bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of glutathione in the formation of the active form of the oxygen sensor FNR ([4Fe‐4S]·FNR) and in the control of FNR function
Authors: Tran Quang Hon , Arras Tanja , Becker Sabine , Holighaus Gaby , Ohlberger Gabi , Unden Gottfried
Published: March 2003

The oxygen sensor regulator FNR (fumarate nitrate reductase regulator) of Escherichia coli is known to be inactivated by O2 as the result of conversion of a [4Fe‐4S] cluster of the protein into a [2Fe‐2S] cluster. Further incubation with O2 causes loss of the [2Fe‐2S] cluster and production of apoFNR. The reactions involved in cluster assembly and reductive activation of apoFNR isolated under anaerobic or aerobic conditions were studied in vivo and in vitro. In a gshA mutant of E. coli that was completely devoid of glutathione, the O2 tension for the regulatory switch for FNR‐dependent gene regulation was decreased by a factor of 4–5 compared with the wild‐type, suggesting a role for glutathione in FNR function. In isolated apoFNR, glutathione could be used as the reducing agent for HS− formation required for [4Fe‐4S] assembly by cysteine desulfurase (NifS), and for the reduction of cysteine ligands of the FeS cluster in FNR. Air‐inactivated FNR (apoFNR without FeS) could be reconstituted to [4Fe‐4S]·FNR by the same reaction as used for apoFNR isolated under anaerobic conditions. The in vivo effects of glutathione on FNR function and the role of glutathione in the formation of active [4Fe‐4S]·FNR in vitro suggest an important role for glutathione in the de novo assembly of FNR and in the reductive activation of air‐oxidized FNR under anaerobic conditions.
Generation of catalytically active 6‐phosphofructokinase from Saccharomyces cerevisiae in a cell‐free system
Authors: Edelmann Anke , Kirchberger Jürgen , Naumann Manfred , Kopperschläger Gerhard
Published: March 2003

PFK1 and PFK2 coding for the subunits of 6‐phosphofructokinase from Saccharomyces cerevisiae were cloned into plasmids suitable for runoff transcription. In vitro translation products of both kinds of subunit were obtained using rabbit reticulocyte lysate as the synthesis and folding system. They were monitored by chemiluminescent Western‐blot analysis. Folding and assembly of the α‐subunit and β‐subunit of 6‐phosphofructokinase were found to occur in the cell‐free system resulting in an enzymatically active protein. The in vitro generated enzyme exhibits a folding state that is similar to that of the heterooctameric form of 6‐phosphofructokinase in the presence of fructose 6‐phosphate, ATP and ammonium sulfate, as demonstrated by size‐exclusion HPLC followed by ELISA.
Cloning and characterization of a monocot mannose‐binding lectin from Crocus vernus (family Iridaceae)
Authors: Van Damme Els J. M. , Astoul Corinne Houles , Barre Annick , Rougé Pierre , Peumans Willy J.
Published: March 2003

The molecular structure and carbohydrate‐binding activity of the lectin from bulbs of spring crocus (Crocus vernus) has been determined unambiguously using a combination of protein analysis and cDNA cloning. Molecular cloning revealed that the lectin called C. vernus agglutinin (CVA) is encoded by a precursor consisting of two tandemly arrayed lectin domains with a reasonable sequence similarity to the monocot mannose‐binding lectins. Post‐translational cleavage of the precursor yields two equally sized polypeptides. Mature CVA consists of two pairs of polypeptides and hence is a heterotetrameric protein. Surface plasmon resonance studies of the interaction of the crocus lectin with high mannose‐type glycans showed that the lectin interacts specifically with exposed α‐1,3‐dimannosyl motifs. Molecular modelling studies confirmed further the close relationships in overall fold and three‐dimensional structure of the mannose‐binding sites of the crocus lectin and other monocot mannose‐binding lectins. However, docking experiments indicate that only one of the six putative mannose‐binding sites of the CVA protomer is active. These results can explain the weak carbohydrate‐binding activity and low specific agglutination activity of the lectin. As the cloning and characterization of the spring crocus lectin demonstrate that the monocot mannose‐binding lectins occur also within the family Iridaceae a refined model of the molecular evolution of this lectin family is proposed.
Characterization of human HtrA2, a novel serine protease involved in the mammalian cellular stress response
Authors: Gray Carol W. , Ward Robin V. , Karran Eric , Turconi Sandra , Rowles Alison , Viglienghi Daniela , Southan Christopher , Barton Amanda , Fantom Kenneth G. , West Andrew , Savopoulos John , Hassan Namir J. , Clinkenbeard Helen , Hanning Charles , Amegadzie Bernard , Davis John B. , Dingwall Colin , Livi George P. , Creasy Caretha L.
Published: March 2003

Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site‐directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves β‐casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in β‐casein turnover when the assay temperature is raised from 37 to 45 °C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.
Biochemical characterization of recombinant subunits of type 2A protein phosphatase overexpressed in Pichia pastoris
Authors: Swiatek Wojciech , Sugajska Ewa , Lankiewicz Leszek , Hemmings Brian A. , Zolnierowicz Stanislaw
Published: March 2003

Methylotrophic yeast Pichia pastoris was used for a medium‐scale expression of structural (PR65/A) and catalytic (PP2Ac) subunits of human type 2A protein phosphatase (PP2A). Constructs encoding these subunits, which were designed to introduce eight histidines at their N‐termini, were introduced into the KM71 Pichia strain by homologous recombination. Recombinant proteins overproduced after methanol induction were purified from cell‐free extracts by anion‐exchange chromatography on DEAE‐Sepharose, and Ni2+/nitrilotriacetate/agarose. In addition, chromatography on ω‐aminohexyl‐Sepharose was applied to purify recombinant (r)PR65/A. This purification scheme yielded approximately 5 mg and 100 µg of rPR65/A and rPP2Ac, respectively, from 1 L of the yeast culture. The specific activity of rPP2Ac measured with [32P]phosphorylase a[1.7 µmol·min−1·(mg protein)−1] and its inhibition by okadaic acid (IC50 = 0.66 nm) were similar to PP2A isolated from rabbit skeletal muscle. As demonstrated by immunodetection with methylation state‐specific antibodies, recombinant PP2Ac was carboxymethylated at the last C‐terminal leucine residue. Recombinant PP2A subunits were able to form a complex as demonstrated both by activity assays in the presence of protamine and by chromatography on protamine–agarose. In summary, P. pastoris provides a convenient heterologous system for the production of recombinant subunits of PP2A.
Functional characterization of green fluorescent protein–profilin fusion proteins
Authors: Wittenmayer Nina , Rothkegel Martin , Jockusch Brigitte M. , Schlüter Kathrin
Published: March 2003

To clarify the role of profilins in cells, fusion proteins constructed with green fluorescent protein (GFP) should be extremely helpful. As profilins are considerably smaller than the GFP fusion partner (14–17 kDa compared with 27 kDa, respectively), we characterized the fusion proteins in vitro, to ascertain their biological function.

We fused mouse profilin I and II to either the C‐terminus or N‐terminus of GFP. These fusion proteins were expressed in Escherichia coli and affinity‐purified on polyproline–Sepharose. Interaction with vasodilator‐stimulated phosphoprotein, a proline‐rich ligand of profilin, was investigated by ELISA, as was binding to PtdIns(4,5)P2. The affinity for actin was quantitatively determined in polymerization assays. Our results show that fusion of GFP to the C‐terminus of profilin I abolishes polyproline binding. In contrast, the other fusion proteins bound to polyproline–Sepharose and VASP. Binding to PtdIns(4,5)P2 was not significantly altered. Furthermore, fusion of either isoform with GFP did not decrease the affinity for actin. In localization studies with mammalian cells, all fusion proteins showed the localization expected for profilin in areas of high actin dynamics, such as leading lamellae and ruffles induced by epidermal growth factor. However, with regard to our in vitro data, we suspect that only a minor fraction of profilin I carrying the GFP at the C‐terminus can target these sites. Therefore, other constructs should be preferred for further in vivo studies.
Structure and expression of the mouse gene encoding the endozepine‐like peptide from haploid male germ cells
Authors: Valentin Mette , Balvers Marga , Pusch Wolfgang , Weinbauer Gerhard F. , Knudsen Jens , Ivell Richard
Published: March 2003

The endozepine‐like peptide (ELP) represents a testis‐specific isoform of the ubiquitous acyl‐CoA binding protein (ACBP) and is highly expressed in late haploid stages of male germ cell development. The genomic sequence of the functional ELP gene as well as that of a pseudogene were analysed from independent bacteriophage clones of a 129sv mouse genomic library. Unlike the ACBP gene, which comprises four exons, the ELP gene has only a single intron within the region of the 5′ untranslated region, suggesting that, like some other haploid expressed genes, the ELP gene might have evolved by retroposon‐mediated gene duplication. Primer extension analysis was used to define the start site for transcription and hence the 5′ promoter region. Electrophoretic mobility shift analysis was carried out on this region comparing nuclear extracts from adult mouse testis with those from mouse liver. Several testis‐specific DNA–protein complexes were observed throughout 700 bp upstream of the transcription start site. One of these could be identified as corresponding to a steroidogenic factor‐1 (SF‐1) binding element. Further analysis using pure transcription factors showed that this element at position −340 was able to bind specifically to both SF‐1 and to the germ cell nuclear factor (GCNF). Immunohistochemical analysis using an ELP‐specific antibody showed that expression was very restricted within the testis to the postmeiotic germ cells, and in the ovary to interstitial/luteal cells, cell‐types known to express GCNF and SF‐1, respectively. Testes of CREM‐τ knockout mice, lacking all spermatogenic stages later than round spermatids, were devoid of ELP immunoreactivity, whereas in RAD6 knockout mice the few remaining elongated spermatids were clearly defined by this excellent late haploid marker product. The ELP gene and its product thus offer an ideal system with which to investigate the differentiation of late haploid stages of spermatogenesis.
Hypotonic cell swelling induces translocation of the α isoform of cytosolic phospholipase A2 but not the γ isoform in Ehrlich ascites tumor cells
Authors: Pedersen Susanne , Lambert Ian H. , Thoroed Steinar M. , Hoffmann Else K.
Published: March 2003

We demonstrate that two isoforms of the cytosolic phospholipase A2, cPLA2α and cPLA2γ, are present in Ehrlich ascites tumor cells. Both enzymes are almost uniformly distributed throughout the cells under control conditions, as visualized by laser‐scanning confocal microscopy. Stimulation by either hypotonic cell swelling or addition of the Ca2+ ionophore A23187 results in translocation of cPLA2α, but not cPLA2γ, to the nucleus, where it forms hot‐spot‐like clusters. Our group previously showed that release of radioactively labeled arachidonic acid, incorporated into the phospholipids of Ehrlich cells, was immediately and transiently increased on hypotonic cell swelling [Thoroed, S.M., Lauritzen, L., Lambert, I.H., Hansen, H.S. & Hoffmann, E.K. (1997) J. Membr. Biol.160, 47–58]. We now demonstrate that arachidonic acid is released from the nuclear fraction following hypotonic exposure. Stimulation of Ehrlich cells with A23187 also leads to an increase in arachidonic acid release from the nucleus. However, as hypotonic cell swelling is not accompanied by any detectable increase in intracellular concentration of free cytosolic Ca2+ ([Ca2+]i), stimulus‐induced translocation of cPLA2α can also occur without elevation of [Ca2+]i. The stimulus‐induced translocation of cPLA2α appears not to be prevented by inhibition of mitogen‐activated protein (MAP) kinase activation, p38 MAP kinase, tyrosine kinases and protein kinase C, hence, phosphorylation is not crucial for the stimulus‐induced translocation of cPLA2α. Disruption of F‐actin did not affect the translocation process, thus, an intact F‐actin cytoskeleton does not seem to be required for translocation of cPLA2α.
On the kinetics of voltage formation in purple membranes of Halobacterium salinarium
Authors: Hendler Richard W. , Drachev Lel A. , Bose Salil , Joshi Manoj K
Published: March 2003

The kinetics of the bacteriorhodopsin photocycle, measured by voltage changes in a closed membrane system using the direct electrometrical method (DEM) of Drachev, L.A., Jasaitus, A.A., Kaulen, A.D., Kondrashin, A.A., Liberman, E.A., Nemecek, I.B., Ostroumov, S.A., Semenov, Yu, A. & Skulachev, V.P. (1974) Nature249, 321–324 are sixfold slower than the kinetics obtained in optical studies with suspensions of purple membrane patches. In this study, we have investigated the reasons for this discrepancy. In the presence of the uncouplers carbonyl cyanide m‐chlorophenylhydrazone or valinomycin, the rates in the DEM system are similar to the rates in suspensions of purple membrane. Two alternative explanations for the effects of uncouplers were evaluated: (a) the ‘back‐pressure’ of the Δµ˜H+ slows the kinetic steps leading to its formation, and (b) the apparent difference between the two systems is due to slow major electrogenic events that produce little or no change in optical absorbance. In the latter case, the uncouplers would decrease the RC time constant for membrane capacitance leading to a quicker discharge of voltage and concomitant decrease in photocycle turnover time. The experimental results show that the primary cause for the slower kinetics of voltage changes in the DEM system is thermodynamic back‐pressure as described by Westerhoff, H.V. & Dancshazy, Z. (1984) Trends Biochem. Sci.9, 112–117.
Interaction of the targeting sequence of chloroplast precursors with Hsp70 molecular chaperones
Authors: Rial Daniela V. , Arakaki Adrian K. , Ceccarelli Eduardo A.
Published: March 2003

We have analyzed the interaction of DnaK and plant Hsp70 proteins with the wild‐type ferredoxin‐NADP+ reductase precursor (preFNR) and mutants containing amino‐acid replacements in the targeting sequence. Using an algorithm already developed [Rüdiger, S., Germeroth, L., Schneider‐Mergener, J. & Bukau, B. (1997) EMBO J.16, 1501–1507] we observed that 75% of the 727 plastid precursor proteins analyzed contained at least one site with high likelihood of DnaK binding in their transit peptides. Statistical analysis showed a decrease of DnaK binding site frequency within the first 15 amino‐acid residues of the transit peptides. Using fusion proteins we detected the interaction of DnaK with the transit peptide of the folded preFNR but not with the mature region of the protein. Discharge of DnaK from the presequence was favored by addition of MgATP. When a putative DnaK binding site was artificially added at the N‐terminus of the mature protein, we observed formation of complexes with bacterial and plant Hsp70 molecular chaperones. Reducing the likelihood of DnaK binding by directed mutagenesis of the presequence increased the release of bound DnaK. The Hsp70 proteins from plastids and plant cell cytosol also interacted with the preFNR transit peptide. Overall results are discussed in the context of the proposed models to explain the organelle protein import.
Amphioxus alcohol dehydrogenase is a class 3 form of single type and of structural conservation but with unique developmental expression
Authors: Cañestro Cristian , Hjelmqvist Lars , Albalat Ricard , Garcia‐Fernàndez Jordi , Gonzàlez‐Duarte Roser , Jörnvall Hans
Published: March 2003

The coding region of amphioxus alcohol dehydrogenase class 3 (ADH3) has been characterized from two species, Branchiostoma lanceolatum and Branchiostoma floridae. The species variants have residue differences at positions that result in only marginal functional distinctions. Activity measurements show a class 3 glutathione‐dependent formaldehyde dehydrogenase, with kcat/Km values about threefold those of the human class 3 ADH enzyme. Only a single ADH3 form is identified in each of the two amphioxus species, and no ethanol activity ascribed to other classes is detectable, supporting the conclusion that evolution of ethanol‐active ADH classes by gene duplications occurred at early vertebrate radiation after the formation of the amphioxus lineage. Similarly, Southern blot analysis indicated that amphioxus ADH3 is encoded by a single gene present in the methylated fraction of the amphioxus genome and northern blots revealed a single 1.4‐kb transcript. In situ experiments showed that amphioxus Adh3 expression is restricted to particular cell types in the embryos. Transcripts were first evident at the neurula stage and then located at the larval ventral region, in the intestinal epithelium. This tissue‐specific pattern contrasts with the ubiquitous Adh3 expression in mammals.
Developmental and environmental regulation of antifreeze proteins in the mealworm beetle Tenebrio molitor
Authors: Graham Laurie A. , Walker Virginia K. , Davies Peter L.
Published: March 2003

The yellow mealworm beetle, Tenebrio molitor, contains a family of small Cys‐rich and Thr‐rich thermal hysteresis proteins that depress the hemolymph freezing point below the melting point by as much as 5.5 °C (ΔT = thermal hysteresis). Thermal hysteresis protein expression was evaluated throughout development and after exposure to altered environmental conditions. Under favorable growth conditions, small larvae (11–13 mg) had only low levels of thermal hysteresis proteins or thermal hysteresis protein message, but these levels increased 10‐fold and 18‐fold, respectively, by the final larval instar (> 190 mg), resulting in thermal hysteresis > 3 °C. Exposure of small larvae (11–13 mg) to 4 weeks of cold (4 °C) caused an ≈ 20‐fold increase in thermal hysteresis protein concentration, well in excess of the less than threefold developmental increase seen after 4 weeks at 22 °C. Exposure of large larvae (100–120 mg) to cold caused 12‐fold and sixfold increases in thermal hysteresis protein message and protein levels, respectively, approximately double the maximum levels they would have attained in the final larval instar at 22 °C. Thus, thermal hysteresis increased to similar levels (> 4 °C) in the cold, irrespective of the size of the larvae (the overwintering stage). At pupation, thermal hysteresis protein message levels decreased > 20‐fold and remained low thereafter, but thermal hysteresis activity decreased much more slowly. Exposure to cold did not reverse this decline. Desiccation or starvation of larvae had comparable effects to cold exposure, but surprisingly, short daylength photoperiod or total darkness had no effect on either thermal hysteresis or message levels. As all environmental conditions that caused increased thermal hysteresis also inhibited growth, we postulate that developmental arrest is a primary factor in the regulation of T. molitor thermal hysteresis proteins.
A mutation affecting the association equilibrium of formyltransferase from the hyperthermophilic Methanopyrus kandleri and its influence on the enzyme's activity and thermostability
Authors: Shima Seigo , Thauer Rudolf K. , Ermler Ulrich , Durchschlag Helmut , Tziatzios Christos , Schubert Dieter
Published: March 2003

Formyltransferase from Methanopyrus kandleri is composed of only one type of subunit of molecular mass 32 kDa. The enzyme is in a monomer/dimer/tetramer association equilibrium, the association constant being affected by lyotropic salts. Oligomerization is required for enzyme activity and thermostability. We report here on a subunit interface mutation (R261E) which affects the dimer/tetramer part of the association equilibrium of formyltransferase. With the mutant protein it was shown that tetramerization is not required for activity but is necessary for high thermostability.
The expression of Cox17p in rodent tissues and cells
Authors: Kako Koichiro , Tsumori Keiko , Ohmasa Yoshitaka , Takahashi Yoshinori , Munekata Eisuke
Published: March 2003

Previous works have reported the isolation of a novel polypeptide from porcine heart. Structural analysis has shown that it is a mammalian homologue of Cox17p, believed essential for the assembly of functional cytochrome c oxidase and delivery of copper ions to the mitochondrion for insertion into the enzyme in yeast. Although the human, mouse and porcine homologs of this small protein have already been cloned or purified, the function of Cox17p in the mammalian system has not yet been elucidated. To investigate the physiological function of Cox17p in mammals, we performed Northern blot analysis using probes containing the mouse and rat sequences obtained by RT‐PCR. The hybridization signals were detected in all mouse tissues, but notably intense signals were observed in heart, brain and kidney RNA samples. Some of the neuroendocrine and endocrine cell lines showed higher expression levels than fibroblasts. The highest expression level of Cox17p mRNA in mouse brain was observed in the pituitary sample. While in rat heart, Cox17p mRNA expression was detected from early development, in rat brain, embryonic and postnatal changes in the expression were observed. Immunocytochemical analysis showed that Cox17p immunoreactivity was strong in the pituitary cell line, AtT‐20. These findings suggested that Cox17p is not only part of the respiratory chain but also involved in brain and endocrine functions.
Peptide bond formation mediated by substrate mimetics
Authors: Grünberg Raik , Domgall Ines , Günther Robert , Rall Kathrin , Hofmann Hans‐Jörg , Bordusa Frank
Published: March 2003

Substrate mimetics are excellent tools for protease‐mediated peptide synthesis that enable the coupling of peptides independently of the primary specificity of the enzyme without undesired cleavages of the newly formed peptide bonds. However, the synthetic utility of this beneficial approach is limited to reactions with nonspecific amino‐acid‐containing peptides while the coupling of specific ones leads to unwanted cleavages due to the native proteolytic activity of the biocatalyst. This paper reports on the use of site‐directed mutagenesis to design trypsin variants with decreased cleavage activity. Starting from the variant D189S, which is known for its low proteolytic potential, Ser189 and Ser190 were exchanged for Ala to further repress the inherent amidase activity of trypsin D189S. The effect of mutations was analysed by model synthesis reactions using specific amino‐acid‐containing peptides and substrate mimetics as the reactants. Finally, computer‐assisted protein‐ligand docking studies were performed to get closer insight into the molecular basis of the experimental results.
Molecular heterogeneity of the hemocyanin isolated from the king crab Paralithodes camtschaticae
Authors: Molon Annamaria , Di Muro Paolo , Bubacco Luigi , Vasilyev Vadim , Salvato Benedetto , Beltramini Mariano , Conze Werner , Hellmann Nadja , Decker Heinz
Published: March 2003

Native Paralithodes camtschaticae hemocyanin is found as a mixture of dodecamers (24S; 80%) and hexamers (16S; 20%). Removal of Ca2+ ions by dialysis against EDTA‐containing buffer solution at neutral pH induces complete dissociation of the 24S form into the 16S form. Under these conditions, a further increase in pH to 9.2 produces complete dissociation of the hexamers into monomers (5S). In both cases, the dissociation process is reversible. The dodecamer (24S) is composed of two different hexamers which can be discriminated only by ion‐exchange chromatography in the presence of Ca2+ ions. At alkaline pH and in the presence of EDTA, two major monomeric fractions can be separated by ion‐exchange chromatography: ParcI (60%) and ParcII (40%). The reassociation properties of the two fractions were studied separately to define their ability to form hexamers and dodecamers.

The oxygen‐binding properties of the different aggregation states were investigated. Native hemocyanin binds O2 co‐operatively (nH = 3) and with low affinity (p50 ≈ 103 Torr). The two monomeric fractions, ParcI and ParcII, are not co‐operative and the affinity is twice that of the native protein (p50 ≈ 65 and 52 Torr).

Oxygen‐binding measurements of native hemocyanin carried out at different pH values indicate a strong positive Bohr effect within the pH range 6.5–8.0 and an increase in oxygen affinity at pH below 6.5.
Cloning and expression of a distinct subclass of plant thioredoxins
Authors: Juttner Juan , Olde David , Langridge Peter , Baumann Ute
Published: March 2003

mRNAs encoding a novel thioredoxin were isolated from pollen RNA of Lolium perenne (LpTrx), Hordeum bulbosum (HbTrx), Phalaris coerulescens (PTrx) and Secale cereale (ScTrx). The cDNAs contain a single ORF of 393 bp encoding a protein of 131 amino acids. The predicted proteins showed highest homology to plant thioredoxins of the h class yet form a distinct subgroup that is characterized by a high level of sequence conservation (95.4–97.7% identity). GenBank searches revealed additional members of this subclass in tomato, soybean, rice and pine. LpTrx and PTrx were expressed as recombinant proteins in Escherichia coli and tested for thioredoxin activity. Both proteins displayed typical thioredoxin activity in the nonspecific insulin reduction assay, however, were not reduced by E. coli NADPH‐dependant thioredoxin reductase.
Cloning and functional characterization of the 5′ regulatory region of the human mitochondrial glycerol‐3‐phosphate dehydrogenase gene
Authors: Urcelay Elena , Jareño María A. , Menaya Juan , Parrilla Roberto , Ayuso Matilde S. , Martín‐Requero Angeles
Published: March 2003

We report data on the structural and functional characterization of the 5′ flanking region of the human mitochondrial glycerol‐3‐phosphate dehydrogenase (mtGPDH) gene. We found two regions upstream of 5′‐untranslated sequences exhibiting promoter activity in transient transfection assays. Transcription start sites and potential regulatory sites in both promoter regions were defined. The proximal promoter was approximately sevenfold more active than the distal one in most cell lines, but it was only twice as active in a neuroblastoma cell line. These observations seem to indicate that the rate of transcription, as well as the tissue‐specific expression of the human mtGPDH gene, is the result of a combinatorial effect of transcription factors on at least two promoters. 3,5,3′‐Triiodothyronine failed to alter the transcriptional activity of human mtGPDH promoter(s) constructs in transient transfection assays. Although this finding seems to be in conflict with the reported effect of 3,5,3′‐triiodothyronine in rodents, it is consistent with our observation of 3,5,3′‐triiodothyronine stimulation of mtGPDH activity in primary cultures of rat adipocytes, but not human cultured adipocytes, suggesting distinct regulation of this gene in both species.
CREB is involved in mouse annexin A1 regulation by cAMP and glucocorticoids
Authors: Antonicelli Frank , De Coupade Catherine , Russo‐Marie Françoise , Le Garrec Yvonne
Published: March 2003

A cAMP and some glucocorticoid response elements have been underlined in the promoter of mouse annexin A1. To analyse the function of these DNA sequences, the role of cAMP and glucocorticoids, as well as the transcription factors involved in their activation, were investigated. A construct containing 1381 bp of the DNA 5′‐flanking annexin A1 gene fused to LacZ was used. The level of activation of the reporter gene was analysed by transient transfection of the JEG3 cell line. Activation of β‐galactosidase expression was observed with both dibutyryl cAMP and dexamethasone when compared with cells treated with serum only. Simultaneous addition of dexamethasone and dibutyryl cAMP did not result in a synergistic effect but rather in a competitive one. Gel‐shift assays with a probe including the cAMP response element‐like element of the annexin A1 promoter revealed a main specific DNA–protein complex when cells were stimulated with dibutyryl cAMP and/or dexamethasone. In all cases CREB protein was identified by supershift analysis. We therefore conclude that this cAMP response element sequence plays a prominent role in the transactivation of the annexin A1 promoter by dibutyryl cAMP and that it is involved in the response to glucocorticoids.
A chicken hnRNP of the A/B family recognizes the single‐stranded d(CCCTAA)n telomeric repeated motif
Authors: Marsich Eleonora , Bandiera Antonella , Tell Gianluca , Scaloni Andrea , Manzini Giorgio
Published: March 2003

With the aim of identifying proteins able to interact with the C‐rich single‐stranded telomeric repeated motif, three nuclear polypeptides, CBNPα, CBNPβ and CBNPγ, with apparent mobilities in SDS/PAGE of 38, 44 and 55 kDa, respectively, were isolated from mature chicken erythrocytes by affinity chromatography. In situ UV‐cross‐linking experiments demonstrated that CBNPα and CBNPγ interact directly with the telomeric d(CCCTAA)n repeat, whereas CBNPβ does not. Moreover, they provided information on the protein components responsible for each electrophoretic mobility‐shift assay signal. Ion spray and matrix‐assisted laser desorption ionization MS allowed us to identify CBNPα with single‐stranded D‐box‐binding factor (ssDBF), a protein previously characterized as a transcription factor belonging to the A/B family of heterogeneous nuclear ribonucleoproteins, and CBNPβ with an isoform of this protein containing an extra exon. Similarly, CBNPγ was shown to be probably the chicken homolog of hnRNP K, a ribonuclear protein able to bind to polyC oligonucleotides. The relation of CBNPα (i.e. ssDBF), CBNPβ and CBNPγ to a number of similar proteins in the protein and nucleotide sequence databank is discussed. A rather diversified spectrum of functional roles has been assigned to some of these proteins despite the strong sequence homology among them.
Interaction of linear homologous DNA duplexes via Holliday junction formation
Authors: Yakubovskaya Marianna G. , Neschastnova Anna A. , Humphrey Karen E. , Babon Jeff J. , Popenko Vladimir I. , Smith Margaret J. , Lambrinakos Andrianna , Lipatova Zhanna V. , Dobrovolskaia Marina A. , Cappai Roberto , Masters Colin L. , Belitsky Gennady A. , Cotton Richard G.
Published: March 2003

Interaction of linear homologous DNA duplexes by formation of Holliday junctions was revealed by electrophoresis and confirmed by electron microscopy. The phenomenon was demonstrated using a model of five purified PCR products of different size and sequence. The double‐stranded structure of interacting DNA fragments was confirmed using several consecutive purifications, S1‐nuclease analysis, and electron microscopy. Formation of Holliday junctions depends on DNA concentration. A thermodynamic equilibrium between duplexes and Holliday junctions was shown. We propose that homologous duplex interaction is initiated by nucleation of several dissociated terminal base pairs of two fragments. This process is followed by branch migration creating a population of Holliday junctions with the branch point at different sites. Finally, Holliday junctions are resolved via branch migration to new or previously existing duplexes. The phenomenon is a new property of DNA. This type of DNA–DNA interaction may contribute to the process of Holliday junction formation in vivo controlled by DNA conformation and DNA–protein interactions. It is of practical significance for optimization of different PCR‐based methods of gene analysis, especially those involving heteroduplex formation.
Expression of glucagon‐like peptide‐1 (GLP‐1) receptor and the effect of GLP‐1‐(7–36) amide on insulin release by pancreatic islets during rat ontogenic development
Authors: García‐Flores Marta , Zueco José A. , Álvarez Elvira , Blázquez Enrique
Published: March 2003

The expression of glucagon‐like peptide‐1 (GLP‐1) receptor and the effects of GLP‐1‐(7–36) amide (t‐GLP‐1) on glucose metabolism and insulin release by pancreatic islets during rat development were studied. GLP‐1 receptor mRNA was found in significant amounts in pancreatic islets from all age groups studied, GLP‐1 receptor expression being maximal when pancreatic islets were incubated at physiological glucose concentration (5.5 mm), but decreasing significantly when incubated with either 1.67 or 16.7 mm glucose. Glucose utilization and oxidation by pancreatic islets from fetal and adult rats rose as a function of glucose concentration, always being higher in fetal than in adult islets. The addition of t‐GLP‐1 to the incubation medium did not modify glucose metabolism but gastric inhibitory polypeptide and glucagon significantly increased glucose utilization by fetal and adult pancreatic islets at 16.7 mm glucose. At this concentration, glucose produced a significant increase in insulin release by the pancreatic islets from 10‐day‐old and 20‐day‐old suckling rats and adult rats, whereas those from fetuses showed only a significant increase when glucose was raised from 1.67 to 5.5 mm. t‐GLP‐1 elicited an increase in insulin release by pancreatic islets from all the experimental groups when the higher glucose concentrations were used. Our findings indicate that GLP‐1 receptors and the effect of t‐GLP‐1 on insulin release are already present in the fetus, and they therefore exclude the possibility that alterations in the action of t‐GLP‐1 are responsible for the unresponsiveness of pancreatic β cells to glucose in the fetus, but stimulation of t‐GLP‐1 release by food ingestion in newborns may partially confer glucose competence on β cells.
Atypical binding of the neuronal POU protein N‐Oct3 to noncanonical DNA targets
Authors: Millevoi Stefania , Thion Laurent , Joseph Gerard , Vossen Christine , Ghisolfi‐Nieto Laurence , Erard Monique
Published: March 2003

The capacity of POU proteins to recognize different DNA sequences and to bind target DNA in the form of monomers, cooperative dimers or heterodimers is important in relation to their transcriptional regulatory properties. The N‐Oct3 neuron‐specific protein binds to an octamer‐like sequence (AAATAATGC) within the (−102/−72) neuronal promoter region of the human aromatic l‐amino acid decarboxylase (AADC) gene. In this atypical case the POUh and POUs tetrameric subsites are spaced one nucleotide apart and in switched order as compared with the consensus octamer. Moreover this POU binding motif overlaps the hepatocyte nuclear factor HNF‐3β binding site (TGCTCAGTAAA) which itself contains a heptamer‐like sequence (CTCAGTA). Using the isolated DNA binding domains (DBD) of the two proteins, it is shown that, when binding to this unusual recognition sequence, N‐Oct3 either exhibits noncooperative homodimerization or allows the simultaneous binding of the second transcription activator HNF‐3β. CD studies indicate that the binding of N‐Oct3 monomers/dimers and N‐Oct3−HNF‐3β heterodimers to the DNA induces conformational changes of both protein and DNA. Partial proteolysis/MALDI‐MS was used in conjunction with molecular modelling to show that the protein conformational change resulting from binary N‐Oct3/DNA complex formation occurs within the linker peptide joining the POUs and POUh subdomains. Furthermore, modelling the N‐Oct3/HNF‐3β/DNA ternary complex predicts a nucleotide rearrangement in the overlap region and an interaction between both transcription factors. In the light of our findings, which illustrate both site‐dependent and site‐independent protein and DNA conformational changes, general implications for the allosteric function of DNA response elements in transcriptional regulation are discussed.
A novel sequence element is involved in the transcriptional regulation of expression of the ERG1 (squalene epoxidase) gene in Saccharomyces cerevisiae
Authors: Leber Regina , Zenz Rainer , Schröttner Klarissa , Fuchsbichler Sandra , Pühringer Brigitte , Turnowsky Friederike
Published: March 2003

Squalene epoxidase is an essential enzyme in the ergosterol‐biosynthesis pathway. It catalyzes the epoxidation of squalene to 2,3‐oxidosqualene and is the specific target of the antifungal drug terbinafine. Treatment of yeast cells with this inhibitor leads to squalene accumulation and sterol depletion. As ergosterol fulfils several essential functions, each requiring optimal sterol concentrations, synthesis of sterols in yeast must be tightly regulated. This study focuses on the sterol‐mediated regulation of expression of the ERG1 gene, which codes for squalene epoxidase in Saccharomyces cerevisiae. Inhibition of ergosterol biosynthesis with terbinafine increases the expression of ERG1 in a concentration‐dependent manner to a maximum of sevenfold. Inhibition of later steps in the ergosterol‐biosynthetic pathway by ketoconazol, an inhibitor of the lanosterol‐14α‐demethylase, and U18666A, an inhibitor of the squalene‐2,3‐epoxide–lanosterol cyclase, also induce expression of ERG1, suggesting that ERG1 expression is positively regulated by diminished intracellular ergosterol levels. The regulatory effect of sterols is manifested at the level of transcription. Deletion analysis of the ERG1 promoter identified a novel regulatory DNA sequence element. Two 6‐bp direct repeats, separated by 4 bp, AGCTCGGCCGAGCTCG, are unique to the ERG1 promoter. A DNA fragment containing this region confers ergosterol‐regulated expression on an otherwise unregulated CYC1 promoter construction. One copy of the 6‐bp element, AGCTCG, is sufficient to confer regulation, albeit less effectively than when both elements are present, whereas the removal of both elements from the ERG1 promoter leads to the loss of sterol‐dependent ERG1 regulation.
A comparison of kinetic and regulatory properties of the tetrameric and dimeric forms of wild‐type and Thr427→Pro mutant human phenylalanine hydroxylase
Authors: Bjørgo Elisa , de Carvalho Raquel Margarida Negrão , Flatmark Torgeir
Published: March 2003

Recombinant human phenylalanine hydroxylase (hPAH, phenylalanine 4‐monooxygenase EC 1.14.16.1) is catalytically active both as a tetramer and a dimer [Knappskog, P.M., Flatmark, T., Aarden, J.M., Haavik, J. and Martínez, A. (1996) Eur. J. Biochem.242, 813–821]. In the present study we have further characterized the differences in kinetic and regulatory properties of the two oligomeric forms when expressed in Escherichia coli. The positive cooperativity of l‐Phe binding to the tetrameric form both in enzyme kinetic studies (h = 1.6) and intrinsic tryptophan fluorescence measurements (h = 2.3) was abolished in the dimer, which also revealed a catalytic efficiency (Vmax/[S]0.5) of only 35% of the tetramer. Whereas the catalytic activity of the tetramer was activated fivefold to sixfold by preincubation with l‐Phe, the dimer revealed only a 1.6‐fold activation. The crystal structure has identified a five‐residue flexible hinge region (Asp425–Gln429) that links the β‐strand Tβ2 (Ile421–Leu424) and the 24 residue amphipathic α‐helix Tα1 (Gln428–Lys452) at the C‐terminus which forms an antiparallel coiled‐coil structure in the center of the tetramer [Fusetti, F., Erlandsen, H., Flatmark, T. & Stevens, R.C. (1998) J. Biol. Chem.273, 16962–16967]. The potential role of this flexible hinge in the tetramerization and the conformational transition of wt‐hPAH on the cooperative binding of l‐Phe was examined by site‐specific mutagenesis. Substitution of Thr427 by a Pro (as in tyrosine hydroxylase) resulted in a mutant protein which was isolated mainly (about 95%) as a dimer. The isolated tetramer of T427P revealed no kinetic cooperativity of l‐Phe binding, the catalytic efficiency (Vmax/[S]0.5) was decreased to about 39% of the wild‐type tetramer and it was not activated by l‐Phe preincubation. The dimeric forms of T427P and wt‐hPAH revealed rather similar kinetic properties. The lack of kinetic cooperativity of the T427P tetramer was associated with a corresponding change in the binding isotherm for l‐Phe as studied by intrinsic tryptophan fluorescence measurements. Protein stability was also reduced both for the E. coli expressed and the in vitro synthesized mutant enzyme. Collectively, these results indicate that the positive cooperativity of l‐Phe binding to wt‐hPAH requires a tetrameric enzyme with a C‐terminal flexibel hinge region (Asp425–Gln429) which has a structural role in the formation of the enzyme tetramer. Furthermore, this hinge region represents a motif in the PAH structure that is involved in the conformational change transmitted through the protein on the cooperative binding of l‐Phe to tetrameric wt‐hPAH. This conclusion is further supported by studies on two disease (phenylketonuria)‐associated mutant forms.
S1 subsite specificity of a recombinant cysteine proteinase, CPB, of Leishmania mexicana compared with cruzain, human cathepsin L and papain using substrates containing non‐natural basic amino acids
Authors: Alves Lira C. , Melo Robson L. , Sanderson Sanya J. , Mottram Jeremy C. , Coombs Graham H. , Caliendo Giuseppe , Santagada Vincenzo , Juliano Luiz , Juliano Maria A.
Published: March 2003

We have explored the substrate specificity of a recombinant cysteine proteinase of Leishmania mexicana (CPB2.8ΔCTE) in order to obtain data that will enable us to design specific inhibitors of the enzyme. Previously we have shown that the enzyme has high activity towards substrates with a basic group at the P1 position [Hilaire, P.M.S., Alves, L.C., Sanderson, S.J., Mottram, J.C., Juliano, M.A., Juliano, L., Coombs, G.H. & Meldal M. (2000) Chem. Biochem.1, 115–122], but we have also observed high affinity for peptides with hydrophobic residues at this position. In order to have substrates containing both features, we synthesized one series of internally quenched fluorogenic peptides derived from the sequence ortho‐amino‐benzoyl‐FRSRQ‐N‐[2,4‐dinitrophenyl]‐ethylenediamine, and substituted the Arg at the P1 position with the following non‐natural basic amino acids: 4‐aminomethyl‐phenylalanine (Amf), 4‐guanidine‐phenylalanine (Gnf), 4‐aminomethyl‐N‐isopropyl‐phenylalanine (Iaf), 3‐pyridyl‐alanine (Pya), 4‐piperidinyl‐alanine (Ppa), 4‐aminomethyl‐cyclohexyl‐alanine (Ama), and 4‐aminocyclohexyl‐alanine (Aca). For comparison, the series derived from ortho‐amino‐benzoyl‐FRSRQ‐N‐[2,4‐dinitrophenyl]‐ethylenediamine was also assayed with cruzain (the major cysteine proteinase of Trypanosoma cruzi), human cathepsin L and papain. The peptides ortho‐amino‐benzoyl‐FAmfSRQ‐N‐[2,4‐dinitrophenyl]‐ethylenediamine (kcat/Km = 12 000 mm−1·s−1) and ortho‐amino‐benzoyl‐FIafSRQ‐N‐[2,4‐dinitrophenyl]‐ethylenediamine (kcat/Km = 27 000 mm−1·s−1) were the best substrates for CPB2.8ΔCTE. In contrast, ortho‐amino‐benzoyl‐FAmaSRQ‐N‐[2,4‐dinitrophenyl]‐ethylenediamine and ortho‐amino‐benzoyl‐FAcaSRQ‐N‐[2,4‐dinitrophenyl]‐ethylenediamine were very resistant and inhibited this enzyme with Ki values of 23 nm and 30 nm, respectively. Cruzain hydrolyzed quite well the substrates in this series with Amf, Ppa and Aca, whereas the peptide with Ama was resistant and inhibited cruzain with a Ki of 40 nm. Human cathepsin L presented an activity on these peptides very similar to that of CPB2.8ΔCTE and papain hydrolyzed all the peptides with high efficiency. In conclusion, we have demonstrated that CPB2.8ΔCTE has more restricted specificity at the S1 subsite and it seems possible to design efficient inhibitors with amino acids such as Ama or Aca at the P1 position.
The rainbow trout (Oncorhynchus mykiss) Mx1 promoter
Authors: Collet Bertrand , Secombes Chris J.
Published: March 2003

Mx1 gene expression was studied in the rainbow trout (Oncorhynchus mykiss) gonad (RTG) (fibroblast) cell line. RT‐PCR analyses showed that both poly I:C and interferon containing supernatants induced expression of Mx1 in RTG cells. Kinetic analyses suggest that poly I:C acts indirectly through the production of interferons (IFN), as shown in other studies. By gene walking with trout genomic DNA the regulatory sequence of the Mx1 gene was cloned and sequenced. Sequence analysis showed that the 5′ flanking region has a structure, which is typical for an interferon‐induced gene promoter. Relative to the transcription start, it has a TATA box at −29 to −25, a 13 nucleotide interferon response element (ISRE) between −101 to −89, and a Sp1 binding site at −382 to −374. This region, with a single ISRE, is enough to induce strong expression of a luciferase reporter gene after stimulation of RTG cells with poly I:C. A time‐course of induction of this reporter construct showed maximal expression (22‐fold increase) after incubation with 100 µg mL−1 poly I:C for 48 h. An optimized method of transient transfection of RTG cells is also described.
Palmitoylation of the luteinizing hormone/human chorionic gonadotropin receptor regulates receptor interaction with the arrestin‐mediated internalization pathway
Authors: Munshi Utpal M. , Peegel Helle , Menon K. M. J.
Published: March 2003

The luteinizing hormone/human chorionic gonadotropin receptor (LH/hCGR) undergoes palmitoylation at cysteine residues 621 and 622 located in the carboxyl terminal tail of the receptor. This study examined the biological function of palmitoylation with respect to its effect on receptor internalization. Coexpression of wild‐type (WT) or C621/622G mutant receptors with arrestin‐2 increased receptor internalization in 293T cells. Furthermore, measurements of rate enhancement upon overexpression of arrestin indicate that the palmitoylation deficient mutant receptor is more prone to utilizing the arrestin mediated internalization pathway than the WT receptor. Coexpression of G‐protein‐coupled receptor kinase 4 (GRK4) with wild type receptor resulted in an increase in internalization, while coexpression with the mutant receptor did not result in further enhancement of internalization. Additionally, 293T cells expressing mutant receptor were responsive to hCG with respect to production of inositol phosphates. Taken together, these results suggest that the palmitoylation state of the receptor governs internalization by regulating the accessibility of the receptor to the arrestin‐mediated internalization pathway.