bluuebunny/crossref_metadata_2025 huggingface dataset:
Ubiquitination of tissue transglutaminase is modulated by interferon alpha in human lung cancer cells
Authors: ESPOSITO Carla , MARRA Monica , GIUBERTI Gaia , D'ALESSANDRO Anna Maria , PORTA Raffaele , COZZOLINO Anna , CARAGLIA Michele , ABBRUZZESE Alberto
Published: February 2003

The addition of 2500i.u./ml interferon α (IFNα) for 48h induced apoptosis, and caused an approx. 4-fold increase in the activity and expression of tissue transglutaminase (tTG), in human lung cancer H1355 cells. However, the increase in mRNA levels for tTG was just 1.6-fold. On the basis of these data, we investigated whether tTG levels may be regulated through regulation of its degradation via ubiquitination. It was found that 2500i.u./ml IFNα induced a time-dependent decrease in tTG ubiquitination. On the other hand, addition of the proteasome inhibitor lactacystin led to accumulation of the ubiquitinated form of the enzyme and to a consequent increase in its expression. Treatment of the cells with the two agents combined antagonized the accumulation of the ubiquitinated isoforms of tTG induced by lactacystin and caused a potentiation of tTG expression. Moreover, the tTG inducer retinoic acid was also able to cause increased expression and ubiquitination of tTG in H1355 cells. The addition of monodansylcadaverine (a tTG inhibitor) to IFNα-treated H1355 cells completely antagonized growth inhibition and apoptosis induced by the cytokine. In conclusion, we demonstrate for the first time that tTG is ubiquitinated and degraded by a proteasome-dependent pathway. Moreover, IFNα can, at least in part, induce apoptosis through the modulation of this pathway.
Expression and characterization of a human pyruvate carboxylase variant by retroviral gene transfer
Authors: CARBONE Mary Anna , ROBINSON Brian H.
Published: February 2003

Type A pyruvate carboxylase (PC) deficiency presents mainly in the Amerindian population, specifically the Ojibwa, Cree and Micmac tribes of the Algonquin-speaking peoples. The gene for PC contains a homozygous founder mutation (G1828→A) that results in an Ala610→Thr amino acid substitution in Ojibwa with Type A PC deficiency. The mutation is located in the highly conserved pyruvate-binding domain of PC. The present paper describes a retroviral expression system for human PC used to analyse the effects of this mutation. We show, through immunoblot analysis, PC enzyme activity assays, reverse-transcription PCR and mitochondrial-import experiments, that this mutation is disease-causing in the Ojibwa population owing to its decreased catalytic activity, decreased steady-state levels of expression and inefficient import into the mitochondria. Our data suggest that this mutation may affect the stability of the protein, resulting in decreased steady-state levels of expression, and that it may also affect the secondary structure of the protein during the import process, thereby inhibiting proper translocation into the mitochondria, where PC is active.
Human meprinbeta: O-linked glycans in the intervening region of the type I membrane protein protect the C-terminal region from proteolytic cleavage and diminish its secretion
Authors: LEUENBERGER Boris , HAHN Dagmar , PISCHITZIS Anastassios , HANSEN Marianne K. , STERCHI Erwin E.
Published: February 2003

Human meprin (hmeprin; N-benzoyl-l-tyrosyl-p-aminobenzoic acid hydrolase; EC 3.4.24.18) is a member of the astacin family of zinc metalloendopeptidases. The major site of expression is the brush border membrane of small intestinal and kidney epithelial cells. The enzyme is a type I integral membrane protein composed of two distinct subunits, α and β, which are linked by disulphide bridges. The enzyme complex is attached to the plasma membrane only via the β-subunit. The α-subunit is cleaved in the endoplasmic reticulum in a constitutive manner to remove the C-terminal membrane anchor which leads to secretion of the protein. While the β-subunit of hmeprin remains largely attached to the brush-border membrane some proteolytic processing occurs intracellularly as well as at the cell surface and results in the release of this subunit from the cell. In the present paper, we report that the β-subunit bears multiple O-linked sugar residues in the intervening domain. In contrast, the α-subunit does not contain O-linked oligosaccharides. Our results show that the O-linked carbohydrate side chains in hmeprinβ are clustered around a 13 amino acid sequence that contains the main cleavage site for proteolytic processing of the subunit. Prevention of O-glycosylation by specific inhibitors leads to enhanced proteolytic processing and the consequence is an increased release of hmeprinβ into the culture medium.
Cytotoxic activity of Bacillus thuringiensis Cry proteins on mammalian cells transfected with cadherin-like Cry receptor gene of Bombyx mori (silkworm)
Authors: TSUDA Yoko , NAKATANI Fumiki , HASHIMOTO Keiko , IKAWA Satoshi , MATSUURA Chikako , FUKADA Takashi , SUGIMOTO Kenji , HIMENO Michio
Published: February 2003

Cry1Aa, an insecticidal protein produced by Bacillus thuringiensis, has been shown to bind to cadherin-like protein, BtR175, in Bombyx mori (silkworm) midgut. We previously reported three variant alleles of BtR175 (BtR175a, b and c). When transiently expressed in COS7 cells, all the three BtR175 variants bound to Cry1Aa. We stably expressed BtR175b in HEK293 cells. These BtR175b-expressing cells swelled and died in the presence of activated Cry1Aa in a dose- and time-dependent manner, showing that BtR175b itself can impart Cry1Aa-susceptibility to mammalian cells. These cells were more susceptible to Cry1Aa than to Cry1Ab and Cry1Ac. Since dispersed B. mori midgut cells were reported to be highly susceptible to Cry1Ac, this result suggested that other Cry1Ac-specific receptor(s) were simultaneously working with BtR175 in the midgut cells. Advantages are also discussed of applying these transfected mammalian cells to toxicity assays of mutant Cry proteins.
Thermodynamic characterization of a tetrahaem cytochrome isolated from a facultative aerobic bacterium, Shewanella frigidimarina: a putative redox model for flavocytochrome c3
Authors: PESSANHA Miguel , LOURO Ricardo O. , CORREIA Ilídio J. , ROTHERY Emma L. , PANKHURST Kate L. , REID Graeme A. , CHAPMAN Stephen K. , TURNER David L. , SALGUEIRO Carlos A.
Published: February 2003

The facultative aerobic bacterium Shewanella frigidimarina produces a small c-type tetrahaem cytochrome (86 residues) under anaerobic growth conditions. This protein is involved in the respiration of iron and shares 42% sequence identity with the N-terminal domain of a soluble flavocytochrome, isolated from the periplasm of the same bacterium, which also contains four c-type haem groups. The thermodynamic properties of the redox centres and of an ionizable centre in the tetrahaem cytochrome were determined using NMR and visible spectroscopy techniques. This is the first detailed thermodynamic study performed on a tetrahaem cytochrome isolated from a facultative aerobic bacterium and reveals that this protein presents unique features. The redox centres have negative and different redox potentials, which are modulated by redox interactions between the four haems (covering a range of 8—56mV) and by redox—Bohr interactions between the haems and an ionizable centre (-4 to −36mV) located in close proximity to haem III. All of the interactions between the five centres are clearly dominated by electrostatic effects and the microscopic reduction potential of haem III is the one most affected by the oxidation of the other haems and by the protonation state of the molecule. Altogether, this study indicates that the tetrahaem cytochrome isolated from S. frigidimarina (Sfc) has the thermodynamic properties to work as an electron wire between its redox partners. Considering the high degree of sequence identity between Sfc and the cytochrome domain of flavocytochrome c3, the structural similarities of the haem core, and that the macroscopic potentials are also identical, the results obtained in this work are rationalized in order to put forward a putative redox model for flavocytochrome c3.
Inhibitors of vacuolar H+-ATPase impair the preferential accumulation of daunomycin in lysosomes and reverse the resistance to anthracyclines in drug-resistant renal epithelial cells
Authors: OUAR Zahia , BENS Marcelle , VIGNES Caroline , PAULAIS Marc , PRINGEL Claudine , FLEURY Jocelyne , CLUZEAUD Françoise , LACAVE Roger , VANDEWALLE Alain
Published: February 2003

It has been suggested that the inappropriate sequestration of weak-base chemotherapeutic drugs in acidic vesicles by multidrug-resistance (MDR) cells contributes to the mechanisms of drug resistance. The function of the acidic lysosomes can be altered in MDR cells, and so we investigated the effects of lysosomotropic agents on the secretion of lysosomal enzymes and on the intracellular distribution of the weak-base anthracycline daunomycin in drug-resistant renal proximal tubule PKSV-PRcol50 cells and their drug-sensitive PKSV-PR cell counterparts. Imaging studies using pH-dependent lysosomotropic dyes revealed that drug-sensitive and drug-resistant cells exhibited a similar acidic lysosomal pH (around 5.6—5.7), but that PKSV-PRcol50 cells contained more acidic lysosomes and secreted more of the lysosomal enzymes N-acetyl-β-hexosaminidase and β-glucuronidase than their parent PKSV-PR cells. Concanamycin A (CCM A), a potent inhibitor of the vacuolar H+-ATPase, but not the P-glycoprotein modulator verapamil, stimulated the secretion of N-acetyl-β-hexosaminidase in both drug-sensitive and drug-resistant cells. Fluorescent studies and Percoll density gradient fractionation studies revealed that daunomycin accumulated predominantly in the lysosomes of PKSV-PRcol50 cells, whereas in PKSV-PR cells the drug was distributed evenly throughout the nucleo-cytoplasmic compartments. CCM A did not impair the cellular efflux of daunomycin, but induced the rapid nucleo-cytoplasmic redistribution of the drug in PKSV-PRcol50 cells. In addition, CCM A and bafilomycin A1 almost completely restored the sensitivity of these drug-resistant cells to daunomycin, doxorubicin and epirubicin. These findings indicate that lysosomotropic agents that impair the acidic-pH-dependent accumulation of weak-base chemotherapeutic drugs may reverse anthracycline resistance in MDR cells with an expanded acidic lysosomal compartment.
Cd2+ and the N-terminal metal-binding domain protect the putative membranous CPC motif of the Cd2+-ATPase of Listeria monocytogenes
Authors: BAL Nathalie , WU Chen Chou , CATTY Patrice , GUILLAIN Florent , MINTZ Elisabeth
Published: February 2003

CadA, the Cd2+-ATPase of Listeria monocytogenes, contains four cysteine residues: two in the CTNC (Cys-Thr-Asn-Cys) sequence in the cytoplasmic metal-binding domain (MBD), and two in the CPC (Cys-Pro-Cys) sequence in the membrane domain. Taking advantage of ΔMBD, a truncated version of CadA that lacks the MBD but which still acts as a functional Cd2+-ATPase [Bal, Mintz, Guillain and Catty (2001) FEBS Lett. 506, 249—252], we analysed the role of the membrane cysteine residues (studied using ΔMBD) separately from that of the cysteine residues of the MBD, which were studied using full-length CadA. The role of the cysteines was assessed by reacting ΔMBD and CadA with N-ethylmaleimide (NEM), an SH-specific reagent, in the presence or absence of Cd2+. We show here that (i) in both ΔMBD and CadA, the cysteine residues in the CPC motif are essential for phosphorylation; (ii) in both proteins, Cd2+ protects against alkylation by NEM; and (iii) in the absence of Cd2+, the MBD of CadA also protects against alkylation by NEM. Our results suggest that the CPC motif is present in the membrane Cd2+ transport site(s) and that the MBD protects these site(s).
Formation of active monomers from tetrameric human beta-tryptase
Authors: FAJARDO Ignacio , PEJLER Gunnar
Published: February 2003

Tryptase is a serine protease that is stored at low pH in the mast cell secretory granules in complex with heparin proteoglycan. When mast cells are activated, e.g. during allergic responses, the tryptase/heparin complexes are released together with a variety of other preformed inflammatory mediators. Previous crystallization of human β-tryptase revealed a unique tetrameric structure with all of the active sites facing a central pore that has a limited accessibility both for potential substrates as well as for protease inhibitors. In this study we examined whether human β-tryptase, in addition, could form active monomers. Incubation of recombinant tetrameric human β-tryptase at neutral pH and 37°C, followed by gel-filtration analysis using a running buffer containing pig mucosal heparin, led to the formation of enzymically active compounds that were of a size compatible with tryptase monomers in complex with heparin. The monomers were, in contrast to tryptase in the tetrameric form, inhibited by bovine pancreatic trypsin inhibitor. Further, the monomers, but not the tetramers, degraded fibronectin. Formation of active monomers was more pronounced at pH7.5 than at pH6.0 and was not detected at room temperature or at high heparin/tryptase ratios. The present findings thus introduce the possibility that human β-tryptase, after mast cell degranulation and exposure to neutral pH in the tissue, may dissociate into active monomers with properties that are distinct from the tetrameric counterpart. Possibly, some of the biological activities of human tryptase may be attributable to active tryptase in its monomeric rather than tetrameric form.
In situ modulation of the human cardiac ryanodine receptor (hRyR2) by FKBP12.6
Authors: GEORGE Christopher H. , SORATHIA Rina , BERTRAND Benedicte M.A. , LAI F. Anthony
Published: February 2003

The ryanodine receptor complex (RyR), a large oligomeric assembly that functions as a Ca2+-release channel in the sarcoplasmic reticulum (SR)/endoplasmic reticulum (ER), comprises four RyR subunits and four FK506-binding proteins (FKBP). The precise mode of interaction and modulation of the cardiac RyR (RyR2) channel by FKBP12/FKBP12.6 remains to be fully defined. We have generated a series of Chinese-hamster ovary (CHO) cell lines stably expressing discrete levels of recombinant human RyR2 (hRyR2) (CHOhRyR2). Confocal microscopy of CHOhRyR2 cells co-expressing either FKBP12 or FKBP12.6 demonstrated that FKBP12.6 was sequestered from the cytoplasm to ER membranes as the cellular levels of hRyR2 increased. There was negligible hRyR2-induced subcellular redistribution of FKBP12. The magnitude of Ca2+ release in CHOhRyR2 cells in response to stimulation by 4-chloro-m-cresol was in direct proportion to the expression levels of hRyR2. However, in CHOhRyR2 cells co-expressing FKBP12.6, Ca2+ release triggered by the addition of 4-chloro-m-cresol was markedly decreased. In contrast, co-expression of FKBP12 did not affect agonist-induced Ca2+ release in CHOhRyR2 cells. Resting cytoplasmic [Ca2+] in CHOhRyR2 remained unaltered after co-expression of FKBP12 or FKBP12.6, but estimation of the ER Ca2+ load status showed that co-expression of FKBP12.6, but not FKBP12, promoted superfilling of the ER Ca2+ store which could not be released by RyR2 after agonist activation. The effects of FKBP12.6 on hRyR2-mediated intracellular Ca2+ handling could be antagonized using rapamycin (5μM). These results suggest that FKBP12.6 associates with hRyR2 in situ to modulate precisely the functionality of hRyR2 Ca2+-release channel.
Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium
Authors: RAUX Evelyne , LEECH Helen K. , BECK Richard , SCHUBERT Heidi L. , SANTANDER Patricio J. , ROESSNER Charles A. , SCOTT A. Ian , MARTENS Jan H. , JAHN Dieter , THERMES Claude , RAMBACH Alain , WARREN Martin J.
Published: February 2003

In Bacillus megaterium, the hemAXBCDL genes were isolated and were found to be highly similar to the genes from Bacillus subtilis that are required for the conversion of glutamyl-tRNA into uroporphyrinogen III. Overproduction and purification of HemC (porphobilinogen deaminase) and -D (uroporphyrinogen III synthase) allowed these enzymes to be used for the in vitro synthesis of uroporphyrinogen III from porphobilinogen. A second smaller cluster of three genes (termed sirABC) was also isolated and found to encode the enzymes that catalyse the transformation of uroporphyrinogen III into sirohaem on the basis of their ability to complement a defined Escherichia coli (cysG) mutant. The functions of SirC and -B were investigated by direct enzyme assay, where SirC was found to act as a precorrin-2 dehydrogenase, generating sirohydrochlorin, and SirB was found to act as a ferrochelatase responsible for the final step in sirohaem synthesis. CbiX, a protein found encoded within the main B. megaterium cobalamin biosynthetic operon, shares a high degree of similarity with SirB and acts as the cobaltochelatase associated with cobalamin biosynthesis by inserting cobalt into sirohydrochlorin. CbiX contains an unusual histidine-rich region in the C-terminal portion of the protein, which was not found to be essential in the chelation process. Sequence alignments suggest that SirB and CbiX share a similar active site to the cobaltochelatase, CbiK, from Salmonella enterica.
An amphibian antimicrobial peptide variant expressed in Nicotiana tabacum confers resistance to phytopathogens
Authors: PONTI Donatella , MANGONI M. LUISA , MIGNOGNA Giuseppina , SIMMACO Maurizio , BARRA Donatella
Published: February 2003

Esculentin-1 is a 46-residue antimicrobial peptide present in skin secretions of Rana esculenta. It is effective against a wide variety of micro-organisms, including plant pathogens with negligible effects on eukaryotic cells. As a possible approach to enhance plant resistance, a DNA coding for esculentin-1, with the substitution Met-28Leu, was fused at the C-terminal end of the leader sequence of endopolygalacturonase-inhibiting protein, under the control of the cauliflower mosaic virus 35S promoter region, and introduced into Nicotiana tabacum. The antimicrobial peptide was isolated from the intercellular fluids of healthy leaves of transgenic plants, suggesting that it was properly processed, secreted outside cells and accumulated in the intercellular spaces. The morphology of transgenic plants was unaffected. Challenging these plants with bacterial or fungal phytopathogens demonstrated enhanced resistance up to the second generation. Moreover, transgenic plants displayed insecticidal properties.
Molecular cloning and characterization of a novel carboxylesterase-like protein that is physiologically present at high concentrations in the urine of domestic cats (Felis catus)
Authors: MIYAZAKI Masao , KAMIIE Katsuyoshi , SOETA Satoshi , TAIRA Hideharu , YAMASHITA Tetsuro
Published: February 2003

Normal mammals generally excrete only small amounts of protein in the urine, thus avoiding major leakage of proteins from the body. Proteinuria is the most commonly recognized abnormality in renal disease. However, healthy domestic cats (Felis catus) excrete proteins at high concentrations (about 0.5mg/ml) in their urine. We investigated the possible cause of proteinuria in healthy cats, and discovered a 70kDa glycoprotein, which was excreted as a major urinary protein in cat urine, irrespective of gender. To elucidate the biochemical functions and the excretion mechanism of this protein, we cloned the cDNA for this protein from a cat kidney cDNA library. The deduced amino acid sequence shared 47% identity with the rat liver carboxylesterase (EC 3.1.1.1), and both the serine hydrolase active site and the carboxylesterase-specific sequence were conserved. Therefore we named this protein cauxin (carboxylesterase-like urinary excreted protein). In contrast to the mammalian carboxylesterases, most of which are localized within the cells of various organs, cauxin was expressed specifically in the epithelial cells of the distal tubules, and was secreted efficiently into the urine, probably because it lacked the endoplasmic reticulum retention sequence (HDEL). Based on our finding that cauxin is not expressed in the immature cat kidney, we conclude that cauxin is involved in physiological functions that are specific for mature cats. Recently, cauxin-like cDNAs were found from human brain and teratocarcinoma cells. These data suggest that cauxin and cauxin-like human proteins are categorized as a novel group of carboxylesterase multigene family.
Transport of leukotriene C4 by a cysteine-less multidrug resistance protein 1 (MRP1)
Authors: LEE Sung H. , ALTENBERG Guillermo A.
Published: February 2003

Overexpression of multidrug resistance protein 1 (MRP1), an ATP-binding cassette protein, causes multidrug resistance. We developed a functional cysteine-less version of MRP1 that provides a framework for detailed biochemical and biophysical studies. The 18 Cys residues of a truncated MRP1 (tMRP1; lacking the first multispanning transmembrane domain) were replaced with Ala to generate Cys-less tMRP1 (CL tMRP1). CL tMRP1 expressed in Saccharomyces cerevisiae membranes displayed high-affinity ATP-dependent transport of the MRP1 substrate leukotriene C4. Compared with full-length MRP1, the Km for leukotriene C4 transport by CL tMRP1 was increased 3-fold, while Vmax was not affected. Thus a functional CL tMRP1 can be expressed using a low-cost and rapid-generation yeast expression system. This Cys-less protein can be used for biochemical, spectroscopic and structural studies to elucidate the mechanism of drug transport by MRP1.
Calcium triggers the refolding of Bacillus subtilis chitosanase
Authors: COLOMER-PALLAS Anne , PEREIRA Yannick , PETIT-GLATRON Marie-Françoise , CHAMBERT Régis
Published: February 2003

We characterized the reversible folding—unfolding transition of Bacillus subtilis exocellular chitosanase from either thermal or urea denaturation of the protein. The transitions were monitored in each case by intrinsic fluorescence changes and resistance to proteolysis. Unfolding and refolding kinetics and differential scanning calorimetry analysis suggested a two-state equilibrium. The equilibrium between the folded and unfolded states was rapidly displaced towards the folded state in the presence of a low concentration of calcium (2—20mM). The binding titration curve indicated that chitosanase possesses one weak Ca2+-binding site (with an equilibrium affinity constant, KA, of 0.3×103M-1). These results support the hypothesis that this metal ion, which is accumulated in the cell wall environment of B. subtilis, is an effector that influences folding and stability of newly translocated proteins.
Role of cdc2 kinase phosphorylation and conserved N-terminal proteolysis motifs in cytoplasmic polyadenylation-element-binding protein (CPEB) complex dissociation and degradation
Authors: THOM George , MINSHALL Nicola , GIT Anna , ARGASINSKA Joanna , STANDART Nancy
Published: February 2003

Cytoplasmic polyadenylation-element-binding protein (CPEB) is a well-characterized and important regulator of translation of maternal mRNA in early development in organisms ranging from worms, flies and clams to frogs and mice. Previous studies provided evidence that clam and Xenopus CPEB are hyperphosphorylated at germinal vesicle breakdown (GVBD) by cdc2 kinase, and degraded shortly after. To examine the conserved features of CPEB that mediate its modification during meiotic maturation, we microinjected mRNA encoding wild-type and mutated clam CPEB into Xenopus oocytes that were subsequently allowed to mature with progesterone. We observed that (i) ectopically expressed clam CPEB is phosphorylated at GVBD and subsequently degraded, mirroring the fate of the endogenous Xenopus CPEB protein, (ii) mutation of nine Ser/Thr Pro-directed kinase sites prevents phosphorylation and degradation and (iii) deletion of the PEST box, and to a lesser extent of the putative cyclin destruction box, generates a stable and phosphorylated version of CPEB. We conclude that phosphorylation of both consensus and non-consensus sites by cdc2 kinase targets clam CPEB for PEST-mediated destruction. We also show that phosphorylation of CPEB mediates its dissociation from ribonucleoprotein complexes, prior to degradation. Our findings reinforce results obtained in Xenopus, and have implications for CPEB from other invertebrates including Drosophila, Caenorhabditis elegans and Aplysia, which lack PEST boxes.
The three-dimensional structural surface of two beta-sheet scorpion toxins mimics that of an alpha-helical dihydropyridine receptor segment
Authors: GREEN Daniel , PACE Suzi , CURTIS Suzanne M. , SAKOWSKA Magdalena , LAMB Graham D. , DULHUNTY Angela F. , CASAROTTO Marco G.
Published: February 2003

An α-helical II—III loop segment of the dihydropyridine receptor activates the ryanodine receptor calcium-release channel. We describe a novel manipulation in which this agonist's activity is increased by modifying its surface structure to resemble that of a toxin molecule. In a unique system, native β-sheet scorpion toxins have been reported to activate skeletal muscle ryanodine receptor calcium channels with high affinity by binding to the same site as the lower-affinity α-helical dihydropyridine receptor segment. We increased the alignment of basic residues in the α-helical peptide to mimic the spatial orientation of active residues in the scorpion toxin, with a consequent 2—20-fold increase in the activity of the α-helical peptide. We hypothesized that, like the native peptide, the modified peptide and the scorpion toxin may bind to a common site. This was supported by (i) similar changes in ryanodine receptor channel gating induced by the native or modified α-helical peptide and the β-sheet toxin, a 10—100-fold reduction in channel closed time, with a ≤2-fold increase in open dwell time and (ii) a failure of the toxin to further activate channels activated by the peptides. These results suggest that diverse structural scaffolds can present similar conformational surface properties to target common receptor sites.
Effect of mutation of two critical glutamic acid residues on the activity and stability of human carboxypeptidase M and characterization of its signal for glycosylphosphatidylinositol anchoring
Authors: TAN Fulong , BALSITIS Scott , BLACK Judy K. , BLÖCHL Andrea , MAO Ji-Fang , BECKER Robert P. , SCHACHT David , SKIDGEL Randal A.
Published: February 2003

Human carboxypeptidase (CP) M was expressed in baculovirus-infected insect cells in a glycosylphosphatidylinositol-anchored form, whereas a truncated form, lacking the putative signal sequence for glycosylphosphatidylinositol anchoring, was secreted at high levels into the medium. Both forms had lower molecular masses (50kDa) than native placental CPM (62kDa), indicating minimal glycosylation. The predicted glycosylphosphatidylinositol-anchor attachment site was investigated by mutation of Ser406 to Ala, Thr or Pro and expression in HEK-293 and COS-7 cells. The wild-type and S406A and S406T mutants were expressed on the plasma membrane in glycosylphosphatidylinositol-anchored form, but the S406P mutant was not and was retained in a perinuclear location. The roles of Glu260 and Glu264 in CPM were investigated by site-directed mutagenesis. Mutation of Glu260 to Gln had minimal effects on kinetic parameters, but decreased heat stability, whereas mutation to Ala reduced the kcat/Km by 104-fold and further decreased stability. In contrast, mutation of Glu264 to Gln resulted in a 10000-fold decrease in activity, but the enzyme still bound to p-aminobenzoylarginine—Sepharose and was resistant to trypsin treatment, indicating that the protein was folded properly. These results show that Glu264 is the critical catalytic glutamic acid and that Glu260 probably stabilizes the conformation of the active site.
Evidence for secretion-like coupling involving pp60src in the activation and maintenance of store-mediated Ca2+ entry in mouse pancreatic acinar cells
Authors: REDONDO Pedro C. , LAJAS Ana I. , SALIDO Ginés M. , GONZALEZ Antonio , ROSADO Juan A. , PARIENTE José A.
Published: February 2003

Store-mediated Ca2+ entry (SMCE) is one of the main pathways for Ca2+ influx in non-excitable cells. Recent studies favour a secretion-like coupling mechanism to explain SMCE, where Ca2+ entry is mediated by an interaction of the endoplasmic reticulum (ER) with the plasma membrane (PM) and is modulated by the actin cytoskeleton. To explore this possibility further we have now investigated the role of the actin cytoskeleton in the activation and maintenance of SMCE in pancreatic acinar cells, a more specialized secretory cell type which might be an ideal cellular model to investigate further the properties of the secretion-like coupling model. In these cells, the cytoskeletal disrupters cytochalasin D and latrunculin A inhibited both the activation and maintenance of SMCE. In addition, stabilization of a cortical actin barrier by jasplakinolide prevented the activation, but not the maintenance, of SMCE, suggesting that, as for secretion, the actin cytoskeleton plays a double role in SMCE as a negative modulator of the interaction between the ER and PM, but is also required for this mechanism, since the cytoskeleton disrupters impaired Ca2+ entry. Finally, depletion of the intracellular Ca2+ stores induces cytoskeletal association and activation of pp60src, which is independent on Ca2+ entry. pp60src activation requires the integrity of the actin cytoskeleton and participates in the initial phase of the activation of SMCE in pancreatic acinar cells.
Investigation of potential mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase isoforms 2 and 4 by fatty acids and thyroid hormone
Authors: HOLNESS Mark J. , BULMER Karen , SMITH Nicholas D. , SUGDEN Mary C.
Published: February 2003

Liver contains two pyruvate dehydrogenase kinases (PDKs), namely PDK2 and PDK4, which regulate glucose oxidation through inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Starvation increases hepatic PDK2 and PDK4 protein expression, the latter occurring, in part, via a mechanism involving peroxisome proliferator-activated receptor-α (PPARα). High-fat feeding and hyperthyroidism, which increase circulating lipid supply, enhance hepatic PDK2 protein expression, but these increases are insufficient to account for observed increases in hepatic PDK activity. Enhanced expression of PDK4, but not PDK2, occurs in part via a mechanism involving PPAR-α. Heterodimerization partners for retinoid X receptors (RXRs) include PPARα and thyroid-hormone receptors (TRs). We therefore investigated the responses of hepatic PDK protein expression to high-fat feeding and hyperthyroidism in relation to hepatic lipid delivery and disposal. High-fat feeding increased hepatic PDK2, but not PDK4, protein expression whereas hyperthyroidism increased both hepatic PDK2 and PDK4 protein expression. Both manipulations decreased the sensitivity of hepatic carnitine palmitoyltransferase I (CPT I) to suppression by malonyl-CoA, but only hyperthyrodism elevated plasma fatty acid and ketone-body concentrations and CPT I maximal activity. Administration of the selective PPAR-α activator WY14,643 significantly increased PDK4 protein to a similar extent in both control and high-fat-fed rats, but WY14,643 treatment and hyperthyroidism did not have additive effects on hepatic PDK4 protein expression. PPARα activation did not influence hepatic PDK2 protein expression in euthyroid rats, suggesting that up-regulation of PDK2 by hyperthyroidism does not involve PPARα, but attenuated the effect of hyperthyroidism to increase hepatic PDK2 expression. The results indicate that hepatic PDK4 up-regulation can be achieved by heterodimerization of either PPARα or TR with the RXR receptor and that effects of PPARα activation on hepatic PDK2 and PDK4 expression favour a switch towards preferential expression of PDK4.
Prostaglandin E2 mediates growth arrest in NFS-60 cells by down-regulating interleukin-6 receptor expression
Authors: SILVA Kumudika I. de , DAUD Asif N. , DENG JiangPing , JONES Stephen B. , GAMELLI Richard L. , SHANKAR Ravi
Published: February 2003

Interleukin-6 (IL-6), a potent myeloid mitogen, and the immunosuppressive prostanoid prostaglandin E2 (PGE2) are elevated following thermal injury and sepsis. We have previously demonstrated that bone marrow myeloid commitment shifts toward monocytopoiesis and away from granulocytopoiesis during thermal injury and sepsis and that PGE2 plays a central role in this alteration. Here we investigated whether PGE2 can modulate IL-6-stimulated growth in the promyelocytic cell line, NFS-60, by down-regulating IL-6 receptor (IL-6r) expression. Exposure of NFS-60 cells to PGE2 suppressed IL-6-stimulated proliferation as well as IL-6r expression. Receptor down-regulation is functionally significant since IL-6-induced signal transduction through activators of transcription (STAT)-3 is also decreased. Down-regulation of IL-6r correlated with the ability of PGE2 to arrest cells in the G0/G1 phase of the cell cycle. PGE2 appears to signal through EP2 receptors. Butaprost (EP2 agonist) but not sulprostone (EP3 agonist) inhibited IL-6-stimulated proliferation. In addition, an EP2 antagonist (AH6809) alleviated the anti-proliferative effects of PGE2. NFS-60 cells express predominantly EP2 and EP4 receptors. While PGE2 down-regulated both the IL-6r protein and mRNA expression, it had no influence on EP2 or EP4 mRNA expression. The present study demonstrates that PGE2 is a potent down-regulator of IL-6r expression and thus may provide a mechanistic explanation for the granulocytopenia seen in thermal injury and sepsis.
Modulation of cytochrome c spin states by lipid acyl chains: a continuous-wave electron paramagnetic resonance (CW-EPR) study of haem iron
Authors: ZUCCHI Maria R. , NASCIMENTO Otaciro R. , FALJONI-ALARIO Adelaide , PRIETO Tatiana , NANTES Iseli L.
Published: February 2003

This work is a systematic study, showing a clear correlation between the nature of the lipid acyl chain and the spin states of cytochrome c interacting with different types of lipid membranes. According to the lipid acyl chain type, and the head group charge present in the bilayer, three spin states of cytochrome c were observed in different proportions: the native cytochrome c low spin state with rhombic symmetry (spin 1/2, g//=3.07 and g⊥=2.23), a low spin state with less rhombic symmetry (spin 1/2, g1 = 2.902, g2 = 2.225, and g3 = 1.510) and the high spin state (spin 5/2, g// = 6.0 and g⊥ = 2.0). The proportion of the spin states of cytochrome c bound to bilayers was also dependent on the lipid/protein ratio, suggesting the existence of two or more protein sites interacting with the lipids. The lipid-induced alterations in the symmetry and spin states of cytochrome c exhibited partial reversibility when the ionic strength was increased, which reinforces the crucial role played by the electrostatic interaction with the lipid bilayer. Different cytochrome c spin states exhibited corresponding modifications in the haemprotein UV/visible spectra, particularly in the Q-band associated with loss of the 695nm band and appearance of a band in the region of 600—650nm. The observed reactivity of cytochrome c with oxidized forms of unsaturated lipids reinforces the possibility of the acyl chain insertion in the haemprotein structure.
Cbl-ArgBP2 complex mediates ubiquitination and degradation of c-Abl
Authors: SOUBEYRAN Philippe , BARAC Ana , SZYMKIEWICZ Iwona , DIKIC Ivan
Published: February 2003

The mechanisms leading to the ubiquitination and degradation of the activated c-Abl kinase have not yet been identified. We found that the multi-adaptor protein ArgBP2 links c-Abl to the ubiquitin ligase Cbl. Phosphorylation of Cbl and ArgBP2 by c-Abl resulted in the stabilization of their interactions, thus facilitating Cbl-induced ubiquitination and subsequent degradation of c-Abl and ArgBP2.
Archaeal protein kinases and protein phosphatases: insights from genomics and biochemistry
Authors: KENNELLY Peter J.
Published: February 2003

Protein phosphorylation/dephosphorylation has long been considered a recent addition to Nature's regulatory arsenal. Early studies indicated that this molecular regulatory mechanism existed only in higher eukaryotes, suggesting that protein phosphorylation/dephosphorylation had emerged to meet the particular signal-transduction requirements of multicellular organisms. Although it has since become apparent that simple eukaryotes and even bacteria are sites of protein phosphorylation/dephosphorylation, the perception widely persists that this molecular regulatory mechanism emerged late in evolution, i.e. after the divergence of the contemporary phylogenetic domains. Only highly developed cells, it was reasoned, could afford the high ‘overhead’ costs inherent in the acquisition of dedicated protein kinases and protein phosphatases. The advent of genome sequencing has provided an opportunity to exploit Nature's phylogenetic diversity as a vehicle for critically examining this hypothesis. In tracing the origins and evolution of protein phosphorylation/dephosphorylation, the members of the Archaea, the so-called ‘third domain of life’, will play a critical role. Whereas several studies have demonstrated that archaeal proteins are subject to modification by covalent phosphorylation, relatively little is known concerning the identities of the proteins affected, the impact on their functional properties, or the enzymes that catalyse these events. However, examination of several archaeal genomes has revealed the widespread presence of several ostensibly ‘eukaryotic’ and ‘bacterial’ protein kinase and protein phosphatase paradigms. Similar findings of ‘phylogenetic trespass’ in members of the Eucarya (eukaryotes) and the Bacteria suggest that this versatile molecular regulatory mechanism emerged at an unexpectedly early point in development of ‘life as we know it'.
The TATA-binding protein is not an essential target of the transcriptional activators Gal4p and Gcn4p in Saccharomyces cerevisiae
Authors: BONGARDS Christine , CHEW Boon Shang , LEHMING Norbert
Published: February 2003

According to the recruitment model, transcriptional activators work by increasing the local concentration of one or several limiting factors for the transcription process at the target promoter. The TATA-binding protein Tbp1 has been considered as a likely candidate for such a limiting factor. We have used a series of Gal4p and Tbp1 mutants to correlate the in vivo interaction between the two proteins with the strength of activation. We find a clear correlation between activation strength and in vivo interaction for the series of Gal4p mutants. Consistently, the weaker activator Gcn4p does not interact with Tbp1. However, a corresponding analysis of the series of Tbp1 mutants revealed that Tbp1 is not an essential target of the acidic activators Gal4p and Gcn4p. Furthermore, detailed analysis of a Tbp1 mutant deficient for transcriptional activation by Gal4p revealed that the mutant is defective in interactions with five other proteins involved in the process of transcription.
Phosphorylation of myosin II regulatory light chain is necessary for migration of HeLa cells but not for localization of myosin II at the leading edge
Authors: FUMOTO Katsumi , UCHIMURA Takashi , IWASAKI Takahiro , UEDA Kozue , HOSOYA Hiroshi
Published: February 2003

To investigate the role of phosphorylated myosin II regulatory light chain (MRLC) in living cell migration, these mutant MRLCs were engineered and introduced into HeLa cells. The mutant MRLCs include an unphosphorylatable form, in which both Thr-18 and Ser-19 were substituted with Ala (AA-MRLC), and pseudophosphorylated forms, in which Thr-18 and Ser-19 were replaced with Ala and Asp, respectively (AD-MRLC), and both Thr-18 and Ser-19 were replaced with Asp (DD-MRLC). Mutant MRLC-expressing cell monolayers were mechanically stimulated by scratching, and the cells were forced to migrate in a given direction. In this wound-healing assay, the AA-MRLC-expressing cells migrated much more slowly than the wild-type MRLC-expressing cells. In the case of DD-MRLC- and AD-MRLC-expressing cells, no significant differences compared with wild-type MRLC-expressing cells were observed in their migration speed. Indirect immunofluorescence staining showed that the accumulation of endogenous diphosphorylated MRLC at the leading edge was not observed in AA-MRLC-expressing cells, although AA-MRLC was incorporated into myosin heavy chain and localized at the leading edge. In conclusion, we propose that the phosphorylation of MRLC is required to generate the driving force in the migration of the cells but not necessary for localization of myosin II at the leading edge.
The transcriptional repressor gene Mad3 is a novel target for regulation by E2F1
Authors: FOX Elizabeth J. , WRIGHT Stephanie C.
Published: February 2003

Mad family proteins are transcriptional repressors that antagonize the activity of the c-Myc proto-oncogene product. Mad3 is expressed specifically during the S-phase of the cell cycle in both proliferating and differentiating cells, suggesting that its biological function is probably linked to processes that occur during this period. To determine the mechanisms that regulate the cell-cycle-specific transcription of Mad3, we used reporter gene assays in stably transfected fibroblasts. We show that the activation of Mad3 at the G1—S boundary is mediated by a single E2F (E2 promoter binding factor)-binding site within the 5′-flanking region of the gene. Mutation of this element eliminated transcriptional activation at S-phase, suggesting that the positively acting E2F proteins play a role in Mad3 regulation. Using electrophoretic mobility-shift assays and chromatin immunoprecipitation, we show that E2F1 binds to the Mad3 5′-flanking region both in vitro and in vivo. We thus identify Mad3 as a novel transcriptional target of E2F1.
Molecular and biochemical characterization of the thermoactive family 1 pectate lyase from the hyperthermophilic bacterium Thermotoga maritima
Authors: KLUSKENS Leon D. , ALEBEEK Gert-Jan W.M. van , VORAGEN Alphons G.J. , VOS Willem M. de , OOST John van der
Published: February 2003

The ability of the hyperthermophilic bacterium Thermotoga maritima to grow on pectin as a sole carbon source coincides with the secretion of a pectate lyase A (PelA) in the extracellular medium. The pelA gene of T. maritima was functionally expressed in Escherichia coli as the first heterologously produced thermophilic pectinase, and purified to homogeneity. Gel filtration indicated that the native form of PelA is tetrameric. Highest activity (422units/mg, with a Km of 0.06mM) was demonstrated on polygalacturonic acid (PGA), whereas pectins with an increasing degree of methylation were degraded at a decreasing rate. In the tradition of pectate lyases, PelA demonstrated full dependency on Ca2+ for stability and activity. The enzyme is highly thermoactive and thermostable, operating optimally at 90°C and pH9.0, with a half-life for thermal inactivation of almost 2h at 95°C, and an apparent melting temperature of 102.5°C. Detailed characterization of the product formation with PGA indicated that PelA has a unique eliminative exo-cleavage pattern liberating unsaturated trigalacturonate as the major product, in contrast with unsaturated digalacturonate for other exopectate lyases known. The unique exo-acting mode of action was supported by progression profiles of PelA on oligogalacturonides (degree of polymerization, 3—8) and the examination of the bond cleavage frequencies.
Reactive oxygen species mediate the down-regulation of mitochondrial transcripts and proteins by tumour necrosis factor-alpha in L929 cells
Authors: SÁNCHEZ-ALCÁZAR José A. , SCHNEIDER Erasmus , HERNÁNDEZ-MUÑOZ Inmaculada , RUIZ-CABELLO Jesús , SILES-RIVAS Eva , TORRE Paz de la , BORNSTEIN Belen , BREA Gloria , ARENAS Joaquín , GARESSE Rafael , SOLÍS-HERRUZO José A. , KNOX Alan J. , NAVAS Plácido
Published: February 2003

In this study, we show that reactive oxygen species production induced by tumour necrosis factor α (TNF-α) in L929 cells was associated with a decrease in the steady-state mRNA levels of the mitochondrial transcript ATPase 6-8. Simultaneously, the transcript levels of two nuclear-encoded glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphofructokinase, were increased. These changes were associated with decreased protein levels of the ATPase subunit a (encoded by the mitochondrial ATPase 6 gene) and cytochrome c oxidase subunit II, and increased protein levels of phosphofructokinase. Since TNF-α had no effect on the amount of mitochondrial DNA, the results suggested that TNF-α acted at the transcriptional and/or post-transcriptional level. Reactive oxygen species scavengers, such as butylated hydroxianisole and butylated hydroxytoluene, blocked the production of free radicals, prevented the down-regulation of ATPase 6-8 transcripts, preserved the protein levels of ATPase subunit a and cytochrome c oxidase subunit II, and attenuated the cytotoxic response to TNF-α, indicating a direct link between these two phenomena.
Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm
Authors: NEWTON Alexandra C.
Published: February 2003

Phosphorylation plays a central role in regulating the activation and signalling lifetime of protein kinases A, B (also known as Akt) and C. These kinases share three conserved phosphorylation motifs: the activation loop segment, the turn motif and the hydrophobic motif. This review focuses on how phosphorylation at each of these sites regulates the maturation, signalling and down-regulation of PKC as a paradigm for how these sites control the function of the ABC kinases.
Evidence that Ca2+-release-activated Ca2+ channels in rat hepatocytes are required for the maintenance of hormone-induced Ca2+ oscillations
Authors: GREGORY Roland B. , BARRITT Gregory J.
Published: February 2003

Store-operated Ca2+ channels in liver cells have been shown previously to exhibit a high selectivity for Ca2+ and to have properties indistinguishable from those of Ca2+-release-activated Ca2+ (CRAC) channels in mast cells and lymphocytes [Rychkov, Brereton, Harland and Barritt (2001) Hepatology 33, 938—947]. The role of CRAC channels in the maintenance of hormone-induced oscillations in the cytoplasmic free Ca2+ concentration ([Ca2+]cyt) in isolated rat hepatocytes was investigated using several inhibitors of CRAC channels. 2-Aminoethyl diphenylborate (2-APB; 75μM), Gd3+ (1μM) and 1-{β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SK&F 96365; 50μM) each inhibited vasopressin- and adrenaline (epinephrine)-induced Ca2+ oscillations (measured using fura-2). The characteristics of this inhibition were similar to those of inhibition caused by decreasing the extracellular Ca2+ concentration to zero by addition of EGTA. The effect of 2-APB was reversible. In contrast, LOE-908 {(R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamidemesylate}(30μM), used commonly to block Ca2+ inflow through intracellular-messenger-activated, non-selective cation channels, did not inhibit the Ca2+ oscillations. In the absence of added extracellular Ca2+, 2-APB, Gd3+ and SK&F 96365 did not alter the kinetics of the increase in [Ca2+]cyt induced by a concentration of adrenaline or vasopressin that induces continuous Ca2+ oscillations at the physiological extracellular Ca2+ concentration. Ca2+ inflow through non-selective cation channels activated by maitotoxin could not restore Ca2+ oscillations in cells treated with 2-APB to block Ca2+ inflow through CRAC channels. Evidence for the specificity of the pharmacological agents for inhibition of CRAC channels under the conditions of the present experiments with hepatocytes is discussed. It is concluded that Ca2+ inflow through CRAC channels is required for the maintenance of hormone-induced Ca2+ oscillations in isolated hepatocytes.