bluuebunny/crossref_metadata_2025 huggingface dataset:
The dual role of thrombin’s anion‐binding exosite‐I in the recognition and cleavage of the protease‐activated receptor 1
Authors: Myles Timothy , Le Bonniec Bernard F. , Stone Stuart R.
Published: March 2003

The role of thrombin anion‐binding exosite‐I in the recognition and cleavage of the extracellular domain of the seven transmembrane domain thrombin receptor (PAR1) was determined using site‐directed mutagenesis. Basic residues in anion‐binding exosite‐I (Arg35, Arg36, Arg67, Arg73, Arg75, Arg77A, Lys81, Lys109, Lys110 and Lys149E) were substituted with glutamines and the resultant recombinant mutant thrombins were used to determine kinetic parameters for the cleavage of a peptide (PAR38–60) based on the PAR1 extracellular domain. Compared with wild‐type thrombin, replacement of Arg67 and Arg73 had a dramatic effect on the cleavage of PAR38–60 (kcat/Km = 1.8 × 106 and 4.6 × 106 vs 9.2 × 107 m−1·s−1), whereas the remaining mutations of the anion‐binding exosite‐I of thrombin had a less pronounced effect, with kcat/Km values ranging from 3.3 × 107 m−1·s−1 (R77aQ) to 5.8 × 107 m−1·s−1 (K109Q). The ability of thrombin mutants to activate platelets paralleled that of PAR38–60 cleavage, whereas their ability to clot fibrinogen differed profoundly, as did their susceptibility to hirudin inhibition. Results are interpreted with respect to known interactions of thrombin with thrombomodulin, hirudin, rhodniin and heparin cofactor II. We conclude that the basic residues of anion‐binding exosite‐I contribute significantly to enhancing the rate of complex formation in two ways; the first (general) ensures electrostatic steering of ligands with complementary electrostatic fields, the second (specific) involves a combination of molecular contacts within the complex that is unique for each ligand.
An isoform–specific interaction of the membrane anchors affects mammalian adenylyl cyclase type V activity
Authors: Seebacher Thomas , Linder Jürgen U. , Schultz Joachim E.
Published: March 2003

The nine membrane‐bound mammalian adenylyl cyclases (ACs) contain two highly diverged membrane anchors, M1 and M2, with six transmembrane spans each and two conserved cytosolic domains which coalesce into a pseudoheterodimeric catalytic unit. Previously, the catalytic segments, bacterially expressed as soluble proteins, were characterized extensively whereas the function of the membrane domains remained unexplored. Using the catalytic C1 and C2 domains of AC type V we employed the membrane anchors from type V and VII ACs for construction of enzymes with duplicated, inverted, fully swapped and chimeric membrane anchors. Further, in the M1 membrane domain individual transmembrane spans were removed or exchanged between type V and VII ACs. The constructs were expressed in HEK293 cells, the expression levels and membrane localization was assessed by Western blotting. Cell‐free basal, forskolin‐, GTPγS‐and Gsα/GTPγS‐stimulated AC activities were determined. The results demonstrate that enzymatic activities were only maintained when the M1 and M2 membrane domains were derived from either AC V or VII. Constructs with chimeric membrane domains, i.e. M1 from type V and M2 from type VII AC or vice versa, were essentially inactive although the expression levels and membrane localization appeared to be normal. The data indicate a functionally important interaction of the membrane domains of ACs in that they seem to interact in a pair‐like, isoform delimited manner. This interaction directly impinges on the formation of the catalytic interface. We propose that protein–protein interactions of the AC membrane domains may constitute another, yet unexplored level of AC regulation.
The ptsH gene from Bacillus thuringiensis israelensis
Authors: Khan Sharik R. , Deutscher Josef , Vishwakarma Ram A. , Monedero Vicente , Bhatnagar Nirupama B.
Published: March 2003

The ptsH gene from Bacillus thuringiensis israelensis (Bti), coding for the phosphocarrier protein HPr of the phosphotransferase system has been cloned and overexpressed in Escherichia coli. Comparison of its primary sequence with other HPr sequences revealed that the conserved His15 and Ser46 residues were shifted by one amino acid and located at positions 14 and 45, respectively. The biological activity of the protein was not affected by this change. When expressed in a Bacillus subtilis ptsH deletion strain, Bti HPr was able to complement the functions of HPr in sugar uptake and glucose catabolite repression of the gnt and iol operons. A modified form of HPr was detected in Bti cells, and also when Bti ptsH was expressed in E. coli or B. subtilis. This modification was identified as phosphorylation, because alkaline phosphatase treatment converted the modified form to unmodified HPr. The phosphoryl bond in the new form of in vivo phosphorylated HPr was resistant to alkali treatment but sensitive to acid treatment, suggesting phosphorylation at a histidine residue. Replacement of His14 with alanine in Bti HPr prevented formation of the new form of phosphorylated HPr. The phosphorylated HPr was stable at 60 °C, in contrast with HPr phosphorylated at the Nδ1 position of His14 with phosphoenolpyruvate and enzyme I. 31P‐NMR spectroscopy was used to show that the new form of P‐HPr carried the phosphoryl group bound to the Nε2 position of His14 of Bti HPr. Phosphorylation of HPr at the novel site did not occur when Bti HPr was expressed in an enzyme I‐deficient B. subtilis strain. In addition, P‐(Nε2)His‐HPr did not transfer its phosphoryl group to the purified glucose‐specific enzyme IIA domain of B. subtilis.
cDNA cloning of the chicken branched‐chain α‐keto acid dehydrogenase complex
Authors: Ono Kazuo , Hakozaki Masatoshi , Suzuki Toshiyuki , Mori Tsutomu , Hata Hiroaki , Kochi Hideo
Published: March 2003

Branched‐chain α‐keto acid dehydrogenase complex is a macromolecule comprising three catalytic components: a dehydrogenase (E1) with α2β2 structure, an acyltransferase (E2) and a dihydrolipoamide dehydrogenase (E3). In the mammalian complex, the E2 component with 24 identical subunits forms a structural core, to which multiple copies of E1 and E3 bind noncovalently. We isolated cDNA clones encoding E1α, E1β and E2 subunits from a chicken‐liver cDNA library and performed nucleotide sequencing. Amino‐acid sequences deduced from the nucleotide sequences revealed that chicken E1α and E1β chains had substantially homologous sequences with the corresponding mammalian polypeptides, except for the N‐terminus. Chicken E2 conserved three functional domains, a lipoyl‐bearing domain, an E1/E3 binding domain and an inner‐core domain, but contrasted strongly with mammalian E2 in respect of containing 11 additional residues in two interdomain linkers: nine sequential residues in one linker and two residues in the other. Replacement of many residues was also observed in the chicken linkers. When E2 activity for catalyzing the overall reaction was measured by activity reconstitution in combination with E1 and E3, chicken E2 was markedly less effective than mammalian E2. The capability of chicken E2 for binding E1 was also reduced when determined by the binding assay using sucrose density gradient centrifugation. Chicken E1 was functionally as well as structurally indistinguishable from mammalian E1. Thus the reduced catalytic activity of chicken E2 must arise from its reduced E1‐binding capacity, which results from the characteristic structure of interdomain linkers in chicken E2.
Major tyrosine identity determinants in Methanococcus jannaschii and Saccharomyces cerevisiae tRNATyr are conserved but expressed differently
Authors: Fechter Pierre , Rudinger‐Thirion Joëlle , Tukalo Mikhail , Giegé Richard
Published: March 2003

Using in vitro tRNA transcripts and minihelices it was shown that the tyrosine identity for tRNA charging by tyrosyl‐tRNA synthetase (TyrRS) from the archaeon Methanococcus jannaschii is determined by six nucleotides: the discriminator base A73 and the first base‐pair C1–G72 in the acceptor stem together with the anticodon triplet. The anticodon residues however, participate only weakly in identity determination, especially residues 35 and 36. The completeness of the aforementioned identity set was verified by its tranfer into several tRNAs which then become as efficiently tyrosylatable as the wild‐type transcript from M. jannaschii. Temperature dependence experiments on both the structure and the tyrosylation properties of M. jannaschii and yeast tRNATyr transcripts show that the archaeal transcript has greater structural stability and enhanced aminoacylation behaviour than the yeast transcript. Tyrosine identity in M. jannaschii is compared to that in yeast, and the conservation of the major determinant in both organisms, namely the C1–G72 pair, gives additional support to the existence of a functional connection between archaeal and eukaryotic aminoacylation systems.
Interaction of Escherichia coli hemolysin with biological membranes
Authors: Schindel Christine , Zitzer Alexander , Schulte Berit , Gerhards Arnd , Stanley Peter , Hughes Colin , Koronakis Vassilis , Bhakdi Sucharit , Palmer Michael
Published: March 2003

Escherichia coli hemolysin (HlyA) is a membrane‐permeabilizing protein belonging to the family of RTX‐toxins. Lytic activity depends on binding of Ca2+ to the C‐terminus of the molecule. The N‐terminus of HlyA harbors hydrophobic sequences that are believed to constitute the membrane‐inserting domain. In this study, 13 HlyA cysteine‐replacement mutants were constructed and labeled with the polarity‐sensitive fluorescent probe 6‐bromoacetyl‐2‐dimethylaminonaphthalene (badan). The fluorescence emission of the label was examined in soluble and membrane‐bound toxin. Binding effected a major blue shift in the emission of six residues within the N‐terminal hydrophobic domain, indicating insertion of this domain into the lipid bilayer. The emission shifts occurred both in the presence and absence of Ca2+, suggesting that Ca2+ is not required for the toxin to enter membranes. However, binding of Ca2+ to HlyA in solution effected conformational changes in both the C‐terminal and N‐terminal domain that paralleled activation. Our data indicate that binding of Ca2+ to the toxin in solution effects a conformational change that is relayed to the N‐terminal domain, rendering it capable of adopting the structure of a functional pore upon membrane binding.
Pumping capacity of bacterial reaction centers and backpressure regulation of energy transduction
Authors: van Rotterdam Bart J. , Westerhoff Hans V. , Visschers Ronald W. , Bloch Dimitri A. , Hellingwerf Klaas J. , Jones Michael R. , Crielaard Wim
Published: March 2003

Transduction of free‐energy by Rhodobacter sphaeroides reaction‐center‐light‐harvesting‐complex‐1 (RCLH1) was quantified. RCLH1 complexes were reconstituted into liposomal membranes. The capacity of the RCLH1 complex to build up a proton motive force was examined at a range of incident light intensities, and induced proton permeabilities, in the presence of artificial electron donors and acceptors. Experiments were also performed with RCLH1 complexes in which the midpoint potential of the reaction center primary donor was modified over an 85‐mV range by replacement of the tyrosine residue at the M210 position of the reaction center protein by histidine, phenylalanine, leucine or tryptophan. The intrinsic driving force with which the reaction center pumped protons tended to decrease as the midpoint potential of the primary donor was increased. This observation is discussed in terms of the control of the energetics of the first steps in light‐driven electron transfer on the thermodynamic efficiency of the bacterial photosynthetic process. The light intensity at which half of the maximal proton motive force was generated, increased with increasing proton permeability of the membrane. This presents the first direct evidence for so‐called backpressure control exerted by the proton motive force on steady‐state cyclic electron transfer through and coupled proton pumping by the bacterial reaction center.
Structure, localization and potential role of a novel molluscan trypsin inhibitor in Lymnaea
Authors: Nagle Gregg T. , de Jong‐Brink Marijke , Painter Sherry D. , Li Ka Wan
Published: March 2003

Eggs and egg masses of the freshwater gastropod mollusc Lymnaea provide a microenvironment for developing embryos. Secretions of the exocrine albumen gland of Lymnaea are packaged in the eggs of an egg mass before the eggs are laid externally. The perivitelline fluid that directly surrounds individual oocytes is the main source of nutrition for developing embryos. During early stages of development, the perivitelline fluid is initially internalized by pinocytosis and degraded by lysosomes; in later stages, the embryo ingests the fluid. We previously found that the albumen gland produces large amounts of Lymnaea epidermal growth factor. The albumen gland also appears to produce significant amounts of a novel Lymnaea trypsin inhibitor (LTI), a second peptide that was purified and characterized from Lymnaea albumen gland extracts. The primary structure was determined by microsequence analysis, mass spectrometry, and C‐terminal sequence analysis, and showed that LTI is a 57‐residue glycosylated peptide. Comparison of the LTI sequence with other known serine protease inhibitors indicates that LTI is a member of the bovine pancreatic trypsin inhibitor family. Reverse phase‐high performance liquid chromatography, microsequence analysis, mass spectrometry, and immunocytochemistry demonstrated that abundant amounts of intact LTI are packaged in egg masses. The presence of a trypsin inhibitor in the perivitelline fluid compartment of the egg mass may minimize digestion of peptides and proteins in the perivitelline fluid that are important for the development of the embryo, for example, Lymnaea epidermal growth factor.
Chromatin structure of the bovine Cyp19 promoter 1.1
Authors: Fürbass Rainer , Said Harun M. , Schwerin Manfred , Vanselow Jens
Published: March 2003

Expression of the Cyp19 gene, encoding aromatase cytochrome P450, is driven by several tissue‐specific promoters. The underlying mechanisms of this complex regulation have not yet been elucidated in detail. In the present report we investigate a possible link between chromatin structure and tissue‐specific regulation of the bovine Cyp19 gene. We analysed the DNA methylation status and mapped DNaseI hypersensitive sites in the region encompassing the Cyp19 promoter 1.1 (P1.1) which controls Cyp19 expression in the bovine placenta. We show that P1.1 is hypomethylated in placental cotyledons (foetal layer) whereas it is methylated in placental caruncles (maternal layer), testis and corpus luteum. Furthermore, two placenta‐specific DNaseI hypersensitive sites, HS1 and HS2, were observed within P1.1. Both DNA hypomethylation and the presence of DNaseI hypersensitive sites correlate with transcriptional activity of P1.1. Sequence analysis of hypersensitive sites revealed potential cis‐regulatory elements, an E‐box in HS1 and a trophoblast‐specific element‐like sequence in HS2. It could be demonstrated by electrophoretic mobility shift assays that both sequence motifs are specific targets for placenta‐derived nuclear factors. In conclusion, observed tissue‐specific differences of the chromatin structure which correlate with tissue‐specific promoter activity suggest that chromatin might be an important regulator of aromatase expression in cattle.
Activator protein‐1 is necessary for angiotensin‐II stimulation of human adrenocorticotropin receptor gene transcription
Authors: Naville Danielle , Bordet Estelle , Berthelon Marie‐Claude , Durand Philippe , Bégeot Martine
Published: March 2003

Previous studies have demonstrated that angiotensin‐II (A‐II) increases the human adrenocorticotropin receptor (hMC2R) gene expression in adrenal cells. In the present study, we have characterized two activator protein‐1 (AP‐1)‐binding sites involved in the A‐II stimulation of hMC2R gene transcription. Vectors containing different fragments of the hMC2R gene promoter inserted upstream of the luciferase gene, have been constructed. After transfection of H295R cells with these constructs and treatment of the cells by A‐II during 48 h, maximal stimulation of the luciferase activity was obtained using the construct p(−263/+22)luc. Using progressively deleted constructs, three regions responsible for the A‐II‐stimulated transcription of hMC2R have been delineated. Inside these regions, two sequences displayed some homology with an AP‐1 binding element (AP‐108 and AP‐203). Mutation of either AP‐108 or AP‐203 site induced a decrease of A‐II‐stimulated luciferase activity by 40% and 25%, respectively. Gel‐shift analysis showed protein binding to these sites which was increased by an A‐II treatment (maximum obtained after 3 h). Moreover, A‐II could rapidly increase mRNA levels of several factors belonging to the Fos and Jun families which may be components of the AP‐1 complex.
The human neutrophil lipocalin supports the allosteric activation of matrix metalloproteinases
Authors: Tschesche Harald , Zölzer Volker , Triebel Susanne , Bartsch Stefan
Published: March 2003

The human neutrophil lipocalin (HNL), a member of the large family of lipocalins that exhibit various physiological functions, is coexpressed in granulocytes with progelatinase B (MMP‐9). Part of it is covalently bound to the proenzyme and therefore may play a possible role in the activation process of promatrix metalloproteinases. We now report that HNL is able to accelerate the direct activation of promatrix metalloproteinases slightly. A significant enhancement of the activity could be demonstrated for the HgCl2‐ and the plasma kallikrein‐induced activation of all three secretory forms of proMMP‐9 and of proMMP‐8. The same activating effects were exerted by HNL isolated from granulocytes as well as by the recombinant forms expressed by the yeast Pichia pastoris or by Escherichia coli. This demonstrates that the carbohydrate moiety is not essential for the biological activity of HNL. Activation and activity enhancement are obviously mediated by entrapping the remaining N‐terminal sequence residues of the partially truncated proenzyme into the hydrophobic binding pocket of the HNL. In conclusion these results document that HNL can exert an enzyme‐activating effect in the regulation of inflammatory and pathophysiological responses of granulocytes in the physiological activation of MMPs that have been subject to limited proteolytic processing.
Cytotoxic mechanism of the ribotoxin α‐sarcin
Authors: Olmo Nieves , Turnay Javier , de Buitrago Gonzalo González , de Silanes Isabel López , Gavilanes José G. , Lizarbe Maria A.
Published: March 2003

α‐Sarcin is a ribosome‐inactivating protein that has been well characterized in vitro, but little is known about its toxicity in living cells. We have analyzed the mechanism of internalization of α‐sarcin into human rhabdomyosarcoma cells and the cellular events that result in the induction of cell death. No specific cell surface receptor for α‐sarcin has been found. The toxin is internalized via endocytosis involving acidic endosomes and the Golgi, as deduced from the ATP requirement and the effects of NH4Cl, monensin and nigericin on its cytotoxicity. Specific cleavage of 28S rRNA in cultured rhabdomyosarcoma cells, associated with protein biosynthesis inhibition, has been detected. α‐Sarcin kills rhabdomyosarcoma cells via apoptosis: incubation of cells with α‐sarcin at a concentration below its IC50 induces internucleosomal genomic DNA fragmentation, reversion of membrane asymmetry, activation of caspase‐3‐like activity and cleavage of poly(ADP‐ribose)polymerase. Apoptosis is not a general direct consequence of protein biosynthesis inhibition, as deduced from the comparative analysis of the effects of α‐sarcin and cycloheximide; the latter does not induce apoptosis even at concentrations far beyond its IC50, where protein biosynthesis is null. Experiments with a catalytically inactive α‐sarcin mutant, neither toxic nor apoptotic, reveal that induced apoptosis is directly related to the effects of catalytic activity of the toxin on the ribosomes. The caspase inhibitor z‐VAD‐fmk does not suppress protein synthesis inhibition by α‐sarcin. Together, these data suggest that α‐sarcin‐induced caspase activation is a pathway downstream of the 28S rRNA catalytic cleavage and consequent protein biosynthesis inhibition.
Structural analysis of the O‐antigen polysaccharide from the Shiga toxin‐producing Escherichia coli O172
Authors: Landersjö Clas , Weintraub Andrej , Widmalm Göran
Published: March 2003

The structure of the O‐antigen polysaccharide from Escherichia coli O172 has been determined. In combination with sugar analysis, NMR spectroscopy shows that the polysaccharide is composed of pentasaccharide repeating units. Sequential information was obtained by mass spectrometry and two‐dimensional NMR techniques. An O‐acetyl group was present as 0.7 equivalent per repeating unit. Treatment of the O‐deacetylated polysaccharide with aqueous 48% hydrofluoric acid rendered cleavage of the phosphodiester in the backbone of the polymer and the pentasaccharide isolated after gel permeation chromatography was structurally characterized. Subsequent NMR experiments on polymeric materials revealed the structure of the repeating unit of the O‐polysaccharide from E. coli O172 as:→P‐4)‐α‐d‐Glcp‐(1→3)‐α‐l‐FucpNAc‐(1→3)‐α‐d‐ GlcpNAc‐(1→3)‐α‐l‐FucpNAc‐(1→4)‐α‐d‐Glcp6Ac‐(1→
Purification, characterization and crystallization of thermostable anthranilate phosphoribosyltransferase from Sulfolobus solfataricus
Authors: Ivens Andreas , Mayans Olga , Szadkowski Halina , Wilmanns Matthias , Kirschner Kasper
Published: March 2003

Anthranilate phosphoribosyltransferase (TrpD; EC 2.4.2.18) from the hyperthermophilic archaeon Sulfolobus solfataricus (ssTrpD) was expressed in Escherichia coli, purified and crystallized. Analytical gel permeation chromatography revealed a homodimeric composition of the enzyme. The steady‐state kinetic characteristics suggest tight binding of the substrate anthranilic acid and efficient catalysis at the physiological growth temperature of S. solfataricus. Crystals of ssTrpD diffract to better than 2.6 Å resolution and preliminary X‐ray characterization was carried out. The crystals are suitable for structure determination.
High affinity binding of paclitaxel to human serum albumin
Authors: Paál Krisztina , Müller József , Hegedûs Lajos
Published: March 2003

Paclitaxel, a very potent antitumor agent is a hydrophobic molecule with low aqueous solubility. Its currently used formula (Taxol®) contains the drug in a 1 : 1 (v/v) mixture of ethanol and Cremophor EL. To minimize vehicle‐related toxicity, we developed a novel, water‐soluble formulation in which paclitaxel is bound noncovalently to human serum albumin. For this purpose, studies of the paclitaxel–albumin binding equilibrium were performed. Paclitaxel dissolved in ethanol was added to the aqueous solution of human serum albumin. Precipitated paclitaxel was removed and unbound drug was separated by ultrafiltration. Paclitaxel concentration was measured by RP‐HPLC. Binding data were evaluated based both on the Scatchard plot and the general binding equation describing binding equilibria with the stepwise stoichiometric binding constants. The Scatchard plot was found to be curvilinear with a slight positive slope of the final part. Parameters of high affinity specific binding were determined from the initial part of the curve (nsp = 1.3 and Ksp = 1.7 × 106 m−1). Stoichiometric binding constants were estimated by fitting the general binding equation to the experimental data (K1 = 2.4 × 106 m−1 and K2 = 1.0 × 105 m−1). Saturation of the protein with paclitaxel, similarly to other ligands of albumin, could not be reached. The greatest observed value of r (number of paclitaxel molecules bound to one albumin molecule) was 6.6.
Solution NMR structure of the cold‐shock protein from the hyperthermophilic bacterium Thermotoga maritima
Authors: Kremer Werner , Schuler Benjamin , Harrieder Stefan , Geyer Matthias , Gronwald Wolfram , Welker Christine , Jaenicke Rainer , Kalbitzer Hans R.
Published: March 2003

Cold‐shock proteins (Csps) are a subgroup of the cold‐induced proteins preferentially expressed in bacteria and other organisms on reduction of the growth temperature below the physiological temperature. They are related to the cold‐shock domain found in eukaryotes and are some of the most conserved proteins known. Their exact function is still not known, but translational regulation, possibly via RNA chaperoning, has been discussed. Here we present the structure of a hyperthermophilic member of the Csp family. The NMR solution structure of TmCsp from Thermotoga maritima, the hyperthermophilic member of this class of proteins, was solved on the basis of 1015 conformational constraints. It contains five β strands combined in two antiparallel β sheets making up a β barrel structure, in which β strands 1–4 are arranged in a Greek‐key topology. The side chain of R2, which is exclusively found in thermophilic members of the Csp family, probably participates in a peripheral ion cluster involving residues D20, R2, E47 and K63, suggesting that the thermostability of TmCsp is based on the peripheral ion cluster around the side chain of R2.
Identification and characterization of the genes for N‐acetylglucosamine kinase and N‐acetylglucosamine‐phosphate deacetylase in the pathogenic fungus Candida albicans
Authors: Yamada‐Okabe Toshiko , Sakamori Yoshimi , Mio Toshiyuki , Yamada‐Okabe Hisafumi
Published: March 2003

Like bacteria and many fungi, the pathogenic fungus Candida albicans can utilize GlcNAc as a carbon source for growth. A cluster of six genes was identified in the C. albicans genome. One of the genes in the cluster was CaNAG1, which is responsible for GlcN6P deaminase and is therefore essential for GlcNAc‐dependent growth. The other five genes were designated CaNAG2, CaNAG3, CaNAG4, CaNAG5 and CaNAG6. The mRNA levels of CaNAG1, CaNAG2 and CaNAG5 were significantly induced by GlcNAc, whereas those of CaNAG3, CaNAG4 and CaNAG6 were not. Neither CaNAG2 nor CaNAG5 was essential for growth, but disruption of CaNAG2 or CaNAG5 greatly retarded the growth of cells using GlcNAc as the sole carbon source. Although no homolog of CaNAG2 or CaNAG5 was found in the Saccharomyces cerevisiae genome, CaNag2p displayed sequence similarities to Escherichia coli nagA, and CaNag5p is homologous to a wide variety of hexose kinases. When expressed as a fusion protein with glutathione S‐transferase (GST), CaNag5p produced GlcNAc‐P from GlcNAc in the presence of ATP, whereas GST alone did not. Furthermore, the recombinant GST–CaNag2p fusion protein converted GlcNAcP, which was produced by CaNag5p, into GlcNP. These results clearly demonstrate that CaNAG2 and CaNAG5 encode GlcNAcP deacetylase and GlcNAc kinase, respectively. CaNag5p recognized glucose and mannose as substrates, whereas the recently identified human GlcNAc kinase was specific to GlcNAc. Deletion of CaNAG2 or CaNAG5 markedly, and that of CaNAG1 moderately, attenuated the virulence of C. albicans in a mouse systemic infection model. Thus, it appears that GlcNAc metabolism of C. albicans is closely associated with its virulence.
Regulation of human and mouse procathepsin E gene expression
Authors: Cook Matthew , Caswell Richard C. , Richards Roy J. , Kay John , Tatnell Peter J.
Published: March 2003

Cathepsin E is an intracellular aspartic proteinase that is considered to have a number of physiological roles including antigen processing. Quantitation of procathepsin E mRNA by LightCycler™ technology indicated that the gene was transcribed in lung but not in kidney of both human and mouse origin. In contrast, the transcript was present in mouse spleen and alveolar macrophages but not in the counterpart tissue/cells from humans. Regulation of human and mouse procathepsin E gene expression was shown not to be influenced by the extent of CpG methylation but depended on the recognition of potential binding motifs in each promoter region by transcription factors such as GATA1, PU1 and YY1, as revealed by functional analysis using a series of promoter/luciferase reporter gene fusion constructs. Thus the extent to which the procathepsin E gene is expressed in a particular cell type may depend on the balance between the effects produced by positive‐acting, cell‐specific transcription factors such as GATA1 and PU1 and the negative influence of the ubiquitous YY1 factor. In this way, the relative abundance and influence of general and cell‐specific transcription factors can govern the production of cathepsin E and thereby account for the sporadic cell and tissue distribution of this enzyme in different species.
The polypeptide chain release factor eRF1 specifically contacts the s4UGA stop codon located in the A site of eukaryotic ribosomes
Authors: Chavatte Laurent , Frolova Ludmila , Kisselev Lev , Favre Alain
Published: March 2003

It has been shown previously [Brown, C.M. & Tate, W.P. (1994) J. Biol. Chem.269, 33164–33170.] that the polypeptide chain release factor RF2 involved in translation termination in prokaryotes was able to photocrossreact with mini‐messenger RNAs containing stop signals in which U was replaced by 4‐thiouridine (s4U). Here, using the same strategy we have monitored photocrosslinking to eukaryotic ribosomal components of 14‐mer mRNA in the presence of

, and 42‐mer mRNA in the presence of tRNAAsp (tRNAAsp gene transcript). We show that: (a) both 14‐mer and 42‐mer mRNAs crossreact with ribosomal RNA and ribosomal proteins. The patterns of the crosslinked ribosomal proteins are similar with both mRNAs and sensitive to ionic conditions; (b) the crosslinking patterns obtained with 42‐mer mRNAs show characteristic modification upon addition of tRNAAsp providing evidence for appropriate mRNA phasing onto the ribosome. Similar changes are not detected with the 14‐mer

pairs; (c) when eukaryotic polypeptide chain release factor 1 (eRF1) is added to the ribosome·tRNAAsp complex it crossreacts with the 42‐mer mRNA containing the s4UGA stop codon located in the A site, but not with the s4UCA sense codon; this crosslink involves the N‐terminal and middle domains of eRF1 but not the C domain which interacts with eukaryotic polypeptide chain release factor 3 (eRF3); (d) addition of eRF3 has no effect on the yield of eRF1–42‐mer mRNA crosslinking and eRF3 does not crossreact with 42‐mer mRNA. These experiments delineate the in vitro conditions allowing optimal phasing of mRNA on the eukaryotic ribosome and demonstrate a direct and specific contact of ‘core’ eRF1 and s4UGA stop codon within the ribosomal A site.
Insulin‐like growth factor binding proteins (IGFBPs) as potential physiological substrates for human kallikreins hK2 and hK3
Authors: Réhault Sophie , Monget Philippe , Mazerbourg Sabine , Tremblay Roland , Gutman Ninette , Gauthier Francis , Moreau Thierry
Published: March 2003

Insulin‐like growth factors (IGFs) are important growth regulators of both normal and malignant prostate cells. Their action is regulated by six insulin‐like growth factor binding proteins (IGFBPs). The proteolytic cleavage of IGFBPs by various proteases decreases dramatically their affinity for their ligands and therefore enhances the bioavailability of IGFs. To elucidate the putative biological role of prostatic kallikreins hK2 and hK3 (prostate‐specific antigen) in tumour progression, we analyzed the degradation of IGFBP‐2, ‐3, ‐4 and ‐5 by these two tissue kallikreins. We found that hK3, already characterized as an IGFBP‐3 degrading protease, cleaved IGFBP‐4 but not IGFBP‐2 and ‐5, whereas hK2 cleaved all of the IGFBPs much more effectively, and at concentrations far lower than those reported for other IGFBP‐degrading proteases. The proteolytic patterns after cleavage of IGFBPs by hK2 and hK3 were similar and were not modified in the presence of IGF‐I. Heparin, but not other glycosaminoglycans, enhanced dramatically the ability of hK3 but not hK2 to degrade IGFBP‐3 and IGFBP‐4. More importantly, the IGFBP fragments generated by hK2 and hK3 had no IGF‐binding capacity, as assessed by Western ligand blotting. Our results suggest that the prostatic kallikreins hK2 and hK3 may influence specifically the tumoral growth of prostate cells through the degradation of IGFBPs, to increase IGF bioavailability.
Biochemical characterization and identification of catalytic residues in α‐glucuronidase from Bacillus stearothermophilus T‐6
Authors: Zaide Galia , Shallom Dalia , Shulami Smadar , Zolotnitsky Gennady , Golan Gali , Baasov Timor , Shoham Gil , Shoham Yuval
Published: March 2003

α‐d‐Glucuronidases cleave the α‐1,2‐glycosidic bond of the 4‐O‐methyl‐d‐glucuronic acid side chain of xylan, as a part of an array of xylan hydrolyzing enzymes. The α‐d‐glucuronidase from Bacillus stearothermophilus T‐6 was overexpressed in Escherichia coli using the T7 polymerase expression system. The purification procedure included two steps, heat treatment and gel filtration chromatography, and provided over 0.3 g of pure enzyme from 1 L of overnight culture. Based on gel filtration, the native protein is comprised of two identical subunits. Kinetic constants with aldotetraouronic acid as a substrate, at 55 °C, were a Km of 0.2 mm, and a specific activity of 42 U·mg−1 (kcat= 54.9 s−1). The enzyme was most active at 65 °C, pH 5.5–6.0, in a 10‐min assay, and retained 100% of its activity following incubation at 70 °C for 20 min. Based on differential scanning calorimetry, the protein denatured at 73.4 °C. Truncated forms of the enzyme, lacking either 126 amino acids from its N‐terminus or 81 amino acids from its C‐terminus, exhibited low residual activity, indicating that the catalytic site is located in the central region of the protein. To identify the potential catalytic residues, site‐directed mutagenesis was applied on highly conserved acidic amino acids in the central region. The replacements Glu392→Cys and Asp364→Ala resulted in a decrease in activity of about five orders of magnitude, suggesting that these residues are the catalytic pair.
Purification and cDNA cloning of nitric oxide reductase cytochrome P450nor (CYP55A4) from Trichosporon cutaneum
Authors: Zhang Li , Takaya Naoki , Kitazume Tatsuya , Kondo Toshihiro , Shoun Hirofumi
Published: March 2003

Cytochrome P450nor is involved in fungal denitrification as nitric oxide (NO) reductase. Although the heme protein has been known to occur in restricted species of fungi that belong to ascomycotina, we have previously suggested that it would also occur in the yeast Trichosporon cutaneum, which is phylogenetically far from those P450nor‐producing ascomycetous fungi. Here we isolated and characterized the heme protein from the basidiomycetous yeast T. cutaneum. P450nor of the yeast (TcP450nor) exhibited properties in terms of catalysis, absorption spectrum and molecular mass that are almost identical to those of its counterparts in ascomycetous fungi. We also isolated and sequenced its cDNA. The predicted primary structure of TcP450nor showed high sequence identities (around 65%) to those of other P450nors, indicating that they belong to the same family. TcP450nor protein cofractionated with cytochrome c oxidase by subcellular fractionation and its predicted primary structure contained an extension on its amino terminus that is characteristic of a mitochondrial‐targeting signal, indicating that it is a mitochondrial protein like some of the isoforms of other fungi. On the other hand, TcP450nor was unique in that inducers such as nitrate, nitrite, or NO were not required for its production in the cells. The occurrence of P450nor across the subdivisions of eumycota suggests that P450nor and denitrification are distributed more universally among fungi than was previously thought.
Samui, a novel cold‐inducible gene, encoding a protein with a BAG domain similar to silencer of death domains (SODD/BAG‐4), isolated from Bombyx diapause eggs
Authors: Moribe Yoriko , Niimi Teruyuki , Yamashita Okitsugu , Yaginuma Toshinobu
Published: March 2003

Cellular responses to cold‐acclimation have not yet been studied in depth. To explore this field, we focussed on insect diapause development. Although embryonic diapause of Bombyx mori is sustained at 25 °C, chilling at 5 °C for 2 months causes diapause termination, a transition that is marked when the sorbitol dehydrogenase gene (SDH) is activated. To clarify the relationship between this activation and incubation at 5 °C, we isolated a novel cold‐inducible gene, Samui. Expression of Samui mRNA and protein was activated after incubation at 5 °C for 5–6 days, lasted for another 30 days and then weakened. Exposure to 25 °C suppressed both mRNA and protein expression. In nondiapause eggs incubated at 5 °C, Samui was also up‐regulated, although the expression was weaker. Samui contained nuclear localization‐signals, a ssDNA‐binding motif and a BAG domain similar to that of SODD/BAG‐4. Because Samui could bind to HSP70, it is a member of BAG protein family. It is proposed that Samui serves to transmit the ‘5 °C signal’ for SDH expression in diapause eggs, while also protecting against cold‐injures in nondiapause eggs, through binding to respective partners. This is the first report that a member of BAG protein family is up‐regulated by cold.
Mechanism of fatty acid desaturation in the green alga Chlorella vulgaris
Authors: Behrouzian Behnaz , Fauconnot Laëtitia , Daligault Franck , Nugier‐Chauvin Caroline , Patin Henri , Buist Peter H.
Published: March 2003

The hypothesis that the Δ9 desaturase of Chlorella vulgaris might operate by a synchronous mechanism has been tested using a kinetic isotope effect (KIE) approach. Thus the intermolecular primary deuterium KIE on the individual C–H bond cleavage steps involved in Δ9 desaturation have been determined by incubating growing cultures of C. vulgaris (strain 211/8K) with mixtures of the appropriate regiospecifically deuterated fatty acid analogues. Our analysis shows that the introduction of a double bond between C‐9 and C‐10 occurs in two discrete steps as the cleavage of the C9–H bond is very sensitive to isotopic substitution (kH/kd = 6.6 ± 0.3) whereas a negligible isotope effect (kH/kd = 1.05 ± 0.05) was observed for the C10–H bond‐breaking step. Similar results were obtained for linoleic acid biosynthesis (Δ12 desaturation). These data clearly rule out a synchronous mechanism for these reactions.
Cleavage of AML1/MTG8 by asymmetric hammerhead ribozymes
Authors: Szyrach Mara , Münchberg Frank E. , Riehle Heidemarie , Nordheim Alfred , Krauter Jürgen , Nagel Stefan , Heil Gerhard , Heidenreich Olaf
Published: March 2003

The chromosomal translocation t(8;21) is one of the most frequent aberrations associated with acute myeloid leukaemia. It joins the 5′ section of the AML1 gene with the almost complete open reading frame of MTG8 (ETO). The resulting fusion RNA represents a leukaemia‐specific target for antisense/ribozyme inhibition. We tested several asymmetric hammerhead ribozymes targeted against the fusion site for their ability to cleave the AML1/MTG8 RNA at low magnesium concentrations. One ribozyme cleaves AML1/MTG8 RNA with high catalytic efficiency without binding or cleaving the wild‐type AML1 transcript. The presence of cellular RNA does not affect the cleavage. Injection of AML1/MTG8 RNA and ribozyme RNA into Xenopus eggs or oocytes causes a specific reduction of AML1/MTG8 protein expression. Asymmetric anti‐AML1/MTG8 ribozymes may be valuable modulators of AML1/MTG8 expression in leukaemic cells.
2‐Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans
Authors: Brock Matthias , Darley Dan , Textor Susanne , Buckel Wolfgang
Published: March 2003

In Escherichia coli and Aspergillus nidulans, propionate is oxidized to pyruvate via the methylcitrate cycle. The last step of this cycle, the cleavage of 2‐methylisocitrate to succinate and pyruvate is catalysed by 2‐methylisocitrate lyase. The enzymes from both organisms were assayed with chemically synthesized threo‐2‐methylisocitrate; the erythro‐diastereomer was not active. 2‐Methylisocitrate lyase from E. coli corresponds to the PrpB protein of the prp operon involved in propionate oxidation. The purified enzyme has a molecular mass of approximately 32 kDa per subunit, which is lower than those of isocitrate lyases from bacterial sources (≈ 48 kDa). 2‐Methylisocitrate lyase from A. nidulans shows an apparent molecular mass of 66 kDa per subunit, almost equal to that of isocitrate lyase of the same organism. Both 2‐methylisocitrate lyases have a native homotetrameric structure as identified by size‐exclusion chromatography. The enzymes show no measurable activity with isocitrate. Starting from 250 mm pyruvate, 150 mm succinate and 10 µm PrpB, the enzymatically active stereoisomer could be synthesized in 1% yield. As revealed by chiral HPLC, the product consisted of a single enantiomer. This isomer is cleaved by 2‐methylisocitrate lyases from A. nidulans and E. coli. The PrpB protein reacted with stoichiometric amounts of 3‐bromopyruvate whereby the activity was lost and one amino‐acid residue per subunit became modified, most likely a cysteine as shown for isocitrate lyase of E. coli. PrpB exhibits 34% sequence identity with carboxyphosphoenolpyruvate phosphonomutase from Streptomyces hygroscopicus, in which the essential cysteine residue is conserved.
The variant tuf3 gene of Streptomyces coelicolor A3(2) encodes a real elongation factor Tu, as shown in a novel Streptomyces in vitro translation system
Authors: Olsthoorn‐Tieleman Lian N. , Plooster Lizanne J. , Kraal Barend
Published: March 2003

In Streptomyces coelicolor, the regular and abundant elongation factor (EF)‐Tu1 is encoded by tuf1, while the actual function of the highly divergent tuf3 gene product is not yet known. Expression of the latter could so far only be detected on the transcriptional level under stress conditions. In this paper we demonstrate the presence of low levels of EF‐Tu3 in strains of the J1501 lineage. Enhanced expression was observed for J1501 glkA and relA deletion mutants, which lack glucose kinase and ribosome‐bound ppGpp synthetase, respectively. To assess the putative translational capacities of EF‐Tu3, a novel Streptomyces in vitro translation assay was designed, based on the full elimination by Ni2+ affinity adsorption of chromosomally encoded (His)6‐tagged EF‐Tu1 from a S. coelicolor cell‐free extract. Translational activity of this system is totally dependent on the addition of purified EF‐Tu species or on the presence of an additional elongation factor Tu in the extract, e.g. encoded by a plasmid‐borne tuf gene. Using this EF‐Tu‐dependent translation system, we have established that S. coelicolor EF‐Tu3 has translational capacities despite its striking deviation from the common prokaryotic EF‐Tu sequence at positions involved in either aminoacyl‐tRNA binding or interaction with the guanine‐nucleotide exchange factor EF‐Ts.
A barley polyamine oxidase isoform with distinct structural features and subcellular localization
Authors: Cervelli Manuela , Cona Alessandra , Angelini Riccardo , Polticelli Fabio , Federico Rodolfo , Mariottini Paolo
Published: March 2003

Two cDNAs encoding polyamine oxidase (PAO) isoforms (BPAO1 and BPAO2) and the corresponding gene copies were isolated from barley cultivar Aura. Gene organization is not conserved between these two nonallelic coding sequences. Both precursor proteins include a cleavable N‐terminal leader of 25 amino acids. N‐terminal sequencing of PAO purified from barley seedlings reveals a unique amino‐acid sequence corresponding to the BPAO2 N‐terminus as predicted from the corresponding cDNA. BPAO2 has been purified, characterized and compared to maize PAO (MPAO), the best characterized member of this enzyme class. The two proteins show different pH optima for catalytic activity, Km and Vmax values with spermidine and spermine as substrates. Molecular modelling of BPAO2 reveals the same global fold as in MPAO. However, substitution of the active site residue Phe403 by a tyrosine, provides a rationale for the different catalytic properties of the two enzymes. In barley leaves PAO‐specific activity is higher in isolated mesophyll protoplasts than in the extracellular fluids, whereas in maize the reverse is true. The C‐terminus of BPAO2 shows homology with the endoplasmic reticulum retention signal that might be responsible for the subcellular localization observed. We conclude that BPAO2 is a symplastic PAO in barley mesophyll cells. Production of BPAO2 mRNA and the corresponding protein is induced by light, and has a different pattern of accumulation in leaves and coleoptiles.
Inactivation of Aeromonas hydrophila metallo‐β‐lactamase by cephamycins and moxalactam
Authors: Zervosen Astrid , Valladares Maria Hernandez , Devreese Bart , Prosperi‐Meys Christelle , Adolph Hans‐Werner , Mercuri Paola Sandra , Vanhove Marc , Amicosante Gianfranco , van Beeumen Jozef , Frère Jean‐Marie , Galleni Moreno
Published: March 2003

Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo‐β‐lactamase CphA leads to enzyme‐catalyzed hydrolysis of both compounds and to irreversible inactivation of the enzyme by the reaction products. As shown by electrospray mass spectrometry, the inactivation of CphA by cefoxitin and moxalactam is accompanied by the formation of stable adducts with mass increases of 445 and 111 Da, respectively. The single thiol group of the inactivated enzyme is no longer titrable, and dithiothreitol treatment of the complexes partially restores the catalytic activity. The mechanism of inactivation by moxalactam was studied in detail. Hydrolysis of moxalactam is followed by elimination of the 3′ leaving group (5‐mercapto‐1‐methyltetrazole), which forms a disulfide bond with the cysteine residue of CphA located in the active site. Interestingly, this reaction is catalyzed by cacodylate.
Complex of Burkholderia cepacia lipase with transition state analogue of 1‐phenoxy‐2‐acetoxybutane
Authors: Luić Marija , Tomić Sanja , Leščić Ivana , Ljubović Edina , Šepac Dragan , Šunjić Vitomir , Vitale Ljubinka , Saenger Wolfram , Kojić‐Prodić Biserka
Published: March 2003

In a series of four racemic phenoxyalkyl‐alkyl carbinols, 1‐phenoxy‐2‐hydroxybutane (1) is enantioselectively acetylated by Burkholderia cepacia (formerly Pseudomonas cepacia) lipase with an E value ≥ 200, whereas for the other three racemates E was found to be ≤ 4. To explain the high preference of B. cepacia lipase for (R)‐(+)‐1, a precursor of its transition state analogue with a tetrahedral P‐atom, (RP,SP)‐O‐(2R)‐(1‐phenoxybut‐2‐yl)methylphosphonic acid chloride was prepared and crystallized in complex with B. cepacia lipase. The X‐ray structure of the complex was determined, allowing to compare the conformation of the inhibitor with results of molecular modelling.
Phosphorylation of seminal vesicle protein IV on Ser58 enhances its peroxidase‐stimulating activity
Authors: Metafora Vittoria , Franco Paola , Massa Ornella , Morelli Francesco , Stiuso Paola , Ferranti Pasquale , Mamone Gianfranco , Malorni Antonio , Stoppelli M. Patrizia , Metafora Salvatore
Published: March 2003

In this study we show that SV‐IV, a major immunomodulatory, anti‐inflammatory, and sperm immunoprotective protein secreted from the rat seminal vesicle epithelium, acts in vitro as a substrate of protein kinase C (PKC) competing efficiently with H1 histone, a very well known PKC substrate. Electrospray mass spectrometry (ES‐MS) analysis demonstrated that approximately 10% of the native SV‐IV molecules were phosphorylated by PKC and that such a modification involved only a single serine residue (Ser58) out of the 22 occurring in the protein. Interestingly, this modification produced a substantial enhancement (approximately 50%) of the native SV‐IV's ability to stimulate the activity of both horseradish peroxidase (POD) and selenium‐dependent glutathione peroxidase (GPX), an enzyme that is known to protect the mammalian spermatozoa from oxidative stress and loss of motility in the female genital tract following ejaculation. In contrast, the phosphorylation of SV‐IV on Ser58 did not produce any effect on the anti‐inflammatory properties of SV‐IV, as measured by its ability to inhibit the phospholipase A2.