bluuebunny/crossref_metadata_2025 huggingface dataset:
NMR investigation of the multidrug transporter EmrE, an integral membrane protein
Authors: Schwaiger Manfred , Lebendiker Mario , Yerushalmi Hagit , Coles Murray , Gröger Adriane , Schwarz Christian , Schuldiner Shimon , Kessler Horst
Published: March 2003

EmrE is an Escherichia coli multidrug transport protein that confers resistance to a wide range of toxicants by active transport across the bacterial cell membrane. The highly hydrophobic polytopic integral membrane protein has been purified and studied in its full‐length form by high‐resolution NMR spectroscopy in a mixture of chloroform/methanol/water (6 : 6 : 1, by vol.). Full activity is maintained after reconstitution of the protein into proteoliposomes from this solvent mixture. A series of heteronuclear (1H‐15N) two‐ and three‐dimensional experiments, as well as triple resonance experiments, were applied to the 110‐residue protein and led to the assignment of the 1H, 15N and a large part of the 13C backbone resonances as well as many of the sidechain resonances. A preliminary analysis of the secondary structure, based on sequential NOE connectivities, deviation of chemical shifts from random coil values and 3JNH‐Hα coupling constants supports a model where the protein forms four α‐helices between residues 4−26 (TM1), 32−53 (TM2), 58−76 (TM3) and 85−106 (TM4). For the residues of helices TM2 and TM3 a significant line broadening occurs due to slow conformational processes.
Biosynthesis of vitamin B12 in anaerobic bacteria
Authors: Schulze Bettina , Vogler Bernhard , Renz Paul
Published: March 2003

In anaerobic bacteria 5‐hydroxybenzimidazole and 5‐hydroxy‐6‐methylbenzimidazole are precursors of the 5,6‐dimethylbenzimidazole moiety of vitamin B12. In order to elucidate the pathway from these bases to vitamin B12, experiments on the transformation of 5‐hydroxy‐6‐methylbenzimidazole, of 5‐hydroxy‐6‐methylbenzimidazole‐α‐
D‐ribofuranoside, of 5‐hydroxybenzimidazolylcobamide and of 5‐hydroxy‐6‐methylbenzimidazolylcobamide into vitamin B12 were carried out.

The vitamin B12 synthesized by the anaerobe Eubacterium limosum in the presence of 5‐hydroxy‐6‐methylbenzimidazole and
L‐[methyl‐13C]methionine was subjected to NMR spectroscopy. It revealed that the methyl group at C5 of the 5,6‐dimethylbenzimidazole moiety was 13C labeled, whereas the methyl group at C6 was unlabeled. This shows that the transformation of 5‐hydroxy‐6‐methylbenzimidazole into the base moiety of vitamin B12 occurs regiospecifically.

5‐Hydroxy‐6‐methylbenzimidazole‐α‐ D‐ribofuranoside as well as 5‐hydroxybenzimidazolylcobamide and 5‐hydroxy‐6‐methylbenzimidazolylcobamide were also transformed into vitamin B12 by E. limosum. When 5‐hydroxy‐6‐methylbenzimidazolylcobamide 13C labeled at C2 of the base part and 14C labeled in the ribose was used for this experiment, the vitamin B12 obtained from this cobamide was 13C and 14C labeled in the same positions. This demonstrates that the α‐glycosidic bond of the precursor cobamide is not split during the formation of vitamin B12.

It can be deduced from these results that the precursor bases are transformed regiospecifically into their α‐nucleotides, and partially into their cobamides. The α‐nucleotides are then transformed into αribazole‐5′‐phosphate and, subsequently, into vitamin B12. Most likely the cobamides are degraded to the α‐nucleotides before being used for the biosynthesis of vitamin B12. A pathway for the latter process is suggested.
Growth‐hormone‐receptor and cytokine‐receptor‐family signaling
Authors: Moutoussamy Soraya , Kelly Paul A. , Finidori Joëlle
Published: March 2003

Structure of growth‐hormone receptor and the class I type of cytokine receptors : common structural features; cytokine‐receptor isoforms; oligomerization of receptor components initiates cytokine signalling.

Role of the Jak kinases in mediating specific functions of growth‐hormone receptor and cytokine receptors.

Role of signal transducer and activator of transcription (Stat) proteins in growth hormone and cytokine functions.

Other pathways activated by cytokine receptors : the mitogen‐activated protein kinase pathway; insulin‐receptor substrates 1 and 2 and phosphatidylinositol‐3‐kinase pathways; the Src pathways and other tyrosine kinase pathways; the phospholipase C/protein kinase C/Ca2+ pathways.

Regulation of growth‐hormone receptor and cytokine receptor signaling : binding sites, internalization and ubiquitination; phosphatases and Janus kinase/Stat inhibitors.

Conclusions and future prospects.
Proteasome inhibition leads to the activation of all members of the heat‐shock‐factor family
Authors: Kawazoe Yoshinori , Nakai Akira , Tanabe Masako , Nagata Kazuhiro
Published: March 2003

Heat‐shock proteins and molecular chaperones are involved in various cellular metabolic processes including protein synthesis and degradation. These expressions are elevated at the level of transcription by the accumulation of abnormal proteins when these metabolic processes are disturbed. Recent works suggest the induction of heat‐shock proteins by the inhibiton of proteasome. To elucidate the mechanism of this induction, we examined the activation of heat‐shock transcription factors by proteasome inhibitors in avian cells. Activation of the two heat‐shock‐inducible factors, HSF1 and HSF3, was produced by the treatment of cells with proteasome inhibitors. This activation was not produced by treatment with various other protease inhibitors. The HSF activation by proteasome inhibitors was completely blocked in the presence of the protein synthesis inhibitor cycloheximide. Unexpectedly, the development‐related factor HSF2 was also activated by proteasome inhibitors, with an increase in its protein level. These results suggest that the ubiqutin‐proteasome pathway may regulate all of the three HSFs by controlling the level of some regulatory factor for HSF or HSF itself, as well as controlling abnormal proteins.
Cathepsin C from Schistosoma japonicum
Authors: Hola‐Jamriska Lubomira , Tort Jose F. , Dalton John P. , Day Sharon R. , Fan Jinjiang , Aaskov John , Brindley Paul J.
Published: March 2003

A cDNA encoding preprocathepsin C was isolated from adults of the asian blood fluke Schistosoma japonicum. The deduced amino acid sequence of S. japonicum cathepsin C comprised 458 amino acid residues; 22 NH2‐terminal residues corresponding to the signal peptide, 199 residues corresponding to the propeptide and 237 COOH‐terminal residues corresponding to the mature enzyme region. The amino acid sequence of this preprocathepsin showed 43 % and 50 % identity to that of human and rat, respectively. The preproenzyme shared only 59 % identity with the sequence for a cathepsin C reported from Schistosoma mansoni, differing from it in active‐site residues and in its potential N‐glycosylation sites. Northern‐blot analysis showed that S. japonicum cathepsin C was expressed in greater quantities in female than in male parasites. Phylogenetic analysis utilizing the mature enzyme sequences of S. japonicum and other cathepsin Cs demonstrated that cathepsin Cs and cathepsin Bs shared a common ancestry. The unusually long prosegment observed in cathepsin C from S. japonicum and from other species was compared to that of cathepsin Bs and cathepsin Ls. The extension contained two blocks of residues which were highly conserved among cathepsin Cs. The COOH terminus of the prosegment exhibited a composite of features present in the prosegments of cathepsin Ls and cathepsin Bs. Most significantly, given the common ancestry of cathepsin B and cathepsin C, the prosegment of cathepsin C included ERFNIN‐like motifs and other residues more characteristic of non‐cathepsin‐B‐like members of the papain superfamily such as cathepsin L.
Translocation of the precursor of α‐amylase into Bacillus subtilis membrane vesicles
Authors: van Wely Karel H. M. , Swaving Jelto , Driessen Arnold J. M.
Published: March 2003

Bacilli vigorously secrete proteins into the extracellular environment, and are therefore used in industry for the bulk production of enzymes such as proteinases and amylases. Studies on the mechanism of protein translocation in these Gram‐positive bacteria have been hampered by the lack of an in vitro system. To establish such a system for Bacillus subtilis, everted membranes were isolated from a strain deficient in the alkaline and neutral protease. Translocation‐competent membrane vesicles were obtained only when a broad range proteinase‐inhibitor cocktail was used during membrane isolation. This method efficiently prevented proteolysis of SecY, one of the core integral membrane components of the preprotein translocase. Translocation of the urea‐denatured precursor of the Bacillus licheniformisα‐amylase, preAmyL, and B. subtilis alkaline phosphatase, prePhoB, into the B. subtilis membrane vesicles require the B. subtilis SecA protein and are driven by ATP hydrolysis and the proton‐motive force. These studies establish an authentic in vitro translocation system for protein secretion in B. subtilis.
An adrenocorticotropin‐regulated phosphoprotein intermediary in steroid synthesis is similar to an acyl‐CoA thioesterase enzyme
Authors: Finkielstein Carla , Maloberti Paula , Mendez Carlos F. , Paz Cristina , Cornejo Maciel Fabiana , Cymeryng Cora , Neuman Isabel , Dada Laura , Mele Pablo G. , Solano Angela , Podestá Ernesto J.
Published: March 2003

We have previously reported the purification of a phosphoprotein (p43) intermediary in steroid synthesis from adrenal zona fasciculata [Paz C., Dada, L. A., Cornejo Maciel, M. F., Mele, P. G., Cymeryng, C. B., Neuman, I., Mendez, C. F., Finkielstein, C. V., Solano, A. R., Park, M., Fischer, W. H., Towbin, H., Scartazzini, R. & Podestá, E. J. (1994) Eur. J. Biochem. 224, 709−716]. Here, we describe the cloning and sequencing of a cDNA encoding p43 as well as the hormonal regulation of the p43 transcript. The protein resulted homologous to a very recently described mitochondrial peroxisome‐proliferator‐induced very‐long‐chain acyl‐CoA thioesterase (MTE‐I). The deduced amino acid sequence of the protein shows consensus sites for phosphorylation by different protein kinases, and a lipase serine motif. Antibodies raised against a synthetic peptide that includes the lipase serine motif and against the N‐terminal region of p43 block the action of the protein. The transcript of p43 was detected in ovary of pseudopregnant rats, rat adrenal zona fasciculata and glomerulosa, mouse Leydig tumor cell line (MA‐10), rat brain and human placenta. Inhibition of adrenocorticotropin hormone (ACTH) release and steroid synthesis by dexamethasone produced a dose‐dependent decrease in the abundance of the adrenal transcript. The transcript was induced by in vivo stimulation of the adrenals with ACTH. The effect had a rapid onset (5 min), reached maximal stimulation (62 %) at 15 min, and returned to basal levels at 30 min. The effect of ACTH on the p43 transcript was inhibited by actinomycin D and enhanced by cycloheximide. Our results provide the first evidence linking acyl‐CoA thioesterases with very‐long‐chain specificities, and a protein intermediary in steroid synthesis, thereby supporting a regulatory role for acyl‐CoA thioesterases in steroidogenic tissues.
Interaction of ribosomal L1 proteins from mesophilic and thermophilic Archaea and Bacteria with specific L1‐binding sites on 23S rRNA and mRNA
Authors: Köhrer Caroline , Mayer Christine , Neumair Oliver , Gröbner Peter , Piendl Wolfgang
Published: March 2003

In Bacteria and Archaea (formerly Archaebacteria) ribosomal protein L1 has a dual function, as a primary rRNA‐binding protein and as a translational repressor which binds to its own mRNA. The L1‐binding site on the mRNA exhibits high similarity in both sequence and secondary structure to the binding site for L1 on the 23 S rRNA. A sensitive membrane‐filter‐binding assay has been used to examine the interactions between ribosomal L1 proteins from different archaeal and bacterial species, and 23S rRNA and mRNA fragments from Methanococcus vannielii containing the MvaL1‐binding site. Under standard conditions (0 +C, pH 7.5, 20 mM Mg2+, 500 mM KCl), the apparent dissociation constant Kd of the homologous MvaL1‐23S rRNA complex is 5 nM, the apparent dissociation constant Kd of the MvaL1‐mRNA complex is 0.15 μM. L1 proteins from Escherichia coli (EcoL1) and from the thermophilic Bacterium Thermus thermophilus (TthL1), and from the thermophilic Archaea Methanococcus thermolithotrophicus (MthL1), Methanococcus jannaschii (MjaL1), and Sulfolobus solfataricus (SsoL1) were tested for their affinity to the specific L1‐binding sites on the 23 S rRNA and mRNA. In general, the affinity of L1 proteins from thermophilic species to the binding sites on both 23 S rRNA and mRNA is about one order of magnitude higher than that of their mesophilic counterparts. This stronger protein‐RNA interaction might make a substantial contribution to the thermal tolerance of ribosomes in thermophilic organisms.
The ATPase and ATP‐binding functions of P‐glycoprotein
Authors: Romsicki Yolanda , Sharom Frances J.
Published: March 2003

P‐glycoprotein functions as an active efflux pump for lipophilic compounds and plays an important role in the resistance of human cancers to chemotherapeutic drugs. Drug transport is powered by ATP hydrolysis at two highly conserved nucleotide‐binding domains, which are proposed to be located at the cytosolic face of the protein. The ATPase activity of P‐glycoprotein depends on the presence of phospholipids, and various lipids affect both basal ATPase activity and its stimulation or inhibition by drug substrates. The modulating effects of the lipid‐phase state and effects on the function of the nucleotide‐binding domains of P‐glycoprotein have been studied in reconstituted vesicles of the synthetic phospholipids 1‐palmitoyl‐2‐myristoylphosphatidylcholine (PamMyrGroPCho) and dimyristoylphosphatidylcholine (Myr2GroPCho). The kinetic parameters for P‐glycoprotein ATPase activity were determined, and a fluorescence‐quenching technique was used to measure the Kd for ATP binding. The values of both the Km for ATP hydrolysis and Kd for ATP binding were significantly different above and below the gel/liquid‐crystalline phase transition temperature (tm) of PamMyrGroPCho and Myr2GroPCho, whereas they were similar at the same temperatures for P‐glycoprotein in detergent solution. A discontinuity at 21−24 +C was observed in the Arrhenius plots of P‐glycoprotein ATPase activity in a membrane environment, but not in detergent solution. In addition, the activation energies for ATP hydrolysis in the gel and liquid‐crystalline phases of the lipid bilayer were significantly different. P‐glycoprotein in PamMyrGroPCho bilayers displayed an unusually low activation energy just below the melting transition. These results indicate that both ATP binding and ATP hydrolysis by P‐glycoprotein are affected by the phase state of the host lipids in which it is reconstituted. Lipids may modulate the function of the nucleotide‐binding domains of P‐glycoprotein by interacting with the transmembrane regions of the protein, or the nucleotide‐binding domains themselves may interact with the surface of the bilayer.
Characterization of the interaction of myosin with ATP analogues having the syn conformation with respect to the adenine‐ribose bond
Authors: Maruta Shinsaku , Ohki Takashi , Kambara Taketoshi , Ikebe Mitsuo
Published: March 2003

Numerous analytical experiments have shown that, in solution, ATP analogues with bulky substitutions at the eighth position of the adenine ring predominantly assume the syn conformation with respect to the adenine−ribose bond. Two such analogues, 3′‐O‐(N‐methylanthraniloyl)‐8‐azido‐ATP (Mant‐8‐N3‐ATP) and 8‐Br‐ATP, were synthesized and used to probe the conformation of the ATP‐binding site of myosin. In the presence of these analogues, actomyosin was rapidly dissociated; Mg2+‐dependent ATP hydrolysis was significantly activated by actin; and Pi bursting was observed. For skeletal myosin, however, these analogues failed to support actin translocation, and they did not significantly enhance the intrinsic tryptophan fluorescence of skeletal muscle myosin subfragment‐1 (SKE S‐1). These results suggest that although myosin**/ADP/Pi intermediates can be formed with these analogues, the crucial conformational changes required for cross‐bridge cycling do not occur in skeletal muscle myosin. The conformations of the ATP‐binding sites of skeletal and smooth‐muscle myosin were compared using the ternary complexes, myosin‐ADP‐beryllium fluoride (BeFn) or myosin‐ADP‐aluminium fluoride (AlF.es−.rb.ei4.rb). In AlF.es−.rb.ei4.rb complexes, Mant‐8‐N3‐ADP affinity labeled the N‐terminal 29‐kDa domain of smooth‐muscle myosin subfragment‐1 (SM S‐1), as did ATP analogues having the anti conformation, whereas it labeled the C‐terminal 20‐kDa domain of skeletal S‐1. In smooth muscle BeFn complexes, Mant‐8‐N3‐ADP was equally likely to cross‐link to the 29‐kDa N‐terminal and the 25‐kDa C‐terminal domains. These analogues induced smooth muscle actomyosin super‐precipitation and increased intrinsic tryptophan fluorescence to the same degree as ATP itself. As was expected from above results, the analogues supported smooth‐muscle‐myosin‐induced actin translocation. These results suggest that smooth‐muscle myosin adopts the eight‐substituted ATP analogue in the normal conformation, but skeletal muscle myosin does not. This reflects the likely differences in the structures of their respective ATPase sites.
An isopeptide bond splitting enzyme from Hirudo medicinalis similar to γ‐glutamyl transpeptidase
Authors: Friedrich Thomas , Kröger Burkhard , Koerwer Wolfgang , Strube Karl‐Hermann , Meyer Thomas , Bialojan Siegfried
Published: March 2003

A new enzyme from Hirudo medicinalis capable of splitting γ‐glutamyl‐p‐nitroanilide and Glu  Lys(N6‐γ‐glutamyllysine) (isopeptidic bond between the ε‐amino group of lysine and the γ‐carboxylic group of glutamic acid) isopeptide bonds was purified. The protein was partially sequenced at the amino acid level, and the complete nucleotide and amino acid sequences were determined after cDNA cloning. The new enzyme has more than 60 % similarity at the amino acid level to vertebrate γ‐glutamyl transpeptidase (γ‐GT). According to the cDNA, the new protein has a molecular mass of 65 521 Da and a length of 600 amino acids.
Probing the inhibitor‐binding site of aldose reductase with site‐directed mutagenesis
Authors: Hohman Thomas C. , El‐Kabbani Ossama , Malamas Michael S. , Lai Kehdih , Putilina Tatiana , McGowan Michelle H. , Wane Yi‐Qun , Carper Deborah A.
Published: March 2003

Aldose reductase (AR) has been implicated in the etiology of the secondary complications of diabetes, and enzyme inhibitors have been proposed as therapeutic agents. While effectively preventing the development of diabetic complications in animals, results from clinical studies of AR inhibitors have been disappointing, possibly due to poor potency in man. To assist in the design of more potent and specific inhibitors, crystallographic studies have attempted to identify enzyme‐inhibitor interactions. Resolution of crystal complexes has suggested that the inhibitors bind to the enzyme active site and are held in place through hydrogen bonding and van der Waals interactions formed within two hydrophobic pockets. To confirm and extend these findings we quantified inhibitor activity with single, site‐directed, mutant, human AR enzymes in which the apolar active‐site residues tryptophan 20, −79, −111 and phenylalanine 115 were replaced with alanine or tyrosine, decreasing the potential for van der Waals interactions.

Consistent with molecular models, the inhibitory activity of Tolrestat, Sorbinil and Zopolrestat decreased 800−2000‐fold when tested with the mutant enzyme in which Trp20 was replaced with alanine. Further, alanine substitution for Trp111 decreased Zopolrestat's activity 400‐fold, while mutations to Trp79 and Phe115 had little effect on the activity of any of the inhibitors. The alanine mutation at Trp111 had no effect on Tolrestat's activity but decreased the activity of Sorbinil by about 1000‐fold. These latter effects were unanticipated based on the number of non‐bonded interactions between the inhibitors, Tolrestat and Sorbinil, and Trp20 and Trp111 that have been identified in the crystal structures. In spite of these unexpected findings, our results are consistent with the hypothesis that AR inhibitors occupy the enzyme active site and that hydrophobic interactions between the enzyme and inhibitor contribute to inhibitor binding stability.
Dipeptidyl‐peptidase IV‐β
Authors: Blanco Julià , Nguyen Coralie , Callebaut Christian , Jacotot Etienne , Krust Bernard , Mazaleyrat Jean‐Paul , Wakselman Michel , Hovanessian Ara G.
Published: March 2003

Dipeptidyl peptidase IV‐β (DPP IV‐β) is a novel protein which shows a peptidase activity similar to the T‐cell‐activation antigen CD26. To further characterize this DPP IV‐β and confirm its cell surface expression, we have developed a purification strategy using the CD26− cell line C8166. The purification process includes biotinylation of cell surface proteins before preparation of cell extracts and processing by gel‐filtration, ion‐exchange and lectin chromatographies. Consistent with the molecular mass of DPP IV‐β estimated by gel‐filtration chromatography, the final purified fraction, manifesting a typical DPP IV activity, showed a major biotinylated 75−80‐kDa band in SDS/PAGE, thus suggesting the monomeric nature of this enzyme. Kinetic parameters of DPP IV‐β and the sensitivity to a new family of irreversible DPP IV inhibitors, were studied in comparison to CD26. Both enzymes followed a Michaelis kinetics with different Km values for Gly‐Pro‐NH‐Np (NH‐Np, para‐nitroanilide) hydrolysis (0.28 ± 0.05 mM and 0.12 ± 0.02 mM). More significant differences were observed in the sensitivity to inhibitors, which exerted a much higher activity on CD26 than on DPP IV‐β. These differences permitted us to study DPP IV‐β expression in CD26‐expressing cells, showing the expression of this new enzyme in all lymphoid cells tested, and a rapid enhancement in phytohemagglutinin‐stimulated or protein‐A‐stimulated peripheral blood mononuclear cells. Our results indicate that, although DPP IV‐β and CD26 are coexpressed and manifest a typical DPP IV activity, there are distinct features in their catalytic activities that may confer to each enzyme a complementary role in peptide processing.
Conserved sequence elements in human main type‐H1 histone gene promoters : their role in H1 gene expression
Authors: Meergans Thomas , Albig Werner , Doenecke Detlef
Published: March 2003

In man, the H1 class of histones consists of seven different isoforms, termed H1.1−H1.5, H1t and H1+. Analysis of the promoters of the respective genes reveals that all seven H1 gene promoters share conserved sequence elements : a TATA box at around position −25 (relative to the transcription start site), a CCAAT motif at about position −50 (except in the H1 promoter), an H1‐box (AAACACA) around position −110 (except in the H1.1 promoter), and the highly conserved motif TGTGT/CTA (TG‐box or CH1UE) at around nucleotide position −450 (except in the H1.1 promoter). Analysis of the H1.3 gene promoter was carried out with reporter gene assays (using the luciferase gene as a reporter gene) including stepwise deletion and site‐directed mutagenesis. In addition, electrophoretic mobility‐shift assays were carried out for the analysis of protein/DNA interactions at conserved promoter motifs. Mutation analysis indicates that the CH1UE motif is involved in mediating the S‐phase‐dependent expression of the H1.3 gene. Comparison of H1 promoters shows that the CCAAT‐box is extended in each case by CA. Mutational analysis indicates that only the CCAATCA heptanucleotide, but not just the CCAAT sequence mediates the effect of this element in H1 gene promoters.
Fusion of two subunits does not impair the function of a [NiFeSe]‐hydrogenase in the archaeon Methanococcus voltae
Authors: Pfeiffer Matthias , Bingemann Ruth , Klein Albrecht
Published: March 2003

[NiFe]‐hydrogenases generally carry the bimetallic Ni‐Fe reaction center on their largest subunit. The [NiFeSe]‐hydrogenase Vhu from Methanococcus voltae has an unusual subunit composition. Some of the amino acids participating in the formation of the reaction center are within a separate, very small subunit, called VhuU. It consists of only 25 amino acids and contains the selenocysteinyl residue, a ligand to the Ni atom. We have tested whether the special configuration of the Vhu‐hydrogenase is of particular biochemical relevance. We have constructed a fusion subunit derived from the VhuA and VhuU subunits by generating a gene fusion which was inserted into the chromosome of M. voltae by gene replacement. The enzyme was purified and shown to be as active as the wild‐type enzyme. M. voltae carries the genetic information for four different [NiFe]‐hydrogenases. In addition to the Vhu‐hydrogenase, a second selenium‐containing enzyme, Fru, has been purified. Two selenium‐free enzymes, Vhc and Frc, are homologues of Vhu and Fru, respectively. Their gene groups, vhc and frc are transcribed only upon selenium depletion. The selenium‐containing subunit VhuU has been implicated in their negative regulation. However, cells containing the fusion hydrogenase still exhibited normal regulation of the vhc and frc promoter activities as tested in reporter gene constructs. This indicates that the free VhuU polypeptide is not required for the negative regulation of the vhc or frc genes.
β‐1,3‐galactosyltransferase and α‐1,2‐fucosyltransferase involved in the biosynthesis of type‐1‐chain carbohydrate antigens in human colon adenocarcinoma cell lines
Authors: Valli Maurizia , Gallanti Angelo , Bozzaro Salvatore , Trinchera Marco
Published: March 2003

We studied the β‐1,3‐galactosyltransferase (GalT) and α‐1,2‐fucosyltansferase (FT) involved in the biosynthesis of type‐1‐chain carbohydrate antigens in human colon adenocarcinoma cell lines. We detected a GalT activity able to use GlcNAc as acceptor and found that lacto‐N‐biose I (Galβ1‐3GlcNAc) is the only reaction product. Such β1,3GalT is kinetically similar to a pig trachea enzyme involved in mucin synthesis. The specific activity is high in cells that react strongly with anti‐Lewis a and anti‐Lewis b antibodies, and undetectable in a cell line that lacks antibody reaction. Reverse‐transcriptase‐mediated PCR analysis followed by DNA sequencing indicated that secretor‐type α1,2FT is expressed in the cells, while the H type α1,2FT is not. The apparent Km values for donor and acceptor substrates determined for α1,2FT are similar to those of secretor‐type α1,2FT and the specific activity measured correlates with Lewis b antigen expression on the cell surface. Moreover, some of the cell lines express Lewis y and H type 2 antigens, indicating that secretor type α1,2FT is responsible for their synthesis. Results suggest that biosynthesis of type‐1‐chain tumor‐associated antigens in human colon carcinoma cells is operated by secretor‐type α1,2FT, as reported in normal mucosa, and that β1,3GalT activity may play a relevant role in its control.
The influence of the central region containing residues 19−25 on the aggregation properties and secondary structure of Alzheimer's β‐amyloid peptide
Authors: El‐Agnaf Omar M. A. , Guthrie David J. S. , Walsh Dominic M. , Irvine G. Brent
Published: March 2003

Alzheimer's β‐amyloid peptide (Aβ) is a 39‐ to 43‐amino‐acid peptide that is the major component of neuritic plaques found in Alzheimer's disease (AD). The central region of Aβ plays a crucial role in many of its properties, including aggregation, neurotoxicity, proteolytic processing and interactions with other proteins, such as apolipoprotein E. Two mutations in this region, Ala21←Gly and Glu22←Gln, give rise to early onset forms of disease. We have studied several peptides based on the central region of Aβ in order to clarify the influence of specific amino acid residues on physicochemical behaviour. To avoid difficulties due to oxidation of Met35, the latter was replaced by the amino acid isostere, norleucine (Ahx), giving [Ahx35]Aβ‐(25−35)‐amide as a prototype structure. To this prototype, addition of pairs of amino acid residues from the sequence of Aβ, forming the corresponding 23‐, 21‐ and 19−35 derivatives, resulted in peptides that aggregated to form fibrils of diameter 6−10 nm. The rate of aggregation was more rapid as peptide length increased. Circular dichroism spectra of aged solutions of peptides revealed that aggregation was accompanied by a transition from random structure to β sheet for some, but not all, peptides. The mutation from Ala to Gly at position 21 increased the rate of aggregation and altered the tendency to adopt secondary structure in the direction away from α helix and towards β sheet. In individuals with the Ala21←Gly mutation, these results would suggest that truncated species with N‐termini in the region containing residues 17−20 would be more amyloidogenic than the wild type homologues.
Asn49 is the unique glycosylation site of the trout red blood cell Na+/H+ exchanger
Authors: Malapert Murielle , Pellissier Bernard , Borgese Franck
Published: March 2003

The Na+/H+ exchanger (NHE) is a plasma membrane transport protein found in a wide range of biological systems. NHE is involved in various functions including pH homeostasis, volume regulation, cell proliferation and transcellular Na+ absorption. This study reports immunodetection results obtained with antibodies generated against the C‐terminus of the NHE of trout red blood cells, βNHE. Immunoblotting of cell membrane preparation reveals that βNHE is a protein with an apparent molecular mass of 95 kDa. Moreover enzymatic glycosidase treatment demonstrates that the antiporter is an N‐glycosylated but not O‐glycosylated protein. The primary structure of βNHE contains three putative N‐glycosylation consensus sites (N‐X‐S/T) at Asn49, Asn338 and Asn378. Expression of βNHE in PS120 fibroblasts, a cell line which lacks an endogenous Na+/H+ exchange, allows to determine the precise sites of glycosylation. The construction of a site‐directed mutated βNHE antiporter, lacking the first predicted motif, shows that βNHE possesses an unique glycosylation site located on the first extracellular loop of the exchanger (Asn49). Expression of this deglycosylated antiporter shows that deglycosylation of the protein modifies neither the pHi dependency of the antiporter nor its hormonal stimulation.
NMR characterization of a single‐cysteine mutant of Escherichia coli thioredoxin and a covalent thioredoxin‐peptide complex
Authors: Jeng Mei‐Fen , Reymond Martine T. , Tennant Linda L. , Holmgren Arne , Dyson H. Jane
Published: March 2003

The mechanism of disulfide reduction by thioredoxin in the cell is thought to occur through the formation and subsequent destruction of a mixed‐disulfide intermediate between thioredoxin and the substrate. In order to model the interaction, we have prepared a mutant of Escherichia coli thioredoxin where the second cysteine residue of the active site has been replaced by an alanine residue. A specific covalent complex has been prepared between the remaining cysteine residue and a short cysteine‐containing peptide. This paper describes the preparation and characterization of the mutant protein both free and in the peptide complex.
Retinoid‐binding proteins in the cerebellum and choroid plexus and their relationship to regionalized retinoic acid synthesis and degradation
Authors: Yamamoto Miyuki , Dräger Ursula C. , Ong David E. , McCaffery Peter
Published: March 2003

The expression of cellular retinoic‐acid‐binding protein (CRABP) and cellular retinol‐binding protein (CRBP), as well as their relationship to retinoic acid (RA) synthesis and degradation were examined in the developing mouse cerebellum and choroid plexus of the fourth ventricle. The choroid plexus, which expresses the RA‐synthesizing retinaldehyde dehydrogenase RALDH‐2, is likely to represent a diffusion source of RA for the closely apposed cerebellum, regulating its development. We found CRBP to be expressed in the choroid plexus and, in an in‐vitro assay, addition of recombinant CRBP to RALDH‐2 increased RA synthesis from retinaldehyde, with the amount of increase depending on the CRBP/retinaldehyde ratio. A technique that characterizes RA‐binding proteins according to their isoelectric point showed both CRABP I and CRABP II to be present in the cerebellum and P19 cells, and only CRABP II to be present in the choroid plexus. With this technique, CRABP I could also be detected in the HL60 cell line. In addition to the two known acidic RA‐binding proteins CRABP I and II, the cerebellum expressed a third RA‐binding protein distinguishable by its neutral isoelectric point; the same binding protein was also detected in the olfactory bulb, kidney and testes. We used the RA‐binding technique to follow the rate of elimination of bound RA from the cerebellum. A systemic injection of 0.3 μmols RA into postnatal day‐1 mice was almost completely removed after 8 hours. These results suggest mechanisms by which the retinoid‐binding protein may regulate the equilibrium of RA synthesis and catabolism in the cerebellum and choroid plexus.
The solution structure of human endothelin‐2 a 1H‐NMR and CD study
Authors: Arvidsson Klas , Nemoto Tadashi , Mitsui Youji , Ohashi Shinichi , Nakanishi Hiroshi
Published: March 2003

The solution structure of the vasoactive endogenous 21‐amino‐acid human endothelin‐2 has been determined by NMR and CD techniques, in a mixed solvent of 100 mM aqueous acetic acid and 25 % (by vol.) 1,1,1,3,3,3‐hexafluoro‐2‐propanol. From NMR‐derived restraints on upper limit distances and dihedral angles, distance geometry structures were calculated using the program DADAS90, and refined by simulated annealing in X‐PLOR. The structure of endothelin‐2 consists of an α‐helix of residues 9 to 17, orientated anti‐parallel to a short β‐strand of residues 1 to 3, linked together by a possible β‐turn type I of residues 5−8. These secondary structural elements are stabilised and positioned by two disulphide bonds between residues 1 and 15, and 3 and 11, respectively. The average root mean square deviation over residues 1−17 of 15 accepted low‐energy conformers chosen to reflect the solution structure of endothelin‐2, was 0.73 Å for the backbone and 1.41 Å for all heavy atoms. The data on endothelin‐2 will be discussed and compared with what has been published on other endothelin/sarafotoxin peptides.
Quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa is a homodimer
Authors: Diehl Annette , v. Wintzingerode Friedrich , Görisch Helmut
Published: March 2003

The gene coding for the periplasmic quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa ATCC 17933 was cloned and sequenced. The deduced amino acid sequence contained a signal peptide of 34 residues and the major protein of 589 amino acids showed high similarities to pyrroloquinoline‐quinone‐dependent periplasmic and membrane‐bound dehydrogenases acting on alcohols, glucose and quinate or shikimate.

It was demonstrated by alignment with the amino acid sequence of the large subunit of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens, whose X‐ray structure is known, that the amino acid residues involved in the binding of pyrroloquinoline quinone and Ca2+ at the active site are conserved in the quinoprotein ethanol dehydrogenase of P. aeruginosa. Also, the glycine/tryptophan docking motifs involved in stabilizing the superbarrel structure of the quinoprotein methanol dehydrogenase of M. extorquens were conserved. The known sequences of pyrroloquinoline‐quinone‐dependent dehydrogenases were used to derive new, more specific sequence motifs for detecting members of this family of enzymes.

Despite the sequence similarity between the large α subunit of quinoprotein methanol dehydrogenase from M. extorquens and the quinoprotein ethanol dehydrogenase from P. aeruginosa, the two enzyme systems were quite different. In the presence of the prosthetic group, pyrroloquinoline quinone expression of the Pseudomonas gene encoding the 60‐kDa subunit of quinoprotein ethanol dehydrogenase in Escherichia coli resulted in formation of active enzyme. The formation of active quinoprotein methanol dehydrogenase, however, is known to require, in addition to the large α subunit, the expression of a small β subunit, and helper proteins [Lidstrom, M. E. (1995) Genetics of bacterial quinoproteins, Methods Enzymol. 258, 217−227].
Active site mutants of pyruvate decarboxylase from Zymomonas mobilis
Authors: Pohl Martina , Siegert P. , Mesch K. , Bruhn H. , Grötzinger J.
Published: March 2003

The homotetrameric pyruvate decarboxylase (PDC) from Zymomonas mobilis requires the cofactors thiamin diphosphate and Mg2+ for catalytic activity. We have investigated the role of various amino acid residues in the direct environment of the active site. The role of residue E473 in the catalytic activity and stability of the enzyme was probed by several mutations. All mutant enzymes were either inactive or failed to give any recombinant protein. The close interaction of E473 and N482, which can be deduced from the X‐ray structure, has been probed by mutagenesis of N482 to D. This mutation has a significant influence especially on the carboligation reaction of PDC, whereas the binding of the cofactors and the thermostability were not affected. These data suggest a specific interaction of N482 and E473 which is essential for coordinating the second aldehyde molecule during carboligation.

Three hydrophobic residues (L112, I472 and I476) in the vicinity of the active centre have been investigated with respect to their potential influence on the transition states during catalysis. In contrast to L112, I472 and I476 influence the decarboxylation and carboligation reactions. The enlarged substrate‐binding site of PDCI472A allows the decarboxylation of longer aliphatic 2‐keto acids (C4‐C6) as well as aromatic 2‐keto acids besides pyruvate. Carboligations using PDCI472A as a catalyst yielded 2‐hydroxypropiophenone, benzoin and phenylacetylcarbinol. The enantioselectivity of PAC formation is impaired by mutations of both I472 and I476. The stereochemistry is most significantly affected with the mutant enzyme PDCI476E, which catalyses predominantly the synthesis of (S)‐phenylacetylcarbinol.
Stimulation of Ca2+‐dependent exocytosis and arachidonic acid release in cultured mast cells (RBL‐2H3) by a GTPase‐deficient mutant of Gαi3
Authors: Zussman Ariella , Hermuet Sylvie , Sagi‐Eisenberg Ronit
Published: March 2003

Gi3, a member of the Gi family of heterotrimeric GTP‐binding proteins, regulates vesicle trafficking along both the constitutive and regulated pathways. In mast cells, specialized secretory cells which secrete a variety of inflammatory mediators by regulated exocytosis, activation of Gi3 provides a sufficient signal for exocytosis [Aridor, M., Rajmilevich, G., Beaven, M. A. & Sagi‐Eisenberg, R. (1993) Science 262, 1569−1572]. Such activation can be achieved in patch‐clamped or streptolysin‐O (SLO)‐permeabilized mast cells by a combination of Ca2+ and nonhydrolyzable analogs of GTP. In contrast, Ca2+‐activated exocytosis in intact cells is Gi3 independent. We show here that overexpression of a GTPase‐deficient mutant (Gαi3Q204L), but not of the wild‐type form of Gαi3, in rat basophilic leukemia cells (RBL‐2H3), a tumor analog of mucosal mast cells, resulted in marked potentiation of exocytosis and release of arachidonic acid in intact cells activated by a Ca2+ ionophore alone or in combination with the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate. In contrast, exocytosis and arachidonic acid release stimulated by aggregation of the cell surface receptors for immunoglobulin E (IgE) were unaffected. These results strongly suggest that the intracellular receptor, responsible for the activation of Gi3, is a low‐affinity Ca2+‐binding protein that can only be activated during Ca2+ ionophore stimulation. Moreover, these results also suggest that the propagation of the Ca2+‐activated and Gi3‐mediated signaling pathway requires the blocking of Gi3 GTPase activity. Finally, our results indicate that release of arachidonic acid is at least one of the downstream effectors of Gi3.
Structural organization and chromosomal localization of the human hepatocyte growth factor activator gene
Authors: Miyazawa Keiji , Wang Yimin , Minoshima Shinsei , Shimizu Nobuyoshi , Kitamura Naomi
Published: March 2003

The organization and structure of the gene coding for hepatocyte growth factor activator (HGFA) have been determined by isolation of unique clones from a human genomic library. These clones were characterized by restriction mapping, Southern blotting and DNA sequencing. The complete sequence of the gene was determined and found to span about 7.5 kilobases of DNA and consist of 14 exons separated by 13 introns. The coding region of HGFA consists of multiple putative domains that are homologous to those observed in blood coagulation factor XII (FXII). These regions were found as separate exons in the gene, and the exon/intron arrangement was similar to that of FXII, suggesting that the genes for HGFA and FXII have arisen through gene duplication events from a common ancestral gene. The major transcription initiation site is located 75 bp upstream of the translational start codon. The gene was mapped to chromosome 4p16, using spot‐blot hybridization on sorted chromosomes and fluorescence in situ hybridization on metaphase chromosome spreads. The phylogenetic and functional relationships between HGFA and FXII as well as urokinase and tissue‐type plasminogen activator are discussed.
Refolding of native and recombinant chicken riboflavin carrier (or binding) protein : evidence for the formation of non‐native intermediates during the generation of active protein
Authors: Pattanaik Priyaranjan , Adiga P. Radhakantha , Visweswariah Sandhya S.
Published: March 2003

Riboflavin carrier (or binding) protein (RCP) is a phosphoglycoprotein originally purified from the egg white, yolk and serum of laying hens. The 18 cysteine residues present in RCP form nine disulfide bridges, allowing the protein to form a compact structure to generate a hydrophobic pocket in which riboflavin sits. We studied the refolding of totally reduced and denatured egg white RCP and found that the protein initially folded to generate a molecule that did not possess riboflavin‐binding activity, despite near‐complete oxidation of the cysteine residues. Riboflavin‐binding activity was then slowly regained, but the final refolded form of the protein was less compact in structure than the native molecule, due to incomplete oxidation of all the cysteine residues. Denatured and reduced dephosphorylated RCP refolded as efficiently as the native protein, with similar rates of disulfide‐bond oxidation and generation of riboflavin binding, showing that the phosphoserine stretch of RCP has little role to play during refolding. In order to study the role of glycosylation in the refolding process, the cDNA for full‐length RCP was expressed in Escherichia coli and purified. Recombinant RCP refolded only in the presence of redox buffers, demonstrating that glycosylation of RCP could allow the formation of high yields of productive intermediates in the folding pathway. Using a panel of conformation‐specific monoclonal antibodies to RCP, it appeared that the folding intermediates of RCP possessed a structure distinctly different to the native protein, indicating that the correct folding pathway of RCP passed through conformation(s) generated by non‐native disulfide bridges.
Disulfide bond mapping and structural characterization of spruce budworm antifreeze protein
Authors: Gauthier Sherry Y. , Kay Cyril M. , Sykes Brian D. , Walker Virginia K. , Davies Peter L.
Published: March 2003

The 9‐kDa, Thr‐, Ser‐, and Cys‐rich thermal hysteresis protein from spruce budworm (sbwTHP) is 10−30 times more effective than fish antifreeze proteins (AFPs) at depressing solution freezing points via ice‐crystal growth inhibition. Since this insect protein is only available in microgram quantities from its natural source, recombinant sbwTHP was produced from inclusion bodies in Escherichia coli by a refolding protocol. Incompletely folded forms were removed during ion‐exchange and reverse‐phase chromatography, resulting in fully active sbwTHP that was indistinguishable in its properties from native sbwTHP. The antifreeze was completely inactivated by reduction, showed no reaction with sulfhydryl reagents, and was not inhibited by EDTA. All eight cysteine residues appear to be involved in disulfide bond formation. Tryptic cleavage and peptide analysis is consistent with linkages between the first and second cysteine residues, the third and fourth, fifth and eighth, and the sixth and seventh. NMR analysis confirmed that the fully folded form of sbwTHP was well structured and had a single conformation. Both NMR and CD spectra indicate the presence of extensive β structure (70−80 %) with little or no α helix. The protein maintains antifreeze activity over a broad range of pH values, and its conformation is independent of both temperature (over the range 0 +C to 20 +C), and the presence of 50 % trifluoroethanol.
Molecular cloning and expression of active Ole e 3, a major allergen from olive‐tree pollen and member of a novel family of Ca2+‐binding proteins (polcalcins) involved in allergy
Authors: Ledesma Amalia , Villalba Mayte , Batanero Eva , Rodríguez Rosalía
Published: March 2003

A cDNA encoding Ole e 3, a major allergen from olive‐tree pollen, has been cloned and sequenced. A strategy based on two‐step PCR amplification towards the 5′ end and 3′ end, with an internal specific primer, has been used. The isolated cDNA contains an open reading frame coding for a polypeptide of 84 amino acids, which is in agreement with the composition and molecular mass of the natural allergen, exhibiting two 12‐residue segments homologous to Ca2+‐binding sites of EF‐hand type. The cDNA was inserted into the pET‐11b expression vector and over‐expressed in Escherichia coli. The purified recombinant protein shows identical secondary structure to that of the natural allergen and is able to bind both IgE from sera of patients allergic to olive pollen and polyclonal antibodies raised against olive‐pollen Ole e 3. The capacity of binding Ca2+ has been demonstrated for both natural and recombinant allergens. RNA transcripts of Ole e 3 were only detected in pollen tissue. Northern‐blot and Western‐blot analyses of poly(A)+RNA and protein extracts, respectively, obtained from a variety of olive‐tree‐related and non‐related mature pollens demonstrated the presence of Ole e 3 homologous proteins. This indicates a sequence conservation and widespread distribution for this family of Ca2+‐binding proteins that can be responsible for allergenic cross‐reactivity. We suggest the tentative generic name of polcalcins for the members of this family of Ca2+‐binding proteins from pollen.