchangeable.
Mammalian cell polyamine homeostasis is altered by the radioprotector WR1065
Authors: MITCHELL John L. A. , RUPERT Jennifer , LEYSER Aviva , JUDD Gary G.
Published: August 2015

Mammalian cells become more susceptible to radiation-induced death and mutagenesis when restricted in their production of the natural polyamines putrescine, spermidine and spermine. The effects of polyamine deprivation are reversed by N-(2-mercaptoethyl)-1,3-diaminopropane (WR1065), a simple aminothiol that has been extensively studied for its radioprotectant properties. Because this compound and its oxidized derivative WR33278 bear some resemblance to the polyamines, it was hypothesized that radioprotection by WR1065 or its metabolites is derived, at least in part, from their ability to supplement the natural polyamines. To evaluate the ability of these aminothiol compounds to emulate polyamine function in intact cells, rat liver hepatoma (HTC) cells were treated with radioprotective doses of WR1065; the ability of this compound to affect various aspects of normal polyamine metabolism was monitored. Although cellular WR1065 was maintained at levels exceeding those of the polyamines, this aminothiol did not have any polyamine-like effect on the initial polyamine biosynthetic enzyme, ornithine decarboxylase, or on polyamine degradative reactions. On the contrary, treatment with relatively low levels of WR1065 resulted in an unexpected increase in putrescine and spermidine synthesis. WR1065 treatment enhanced the stability, and consequently the activity, of ornithine decarboxylase. This stabilization seems to result from a WR1065-induced delay in the synthesis of antizyme, a critical regulatory protein required in the feedback modulation of polyamine synthesis and transport. The increase in cellular spermidine induced by WR1065 might explain its antimutagenic properties, but is probably not a factor in protection against cell killing by radiation. This is the first evidence that compounds can be designed to control polyamine levels by targeting the activity of the regulatory protein antizyme.
Voltammetric studies of the reactions of iron–sulphur clusters ([3Fe-4S] or [M3Fe-4S]) formed in Pyrococcus furiosus ferredoxin
Authors: FAWCETT Sarah E. J. , DAVIS David , BRETON Jacques L. , THOMSON Andrew J. , ARMSTRONG Fraser A.
Published: August 2015

Reactions of the [3Fe-4S] cluster and various metallated [M3Fe-4S] adducts co-ordinated in the ferredoxin from the hyperthermophile Pyrococcus furiosus have been studied by protein-film voltammetry, bulk-solution voltammetry, solution kinetics and magnetic CD (MCD). The [3Fe-4S] cluster exhibits two couples, [3Fe-4S]+/0 and [3Fe-4S]0/2-. Film voltammetry is possible over a wide pH range (2–8), revealing that the [3Fe-4S]+/0 couple shows a complex pH dependence with pKred1 = 2.8, pKox = 4.9 and pKred2 = 6.7. From MCD, pKred1 corresponds with protonation of [3Fe-4S]0 to give a spectroscopically distinct species, as reported for ferredoxins from Azotobacterand Sulfolobus. The status of the disulphide/disulphydryl entity makes no significant difference to the data (given for the -S-S- form). Formation of the hyper-reduced [3Fe-4S]2- state is observed, requiring 3H+ for the overall 3e- reduction of [3Fe-4S]+, the change therefore being electroneutral. By comparison with the ferredoxin from Desulfovibrio africanus, uptake of Fe(II) and other M(II) by [3Fe-4S]0 to give [M3Fe-4S] clusters is slow (t1/2 > 10 min at room temperature, slower still if the protein is adsorbed on the electrode), whereas reaction with Tl(I) to produce [Tl3Fe-4S] is very rapid (t1/2 ≪ 1 s), suggesting that co-ordination of Tl does not require reorganization of the protein structure. Rates of formation of [3Fe-4S] from [M3Fe-4S] adducts increase sharply at high potentials, showing that metal release involves a labile ‘super-oxidized ’ [M3Fe-4S]3+ state.
Purification and biochemical characterization of a poly(ADP-ribose) polymerase-like enzyme from the thermophilic archaeon Sulfolobus solfataricus
Authors: FARAONE-MENNELLA Maria Rosaria , GAMBACORTA Agata , NICOLAUS Barbara , FARINA Benedetta
Published: August 2015

A poly(ADP-ribose) polymerase-like enzyme, detected in a crude homogenate from Sulfolobus solfataricus by means of activity and immunoblot analyses, was purified to electrophoretic homogeneity by a rapid procedure including two sequential affinity chromatographies, on NAD+-agarose and DNA-Sepharose. The latter column selected specifically the poly(ADP-ribosyl)ating enzyme with a 17% recovery of enzymic activity and a purification of more than 15000-fold. The molecular mass (54–55 kDa) assessed by SDS/PAGE and immunoblot was definitely lower than that determined for the corresponding eukaryotic protein. The enzyme was proved to be thermophilic, with a temperature optimum of approx. 80 °C, and thermostable, with a half-life of 204 min at 80 °C, in good agreement with the requirements of a thermozyme. It displayed a Km towards NAD+ of 154±50 µM; in the pH range 6.5–10.0 the activity values were similar, not showing a real optimum pH. The enzyme was able to bind homologous DNA, as evidenced by the ethidium bromide displacement assay. The product of the ADP-ribosylating reaction co-migrated with the short oligomers of ADP-ribose (less than 6 residues) from a eukaryotic source. Reverse-phase HPLC analysis of the products, after digestion with phosphodiesterase I, gave an elution profile reproducing that obtained by the enzymic digestion of the rat testis poly(ADP-ribose). These results strongly suggest that the activities of the purified enzyme include the elongation step.
Roles of the N- and C-terminal domains of carnitine palmitoyltransferase I isoforms in malonyl-CoA sensitivity of the enzymes: insights from expression of chimaeric proteins and mutation of conserved histidine residues
Authors: SWANSON S. Todd , FOSTER Daniel W. , McGARRY J. Denis , BROWN Nicholas F.
Published: August 2015

The mitochondrial outer membrane enzyme carnitine palmitoyltransferase I (CPT I) plays a major role in the regulation of fatty acid entry into the mitochondrial matrix for β-oxidation by virtue of its inhibition by malonyl-CoA. Two isoforms of CPT I, the liver type (L) and muscle type (M), have been identified, the latter being 100 times more sensitive to malonyl-CoA and having a much higher Km for the substrate carnitine. Here we have examined the roles of different regions of the CPT I molecules in their response to malonyl-CoA, etomoxir (an irreversible inhibitor) and carnitine. To this end, we analysed the properties of engineered rat CPT I constructs in which (a) the N-terminal domain of L-CPT I was deleted, (b) the N-terminal domains of L- and M-CPT I were switched, or (c) each of three conserved histidine residues located towards the N-terminus in L-CPT I was mutated. Several novel points emerged: (1) whereas the N-terminal domain is critical for a normal malonyl-CoA response, it does not itself account for the widely disparate sensitivities of the liver and muscle enzymes to the inhibitor; (2) His-5 and/or His-140 probably play a direct role in the malonyl-CoA response, but His-133 does not; (3) the truncated, chimaeric and point- mutant variants of the enzyme all bound the covalent, active-site- directed ligand, etomoxir; and (4) only the most radical alteration of L-CPT I, i.e. deletion of the N-terminal 82 residues, affected the response to carnitine. We conclude that the N-terminal domain of CPT I plays an essential, but permissive, role in the inhibition of the enzyme by malonyl-CoA. By contrast, the larger C-terminal region dictates the degree of sensitivity to malonyl-CoA, as well as the response to carnitine; it is also sufficient for etomoxir binding. Additionally, further weight is added to the notion that one or more histidine residues may be involved in the CPT I–malonyl-CoA interaction.
Comparative resistance of the 20S and 26S proteasome to oxidative stress
Authors: REINHECKEL Thomas , SITTE Nicolle , ULLRICH Oliver , KUCKELKORN Ulrike , DAVIES Kelvin J. A. , GRUNE Tilman
Published: August 2015

Oxidatively modified ferritin is selectively recognized and degraded by the 20S proteasome. Concentrations of hydrogen peroxide (H2O2) higher than 10 µmol/mg of protein are able to prevent proteolytic degradation. Exposure of the protease to high amounts of oxidants (H2O2, peroxynitrite and hypochlorite) inhibits the enzymic activity of the 20S proteasome towards the fluorogenic peptide succinyl-leucine-leucine-valine-tyrosine-methylcoumarylamide (Suc-LLVY-MCA), as well as the proteolytic degradation of normal and oxidant-treated ferritin. Fifty per cent inhibition of the degradation of the protein substrates was achieved using 40 µmol of H2O2/mg of proteasome. No change in the composition of the enzyme was revealed by electrophoretic analysis up to concentrations of 120 µmol of H2O2/mg of proteasome. In further experiments, it was found that the 26S proteasome, the ATP- and ubiquitin-dependent form of the proteasomal system, is much more susceptible to oxidative stress. Whereas degradation of the fluorogenic peptide, Suc-LLVY-MCA, by the 20S proteasome was inhibited by 50% with 12 µmol of H2O2/mg, 3 µmol of H2O2/mg was enough to inhibit ATP-stimulated degradation by the 26S proteasome by 50%. This loss in activity could be followed by the loss of band intensity in the non-denaturing gel. Therefore we concluded that the 20S proteasome was more resistant to oxidative stress than the ATP- and ubiquitin-dependent 26S proteasome. Furthermore, we investigated the activity of both proteases in K562 cells after H2O2 treatment. Lysates from K562 cells are able to degrade oxidized ferritin at a higher rate than non-oxidized ferritin, in an ATP-independent manner. This effect could be followed even after treatment of the cells with H2O2 up to a concentration of 2 mM. The lactacystin-sensitive ATP-stimulated degradation of the fluorogenic peptide Suc-LLVY-MCA declined, after treatment of the cells with 1 mM H2O2, to the same level as that obtained without ATP stimulation. Therefore, we conclude that the regulation of the 20 S proteasome by various regulators takes place during oxidative stress. This provides further evidence for the role of the 20S proteasome in the secondary antioxidative defences of mammalian cells.
Activation of dihydrofolate reductase following thiol modification involves a conformational change at the active site
Authors: FAN Ying-Xin , LI Zhen-Yu , ZHU Li , ZHOU Jun-Mei
Published: August 2015

Compared with the activation of dihydrofolate reductase (DHFR) by protein denaturants and inorganic salts, activation of the enzyme by thiol modification is relatively slow. Thus it is an ideal system for kinetic study of the activation mechanism. We describe here a kinetic study of the activation of DHFRs from chicken liver and Chinese hamster ovary by p-hydroxymercuribenzoate (p-HMB). The conformational changes in the enzyme molecule that result from the modification were monitored by measuring fluorescence enhancement due to the binding of 2-p-toluidinylnaphthalene-6-sulphonate (TNS), and by monitoring changes in the intrinsic fluorescence of the enzyme. Both activation and the conformational change probed by TNS followed pseudo-first-order kinetics, and the rate constants obtained are in good agreement with each other. The change in intrinsic fluorescence is a biphasic process. The rate of the fast phase, which may reflect a change in the microenvironment of Trp-24 at the active site, coincides with the rate of activation and the conformational change probed by TNS. The rate of the slow phase, which reflects a global conformational change, is about one order of magnitude lower than that of activation. The results indicate that the activation of DHFR by p-HMB is due to modification-induced conformational changes at its active site, rather than the modification of the thiol group itself, which is almost complete within the dead-time of the experiment. This study provides kinetic evidence for the proposal that flexibility at the active site is essential for full expression of catalytic activity.
CCAAT/enhancer binding protein regulates the promoter activity of the rat GLUT2 glucose transporter gene in liver cells
Authors: KIM Jae-woo , AHN Yong-ho
Published: August 2015

The liver-specific expression of the GLUT2 glucose transporter gene is suppressed in cultured hepatoma cell lines as well as in hepatocytes in primary culture. To understand the underlying mechanism involved in this process, we analysed the rat GLUT2 promoter region. A DNase I footprinting assay with rat liver nuclear extract revealed eight protected regions within a -500 bp region of the GLUT2 promoter (sites A to H). Three of these sites (B, F and H) were occupied by transcription factors that are considerably enriched in liver cells compared with spleen or kidney. The proteins binding to these sites were investigated by a combination of DNase I footprinting assay and electrophoretic mobility-shift assay with the addition of specific oligonucleotide competitors and specific antibody against known transcription factors. As a result it was revealed that hepatocyte nuclear factor 3 binds to site B (-120 to -70), and CCAAT/enhancer binding protein α (C/EBPα) and C/EBPβ bind to site F (-375 to -356) and site H (-500 to -471). The binding of C/EBP to sites F and H was markedly decreased within 4 h when liver cells were subjected to primary culture, suggesting that C/EBP might be responsible for the decreased expression of GLUT2 in this process. In contrast, Western blot analysis revealed that C/EBPα began to decrease after 1 h of hepatocyte culture, and C/EBPβ was not changed significantly throughout the culture period, suggesting that C/EBP could be regulated at the transcriptional level as well as the post-translational level when hepatocytes were put in culture. To confirm the role of C/EBP in the regulation of GLUT2 promoter activity, sites F and H were ligated to a chloramphenicol acetyltransferase (CAT) reporter gene and co-transfected with a C/EBP expression vector into HepG2 cells. The co-expression of C/EBPα and C/EBPβ resulted in 9.1-fold and 3.8-fold increases of CAT activities in the site F-CAT and site H-CAT constructs respectively. These results indicate that C/EBPα and C/EBPβ regulate the promoter activity of the GLUT2 gene and might be responsible for the down-regulation of the GLUT2 gene when hepatocytes are subjected to primary culture.
Protein tyrosine phosphatase PTP-S binds to the juxtamembrane region of the hepatocyte growth factor receptor Met
Authors: VILLA-MORUZZI Emma , PUNTONI Franca , BARDELLI Alberto , VIGNA Elisa , ROSA Sabrina DE , COMOGLIO Paolo M.
Published: August 2015

We reported previously that a protein tyrosine phosphatase (PTP) activity is associated with the immunoprecipitated hepatocyte growth factor (HGF) receptor, also known as Met. The activity increased reversibly when Met was stimulated by HGF and decreased when Met was inactivated by PMA. To identify the PTP-binding region, we used deletion mutants of the receptor β-subunit. The PTP activity did not associate with Tpr–Met, a construct containing residues 1010–1390 of Met fused to Tpr. In contrast, PTP activity was present when the expressed protein contained the full juxtamembrane region (residues 956–1390 of Met) or part of this region (residues 957–1390 or 995–1390), indicating that the PTP-binding region is between residues 995 and 1009. This region includes Tyr1003, a site involved in Met downstream signalling. Incubation of Met immunoprecipitated from GTL-16 cells with an 8-mer phosphopeptide derived from residues 1003–1010 induced a marked decrease in the associated PTP activity, suggesting that the peptide reproduced the PTP-binding region. Mutation of Glu, Asp or Arg at positions -4, -1 or +1 respectively relative to Tyr1003 in a 9-mer peptide (residues 999–1007) abolished the ability of the peptide to decrease the PTP activity associated with Met. Phosphorylation of Tyr1003 was not required for PTP binding, since: (1) both phospho- and dephospho-peptides on a solid bead bound PTP activity from a GTL-16 cell extract, and (2) PTP activity was associated with a Met deletion mutant lacking residues 1–955 in which Tyr1003 had been changed into Phe. In order to partially purify the PTP from the GTL-16 cell extract, an affinity column was prepared using the Met-derived peptide comprising residues 998–1007. Less than 0.1% of the total cellular PTP was retained by the column, and was eluted with low salt concentrations. Using antibodies, this PTP was identified as PTP-S, a soluble PTP present in epithelial cells and fibroblasts.
Groucho/transducin-like Enhancer of split (TLE) family members interact with the yeast transcriptional co-repressor SSN6 and mammalian SSN6-related proteins: implications for evolutionary conservation of transcription repression mechanisms
Authors: GRBAVEC Diane , LO Rita , LIU Yanling , GREENFIELD Andy , STIFANI Stefano
Published: August 2015

The yeast proteins TUP1 and SSN6 form a transcription repressor complex that is recruited to different promoters via pathway-specific DNA-binding proteins and regulates the expression of a variety of genes. TUP1 is functionally related to invertebrate and vertebrate transcriptional repressors of the Groucho/transducin-like Enhancer of split (TLE) family. The aim was to examine whether similar mechanisms underlie the transcription repression functions of TUP1 and Groucho/TLEs by determining whether TLE family members can interact with yeast SSN6 and mammalian SSN6-like proteins. It is shown in the present work that SSN6 binds to TLE1 and mediates transcriptional repression when expressed in mammalian cells. Moreover, TLE1 and TLE2 interact with two mammalian proteins related to SSN6, designated as the products of the ubiquitously transcribed tetratricopeptide-repeat genes on the Y (or X) chromosomes (UTY/X). These findings suggest that mammalian TLE and UTY/X proteins may mediate repression mechanisms similar to those performed by TUP1–SSN6 in yeast.
Dual coupling of the α-thrombin receptor to signal-transduction pathways involving phosphatidylinositol and phosphatidylcholine metabolism
Authors: CHENG Jie , BALDASSARE Joseph J. , RABEN Daniel M.
Published: August 2015

Addition of α-thrombin to quiescent IIC9 cells results in the activation of lipid-metabolizing enzymes associated with signal-transduction cascades. These enzymes include phosphatidylinositol (PI)-specific phospholipase C (PI-PLC), phosphatidylcholine (PC)-specific phospholipases C and D and phospholipase A2 (PLA2). Whereas the α-thrombin receptor has been shown to couple with PI-PLCs, it is not clear whether this receptor, or a putative second receptor, couples to one or more of the other phospholipases. In this report we determine whether the cloned receptor couples to all or a subset of these enzymes. We show that (i) an α-thrombin-receptor-activating peptide also elicits the above responses and (ii) addition of enterokinase to IIC9 cells, stably transfected with an α-thrombin receptor (enterokinase- responsive α-thrombin receptor, EKTR) containing an enterokinase cleavage site in place of an α-thrombin cleavage site, stimulates both PI and PC hydrolysis, including PLA2. Enterokinase also induces mitogenesis in the IIC9s transfected with EKTR. These results indicate that, in addition to initiating a mitogenic signalling cascade, the cloned α-thrombin receptor couples to enzymes involved in generating PC-derived, as well as PI-derived, second-messenger molecules in IIC9s. Additionally, using the cells transfected with EKTR, we further demonstrate that only activated, i.e. cleaved, receptors are desensitized.
Biochemical characterization of a variant human medium-chain acyl-CoA dehydrogenase with a disease-associated mutation localized in the active site
Authors: KÜCHLER Burkhard , ABDEL-GHANY Abdel-Ghany , BROSS Peter , NANDY Andreas , RASCHED Ihab , GHISLA Sandro
Published: August 2015

Medium-chain acyl-CoA dehydrogenase (MCADH) deficiency, an autosomal recessive inherited disorder, is the most common genetic disorder in mitochondrial β-oxidation in humans. In addition to one prevalent disease-causing mutation (K304E), a series of rarer mutations has been reported, but none of these has yet been characterized in detail. We report here on the biochemical characterization of the purified recombinant mutant protein in which threonine is replaced by alanine at position 168 of the mature protein (T168A-MCADH). It is the first mutation to be found in patients that is located in the active site of the enzyme. Thr-168 is hydrogen-bonded to the flavin N(5) of the cofactor FAD. The thermostability of T168A-MCADH is markedly decreased compared with human wild-type MCADH (hwt-MCADH). Catalytic activity with ferricenium as acceptor is lowered by 80% and with the natural acceptor electron-transferring flavoprotein by over 90% compared with hwt-MCADH. In the mutant the extent of flavin semiquinone formed on reduction is approx. 50% that of hwt-MCADH. The pK reflected by the pH-dependence of Vmax is shifted from approx. 8.2 (hwt-MCADH) to approx. 7 (T168A-MCADH) and the rates of enzyme flavin reduction (stopped-flow measurements) are only approx. 1/10 those of the parent enzyme. These properties are discussed in the light of the possible mechanisms leading to disease in humans.
Expression and subcellular localization of the ryanodine receptor in rat pancreatic acinar cells
Authors: LEITE M. Fatima , DRANOFF Jonathan A. , GAO Ling , NATHANSON Michael H.
Published: August 2015

The ryanodine receptor (RyR) is the principal Ca2+-release channel in excitable cells, whereas the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) is primarily responsible for Ca2+ release in non-excitable cells, including epithelia. RyR also is expressed in a number of non-excitable cell types, but is thought to serve as an auxiliary or alternative Ca2+-release pathway in those cells. Here we use reverse transcription PCR to show that a polarized epithelium, the pancreatic acinar cell, expresses the type 2, but not the type 1 or 3, isoform of RyR. We furthermore use immunochemistry to demonstrate that the type 2 RyR is distributed throughout the basolateral and, to a lesser extent, the apical region of the acinar cell, but is excluded from the trigger zone, where cytosolic Ca2+ signals originate in this cell type. Since propagation of Ca2+ waves in acinar cells is sensitive to ryanodine, caffeine and Ca2+, these findings suggest that Ca2+ waves in this cell type result from the co-ordinated release of Ca2+, first from InsP3Rs in the trigger zone, then from RyRs elsewhere in the cell. RyR may play a fundamental role in Ca2+ signalling in polarized epithelia, including for Ca2+ signals initiated by InsP3.
Biochemical characterization of Bacillus thuringiensis cytolytic toxins in association with a phospholipid bilayer
Authors: DU Jinping , KNOWLES Barbara H. , LI Jade , ELLAR David J.
Published: August 2015

The interaction of two Bacillus thuringiensiscytolytic toxins, CytA and CytB, with a phospholipid bilayer and their structure in the membrane-bound state were investigated by proteolysis using phospholipid vesicles as a model system. A toxin conformational change upon membrane binding was detected by comparing the proteolytic profile of membrane-bound toxin and saline-solubilized toxin. When membrane-bound toxin was exposed to protease K or trypsin, novel cleavage sites were found between the α-helical N-terminal half and β-strand C-terminal half of the structure at K154 and N155 in CytA and at I150 and G141 in CytB. N-terminal sequencing of membrane-protected fragments showed that the C-terminal half of the toxin structure comprising mainly β-strands was inserted into the membrane, whereas the N-terminal half comprising mainly α-helices was exposed on the outside of the liposomes and could be removed when liposomes with bound toxin were washed extensively after proteolysis. The C-termini of the membrane-inserted proteolytic fragments were also located by a combination of N-terminal sequencing and measurement of the molecular masses of the fragments by electrospray MS. Using a liposome glucose-release assay, the membrane-inserted structure was seen to retain its function as a membrane pore even after removal of exposed N-terminal segments by proteolysis. These data strongly suggest that the pores for glucose release are assembled from the three major β-strands (β-5, β-6 and β-7) in the C-terminal half of the toxin.
Overexpression of CuZn superoxide dismutase protects RAW 264.7 macrophages against nitric oxide cytotoxicity
Authors: BROCKHAUS Florian , BRÜNE Bernhard
Published: August 2015

Initiation of nitric oxide (NO•)-mediated apoptotic cell death in RAW 264.7 macrophages is associated with up-regulation of mitochondrial manganese superoxide dismutase (MnSOD; SOD2) and down-regulation of cytosolic copper zinc superoxide dismutase (CuZnSOD; SOD1) at their individual mRNA and protein levels. To evaluate the decreased CuZnSOD expression and the initiation of apoptosis we stably transfected macrophages to overexpress human CuZnSOD. Individual clones revealed a 2-fold increase in CuZnSOD activity. Expression of a functional and thus protective CuZnSOD was verified by attenuated superoxide (O2•-)-mediated apoptotic as well as necrotic cell death. In this study we showed that SOD-overexpressing macrophages (R-SOD1-12) were also protected against NO•-initiated programmed cell death. Protection was substantial towards NO• derived from exogenously added NO donors or when NO• was generated by inducible NO synthase activation, and was evident at the level of p53 accumulation, caspase activation and DNA fragmentation. Stimulation of parent and SOD-overexpressing cells with a combination of lipopolysaccharide and murine interferon γ produced equivalent amounts of nitrite/nitrate, which ruled out attenuated inducible NO• synthase activity during protection. Because protection by a O2•--scavenging system during NO•-intoxication implies a role of NO• and O2•- in the progression of cell damage, we used uric acid to delineate the role of peroxynitrite during NO•-elicited apoptosis. The peroxynitrite scavenger uric acid left S-nitrosoglutathione or spermine-NO-elicited apoptosis unaltered, blocking only 3-morpholinosydnonimine-mediated cell death. As a result we exclude peroxynitrite from contributing, to any major extent, to NO•-mediated apoptosis. Therefore protection observed with CuZnSOD overexpression is unlikely to stem from interference with peroxynitrite formation and/or action. Unequivocally, the down-regulation of CuZnSOD is associated with NO• cytotoxicity, whereas CuZnSOD overexpression protects macrophages from apoptosis.
Intestinal trefoil factor binds to intestinal epithelial cells and induces nitric oxide production: priming and enhancing effects of mucin
Authors: TAN Xiao-Di , LIU Qian-Ping , HSUEH Wei , CHEN Yi-Hua , CHANG Hong , GONZALEZ-CRUSSI Frank
Published: August 2015

Intestinal trefoil factor (ITF or TFF3), NO and epithelium-associated mucin have important roles in sustaining mucosal integrity in the gastrointestinal tract. In the present study we examined ITF-binding molecules on IEC-18 cells (an intestinal epithelial cell line) with the use of flow cytometry and localized these molecules on the cell surface by confocal microscopy. Furthermore, we studied the interaction of mucin and ITF and their co-operative effect on NO production by the epithelium. Stimulation of cells with mucin (5 mg/ml) for 90 min resulted in a 5-fold increase in ITF binding. Treatment of IEC-18 cells with actinomycin D or cycloheximide attenuated mucin-enhanced ITF binding. Ligand blot analysis confirmed the induction of ITF-binding protein in IEC-18 cells by mucin. These results indicate that transcriptional and translational mechanisms are involved in the effect of mucin. Treatment with ITF overnight resulted in a low level of nitrite production by the cells, a 5-fold increase over control, in a concentration-dependent manner. ITF-induced NO production was attenuated by 1400W, a selective type II nitric oxide synthase (NOS2) inhibitor. By immunoblotting we found that NOS2 was up-regulated by ITF treatment. Priming IEC-18 cells with mucin for 90 min enhanced the effect of ITF on NO production, suggesting that the up-regulation of ITF-binding molecules by mucin might be physiologically relevant. Taken together, these observations indicate (1) that ITF-binding molecules that are up-regulated by mucin exist on the intestinal epithelial surface, and (2) that ITF modulates epithelial NO production via the NOS2 pathway, which is enhanced by mucin.
Amyloid precursor protein metabolism in fibroblasts from individuals with one, two or three copies of the amyloid precursor protein (APP) gene
Authors: RACCHI Marco , JOHNSTON Janet A. , FLOOD Fiona M. , COWBURN Richard F. , GOVONI Stefano
Published: August 2015

Protein kinase C (PKC)-activated modulation of amyloid precursor protein (APP) metabolism has been investigated in natural models of altered APP expression due to the presence of one, two or three copies of the APP gene. We show that levels of APP present in human skin fibroblasts strongly influence the effect of PKC activation of soluble APP (sAPP) release. Thus fibroblasts derived from a patient with a deletion in chromosome 21 including the APP locus (Δ21) had lower levels of both APP mRNA and cell-associated APP, and showed an exaggerated phorbol-ester-induced sAPP release, when compared with fibroblasts from control individuals. In contrast, fibroblasts from chromosome 21 trisomic Down's syndrome patients failed to show a concentration-dependent response to phorbol ester treatment. These results suggest that the levels of APP expression can affect the degree of response to PKC-mediated modulation of the metabolism of this protein.
Overexpression of an enzymically inactive interleukin-1-receptor-associated kinase activates nuclear factor-κB
Authors: MASCHERA Barbara , RAY Keith , BURNS Kimberly , VOLPE Filippo
Published: August 2015

Upon interleukin 1 (IL-1) stimulation, the IL-1-receptor (IL-1R)-associated kinase (IRAK) is rapidly recruited to the IL-1R complex and undergoes phosphorylation. Here we demonstrate that recombinant wild-type IRAK (IRAK-WT), but not a kinase-defective mutant with Asp340 replaced by an asparagine residue (IRAK-Asp340Asn), is highly phosphorylated and is capable of auto-phosphorylation in vitro. Overexpression of both IRAK-WT and IRAK-Asp340Asn caused activation of nuclear factor κB, suggesting that the kinase activity of IRAK is not required outside of the IL-1R complex.
Inhibition of the peroxidative degradation of haem as the basis of action of chloroquine and other quinoline antimalarials
Authors: LORIA Paul , MILLER Susanne , FOLEY Michael , TILLEY Leann
Published: August 2015

The malaria parasite feeds by degrading haemoglobin in an acidic food vacuole, producing free haem moieties as a by-product. The haem in oxyhaemoglobin is oxidized from the Fe(II) state to the Fe(III) state with the consequent production of an equimolar concentration of H2O2. We have analysed the fate of haem molecules in Plasmodium falciparum-infected erythrocytes and have found that only about one third of the haem is polymerized to form haemozoin. The remainder appears to be degraded by a non-enzymic process which leads to an accumulation of iron in the parasite. A possible route for degradation of the haem is by reacting with H2O2, and we show that, under conditions designed to resemble those found in the food vacuole, i.e., at pH 5.2 in the presence of protein, free haem undergoes rapid peroxidative decomposition. Chloroquine and quinacrine are shown to be efficient inhibitors of the peroxidative destruction of haem, while epiquinine, a quinoline compound with very low antimalarial activity, has little inhibitory effect. We also show that chloroquine enhances the association of haem with membranes, while epiquinine inhibits this association, and that treatment of parasitized erythrocytes with chloroquine leads to a build-up of membrane-associated haem in the parasite. We suggest that chloroquine exerts its antimalarial activity by causing a build-up of toxic membrane-associated haem molecules that eventually destroy the integrity of the malaria parasite. We have further shown that resistance-modulating compounds, such as chlorpromazine, interact with haem and efficiently inhibit its degradation. This may explain the weak antimalarial activities of these compounds.
Association of polo-like kinase with α-, β- and γ-tubulins in a stable complex
Authors: FENG Yang , HODGE David R. , PALMIERI Giuseppe , CHASE Dan L. , LONGO Dan L. , FERRIS Douglas K.
Published: August 2015

The polo-like kinase (Plk) family has been shown to have an important role in the regulation of the cell-division cycle, especially in organization of the spindle structure, in species from fungi to humans. Recent reports have demonstrated that in mammalian cells Plk is associated with components of the anaphase-promoting complex and a peptidyl-prolyl isomerase, Pin1. To characterize a putative Plk-containing complex, we fractionated mitotic cell lysates on a gel-filtration column. The Plk complex was eluted from the column at molecular sizes ranging from 669 to 2500 kDa in the presence of detergent and high concentrations of salt. Specific associations of Plk with α-, β- and γ-tubulins in both interphase and mitotic cells were shown by reciprocal immunoprecipitations and immunoblottings and were independent of the microtubule polymerization state, whereas binding assays in vitro indicated that Plk interacts with α- and β-tubulins directly. In addition, mitotic Plk was able to phosphorylate associated tubulins in vitro. Finally, we show that the kinase domain of the Plk molecule is both required and sufficient for its binding to tubulins in vivo. The specific interaction between Plk and tubulins might provide a molecular basis for the physiological functions of Plk in regulating the cell cycle, particularly in establishing the normal bipolar spindle.
Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain
Authors: SUCIC Joseph F. , MOEHRING Joan M. , INOCENCIO Noel M. , LUCHINI Jason W. , MOEHRING Thomas J.
Published: August 2015

PACE4 is a member of the eukaryotic subtilisin-like endoprotease family. The expression of human PACE4 in RPE.40 cells (furin-null mutants derived from Chinese hamster ovary K1 cells) resulted in the rescue of a number of wild-type characteristics, including sensitivity to Sindbis virus and the ability to process the low-density-lipoprotein receptor-related protein. Expression of PACE4 in these cells failed to restore wild-type sensitivity to Pseudomonas exotoxin A. Co-expression of human PACE4 in these cells with either a secreted form of the human insulin pro-receptor or the precursor form of von Willebrand factor resulted in both proproteins being processed; RPE.40 cells were unable to process either precursor protein in the absence of co-expressed PACE4. Northern analysis demonstrated that untransfected RPE.40 cells express mRNA species for four PACE4 isoforms, suggesting that any endogenous PACE4 proteins produced by these cells are either non-functional or sequestered in a compartment outside of the secretory pathway. In experiments in vitro, PACE4 processed diphtheria toxin and anthrax toxin protective antigen, but not Pseudomonas exotoxin A. The activity of PACE4 in vitro was Ca2+-dependent and, unlike furin, was sensitive to temperature changes between 22 and 37 °C. RPE.40 cells stably expressing human PACE4 secreted an endoprotease with the same Ca2+ dependence and temperature sensitivity as that observed in membrane fractions of these cells assayed in vitro. These results, in conjunction with other published work, demonstrate that PACE4 is an endoprotease with more stringent substrate specificity and more limited operating parameters than furin.
Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor
Authors: LEE Young Bok , JOE Young Ae , WOLFF Edith C. , DIMITRIADIS Emilios K. , PARK Myung Hee
Published: August 2015

Deoxyhypusine synthase catalyses the first step in the post-translational synthesis of hypusine [Nϵ-(4-amino-2-hydroxybutyl) lysine] in a single cellular protein, the precursor of eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase exists as a tetramer with four potential active sites. The formation of a stable complex between human deoxyhypusine synthase and its protein substrate, human recombinant eIF5A precursor (ec-eIF5A), was examined by affinity chromatography using polyhistidine-tagged (His·Tag) ec-eIF5A, by a gel mobility-shift method, and by analytical ultracentrifugation. Deoxyhypusine synthase was selectively retained by His·Tag-ec-eIF5A immobilized on a resin. The complex of deoxyhypusine synthase and ec-eIF5A was separated from the free enzyme and protein substrate by electrophoresis under non-denaturing conditions. The stoichiometry of the two components in the complex was estimated to be 1 deoxyhypusine synthase tetramer to 1 ec-eIF5A monomer by N-terminal amino acid sequencing of the complex. Equilibrium ultracentrifugation data further supported this 1:1 ratio and indicated a very strong interaction of the enzyme with ec-eIF5A (Kd≤ 0.5 nM). Formation of the complex was not dependent on NAD+ or spermidine and occurred at pH 7.0-9.2. An enzyme-product complex, as well as the deoxyhypusine-containing product (modified ec-eIF5A), was also detected at pH 7.0-9.2 in a complete reaction mixture containing 1 mM spermidine.
Isolation and characterization of a novel antifreeze protein from carrot (Daucus carota)
Authors: SMALLWOOD Maggie , WORRALL Dawn , BYASS Louise , ELIAS Luisa , ASHFORD David , DOUCET Charlotte J. , HOLT Chris , TELFORD Julia , LILLFORD Peter , BOWLES Dianna J.
Published: August 2015

A modified assay for inhibition of ice recrystallization which allows unequivocal identification of activity in plant extracts is described. Using this assay a novel, cold-induced, 36 kDa antifreeze protein has been isolated from the tap root of cold-acclimated carrot (Daucus carota) plants. This protein inhibits the recrystallization of ice and exhibits thermal-hysteresis activity. The polypeptide behaves as monomer in solution and is N-glycosylated. The corresponding gene is unique in the carrot genome and induced by cold. The antifreeze protein appears to be localized within the apoplast.
Sepiapterin reductase producing l-threo-dihydrobiopterin from Chlorobium tepidum
Authors: CHO Seung-Hyun , NA Jong-Uk , YOUN Hwan , HWANG Cheol-Sang , LEE Chang-Hun , KANG Sa-Ouk
Published: August 2015

A novel type of NADPH-dependent sepiapterin reductase, which catalysed uniquely the reduction of sepiapterin to L-threo-dihydrobiopterin, was purified 533-fold from the cytosolic fraction of Chlorobium tepidum, with an overall yield of 3%. The native enzyme had a molecular mass of 55 kDa and SDS/PAGE revealed that the enzyme consists of two subunits with a molecular mass of 26 kDa. The enzyme was optimally active at pH 8.8 and 50 °C. Apparent Km values for sepiapterin and NADPH were 21 and 6.2 μM, respectively, and the kcat value was 5.0 s-1. Diacetyl could also serve as a substrate, with a Km of 4.0 mM. The inhibitory effects of N-acetylserotonin, N-acetyldopamine and melatonin were very weak. The Ki value of N-acetyldopamine was measured as 400 μM. The N-terminal amino acid sequence was revealed as Met-Lys-His-Ile-Leu-Leu-Ile-Thr-Gly-Ala-Xaa-Lys - Lys - Ile - Xaa - Arg - Ala - Ile - Ala - Leu - Glu - Xaa - Ala - Arg - Xaa-Xaa-Xaa-His-His-His-, which shared relatively high sequence similarity with other sepiapterin reductases.
Lipid phosphate phosphohydrolase-1 degrades exogenous glycerolipid and sphingolipid phosphate esters
Authors: JASINSKA Renata , ZHANG Qiu-Xia , PILQUIL Carlos , SINGH Indrapal , XU James , DEWALD Jay , DILLON Deirdre A. , BERTHIAUME Luc G. , CARMAN George M. , WAGGONER David W. , BRINDLEY David N.
Published: August 2015

Lipid phosphate phosphohydrolase (LPP)-1 cDNA was cloned from a rat liver cDNA library. It codes for a 32-kDa protein that shares 87 and 82% amino acid sequence identities with putative products of murine and human LPP-1 cDNAs, respectively. Membrane fractions of rat2 fibroblasts that stably expressed mouse or rat LPP-1 exhibited 3.1-3.6-fold higher specific activities for phosphatidate dephosphorylation compared with vector controls. Increases in the dephosphorylation of lysophosphatidate, ceramide 1-phosphate, sphingosine 1-phosphate and diacylglycerol pyrophosphate were similar to those for phosphatidate. Rat2 fibroblasts expressing mouse LPP-1 cDNA showed 1.6-2.3-fold increases in the hydrolysis of exogenous lysophosphatidate, phosphatidate and ceramide 1-phosphate compared with vector control cells. Recombinant LPP-1 was located partially in plasma membranes with its C-terminus on the cytosolic surface. Lysophosphatidate dephosphorylation was inhibited by extracellular Ca2+ and this inhibition was diminished by extracellular Mg2+. Changing intracellular Ca2+ concentrations did not alter exogenous lysophosphatidate dephosphorylation significantly. Permeabilized fibroblasts showed relatively little latency for the dephosphorylation of exogenous lysophosphatidate. LPP-1 expression decreased the activation of mitogen-activated protein kinase and DNA synthesis by exogenous lysophosphatidate. The product of LPP-1 cDNA is concluded to act partly to degrade exogenous lysophosphatidate and thereby regulate its effects on cell signalling.
NO synthase II in mouse skeletal muscle is associated with caveolin 3
Authors: GATH Ingolf , EBERT Jutta , GÖDTEL-ARMBRUST Ute , ROSS Ralf , RESKE-KUNZ Angelika B. , FÖRSTERMANN Ulrich
Published: August 2015

The inducible-type NO synthase (NOS II; iNOS) is constitutively expressed in slow-twitch skeletal muscle fibres of guinea-pigs [Gath, Closs, Gödtel-Armbrust, Schmitt, Nakane, Wessler and Förstermann (1996) FASEB J. 10, 1614-1620]. Here we studied the expression of NOS II in skeletal muscle of wild-type and NOS II-deficient mice and investigated the molecular basis for the membrane association of this NOS in muscle. A basal expression of NOS II mRNA and protein was detected in skeletal muscle from untreated wild-type mice; expression increased when mice were treated with bacterial lipopolysaccharide (LPS). No NOS II was found in any tissue of untreated or LPS-treated NOS II-deficient mice. Immunoprecipitation experiments were performed with homogenates of gastrocnemius muscle from untreated or LPS-treated wild-type mice. A NOS II-specific antibody precipitated caveolin 3 in all homogenates investigated, the effect being most pronounced in skeletal muscle from LPS-treated animals. Conversely, an antibody against caveolin 3 co-precipitated NOS II in muscle homogenates. Similarly, a weak co-precipitation of NOS II and caveolin 3 was seen in homogenates of untreated murine C2C12 myotubes; co-precipitation was markedly enhanced in cells stimulated with LPS/interferon γ. The association of NOS II with caveolin 3 might have implications for the regulation of contraction of, and/or glucose uptake by, slow-twitch muscle fibres.
Digestion of crystalline cellulose substrates by the Clostridium thermocellum cellulosome: structural and morphological aspects
Authors: BOISSET Claire , CHANZY Henri , HENRISSAT Bernard , LAMED Raphael , SHOHAM Yuval , BAYER Edward A.
Published: August 2015

The action of cellulosomes from Clostridium thermocellum on model cellulose microfibrils from Acetobacter xylinum and cellulose microcrystals from Valoniaventricosa was investigated. The biodegradation of these substrates was followed by transmission electron microscopy, Fourier-transform IR spectroscopy and X-ray diffraction analysis, as a function of the extent of degradation. The cellulosomes were very effective in catalysing the complete digestion of bacterial cellulose, but the total degradation of Valonia microcrystals was achieved more slowly. Ultrastructural observations during the digestion process suggested that the rapid degradation of bacterial cellulose was the result of a very efficient synergistic action of the various enzymic components that are attached to the scaffolding protein of the cellulosomes. The degraded Valonia sample assumed various shapes, ranging from thinned-down microcrystals to crystals where one end was pointed and the other intact. This complexity may be correlated with the multi-enzyme content of the cellulosomes and possibly to a diversity of the cellulosome composition within a given batch. Another aspect of the digestion of model celluloses by cellulosomes is the relative invariability of their crystallinity, together with their Iα/Iβ composition throughout the degradation process. Comparison of the action of cellulosomes with that of fungal enzymes indicated that the degradation of cellulose crystals by cellulosomes occurred with only limited levels of processivity, in contrast with the observations reported for fungal enzymes. The findings were consistent with a mechanism whereby initial attack by a cellulosome of an individual cellulose crystal results in its ‘commitment’ towards complete degradation.