bluuebunny/crossref_metadata_2025 huggingface dataset:
Copper induces increased beta-sheet content in the scrapie-susceptible ovine prion protein PrPVRQ compared with the resistant allelic variant PrPARR
Authors: WONG Edmond , THACKRAY Alana M. , BUJDOSO Raymond
Published: June 2004

Prion diseases are characterized by conformational change in the copper-binding protein PrP (prion protein). Polymorphisms in ovine PrP at amino acid residues 136, 154 and 171 are associated with variation in susceptibility to scrapie. PrPVRQ [PrP(Val136/Arg154/Gln171)] or PrPARQ [PrP(Ala136/Arg154/Gln171)] animals show susceptibility to scrapie, whereas those that express Ala136/Arg154/Arg171 (PrPARR) show resistance. Results are presented here that show PrPVRQ and PrPARR display different conformational responses to metal-ion interaction. At 37 °C copper induced different levels of β-sheet content in the allelic variants of ovine full-length prion protein (amino acid 25–232). PrPVRQ showed a significant increase in β-sheet content when exposed to copper at 37 °C, whereas PrPARR remained relatively unchanged. The conversion of α-helical PrPVRQ to β-sheet form was shown by CD spectroscopy and the decreased binding of C-terminal specific monoclonal anti-PrP antibodies. This conversion to an increased β-sheet form did not occur with truncated PrPVRQ (amino acids 89–233), which demonstrates that additional metal-binding sites outside of the N-terminus may not overtly influence the overall structure of ovine PrP. Despite the difference in β-sheet content, both the scrapie-susceptible and -resistant allelic forms of ovine PrP acquired resistance to proteinase K digestion following exposure to copper at 37 °C, suggesting the potential for disease-associated PrPARR to accumulate in vivo. Our present study demonstrates that allelic variants of ovine PrP differ in their structure and response to the interaction with copper. These observations will contribute to a better understanding of the mechanism of susceptibility and resistance to prion disease.
The T-cell protein tyrosine phosphatase is phosphorylated on Ser-304 by cyclin-dependent protein kinases in mitosis
Authors: BUKCZYNSKA Patricia , KLINGLER-HOFFMANN Manuela , MITCHELHILL Kenneth I. , LAM Mark H. C. , CICCOMANCINI Melissa , TONKS Nicholas K. , SARCEVIC Boris , KEMP Bruce E. , TIGANIS Tony
Published: June 2004

Two alternatively spliced forms of the human protein tyrosine phosphatase TCPTP (T-cell protein tyrosine phosphatase) exist: a 48 kDa form that is targeted to the endoplasmic reticulum (TC48) and a shorter 45 kDa form that is targeted to the nucleus (TC45). In this study we have identified Ser-304 (Phe301-Asp-His-Ser304-Pro-Asn-Lys307) as a major TCPTP phosphory-lation site and demonstrate that TC45, but not TC48, is phosphorylated on this site in vivo. Phosphorylation of TC45 on Ser-304 was cell cycle-dependent, and increased as cells progressed from G2 into mitosis, but subsided upon mitotic exit. Ser-304 phosphorylation was increased when cells were arrested in mitosis by microtubule poisons such as nocodazole, but remained unaltered when cells were arrested at the G2/M checkpoint by adriamycin. Phosphorylation of Ser-304 did not alter significantly the phosphatase activity or the protein stability of TC45, and had no apparent effect on TC45 localization. Ser-304 phosphorylation was ablated when cells were treated with the CDK (cyclin-dependent protein kinase) inhibitors roscovitine or SU9516, but remained unaltered when ERK1/2 activation was inhibited with the MEK (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase) inhibitor PD98059. In addition, recombinant CDKs, but not the Polo-like kinase Plk1, phosphorylated Ser-304 in vitro. Our studies identify Ser-304 as a major phosphorylation site in human TCPTP, and the TC45 variant as a novel mitotic CDK substrate.
The C-terminus of prenylin is important in forming a dimer conformation necessary for endoplasmic-reticulum-to-Golgi transport
Authors: LIANG Zhimin , VEERAPRAME Helga , BAYAN Nami , LI Guangpu
Published: June 2004

Prenylin [or prenylated Rab acceptor 1 (PRA1)] is a multi-pass transmembrane protein that initially inserts into the ER (endoplasmic reticulum) membrane, followed by vesicular transport along the exocytic pathway to the Golgi complex where it may regulate the functions of prenylated proteins. Deletion of the C-terminal 10 amino acid residues of prenylin blocks its export from the ER. We have employed site-directed mutagenesis to investigate the role of each of the C-terminal 10 residues in the ER export of prenylin. This region contains a di-acidic motif (Asp176-Xaa-Glu), but changing either acidic residue to alanine has no effect on the ER export of prenylin. Alanine-scanning mutagenesis of the entire C-terminal region reveals that only the very C-terminal Val185 residue is crucial for the ER export of prenylin. Changing the C-terminal Val185 to most other amino acids effectively prevents prenylin from exiting the ER. However, deletion of Val185 has only moderate effect on the ER export of prenylin, suggesting that this valine residue is not part of an export signal itself; instead, it may affect the folding and conformation of prenylin. We show that the wild-type prenylin can efficiently form a homodimer in the cell by using a cell-permeant cross-linker, whereas the large C-terminal truncation and Val185 mutants are defective in forming such a dimer. Thus we have identified a single C-terminal valine residue that is essential for the proper dimerization and ER export of prenylin.
Interaction(s) of rotavirus non-structural protein 4 (NSP4) C-terminal peptides with model membranes
Authors: HUANG Huan , SCHROEDER Friedhelm , ESTES Mary K. , McPHERSON Tanya , BALL Judith M.
Published: June 2004

Rotavirus is the major cause of dehydrating gastroenteritis in children and young animals. NSP4 (non-structural protein 4), a rotaviral non-structural glycoprotein and a peptide NSP4114–135 (DKLTTREIEQVELLKRIYDKLT), corresponding to NSP4 amino acids 114–135, induce diarrhoeal disease in a neonatal mouse model and interact with model membranes that mimic caveolae. Correlation of the mechanisms of diarrhoea induction and membrane interactions by NSP4 protein and peptide remain unclear. Several additional NSP4 peptides were synthesized and their interactions with membranes studied by (i) CD, (ii) a filtration-binding assay and (iii) a fluorescent molecule leakage assay. Model membranes that varied in lipid compositions and radius of curvature were utilized to determine the compositional and structural requirements for optimal interaction with the peptides of NSP4. Similar to the intact protein and NSP4114–135, peptides overlapping residues 114–135 had significantly higher affinities to membranes rich in negatively charged lipids, rich in cholesterol and with a high radius of curvature. In the leakage assay, small and large unilamellar vesicles loaded with the fluorophore/quencher pair 8-aminonaphthalene-1,3,6-trisulphonic acid disodium salt/p-xylene-bis-pyridinium bromide were incubated with the NSP4 peptides and monitored for membrane disruption by lipid reorganization or by pore formation. At a peptide concentration of 15 µM, none of the NSP4 peptides caused leakage. These results confirm that NSP4 interacts with caveolae-like membranes and the α-helical region of NSP4114–135 comprises a membrane interaction domain that does not induce membrane disruption at physiological concentrations.
A mechanistic insight into a proteasome-independent constitutive inhibitor kappaBalpha (IkappaBalpha) degradation and nuclear factor kappaB (NF-kappaB) activation pathway in WEHI-231 B-cells
Authors: SHUMWAY Stuart D. , MIYAMOTO Shigeki
Published: June 2004

Inducible activation of the transcription factor NF-κB (nuclear factor κB) is classically mediated by proteasomal degradation of its associated inhibitors, IκBα (inhibitory κBα) and IκBβ. However, certain B-lymphocytes maintain constitutively nuclear NF-κB activity (a p50–c-Rel heterodimer) which is resistant to inhibition by proteasome inhibitors. This activity in the WEHI-231 B-cell line is associated with continual and preferential degradation of IκBα, which is also unaffected by proteasome inhibitors. Pharmacological studies indicated that there was a correlation between inhibition of IκBα degradation and constitutive p50–c-Rel activity. Domain analysis of IκBα by deletion mutagenesis demonstrated that an N-terminal 36-amino-acid sequence of IκBα represented an instability determinant for constitutive degradation. Moreover, domain grafting studies indicated that this sequence was sufficient to cause IκBβ, but not chloramphenicol acetyltransferase, to be rapidly degraded in WEHI-231 B-cells. However, this sequence was insufficient to target IκBβ to the non-proteasome degradation pathway, suggesting that there was an additional cis-element(s) in IκBα that was required for complete targeting. Nevertheless, the NF-κB pool associated with IκBβ now became constitutively active by virtue of IκBβ instability in these cells. These findings further support the notion that IκB instability governs the maintenance of constitutive p50–c-Rel activity in certain B-cells via a unique degradation pathway.
Intracellular trafficking of the human Wilson protein: the role of the six N-terminal metal-binding sites
Authors: CATER Michael A. , FORBES John , La FONTAINE Sharon , COX Diane , MERCER Julian F. B.
Published: June 2004

The Wilson protein (ATP7B) is a copper-transporting CPx-type ATPase defective in the copper toxicity disorder Wilson disease. In hepatocytes, ATP7B delivers copper to apo-ceruloplasmin and mediates the excretion of excess copper into bile. These distinct functions require the protein to localize at two different subcellular compartments. At the trans-Golgi network, ATP7B transports copper for incorporation into apo-ceruloplasmin. When intracellular copper levels are increased, ATP7B traffics to post-Golgi vesicles in close proximity to the canalicular membrane to facilitate biliary copper excretion. In the present study, we investigated the role of the six N-terminal MBSs (metal-binding sites) in the trafficking process. Using site-directed mutagenesis, we mutated or deleted various combinations of the MBSs and assessed the effect of these changes on the localization and trafficking of ATP7B. Results show that the MBSs required for trafficking are the same as those previously found essential for the copper transport function. Either MBS 5 or MBS 6 alone was sufficient to support the redistribution of ATP7B to vesicular compartments. The first three N-terminal motifs were not required for copper-dependent intracellular trafficking and could not functionally replace sites 4–6 when placed in the same sequence position. Furthermore, the N-terminal region encompassing MBSs 1–5 (amino acids 64–540) was not essential for trafficking, with only one MBS close to the membrane channel, necessary and sufficient to support trafficking. Our findings were similar to those obtained for the closely related ATP7A protein, suggesting similar mechanisms for trafficking between copper-transporting CPx-type ATPases.
Catalytic reaction of cytokinin dehydrogenase: preference for quinones as electron acceptors
Authors: FRÉBORTOVÁ Jitka , FRAAIJE Marco W. , GALUSZKA Petr , ŠEBELA Marek , PEČ Pavel , HRBÁČ Jan , NOVÁK Ondřej , BILYEU Kristin D. , ENGLISH James T. , FRÉBORT Ivo
Published: June 2004

The catalytic reaction of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) was studied in detail using the recombinant flavoenzyme from maize. Determination of the redox potential of the covalently linked flavin cofactor revealed a relatively high potential dictating the type of electron acceptor that can be used by the enzyme. Using 2,6-dichlorophenol indophenol, 2,3-dimethoxy-5-methyl-1,4-benzoquinone or 1,4-naphthoquinone as electron acceptor, turnover rates with N6-(2-isopentenyl)adenine of approx. 150 s−1 could be obtained. This suggests that the natural electron acceptor of the enzyme is quite probably a p-quinone or similar compound. By using the stopped-flow technique, it was found that the enzyme is rapidly reduced by N6-(2-isopentenyl)adenine (kred=950 s−1). Re-oxidation of the reduced enzyme by molecular oxygen is too slow to be of physiological relevance, confirming its classification as a dehydrogenase. Furthermore, it was established for the first time that the enzyme is capable of degrading aromatic cytokinins, although at low reaction rates. As a result, the enzyme displays a dual catalytic mode for oxidative degradation of cytokinins: a low-rate and low-substrate specificity reaction with oxygen as the electron acceptor, and high activity and strict specificity for isopentenyladenine and analogous cytokinins with some specific electron acceptors.
Kinetic and structural characterization of a product complex of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Escherichia coli
Authors: GARÇON Arnaud , BERMINGHAM Alun , LIAN Lu-Yun , DERRICK Jeremy P.
Published: June 2004

HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) catalyses the transfer of pyrophosphate from ATP to HMDP (6-hydroxymethyl-7,8-dihydropterin), to form AMP and DHPPP (6-hydroxymethyl-7,8-dihydropterin pyrophosphate). This transformation is a key step in the biosynthesis of folic acid, and HPPK is consequently a target for antimicrobial drugs. The substrates are known to bind to HPPK in an ordered manner, with ATP binding first followed by HMDP. In the present study we show by isothermal titration calorimetry that the product, DHPPP, can bind to the HPPK apoenzyme with high affinity (equilibrium dissociation constant, Kd=0.2 µM), but without the enhancement of pterin fluorescence that occurs on binding of HMDP. The transient kinetics of the enzyme can be monitored by measuring the change in the fluorescence of the pterin ring using stopped-flow methods. The fluorescence exhibits a pronounced biphasic behaviour: it initially rises and then declines back to its original level. This behaviour is in agreement with a two-state kinetic model, with the first phase of fluorescence increase associated with HMDP binding to the enzyme, and the second phase with a slow event that occurs after the reaction has taken place. The HPPK–DHPPP and HPPK–DHPPP–AMP complexes were examined by NMR, and the binding site for DHPPP partially mapped from changes in chemical shifts identified from two dimensional 1H/15N heteronuclear single-quantum coherence spectra. The results demonstrate that DHPPP, in contrast to HMDP, is able to bind to the HPPK apoenzyme and suggest that the pyrophosphate moieties on the ligand play an important role in establishment of a high affinity binding site for the pterin ring.
Contribution of the active-site metal cation to the catalytic activity and to the conformational stability of phosphotriesterase: temperature- and pH-dependence
Authors: ROCHU Daniel , VIGUIÉ Nathalie , RENAULT Frédérique , CROUZIER David , FROMENT Marie-Thérèse , MASSON Patrick
Published: June 2004

Phosphotriesterase (PTE) detoxifies nerve agents and organophosphate pesticides. The two zinc cations of the PTE active centre can be substituted by other transition metal cations without loss of activity. Furthermore, metal-substituted PTEs display differences in catalytic properties. A prerequisite for engineering highly efficient mutants of PTE is to improve their thermostability. Isoelectric focusing, capillary electrophoresis and steady-state kinetics analysis were used to determine the contribution of the active-site cations Zn2+, Co2+ or Cd2+ to both the catalytic activity and the conformational stability of the corresponding PTE isoforms. The three isoforms have different pI values (7.2, 7.5 and 7.1) and showed non-superimposable electrophoretic titration curves. The overall structural alterations, causing changes in functional properties, were found to be related to the nature of the bound cation: ionic radius and ion electronegativity correlate with Km and kcat respectively. In addition, the pH-dependent activity profiles of isoforms were different. The temperature-dependent profiles of activity showed maximum activity at T≤35 °C, followed by an activation phase near 45–48 °C and then inactivation which was completed at 60 °C. Analysis of thermal denaturation of the PTEs provided evidence that the activation phase resulted from a transient intermediate. Finally, at the optimum activity between pH 8 and 9.4, the thermostability of the different PTEs increased as the pH decreased, and the metal cation modulated stability (Zn2+-, Co2+- and Cd2+-PTE showed different Tm values of 60.5–67 °C, 58–64 °C and 53–64 °C respectively). Requirements for optimum activity of PTE (displayed by Co2+-PTE) and maximum stability (displayed by Zn2+-PTE) were demonstrated.
Complex changes in cellular inositol phosphate complement accompany transit through the cell cycle
Authors: BARKER Christopher J. , WRIGHT Joanne , HUGHES Philip J. , KIRK Christopher J. , MICHELL Robert H.
Published: June 2004

Inositol polyphosphates other than Ins(1,4,5)P3 are involved in several aspects of cell regulation. For example, recent evidence has implicated InsP6, Ins(1,3,4,5,6)P5 and their close metabolic relatives, which are amongst the more abundant intracellular inositol polyphosphates, in chromatin organization, DNA maintenance, gene transcription, nuclear mRNA transport, membrane trafficking and control of cell proliferation. However, little is known of how the intracellular concentrations of inositol polyphosphates change through the cell cycle. Here we show that the concentrations of several inositol polyphosphates fluctuate in synchrony with the cell cycle in proliferating WRK-1 cells. InsP6, Ins(1,3,4,5,6)P5 and their metabolic relatives behave similarly: concentrations are high during G1-phase, fall to much lower levels during S-phase and rise again late in the cycle. The Ins(1,2,3)P3 concentration shows especially large fluctuations, and PP-InsP5 fluctuations are also very marked. Remarkably, Ins(1,2,3)P3 turns over fastest during S-phase, when its concentration is lowest. These results establish that several fairly abundant intracellular inositol polyphosphates, for which important biological roles are emerging, display dynamic behaviour that is synchronized with cell-cycle progression.
Mutation of leucine-92 selectively reduces the apparent affinity of inosine, guanosine, NBMPR [S6-(4-nitrobenzyl)-mercaptopurine riboside] and dilazep for the human equilibrative nucleoside transporter, hENT1
Authors: ENDRES Christopher J. , SENGUPTA Dhruba J. , UNADKAT Jashvant D.
Published: June 2004

We developed a yeast-based assay for selection of hENT1 (human equilibrative nucleoside transporter 1) mutants that have altered affinity for hENT1 inhibitors and substrates. In this assay, expression of hENT1 in a yeast strain deficient in adenine biosynthesis (ade2) permits yeast growth on a plate lacking adenine but containing adenosine, a hENT1 substrate. This growth was prevented when inhibitors of hENT1 {e.g. NBMPR [S6-(4-nitrobenzyl)-mercaptopurine riboside], dilazep or dipyridamole} were included in the media. To identify hENT1 mutants resistant to inhibition by these compounds, hENT1 was randomly mutagenized and introduced into this strain. Mutation(s) that allowed growth of yeast cells in the presence of these inhibitors were then identified and characterized. Mutants harbouring amino acid changes at Leu92 exhibited resistance to NBMPR and dilazep but not dipyridamole. The IC50 values of NBMPR and dilazep for [3H]adenosine transport by one of these mutants L92Q (Leu92→Gln) were approx. 200- and 4-fold greater when compared with the value for the wild-type hENT1, whereas that for dipyridamole remained unchanged. Additionally, when compared with the wild-type transporter, [3H]adenosine transport by L92Q transporter was significantly resistant to inhibition by inosine and guanosine but not by adenosine or pyrimidines. The Km value for inosine transport was approx. 4-fold greater for the L92Q mutant (260±16 µM) when compared with the wild-type transporter (65±7.8 µM). We have identified for the first time an amino acid residue (Leu92) of hENT1 that, when mutated, selectively alters the affinity of hENT1 to transport the nucleosides inosine and guanosine and its sensitivity to the inhibitors NBMPR and dilazep.
Hypoxia/re-oxygenation-induced, redox-dependent activation of STAT1 (signal transducer and activator of transcription 1) confers resistance to apoptotic cell death via hsp70 induction
Authors: TERUI Keita , HAGA Sanae , ENOSAWA Shin , OHNUMA Naomi , OZAKI Michitaka
Published: June 2004

STAT1 (signal transducer and activator of transcription 1) is potentially involved in cell survival, as well as cell death, in different types of cells. The present study was designed to examine the effects of STAT1 on hypoxia/re-oxygenation (H/R)-induced cell death and/or survival, and the underlying mechanisms of any such effects. H/R was shown to induce apoptotic cell death of rat hepatocytes. The addition of a STAT1-specific inhibitor, fludarabine, significantly increased the fraction of apoptotic cells after H/R. Following H/R, STAT1 was activated and sequential phosphorylation of Tyr701 and Ser727 was observed, which could be inhibited by the antioxidant N-acetyl-l-cysteine. Tyrosine and serine phosphorylation of STAT1 was mediated by Janus kinase 2 and phosphoinositide 3-kinase/Akt kinase respectively in a redox-dependent manner following H/R. STAT1-induced HSP70 (heat-shock protein 70) expression and the suppression of apoptosis occurred concomitantly. In conclusion, STAT1 activation, in a redox-dependent manner, following H/R may play crucial roles in cell survival, at least partly via HSP70 induction.
Human aldolase A natural mutants: relationship between flexibility of the C-terminal region and enzyme function
Authors: ESPOSITO Gabriella , VITAGLIANO Luigi , COSTANZO Paola , BORRELLI Loredana , BARONE Rita , PAVONE Lorenzo , IZZO Paola , ZAGARI Adriana , SALVATORE Francesco
Published: June 2004

We have identified a new mutation in the FBP (fructose 1,6-bisphosphate) aldolase A gene in a child with suspected haemolytic anaemia associated with myopathic symptoms at birth and with a subsequent diagnosis of arthrogryposis multiplex congenita and pituitary ectopia. Sequence analysis of the whole gene, also performed on the patient's full-length cDNA, revealed only a Gly346→Ser substitution in the heterozygous state. We expressed in a bacterial system the new aldolase A Gly346→Ser mutant, and the Glu206→Lys mutant identified by others, in a patient with an aldolase A deficit. Analysis of their functional profiles showed that the Gly346→Ser mutant had the same Km as the wild-type enzyme, but a 4-fold lower kcat. The Glu206→Lys mutant had a Km approx. 2-fold higher than that of both the Gly346→Ser mutant and the wild-type enzyme, and a kcat value 40% less than the wild-type. The Gly346→Ser and wild-type enzymes had the same Tm (melting temperature), which was approx. 6–7 °C higher than that of the Glu206→Lys enzyme. An extensive molecular graphic analysis of the mutated enzymes, using human and rabbit aldolase A crystallographic structures, suggests that the Glu206→Lys mutation destabilizes the aldolase A tetramer at the subunit interface, and highlights the fact that the glycine-to-serine substitution at position 346 limits the flexibility of the C-terminal region. These results also provide the first evidence that Gly346 is crucial for the correct conformation and function of aldolase A, because it governs the entry/release of the substrates into/from the enzyme cleft, and/or allows important C-terminal residues to approach the active site.
Iron-regulatory proteins DmdR1 and DmdR2 of Streptomyces coelicolor form two different DNA-protein complexes with iron boxes
Authors: FLORES Francisco J. , MARTÍN Juan F.
Published: June 2004

In high G+C Gram-positive bacteria, the control of expression of genes involved in iron metabolism is exerted by a DmdR [divalent (bivalent) metal-dependent regulatory protein] in the presence of Fe2+ or other bivalent ions. The dmdR1 and dmdR2 genes of Streptomyces coelicolor were overexpressed in Escherichia coli and the DmdR1 and DmdR2 proteins were purified to homogeneity. Electrophoretic mobility-shift assays showed that both DmdR1 and DmdR2 bind to the 19-nt tox and desA iron boxes forming two different complexes in each case. Increasing the concentrations of DmdR1 or DmdR2 protein shifted these complexes from their low-molecular-mass form to the high-molecular-mass complexes. Formation of the DNA–protein complexes was prevented by the bivalent metal chelating agent 2,2´-dipyridyl and by antibodies specific against the DmdR proteins. Cross-linking with glutaraldehyde of pure DmdR1 or DmdR2 proteins showed that DmdR1 forms dimers, whereas DmdR2 is capable of forming dimers and probably tetramers. Ten different iron boxes were found in a search for iron boxes in the genome of S. coelicolor. Most of them correspond to putative genes involved in siderophore biosynthesis. Since the nucleotide sequence of these ten boxes is identical (or slightly different) with the synthetic DNA fragment containing the desA box used in the present study, it is proposed that DmdR1 and DmdR2 bind to the iron boxes upstream of at least ten different genes in S. coelicolor.
Transcription factor Nrf2/MafK regulates rat placental glutathione S-transferase gene during hepatocarcinogenesis
Authors: IKEDA Hiromi , NISHI Shinzo , SAKAI Masaharu
Published: June 2004

The rat GST-P (placental glutathione S-transferase), a phase II detoxifying enzyme, is not expressed in normal liver cells, but is highly and specifically induced during early hepatocarcinogenesis as well as in hepatocellular carcinoma cells. Results of previous studies indicated that GST-P gene activation was mainly controlled by an enhancer element, GPE1 (GST-P enhancer 1), but the specific activation mechanism of the GST-P gene was not fully understood [Morimura, Suzuki, Hochi, Yuki, Nomura, Kitagawa, Nagatsu, Imagawa and Muramatsu (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2065–2068; Suzuki, Imagawa, Hirabayashi, Yuki, Hisatake, Nomura, Kitagawa and Muramatsu (1995) Cancer Res. 55, 2651–2655]. In the present study, we investigate the transcription factor Nrf2/MafK heterodimer (where Nrf2 stands for NF-E2 p45-related factor 2) as an activator of the GST-P gene through the action of GPE1 during hepatocarcinogenesis. Electrophoretic mobility-shift assay and footprinting analysis with wild-type GPE1 and GPE1 point mutants showed that the Nrf2/MafK heterodimer specifically bound GPE1. Reporter transfection assays indicated that Nrf2 strongly stimulated GST-P gene expression in mouse F9 embryonal carcinoma cells and H4IIE rat hepatoma cells. Northern-blot analysis indicated that GST-P and Nrf2 mRNA increased in parallel with development of precancerous lesions and hepatocellular carcinoma. Keap1 (Kelch-like ECH-associated protein 1), an inhibitory factor of Nrf2, decreased the activation of GPE1 by Nrf2 and this suppression was restored after treatment with electrophilic compounds. GST-P mRNA expression in H4IIE cells was induced by electrophilic compounds, as was the expression of mRNAs of other phase II detoxifying enzymes. Chromatin immunoprecipitation analyses showed that antibodies both against Nrf2 and against MafK precipitated GPE1 from the chromatin of the pre-neoplastic hepatocytes and rat hepatoma cells (H4IIE and dRLh84), but not from normal hepatocytes. These results indicate that the Nrf2/MafK heterodimer regulates GST-P gene expression during early hepatocarcinogenesis and in hepatoma cells.
Effects of the antimicrobial peptide temporin L on cell morphology, membrane permeability and viability of Escherichia coli
Authors: MANGONI Maria Luisa , PAPO Niv , BARRA Donatella , SIMMACO Maurizio , BOZZI Argante , DI GIULIO Antonio , RINALDI Andrea C.
Published: June 2004

Antimicrobial peptides are produced by all organisms in response to microbial invasion and are considered as promising candidates for future antibiotics. There is a wealth of evidence that many of them interact and increase the permeability of bacterial membranes as part of their killing mechanism. However, it is not clear whether this is the lethal step. To address this issue, we studied the interaction of the antimicrobial peptide temporin L with Escherichia coli by using fluorescence, confocal and electron microscopy. The peptide previously isolated from skin secretions of the frog Rana temporaria has the sequence FVQWFSKFLGRIL-NH2. With regard to fluorescence microscopy, we applied, for the first time, a triple-staining method based on the fluorochromes 5-cyano-2,3-ditolyl tetrazolium chloride, 4´,6-diamidino-2-phenylindole and FITC. This technique enabled us to identify, in the same sample, both living and total cells, as well as bacteria with altered membrane permeability. These results reveal that temporin L increases the permeability of the bacterial inner membrane in a dose-dependent manner without destroying the cell's integrity. At low peptide concentrations, the inner membrane becomes permeable to small molecules but does not allow the killing of bacteria. However, at high peptide concentrations, larger molecules, but not DNA, leak out, which results in cell death. Very interestingly, in contrast with many antimicrobial peptides, temporin L does not lyse E. coli cells but rather forms ghost-like bacteria, as observed by scanning and transmission electron microscopy. Besides shedding light on the mode of action of temporin L and possibly that of other antimicrobial peptides, the present study demonstrates the advantage of using the triple-fluorescence approach combined with microscopical techniques to explore the mechanism of membrane-active peptides in general.
A novel 110 kDa form of myosin XVIIIA (MysPDZ) is tyrosine-phosphorylated after colony-stimulating factor-1 receptor signalling
Authors: CROSS Maddalena , CSAR Xavier F. , WILSON Nicholas J. , MANES Gaël , ADDONA Theresa A. , MARKS Denese C. , WHITTY Genevieve A. , ASHMAN Keith , HAMILTON John A.
Published: June 2004

Macrophage colony-stimulating factor (M-CSF or CSF-1) controls the development of macrophage lineage cells via activation of its tyrosine kinase receptor, c-Fms. After adding CSF-1 to M1 myeloid cells expressing CSF-1R (CSF-1 receptor), tyrosine phosphorylation of many cellular proteins occurs, which might be linked to subsequent macrophage differentiation. The biological significance and characterization of such proteins were explored by a dual strategy comprising two-dimensional SDS/PAGE analysis of cell lysates of CSF-1-treated M1 cells expressing the wild-type or a mutated receptor, together with an enrichment strategy involving a tyrosine-phosphorylated receptor construct. In the present study, we report the identification by MS of a novel, low-abundance, 110 kDa form of myosin XVIIIA (MysPDZ, myosin containing PDZ domain), which appears to be preferentially tyrosine-phosphorylated after CSF-1R activation when compared with other known isoforms. Receptor mutation studies indicate that CSF-1R-dependent tyrosine phosphorylation of p110myosin XVIIIA requires Tyr-559 in the cytoplasmic domain of the receptor and is therefore Src-family kinase-dependent. Gelsolin, Erp61 protein disulphide-isomerase and possibly non-muscle myosin IIA were also tyrosine-phosphorylated under similar conditions. Similar to the more abundant p190 isoform, p110 myosin XVIIIA lacks a PDZ domain and, in addition, it may lack motor activity. The phosphorylation of p110 myosin XVIIIA by CSF-1 may alter its cellular localization or target its association with other proteins.
Partial reconstruction of in vitro gluconeogenesis arising from mitochondrial l-lactate uptake/metabolism and oxaloacetate export via novel l-lactate translocators
Authors: de BARI Lidia , ATLANTE Anna , VALENTI Daniela , PASSARELLA Salvatore
Published: June 2004

In the light of the occurrence of l-lactate dehydrogenase inside the mitochondrial matrix, we looked at whether isolated rat liver mitochondria can take up and metabolize l-lactate, and provide oxaloacetate outside mitochondria, thus contributing to a partial reconstruction of gluconeogenesis in vitro. We found that: (1) l-lactate (10 mM), added to mitochondria in the presence of a cocktail of glycolysis/gluconeogenesis enzymes and cofactors, can lead to synthesis of glyceraldehyde-3-phosphate at a rate of about 7 nmol/min per mg mitochondrial protein. (2) Three novel translocators exist to mediate l-lactate traffic across the inner mitochondrial membrane. An l-lactate/H+ symporter was identified by measuring fluorimetrically the rate of endogenous pyridine nucleotide reduction. Consistently, l-lactate oxidation was found to occur with P/O ratio=3 (where P/O ratio is the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation) and with generation of membrane potential. Proton uptake, which occurred as a result of addition of l-lactate to RLM together with electron flow inhibitors, and mitochondrial swelling in ammonium l-lactate solutions were also monitored. l-Lactate/oxaloacetate and l-lactate/pyruvate anti-porters were identified by monitoring photometrically the appearance of l-lactate counter-anions outside mitochondria. These l-lactate translocators, which are distinct from the monocarboxylate carrier, were found to differ from each other in Vmax values and in inhibition and pH profiles, and proved to regulate mitochondrial l-lactate metabolism in vitro. The role of lactate/mitochondria interactions in gluconeogenesis is discussed.
Phe120 contributes to the regiospecificity of cytochrome P450 2D6: mutation leads to the formation of a novel dextromethorphan metabolite
Authors: FLANAGAN Jack U. , MARÉCHAL Jean-Didier , WARD Richard , KEMP Carol A. , McLAUGHLIN Lesley A. , SUTCLIFFE Michael J. , ROBERTS Gordon C. K. , PAINE Mark J. I. , WOLF C. Roland
Published: June 2004

Although the residues that determine the preference of CYP2D6 (cytochrome P450 2D6) for compounds containing a basic nitrogen are well characterized, the contribution of other active site residues to substrate binding and orientation is less well understood. Our structural model of CYP2D6 identifies the aromatic residue Phe120 as a likely major feature of the active site. To examine the role of Phe120, mutants of CYP2D6 in which this residue has been substituted by alanine, leucine, tyrosine, serine, histidine, tryptophan or methionine residues have been prepared in bacterial membranes co-expressing human cytochrome NADPH cytochrome P450 oxidoreductase. The mutants have been characterized using the prototypical bufuralol 1´ hydroxylase and dextromethorphan O- and N-demethylase activities of CYP2D6. Larger effects on Km values are observed for dextromethorphan O-demethylation than for bufuralol 1´ hydroxylation, indicating that the Phe120 side chain is more important in dextromethorphan than in bufuralol binding. A role for this side chain in determining the regiospecificity of substrate oxidation was indicated by changes in the relative rates of O- and N-demethylation of dextromethorphan and, notably, by the formation of 7-hydroxy dextromethrophan, a novel dextromethorphan metabolite, in mutants in which it had been substituted. Computational studies of dextromethorphan binding to the active site of the Phe120→Ala mutant were carried out to throw light on the way in which the removal of this side chain leads to different modes of ligand binding.
Regulation of desmocollin gene expression in the epidermis: CCAAT/enhancer-binding proteins modulate early and late events in keratinocyte differentiation
Authors: SMITH Conrad , ZHU Kuichun , MERRITT Anita , PICTON Rhian , YOUNGS Denise , GARROD David , CHIDGEY Martyn
Published: June 2004

Desmocollins (Dscs) are desmosomal cadherins that exhibit differentiation-specific patterns of expression in the epidermis. Dsc3 expression is strongest in basal cell layers, whereas Dsc1 is largely confined to upper, terminally differentiating strata. To understand better the processes by which Dsc expression is regulated in the epidermis, we have isolated Dsc3 and Dsc1 5´-flanking DNAs and analysed their activity in primary keratinocytes. In the present study, we found that transcription factors of the CCAAT/enhancer-binding protein family play a role in the regulation of expression of both Dscs and, in so doing, implicate this class of transcription factors in both early and late events in keratinocyte differentiation. We show that Dscs are differentially regulated by C/EBP (CCAAT/enhancer-binding protein) family members, with Dsc3 expression being activated by C/EBPβ but not C/EBPα, and the reverse being the case for Dsc1. Expression of both Dscs is activated by another family member, C/EBPδ. These results show for the first time how desmosomal cadherin gene expression is regulated and provide a mechanism for the control of other differentiation-specific genes in the epidermis.
The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation
Authors: van der HEIDE Lars P. , HOEKMAN Marco F. M. , SMIDT Marten P.
Published: June 2004

FoxO (forkhead box O; forkhead members of the O class) are transcription factors that function under the control of insulin/insulin-like signalling. FoxO factors have been associated with a multitude of biological processes, including cell-cycle, cell death, DNA repair, metabolism and protection from oxidative stress. Central to the regulation of FoxO factors is a shuttling system, which confines FoxO factors to either the nucleus or the cytosol. Shuttling of FoxO requires protein phosphorylation within several domains, and association with 14-3-3 proteins and the nuclear transport machinery. Description of the FoxO-shuttling mechanism contributes to the understanding of FoxO function in relation to signalling and gene regulation.
Signalling to actin assembly via the WASP (Wiskott-Aldrich syndrome protein)-family proteins and the Arp2/3 complex
Authors: MILLARD Thomas H. , SHARP Stewart J. , MACHESKY Laura M.
Published: June 2004

The assembly of a branched network of actin filaments provides the mechanical propulsion that drives a range of dynamic cellular processes, including cell motility. The Arp2/3 complex is a crucial component of such filament networks. Arp2/3 nucleates new actin filaments while bound to existing filaments, thus creating a branched network. In recent years, a number of proteins that activate the filament nucleation activity of Arp2/3 have been identified, most notably the WASP (Wiskott–Aldrich syndrome protein) family. WASP-family proteins activate the Arp2/3 complex, and consequently stimulate actin assembly, in response to extracellular signals. Structural studies have provided a significant refinement in our understanding of the molecular detail of how the Arp2/3 complex nucleates actin filaments. There has also been much progress towards an understanding of the complicated signalling processes that regulate WASP-family proteins. In addition, the use of gene disruption in a number of organisms has led to new insights into the specific functions of individual WASP-family members. The present review will discuss the Arp2/3 complex and its regulators, in particular the WASP-family proteins. Emphasis will be placed on recent developments in the field that have furthered our understanding of actin dynamics and cell motility.
Endothelin-converting enzyme-like 1 (ECEL1) is present both in the plasma membrane and in the endoplasmic reticulum
Authors: BENOIT Alexandre , VARGAS Miguel Angel , DesGROSEILLERS Luc , BOILEAU Guy
Published: June 2004

Enzymes of the M13 family of zinc-containing endopeptidases are recognized as important regulators of neuropeptide and peptide hormone activity. Peptidases of this family are type II integral-membrane proteins characterized by short cytosolic domains and large extracellular domains containing the active site. The M13 family has, at present, seven members, including ECEL1 (endothelin-converting enzyme-like 1), one of the newest members. ECEL1 is expressed predominantly in the central nervous system. It has been proposed that the enzyme has a role in the nervous regulation of the respiratory system. No physiological substrate has been identified yet. To better understand the function(s) of this enzyme, we have expressed human ECEL1 in cultured cells and monitored its biosynthesis and subcellular localization. Immunoblot and cell-surface biotinylation analysis of transfected cells expressing ECEL1 showed that only a fraction of the protein travelled to the cell surface, while most of the enzyme was present in an intracellular compartment identified by confocal immunofluorescence microscopy and cell fractionation as the ER (endoplasmic reticulum). Pulse–chase experiments showed that ER-localized ECEL1 was stable, with a half-life of more than 3 h. Endogenous ECEL1 from mouse pituitary gland had a similar distribution between the cell surface and the ER. Finally, using domain-swapping experiments with neprilysin, another member of the M13 family, we showed that localization of ECEL1 to the ER requires both the transmembrane and cytoplasmic domains. It thus appears that ECEL1 may have functions both at the cell surface and in the ER.
Mechanical stimuli regulate rapamycin-sensitive signalling by a phosphoinositide 3-kinase-, protein kinase B- and growth factor-independent mechanism
Authors: HORNBERGER Troy A. , STUPPARD Rudy , CONLEY Kevin E. , FEDELE Mark J. , FIOROTTO Marta L. , CHIN Eva R. , ESSER Karyn A.
Published: June 2004

In response to growth factors, mTOR (mammalian target of rapamycin) has been identified as a central component of the signalling pathways that control the translational machinery and cell growth. Signalling through mTOR has also been shown to be necessary for the mechanical load-induced growth of cardiac and skeletal muscles. Although the mechanisms involved for mechanically induced activation of mTOR are not known, it has been suggested that activation of PI3K (phosphoinositide 3-kinase) and protein kinase B (Akt), via the release of locally acting growth factors, underlies this process. In the present study, we show that mechanically stimulating (passive stretch) the skeletal muscle ex vivo results in the activation of mTOR-dependent signalling events. The activation of mTOR-dependent signalling events was necessary for an increase in translational efficiency, demonstrating the physiological significance of this pathway. Using pharmacological inhibitors, we show that activation of mTOR-dependent signalling occurs through a PI3K-independent pathway. Consistent with these results, mechanically induced signalling through mTOR was not disrupted in muscles from Akt1−/− mice. In addition, ex vivo co-incubation experiments, along with in vitro conditioned-media experiments, demonstrate that a mechanically induced release of locally acting autocrine/paracrine growth factors was not sufficient for the activation of the mTOR pathway. Taken together, our results demonstrate that mechanical stimuli can activate the mTOR pathway independent of PI3K/Akt1 and locally acting growth factors. Thus mechanical stimuli and growth factors provide distinct inputs through which mTOR co-ordinates an increase in the translational efficiency.
Regulation of expression of the stress response gene, Osp94: identification of the tonicity response element and intracellular signalling pathways
Authors: KOJIMA Ryoji , RANDALL Jeffrey D. , ITO Eri , MANSHIO Hiroyuki , SUZUKI Yoshio , GULLANS Steven R.
Published: June 2004

Osp94 (osmotic stress protein of 94 kDa) is known to be up-regulated by hypertonic and heat-shock stresses in mouse renal inner medullary collecting duct (mIMCD3) cells. To investigate the molecular mechanism of transcriptional regulation of the Osp94 gene under these stresses, we cloned and characterized the 5´-flanking region of the gene. Sequence analysis of the proximal 4 kb 5´-flanking region revealed a TATA-less G/C-rich promoter region containing a cluster of Sp1 sites. We also identified upstream sequence motifs similar to the consensus TonE/ORE (tonicity-response element/osmotic response element) as well as the consensus HSE (heat-shock element). Luciferase activities in cells transfected with reporter constructs containing a TonE/ORE-like element (Osp94-TonE; 5´-TGGAAAGGACCAG-3´) and HSE enhanced reporter gene expression under hypertonic stress and heat-shock stress respectively. Electrophoretic gel mobility-shift assay showed a slowly migrating band binding to the Osp94-TonE probe, probably representing binding of TonEBP (TonE binding protein) to this enhancer element. Furthermore, treatment of mIMCD3 cells with MAPK (mitogen-activated protein kinase) inhibitors (SB203580, PD98059, U0126 and SP600125) and a proteasome inhibitor (MG132) suppressed the increase in Osp94 gene expression caused by hypertonic NaCl. These results indicate that the 5´-flanking region of Osp94 gene contains a hypertonicity sensitive cis-acting element, Osp94-TonE, which is distinct from a functional HSE. Furthermore, the MAPK and proteasome systems appear to be, at least in part, involved in hypertonic-stressmediated regulation of Osp94 through Osp94-TonE.
Transcriptomic analysis in the leech Theromyzon tessulatum: involvement of cystatin B in innate immunity
Authors: LEFEBVRE Christophe , COCQUERELLE Claude , VANDENBULCKE Franck , HOT David , HUOT Ludovic , LEMOINE Yves , SALZET Michel
Published: June 2004

At the present time, there is little information on mechanisms of innate immunity in invertebrate groups other than insects, especially annelids. In the present study, we have performed a transcriptomic study of the immune response in the leech Theromyzon tessulatum after bacterial challenge, by a combination of differential display RT (reverse transcriptase)–PCR and cDNA microarrays. The results show relevant modulations concerning several known and unknown genes. Indeed, threonine deaminase, malate dehydrogenase, cystatin B, polyadenylate-binding protein and α-tubulin-like genes are up-regulated after immunostimulation. We focused on cystatin B (stefin B), which is an inhibitor of cysteine proteinases involved in the vertebrate immune response. We have cloned the full-length cDNA and named the T. tessulatum gene as Tt-cysb. Main structural features of cystatins were identified in the derived amino acid sequence of Tt-cysb cDNA; namely, a glycine residue in the N-terminus and a consensus sequence of Gln-Xaa-Val-Xaa-Gly (QXVXG) corresponding to the catalytic site. Moreover, Tt-cysb is the first cystatin B gene characterized in invertebrates. We have determined by in situ hybridization and immunocytochemistry that Tt-cysb is only expressed in large coelomic cells. In addition, this analysis confirmed that Tt-cysb is up-regulated after bacterial challenge, and that increased expression occurs only in coelomic cells. These data demonstrate that the innate immune response in the leech involves a cysteine proteinase inhibitor that is not found in ecdysozoan models, such as Drosophila melanogaster or Caenorhabditis elegans, and so underlines the great need for information about innate immunity mechanisms in different invertebrate groups.
Actions by actin: reciprocal regulation of cortactin activity by tyrosine kinases and F-actin
Authors: GUNST Susan J.
Published: June 2004

The polymerization of actin is catalysed by the Arp (actin-related protein) 2/3 complex, which acts downstream of a variety of receptors and signalling cascades. Intermediary molecules such as cortactin bind to the Arp2/3 complex and stimulate its activity, thus promoting actin polymerization and actin filament stabilization. New data in this issue of the Biochemical Journal by the Kapus group suggest that cortactin is reciprocally regulated by filamentous (F) actin and tyrosine kinases. This suggests a new paradigm for considering the cellular processes that regulate the dynamic organization of the actin cytoskeleton.