bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of type V phospholipase A2 expression and function by proinflammatory stimuli
Authors: Sawada Hisashi , Murakami Makoto , Enomoto Ayako , Shimbara Satoko , Kudo Ichiro
Published: March 2003

Types IIA and V secretory phospholipase A2 (sPLA2) are structurally related to each other and their genes are tightly linked to the same chromosome locus. An emerging body of evidence suggests that sPLA2‐IIA plays an augmentative role in long‐term prostaglandin (PG) generation in cells activated by proinflammatory stimuli; however, the mechanism underlying the functional regulation of sPLA2‐V remains largely unknown. Here we show that sPLA2‐V is more widely expressed than sPLA2‐IIA in the mouse, in which its expression is elevated by proinflammatory stimuli such as lipopolysaccharide. In contrast, proinflammatory stimuli induced sPLA2‐IIA in marked preference to sPLA2‐V in the rat. Cotransfection of sPLA2‐V with cyclooxygenase (COX)‐2, but not with COX‐1, into human embryonic kidney 293 cells dramatically increased the interleukin‐1‐dependent PGE2 generation occurring over a 24 h of culture period. Rat mastocytoma RBL‐2H3 cells overexpressing sPLA2‐V exhibited increased IgE‐dependent PGD2 generation and accelerated β‐hexosaminidase exocytosis. These results suggest that sPLA2‐V acts as a regulator of inflammation‐associated cellular responses. This possible compensation of sPLA2‐V for sPLA2‐IIA in many, if not all, tissues may also explain why some mouse strains with natural disruption of the sPLA2‐IIA gene exhibit few abnormalities during their life‐spans.
The carbohydrates of the isoforms of three avian riboflavin‐binding proteins
Authors: Amoresano Angela , Brancaccio Anna , Andolfo Annapaola , Perduca Massimiliano , Monaco Hugo L. , Marino Gennaro
Published: March 2003

The carbohydrate chains of nine isoforms of chicken egg‐white riboflavin‐binding protein (RfBP) and six isoforms each of quail egg‐white and yolk RfBP have been structurally characterized. The two N‐glycosylation sites, Asn36 and Asn147, of the most abundant isoform of each of the three proteins were analyzed in further detail leading to the identification of different glycosylation patterns. In both chicken and quail egg‐white RfBP the carbohydrates attached to position 36 had a lower degree of branching and, in the case of the quail protein, this site was only partially glycosylated. A very heterogeneous mixture of complex structures was characteristic of the other glycosylation site. Analysis of the two sites in quail yolk RfBP confirmed this result which agrees with what has been established for hen yolk RfBP. The presence in the three proteins of a highly heterogeneous mixture of differently branched glycans suggests that the differences in isoelectric points, which is a peculiarity of the different isoforms, are probably indeed due to differences in carbohydrate structure.
Cu/Zn superoxide dismutase in excretory–secretory products of the human hookworm Necator americanus
Authors: Taiwo Fatai A. , Brophy Peter M. , Pritchard David I. , Brown Alan , Wardlaw Andrew , Patterson Laurence H.
Published: March 2003

EPR spectrometry was used to investigate the effect of excretory/secretory product from Necator americanus on superoxide radical anions generated by xanthine/xanthine oxidase as a measure of excretory/secretory product superoxide dismutase activity. Using 1,1′,5,5′‐dimethylpyrollidine‐N‐oxide (DMPO) as a superoxide spin‐trapping agent a 12‐line EPR spectrum characteristic of the DMPO‐OOH adduct was observed to decrease in the presence of excretory/secretory product. Superoxide dismutase activity was proportional to excretory/secretory protein concentration, was inhibited with cyanide treatment and was progressively destroyed with increasing time of heat denaturation of excretory/secretory product. Using a purpose‐built chamber the superoxide dismutase activity of excretory/secretory product from live worms in culture was shown to accumulate with time to a maximum at 4 h. The electron paramagnetic resonance spectrum obtained for the frozen excretory/secretory product of N. americanus recorded at 77 K is typical of Cu(II) in a protein matrix. The results are consistent with the presence of an active Cu/Zn superoxide dismutase in excretory/secretory product from N. americanus and demonstrate a method for the unequivocal determination of the fate of superoxide anions in the presence of live worms.
Heterogeneous motions within human apohemoglobin
Authors: Haouz Ahmed , Mohsni Sadok El , Zentz Christian , Merola Fabienne , Alpert Bernard
Published: March 2003

Conservation of the secondary and tertiary protein organization of human apohemoglobin was observed at temperatures ranging from 7 to 25 °C using CD spectra in the far‐UV (200–250 nm) and near‐UV (250–300 nm) regions. The dynamics of apohemoglobin were probed using fluorescence quenching experiments on the Trp residues and an extrinsic dye (ANS or bis‐ANS) located in the heme cavities. The long decay time of the dye emission (> 10 ns) reveals the dynamics of the protein matrix averaged over the whole molecule. The short decay time of the Trp residue emission (≅3 ns) probes the dynamics of their close vicinities. When the temperature rises from 10 to 20 °C, the average intraproteic motions throughout the whole apohemoglobin matrix are greatly accelerated, whereas the hydrophobic protein regions around the α14, β15 and β37 Trp residues appear much less animated. These dynamic differences between the behavior of the softer matrix and the packed rigid regions containing the tryptophans could be one of the requisites for apohemoglobin stability. We suspect that the highly rigid tryptophan domains in human apohemoglobin are likely to be knots.
Properties of the methylcobalamin:H4folate methyltransferase involved in chloromethane utilization by Methylobacterium sp. strain CM4
Authors: Studer Alex , Vuilleumier Stéphane , Leisinger Thomas
Published: March 2003

Methylobacterium sp. strain CM4 is a strictly aerobic methylotrophic proteobacterium growing with chloromethane as the sole carbon and energy source. Genetic evidence and measurements of enzyme activity in cell‐free extracts have suggested a multistep pathway for the conversion of chloromethane to formate. The postulated pathway is initiated by a corrinoid‐dependent methyltransferase system involving methyltransferase I (CmuA) and methyltransferase II (CmuB), which transfer the methyl group of chloromethane onto tetrahydrofolate (H4folate) [Vannelli et al. (1999) Proc. Natl Acad. Sci. USA96, 4615–4620]. We report the overexpression in Escherichia coli and the purification to apparent homogeneity of methyltransferase II. This homodimeric enzyme, with a subunit molecular mass of 33 kDa, catalyzed the conversion of methylcobalamin and H4folate to cob(I)alamin and methyl‐H4folate with a specific activity of 22 nmol·min−1·(mg protein)−1. The apparent kinetic constants for H4folate were: Km = 240 µM, Vmax = 28.5 nmol·min−1·(mg protein)−1. The reaction appeared to be first order with respect to methylcobalamin at concentrations up to 2 mm, presumably reflecting the fact that methylcobalamin is an artificial substitute for the methylated methyltransferase I, the natural substrate of the enzyme. Tetrahydromethanopterin, a coenzyme also present in Methylobacterium, did not serve as a methyl group acceptor for methyltransferase II. Purified methyltransferase II restored chloromethane dehalogenation by a cell free extract of a strain CM4 mutant defective in methyltransferase II.
Biosynthesis and inactivation of the endocannabinoid 2‐arachidonoylglycerol in circulating and tumoral macrophages
Authors: Di Marzo Vincenzo , Bisogno Tiziana , De Petrocellis Luciano , Melck Dominique , Orlando Pierangelo , Wagner Jens A. , Kunos George
Published: March 2003

The stimulus‐induced biosynthesis of the endocannabinoid 2‐arachidonoylglycerol (2‐AG) in intact mouse J774 macrophages and the inactivation of 2‐AG by the same cells or by rat circulating macrophages was studied. By using gas chromatography‐mass spectrometry, we found that ionomycin (5 µm) and lipopolysaccharide (LPS, 200 µg·mL−1) cause a 24‐fold and 2.5‐fold stimulation of 2‐AG levels in J774 cells, respectively, thus providing unprecedented evidence that this cannabimimetic metabolite can be synthesized by macrophages. In J774 cells, LPS also induced a 7.8‐fold increase of the levels of the other endocannabinoid, anandamide, and, in rat circulating macrophages, an almost twofold increase of 2‐AG levels. Extracellular [3H]2‐AG was cleared from the medium of intact J774 macrophages (t1/2 = 19–28 min) and esterified to phospholipids, diacylglycerols and triglycerides or hydrolyzed to [3H]arachidonic acid and glycerol. These catabolic processes were attenuated differentially by various enzyme inhibitors. Rat circulating macrophages were shown to contain enzymatic activities for the hydrolysis of 2‐AG, including: (a) fatty acid amide hydrolase (FAAH), the enzyme responsible for anandamide breakdown and previously shown to catalyse also 2‐AG hydrolysis, and (b) a 2‐AG hydrolase activity different from FAAH and down‐regulated by LPS. High levels of FAAH mRNA were found in circulating macrophages but not platelets, which, however, contain a 2‐AG hydrolase. Both platelets and macrophages were shown to express the mRNA for the CB1 cannabinoid receptor. A macrophage 2‐AG hydrolase with apparentKm = 110 µmandVmax = 7.9 nmol·min−1·(mg protein)−1was partially characterized in J774 cells and found to exhibit an optimal pH of 6–7 and little or no sensitivity to typical FAAH inhibitors. These findings demonstrate for the first time that macrophages participate in the homeostasis of the hypotensive and immunomodulatory endocannabinoid 2‐AG through metabolic mechanisms that are subject to regulation.
New insights into enzyme catalysis
Authors: Scrutton Nigel S. , Basran Jaswir , Sutcliffe Michael J.
Published: March 2003

The wave‐particle duality of matter suggests that quantum tunnelling may have a prominent role in enzymatic H‐transfer. However, unlike for electron tunnelling, evidence for H‐tunnelling in enzyme molecules is extremely limited. The theoretical development, and verification by experiment, of a role for protein dynamics in driving enzymatic H‐tunnelling is presented. Dynamic theories of H‐tunnelling suggest that the kinetic isotope effect, during rupture of a C–H/C–D bond, for example, can assume values interpreted previously as indicating classical transfer. Vibrationally enhanced ground state tunnelling has been demonstrated for enzymes that cleave stable C–H bonds. This is an attractive mechanism as large activation energies make it energetically unfavourable for a classical, over‐the‐barrier mode of cleavage. Furthermore, it may be a general strategy used by enzymes for catalysing these ‘difficult’ transformations.
Structure–function studies of ω‐atracotoxin, a potent antagonist of insect voltage‐gated calcium channels
Authors: Wang Xiu‐hong , Smith Ross , Fletcher Jamie I. , Wilson Harry , Wood Chris J. , Howden Merlin E. H. , King Glenn F.
Published: March 2003

The ω‐atracotoxins are a family of 36 to 37‐residue peptide neurotoxins that block insect but not mammalian voltage‐gated calcium channels. The high phylogenetic specificity of these toxins recommends them as lead compounds for targeting insects that have developed resistance to chemical pesticides. We have begun to examine structure–function relationships in the ω‐atracotoxins in order to explore the molecular basis of their activity and phylogenetic specificity. By probing the venom of the Blue Mountains funnel‐web spider, Hadronyche versuta, for insecticidal toxins with masses close to that of ω‐atracotoxin‐Hv1a (ω‐ACTX‐Hv1a), we have isolated and sequenced five additional ω‐atracotoxins. Five of the six ω‐atracotoxins isolated from the venom of H. versuta (ω‐ACTX‐Hv1a to ‐Hv1e) differ from one another by only 1–3 residues and have similar insecticidal potencies. In contrast, ω‐ACTX‐Hv1f differs from the other toxins by up to 10 residues and it has markedly reduced insecticidal potency, thus providing information on key functional residues. The new atracotoxin sequences have revealed that the three N‐terminal residues are highly conserved. Despite the fact that these residues are structurally disordered in solution we show here, by a series of N‐terminal truncations, that they contribute significantly to insecticidal potency. However, loss of activity does not correlate with deletion of highly conserved residues, which leads us to propose that the disposition of the N‐terminal charge, rather than the chemical properties of the N‐terminal residues themselves, may be critical for the activity of ω‐atracotoxin on insect calcium channels.
Effect of substrate residues on the P2′ preference of retroviral proteinases
Authors: Boross Péter , Bagossi Péter , Copeland Terry D. , Oroszlan Stephen , Louis John M. , Tözsér József
Published: March 2003

The substrate sequence requirements for preference toward P2′ Glu residue by human immunodeficiency virus type 1 (HIV‐1) proteinase were studied in both the matrix protein/ capsid protein (MA/CA) and CA/p2 cleavage site sequence contexts. These sequences represent typical type 1 (–aromatic*Pro–) and type 2 (–hydrophobic* hydrophobic–) cleavage site sequences, respectively. While in the type 1 sequence context, the preference for P2′ Glu over Ile or Gln was found to be strongly dependent on the ionic strength and the residues being outside the P2–P2′ region of the substrate, it remained preferable in the type 2 substrates when typical type 1 substrate sequence residues were substituted into the outside regions. The pH profile of the specificity constants suggested a lower pH optimum for substrates having P2′ Glu in contrast to those having uncharged residues, in both sequence contexts. The very low frequency of P2′ Glu in naturally occurring retroviral cleavage sites of various retroviruses including equine infectious anemia virus (EIAV) and murine leukemia virus (MuLV) suggests that such a residue may not have a general regulatory role in the retroviral life cycle. In fact, unlike HIV‐1 and HIV‐2, EIAV and MuLV proteinases do not favor P2′ Glu in either the MA/CA or CA/p2 sequence contexts.
Location of the binding site for chloride ion activation of cathepsin C
Authors: Cigić Blaž , Pain Roger H.
Published: March 2003

Cathepsin C, a tetrameric lysosomal dipeptidyl‐peptide hydrolase, is activated by chloride ion. The activation is shown here to be specific and pH‐dependent, dissociation constants for chloride being lower at low pH. Bound chloride decreases the Km for the hydrolysis of chromophore labelled substrates without any significant change in Vmax, confirming its involvement in substrate binding. Determination of the kinetic parameters of chloride activation, using unlabelled substrates, has enabled its site of action to be located. The lower Km for the hydrolysis of simple amide substrates in the presence of Cl− shows that the S sites are involved. Possible involvement of the S′ sites is excluded by the finding that the Km for the nucleophile in the transferase reaction is unaffected by chloride. The rates of inhibition by E‐64 and iodoacetate are both chloride‐dependent and, from the structure of the papain–E‐64 complex, it is concluded that chloride binds close to the S2 site. The binding of guanidinium ion, a positively charged inhibitor, to the S site is dependent on chloride. Based on these results, a model is proposed to explain the chloride activation of cathepsin C. The possible physiological role of chloride in the regulation of proteolysis in the lysosome is discussed.
Isolation, amino acid sequence and functional assays of SGTx1
Authors: Marvin Laure , De Emmanuelle , Cosette Pascal , Gagnon Jean , Molle Gérard , Lange Catherine
Published: March 2003

A new toxin (SGTx1) was purified from the venom of the spider Scodra griseipes by a combination of gel filtration and reverse‐phase chromatography. The complete amino acid sequence of SGTx1, TCRYLFGGCKTTADCCKHLACRSDGKYCAWDGTF, was established by direct automated Edman degradation, and is in perfect agreement with the molecular mass of 3775 Da found by mass spectrometry. The primary structure of SGTx1 exhibited sequence identity with other spider toxins such as hanatoxin (76%), TxP5 toxin (32%) and huwentoxin (26%). The six cysteines in the sequence suggested three disulfide bridges, the presence of which was demonstrated by mass spectrometry after dithiothreitol reduction. Analysis of secondary structure using circular dichroism spectrometry yielded more than 50% β‐sheet and about 15–20% β‐turn. The extent of the β‐content and the presence of disulfide bridges suggest a structure of interconnected β‐strands. In addition, a study of membrane/toxin interactions was carried out by reconstitution in planar lipid bilayers and by antibacterial assays. SGTx1 displays moderate pore‐forming ability (conductance of about 100 pS in 1 m NaCl), but antibacterial activity was not observed against Gram‐positive or Gram‐negative strains. As a preliminary assay, the activity of SGTx1 was investigating using electrophysiological measurements. At 0.15 µm, SGTx1 reversibly inhibits more than 40% of outward potassium currents in rat cerebellum granular cells. This result is reminiscent with the effect described for hanatoxin extracted from the venom of Grammostola spatulata.
Molecular cloning and expression of Galβ1,3GalNAc α2,3‐sialyltransferase from human fetal liver
Authors: Shang Jie , Qiu Ruolun , Wang Junqi , Liu Junjian , Zhou Rouli , Ding Huiping , Yang Shoujun , Zhang Shuzheng , Jin Cheng
Published: March 2003

Based on the sequences of the highly conserved segments in the previously cloned sialyltransferases, a cDNA encoding Galβ1,3GalNAc α2,3‐sialyltransferase (SIATFL) has been isolated from human fetal liver. Expression analysis of the gene has been performed with various carcinoma cell lines, fetal tissues, fetal and adult liver and both hepatoma and the surrounding tissue from the same liver. The SIATFL gene was expressed poorly in fetal liver and in adult liver, slightly in hepatoma and highly in the surrounding tissue of hepatoma. The cDNA encoding the putative active domain was expressed in COS‐1, Escherichia coli, and Pichia pastoris. The recombinant protein expressed in COS‐1 could catalyse the transfer of NeuAc from CMP‐NeuAc to asialo‐fetuin. No enzyme activity was detected with a 32‐kDa protein in E. coli and both 32‐kDa and 41‐kDa proteins in P. pastoris. These results suggested that correct glycosylation of the enzyme might play a key role in its folding that may be directly related to the enzymatic activity.
2‐Hydroxyglutaryl‐CoA dehydratase from Clostridium symbiosum
Authors: Hans Marcus , Sievers Jörg , Müller Uta , Bill Eckhard , Vorholt Julia A. , Linder Dietmar , Buckel Wolfgang
Published: March 2003

Component D (HgdAB) of 2‐hydroxyglutaryl‐CoA dehydratase from Clostridium symbiosum was purified to homogeneity. It is able to use component A from Acidaminococcus fermentans (HgdC) to initiate catalysis together with ATP, Mg2+ and a strong reducing agent such as Ti(III)citrate. Component D from C. symbiosum has a 6× higher specific activity compared with that from A. fermentans and contains a second [4Fe–4S] cluster but the same amount of riboflavin 5′‐phosphate (1.0 per heterodimeric enzyme, m = 100 kDa). Mössbauer spectroscopy revealed symmetric cube‐type structures of the two [4Fe–4S]2+ clusters. EPR spectroscopy showed the resistance of the clusters to reducing agents, but detected a sharp signal at g = 2.004 probably due to a stabilized flavin semiquinone. Three genes from C. symbiosum coding for components D (hgdA and hgdB) and A (hgdC) were cloned and sequenced. Primer extension experiments indicated that the genes are transcribed in the order hgdCAB from an operon only half the size of that from A. fermentans. Sequence comparisons detected a close relationship to the dehydratase system from A. fermentans and HgdA from Fusobacterium nucleatum, as well as to putative proteins of unknown function from Archaeoglobus fulgidus. Lower, but significant, identities were found with putative enzymes from several methanogenic Archaea and Escherichia coli, as well as with the mechanistically related benzoyl‐CoA reductases from the Proteobacteria Rhodopseudomonas palustris and Thauera aromatica.
Effect of serine and tyrosine phosphorylation on retroviral proteinase substrates
Authors: Tözsér József , Bagossi Péter , Boross Péter , Louis John M. , Majerova Eva , Oroszlan Stephen , Copeland Terry D.
Published: March 2003

Vimentin, a cellular substrate of HIV type 1 (HIV‐1) proteinase, contains a protein kinase C (PKC) phosphorylation site at one of its cleavage sites. Peptides representing this site were synthesized in P2 Ser‐phosphorylated and nonphosphorylated forms. While the nonphosphorylated peptide was a fairly good substrate of the enzyme, phosphorylation prevented hydrolysis. Phosphorylation of human recombinant vimentin by PKC prevented its processing within the head domain, where the phosphorylation occurred. Oligopeptides representing naturally occurring cleavage sites at the C‐terminus of the Rous sarcoma virus integrase were assayed as substrates of the avian proteinase. Unlike the nonphosphorylated peptides, a Ser‐phosphorylated peptide was not hydrolyzed by the enzyme at the Ser–Pro bond, suggesting the role of previously established phosphorylation in processing at this site. Ser‐phosphorylated and Tyr‐phosphorylated forms of model substrates were also tested as substrates of the HIV‐1 and the avian retroviral proteinases. In contrast to the moderate effect of P4 Ser phosphorylation, phosphorylation of P1 Tyr prevented substrate hydrolysis by HIV‐1 proteinase. Substrate phosphorylation had substantially smaller effects on the hydrolysis by the avian retroviral proteinase. As the active retroviral proteinase as well as various protein kinases are incorporated into mature virions, substrate phosphorylation resulting in attenuation or prevention of proteolytic processing may have important consequences in the regulation of the retroviral life cycle as well as in virus–host cell interactions.
The effects of pH and semiquinone formation on the oxidation–reduction potentials of flavin mononucleotide
Authors: Mayhew Stephen G.
Published: March 2003

Calculation shows that there is poor agreement between frequently cited values for the midpoint redox potentials of the two one‐electron steps in the reduction of flavin mononucleotide and equations for the lines that relate these potentials to pH and that use the published pKa values for the three redox states of the flavin [Draper, R. & Ingraham, L.L. (1969) Arch. Biochem. Biophys. 125, 802–808]. Equilibrium data for the first step in the reduction obtained by pulse radiolysis [Anderson, R_f. (1983) Biochim. Biophys. Acta722, 158–162] show much closer agreement with theory and lead to values for the semiquinone formation constant of flavin mononucleotide that are close to those derived from measurements of the radical concentration using ESR spectroscopy. It is concluded that the data from the second method are more reliable. The redox potentials for flavin mononucleotide at pH 7.0 and 20 °C are calculated to be −0.207 V for the overall two‐electron reduction (Em), −0.313 V for reduction of the oxidized flavin to the semiquinone (E2) and −0.101 V for the reduction of the semiquinone to the hydroquinone (E1). Information is provided to allow calculation of the three redox potentials at other pH values in the physiological range.
The two subunits of the interleukin‐4 receptor mediate independent and distinct patterns of ligand endocytosis
Authors: Friedrich Karlheinz , Kammer Winfried , Erhardt Ingrid , Brändlein Stephanie , Arnold Stefan , Sebald Walter
Published: March 2003

Interleukin‐4 (IL‐4) triggers cellular responses by interaction with the bipartite interleukin‐4 receptor (IL‐4R). IL‐4‐responsive cells specifically endocytose IL‐4. We studied the ligand internalization properties of the human IL‐4R and analyzed the specific functions of its two subunits IL‐4Rα and γc in this process. IL‐4 mutant RY, which binds to IL‐4Rα but does not recruit γc into the receptor complex was used as a tool to show that IL‐4Rα can promote independent ligand uptake in human T cells. Internalization was limited, however, by rapid IL‐4 dissociation, suggesting that one important function of γc in IL‐4 endocytosis is to retain the ligand sufficiently long within the ternary receptor complex. We then measured IL‐4 internalization by murine Ba/F3 cells that were stably transfected with various human IL‐4R constructs. Efficient IL‐4 uptake required the cytoplasmic section of the receptor. The intracellular domains of IL‐4Rα and γc were responsible for independent endocytosis processes with distinct kinetics. IL‐4Rα‐mediated internalization resulted in long‐term intracellular maintainance of IL‐4, whereas γc directed the associated radioligand to intracellular breakdown and rapid release in the form of degraded protein. Mutants of either IL‐4R subunit deficient in Janus kinase activation were not impaired in internalization, indicating that IL‐4 endocytosis is not functionally connected to signal transduction.
Folding of coliphage T4 short tail fiber in vitro
Authors: Burda Martin R. , Miller Stefan
Published: March 2003

The morphogenesis of the Escherichia coli bacteriophage T4 depends on the presence of helper proteins which are not components of the mature virion. Two bacteriophage‐encoded proteins, p57 and p38, are required for the assembly of the bacteriophage T4 tail fibers. In the absence of p57, two polypeptides of the long fiber (p34 and p37) and that of the short tail fiber (p12) fail to trimerize. Instead they form water‐insoluble aggregates. Co‐expression of the genes 12 and 57 in vivo caused the formation of only trimeric, water‐soluble p12. The function of g57 cannot be replaced by overexpression of the host proteins GroEL/ES or parvulin. The mechanism of action of this helper protein has remained unknown, mainly because it has not been possible to determine its activity in vitro. Purified p12, denatured in 7 m urea, trimerized spontaneously in a slow reaction (half‐time ≈ 6 h) and with low yield. Upon renaturation, p12 forms native SDS‐resistant trimers as indicated by spectroscopic and hydrodynamic measurements. Addition of p57 increased the rate of folding threefold and nearly doubled the yield. These experiments demonstrate that p57 acts as a molecular chaperone during folding of T4 tail fibers.
N‐terminal and C‐terminal plasma membrane anchoring modulate differently agonist‐induced activation of cytosolic phospholipase A2
Authors: Klapisz Elsa , Ziari Mouloud , Wendum Dominique , Koumanov Kamen , Brachet‐Ducos Corinne , Olivier Jean‐Luc , Béréziat Gilbert , Trugnan Germain , Masliah Joëlle
Published: March 2003

The 85 kDa cytosolic phospholipase A2 (cPLA2) plays a key role in liberating arachidonic acid from the sn‐2 position of membrane phospholipids. When activated by extracellular stimuli, cPLA2 undergoes calcium‐dependent translocation from cytosol to membrane sites which are still a matter of debate. In order to evaluate the effect of plasma membrane association on cPLA2 activation, we constructed chimeras of cPLA2 constitutively targeted to the plasma membrane by the N‐terminal targeting sequence of the protein tyrosine kinase Lck (Lck‐cPLA2) or the C‐terminal targeting signal of K‐Ras4B (cPLA2‐Ras). Constitutive expression of these chimeras in Chinese hamster ovary cells overproducing the α2B adrenergic receptor (CHO‐2B cells) did not affect the basal release of [3H]arachidonic acid, indicating that constitutive association of cPLA2 with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck‐cPLA2 increased [3H]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA2‐Ras inhibited it, compared with parental CHO‐2B cells and CHO‐2B cells producing comparable amounts of recombinant wild‐type cPLA2. The lack of stimulation of cPLA2‐Ras was not due to a decreased enzymatic activity as measured using an exogenous substrate, or to a decreased phosphorylation of the protein. These results show that the plasma membrane is a suitable site for cPLA2 activation when orientated correctly.
Pore‐forming peptides of Entamoeba dispar
Authors: Nickel Rosa , Ott Claudia , Dandekar Thomas , Leippe Matthias
Published: March 2003

Amoebapore, a 77‐residue peptide with pore‐forming activity from the human pathogen Entamoeba histolytica, is implicated in the killing of phagocytosed bacteria and in the cytolytic reaction of the amoeba against host cells. Previously, we structurally and functionally characterized three amoebapore isoforms in E. histolytica but recognized only one homolog in the closely related but non‐pathogenic species Entamoeba dispar. Here, we identified two novel amoebapore homologs from E. dispar by molecular cloning. Despite strong resemblance of the primary structures of the homologs, molecular modeling predicts a species‐specific variance between the peptide structures. Parallel isolation from trophozoite extracts of the two species revealed a lower amount of pore‐forming peptides in E. dispar and substantially higher activity of the major isoform from E. histolytica towards natural membranes than that from E. dispar. Differences in abundance and activity of the lytic polypeptides may have an impact on the pathogenicity of amoebae.
Analysis of a DNase I hypersensitive site located −20.9 kb upstream of the CFTR gene
Authors: Nuthall Hugh N. , Vassaux Georges , Huxley Clare , Harris Ann
Published: March 2003

The cystic fibrosis transmembrane conductance regulator gene (CFTR) shows a tightly regulated pattern of expression with spatial and temporal control. The regulatory elements achieving this appear to lie outside the basal promoter of the gene. We previously identified DNase I hypersensitive sites (DHSs) at −79.5 kb and −20.5 kb with respect to the CFTR translational start site which may contain important regulatory elements. We have now investigated further the DHS at −20.5 kb to evaluate its potential function in the regulation of CFTR expression. Finer mapping revealed that the DHS lies at −20.9 kb. Deletion of the DHS from a 310‐kb yeast artificial chromosome (YAC) containing the human CFTR gene has shown that this site may be responsible for about 60% of wild‐type levels of transcription from the YAC transgene when expressed in Caco2 cells. DNase I footprinting showed several regions of protection within the −20.9 kb region with nuclear extracts from Caco2 cells, but not with extracts from lymphoblastoid cells, which do not show the DHS. Matches to several transcription factor‐binding sites were found, but supershift analysis with specific antibodies did not identify the transcription factors involved. Two purine/pyrimidine mirror repeat elements within the −20.9‐kb DHS were shown not to adopt non‐B‐DNA conformations. Thus, we provide evidence for a role for the −20.9 kb DHS in the transcriptional regulation of the CFTR gene, although the mechanisms mediating this effect remain unclear.
In vitro translation in a cell‐free system from Trypanosoma brucei yields glycosylated and glycosylphosphatidylinositol‐anchored proteins
Authors: Duszenko Michael , Kang Xuedong , Böhme Ulrike , Hömke Rolf , Lehner Manfred
Published: March 2003

African trypanosomes escape many cellular and unspecific immune reactions by the expression of a protective barrier formed from a repertoire of several hundred genes encoding immunologically distinct variant surface glycoproteins (VSGs). All mature VSGs are glycosylphosphatidylionositol‐anchored and N‐glycosylated.

To study trypanosome‐specific post‐translational modifications of VSG, a cell‐free system capable of in vitro translation, translocation into the rough endoplasmic reticulum, N‐glycosylation and glycosylphosphatidylinositol‐anchor addition was established using lysates of the bloodstream form of Trypanosoma brucei. Monitoring protein synthesis by [35S]methionine incorporation, labeled protein bands were readily detected by fluorography following SDS/PAGE. Appearance of these bands increased during a time‐course of 45 min and was sensitive to cycloheximide but not chloramphenicol treatment. Efficiency of this system, in terms of incorporation of radiolabeled amino acids into newly formed proteins, is similar to reticulocyte lysates. The system does not, however, allow initiation of protein synthesis.

Depending on the clone used, immunoprecipitation revealed one or two newly formed VSG bands. Upon digestion with N‐glycosidase F these bands resulted in a single band of a lower apparent molecular mass, indicating that newly synthesized VSG underwent translocation and glycosylation in the cell‐free system. Biotinylation of VSG and a combination of precipitation with immobilized avidin and detection of VSG using antibodies specific for clones and cross‐reacting determinants revealed that newly formed VSG contained the glycosylphosphatidylinositol anchor.
Activation of the rod G‐protein Gt by the thrombin receptor (PAR1) expressed in Sf9 cells
Authors: Seibert Christoph , Harteneck Christian , Ernst Oliver P , Schultz Günter , Hofmann Klaus P
Published: March 2003

Functional coupling of the human thrombin receptor PAR1 (protease‐activated receptor 1) with the retinal rod G‐protein transducin (Gt, a member of the Gi family) was studied in a reconstituted system of membranes from Sf9 cells expressing the thrombin receptor and purified Gt from bovine rod outer segments. TRAP6‐agonist‐activated PAR1 interacts productively with the distant G‐protein. Agonist‐dependent Gt activation was measured using a real‐time fluorimetric GTP[S]‐binding assay and membranes from Sf9 cells. To characterize nucleotide‐exchange catalysis by PAR1, we analyzed dependence on nucleotides, temperature and pH. Activation was inhibited by low GDP concentrations (IC50 = 5.2 ± 1.5 µm at 5 µm GTP[S]), indicating that receptor–Gt coupling, followed by instantaneous GDP release, is rate limiting under the conditions (25 °C). Arrhenius plots of the temperature dependence reflect an apparent Ea of 60 ± 3.5 kJ·mol−1. Evaluation of the pH/rate profiles of Gt activation indicates that the activating conformation of the receptor is determined by protonation of a titratable group with an apparent pKa of 6.4. This supports the idea that the active state of agonist‐bound PAR1 depends on forced protonation, indicating possible analogies to the scheme established for rhodopsin.
Both G‐type domains of protein S are required for the high‐affinity interaction with C4b‐binding protein
Authors: Evenäs Petra , García de Frutos Pablo , Linse Sara , Dahlbäck Björn
Published: March 2003

Anticoagulant protein S interacts with the complement regulatory protein C4b‐binding protein (C4BP) via its sex‐hormone‐binding globulin (SHB6)‐like region, which contains two globular (G) domains. Similar G domains are found in Gas6, a protein homologous to protein S, which is not known to bind C4BP or to have any anticoagulant activity. To determine the relative importance of the two G domains in protein S for C4BP protein binding, three recombinant protein S chimeras were produced having either of the two globular domains, or the whole SHB6‐like globulin region, replaced by corresponding parts from Gas6. The chimeras were tested for binding to immobilized C4BP using surface‐plasmon‐resonance technology and microtiter plate‐based assays. In both systems, chimeras containing either only globular domains G1 or G2 from protein S were found to bind C4BP. Binding was stimulated by Ca2+ in a manner similar to that found for wild‐type protein S. The affinities for C4BP of both chimeras containing individual G domains from protein S, were lower than that of wild‐type protein S. Chimera II, containing the G1 domain from protein S, consistently bound C4BP more efficiently than chimera I, which had the protein S‐derived G2 domain. The chimera containing the whole SHB6‐like globulin region from Gas6 interacted considerably more weakly with C4BP. Our results demonstrate that both G domains of protein S are involved in the interaction between protein S and C4BP and that full affinity binding is dependent on contributions from both domains.
600 MHz NMR studies of human articular cartilage keratan sulfates
Authors: Huckerby Thomas N. , Nieduszynski Ian A. , Bayliss Michael T. , Brown Gavin M.
Published: March 2003

The use of high‐field two‐dimensional 1H‐correlation data is described for the detailed comparison of intact keratan sulfate polymer chains derived from human articular cartilage sources as a function of age. For fetal material the nonreducing chain termini are shown to be sparsely capped by sialyl groups which, if present, are exclusively (α2–3)‐linked to an unsulfated galactose residue. The asialo capping segment has the structure: Gal‐GlcNAc6S‐Gal‐GlcNAc6S‐. Examination of keratan sulfate from 10‐year‐old cartilage shows that capping by sialyl groups is complete, with (α2–3)‐linkages predominant; for both this and the 38‐year‐old cartilage the three capping structures: NeuAc(α2–3)‐Gal‐GlcNAc6S‐Gal‐GlcNAc6S‐, NeuAc(α2–3)‐Gal‐GlcNAc6S‐Gal6S‐GlcNAc6S‐, and NeuAc(α2–3)‐Gal6S‐GlcNAc6S‐Gal6S‐GlcNAc6S‐ are clearly recognizable. The level of (α2–6)‐linked chain capping sialyl groups is significant for 38‐year‐old cartilage keratan sulfate. Structural information concerning the linkage region to protein and the distribution of galactose environments is readily obtained from the spectra. Signal complexities severely limit the usefulness of two‐dimensional correlation spectroscopy at 600 MHz for the examination of N‐acetylglucosamine residues within the poly(N‐acetyllactosamine) repeat sequence and signals representing fucose placements remain undifferentiated. This nondestructive approach complements current degradative methods for the structural examination of keratan sulfates.
Slender and stumpy bloodstream forms of Trypanosoma brucei display a differential response to extracellular acidic and proteolytic stress
Authors: Nolan Derek P , Rolin Sylvie , Rodriguez Jesus R. , Van Den Abbeele Jan , Pays Etienne
Published: March 2003

Natural infections of mammals with African trypanosomes, such as Trypanosoma brucei, are generally pleomorphic, the population consisting of different forms, termed slender and stumpy forms, that vary in number as the parasitaemia develops. We show that the differentiation of slender into stumpy forms is characterized by the acquisition by the parasite of the ability to regulate its internal pH, even in the face of a large, inwardly directed gradient of H+, as well as a tolerance towards external proteolytic stress. These adaptations effectively abbrogate cellular stress‐activated signalling pathways involving adenylate cyclase and glycosylphosphoinositol‐specific phospholipase‐C mediated release of the surface coat. Although in metabolic terms stumpy forms of the parasite are considered to be preadapted to life in the arthropod vector, these data clearly demonstrate that these forms also possess additional cellular adaptations designed to deal with the immediate and potentially harmful changes in the extracellular environment that occur upon ingestion of a bloodmeal by the tsetse fly vector.
Tobacco budworm dihydrofolate reductase is a promising target for insecticide discovery
Authors: Walker Virginia K. , Tyshenko Michael G. , Kuiper Michael J. , Dargar Ratna V. , Yuhas Debra A. , Cruickshank Philip A. , Chaguturu Rathnam
Published: March 2003

Structural differences in dihydrofolate reductases from different species have been exploited to develop specific inhibitory molecules, such as chemotherapeutic agents, antibiotics or antihelminthics, that show species specificity or selectivity. As dihydrofolate reductase (DHFR) is a crucial enzyme for the synthesis of purines, pyrimidines and some amino acids, and also because developing insects show a remarkably rapid rate of cell division, DHFR is a potentially promising target for the discovery of novel insecticides. We have thus isolated and characterized the enzyme from a serious agricultural pest, Heliothis (Helicoverpa) virescens, the tobacco budworm. Sequencing tryptic peptides of the 35 000‐fold purified DHFR allowed the subsequent isolation of a partial cDNA, with the full Dhfr gene sequence obtained from a genomic library. The H. virescens Dhfr spans 4 kb, with three introns, and encodes 185 amino acids. The enzyme shows an overall similarity of ≈ 68% with DHFR from other metazoans, which has facilitated the molecular modeling of the protein. DHFRs from insects appear to have strikingly reduced sensitivity to inhibition by methotrexate, compared with the vertebrate enzymes, and this reduction was also reflected in the total binding energy seen after modeling experiments. Four residues that may be characteristic of insect DHFR, as well as a unique cysteine in the H. virescens DHFR active site, offer insight into the nature of inhibitor selectivity and provide suitable target sites for insecticide discovery.
Polypeptides differentially expressed in imaginal discs define the peroxiredoxin family of genes in Drosophila
Authors: Rodriguez Javier , Agudo Marta , Van Damme Jozef , Vandekerckhove Joel , Santarén Juan F.
Published: March 2003

2D gel electrophoresis followed by microsequencing has been used to purify and identify a protein (catalogued in the database as SSP5111) from Drosophila wing imaginal discs of third instar larvae that showed significant differences in their level of expression when compared with other imaginal discs of the same age. The microsequence data showed identity with amino acids encoded by the human proliferation association gene, pag, which is a thiol‐specific antioxidant. By virtue of this homology we have cloned and sequenced two cDNAs that appear to define the peroxiredoxin family of Drosophila. One of them, Jafrac1, encodes the SSP5111 protein searched, had 194 amino acids and mapped in the region 11E in the X chromosome. The other, Jafrac2, encodes a protein of 242 amino acids and mapped in the region 62F in the 3 L chromosome. Both new peroxidases contain two conserved cysteines and share homology with other peroxidases that extends over the entire sequence and ranges between 47% and 76%. An antiserum raised against the SSP5111 protein showed significant changes in the amount of protein in different stages of Drosophila development, being a major product in early embryos. In 2D gels the antibody not only recognizes the SSP5111 polypeptide but also a related one (catalogued in the database as SSP6107) that exhibits identical amino‐acid sequence over at least 85% of its sequence. The data also suggest that the SSP5111 polypeptide could be a maternal‐effect product.
Overexpression of the crgA gene abolishes light requirement for carotenoid biosynthesis in Mucor circinelloides
Authors: Navarro Eusebio , Ruiz‐Pérez Victor L. , Torres‐Martínez Santiago
Published: March 2003

This work describes the isolation and characterization of crgA, a Mucor circinelloides gene, which has a dominant‐positive effect on light‐regulated carotenogenesis. The crgA gene was originally identified in a transformation experiment as a 3′‐truncated open reading frame which caused carotenoid overaccumulation in the dark. The complete cloning and sequencing of crgA revealed that its putative product presented several recognizable structural domains: a RING‐finger zinc binding domain near the N‐terminus, a putative nuclear localization signal, two stretches of acidic amino acids, glutamine‐rich regions and a putative isoprenylation motif. The expression of exogenous copies of the complete crgA gene or two different 3′‐truncated versions, produced a similar dominant‐positive effect on the light‐inducible carotenogenesis of M. circinelloides. The presence of these exogenous sequences also caused a missregulation of the endogenous crgA gene, resulting in its overexpression. Collectively, these observations suggest that crgA is involved in the regulation of carotenoid biosynthesis by light.
The structural basis of substrate activation in yeast pyruvate decarboxylase
Authors: Lu Guoguang , Dobritzsch Doreen , Baumann Sabine , Schneider Gunter , König Stephan
Published: March 2003

The crystal structure of the complex of the thiamine diphosphate dependent tetrameric enzyme pyruvate decarboxylase (PDC) from brewer's yeast strain with the activator pyruvamide has been determined to 2.4 Å resolution. The asymmetric unit of the crystal contains two subunits, and the tetrameric molecule is generated by crystallographic symmetry. Structure analysis revealed conformational nonequivalence of the active sites. One of the two active sites in the asymmetric unit was found in an open conformation, with two active site loop regions (residues 104–113 and 290–304) disordered. In the other subunit, these loop regions are well‐ordered and shield the active site from the bulk solution. In the closed enzyme subunit, one molecule of pyruvamide is bound in the active site channel, and is located in the vicinity of the thiazolium ring of the cofactor. A second pyruvamide binding site was found at the interface between the Pyr and the R domains of the subunit in the closed conformation, about 10 Å away from residue C221. This second pyruvamide molecule might function in stabilizing the unique orientation of the R domain in this subunit which in turn is important for dimer–dimer interactions in the activated tetramer. No difference electron density in the close vicinity of the side chain of residue C221 was found, indicating that this residue does not form a covalent adduct with an activator molecule. Kinetic experiments showed that substrate activation was not affected by oxidation of cysteine residues and therefore does not seem to be dependent on intact thiol groups in the enzyme. The results suggest that a disorder–order transition of two active‐site loop regions is a key event in the activation process triggered by the activator pyruvamide and that covalent modification of C221 is not required for this transition to occur. Based on these findings, a possible mechanism for the activation of PDC by its substrate, pyruvate, is proposed.
High resolution solution structure of the protein part of Cu7 metallothionein
Authors: Bertini Ivano , Hartmann Hans‐Jürgen , Klein Tino , Liu Gaohua , Luchinat Claudio , Weser Ulrich
Published: March 2003

The three‐dimensional solution structure of the protein part of Cu7 metallothionein (Cu7MT) of Saccharomyces cerevisiae has been attempted by 1H two‐dimensional NMR spectroscopy at 800 MHz. The protein part constitutes 53 amino acids. A total of 1192 NOEs, of which 1048 are meaningful, were used to determine the solution structure of the first 40 residues, the last 13 residues being disordered. A family of 30 structures was generated. Root‐mean‐square deviation (rmsd) values from the average structure of 0.32 ± 0.13 Å and 0.61 ± 0.15 Å for backbone and all heavy atoms, respectively, were obtained for the residues 2–40. The ten copper‐coordinating cysteine sulfurs and the empty spaces around them are well defined. The structure of the protein part is similar but not identical to the available ones of the same holoprotein and of the Ag7 metallothionein, and is qualitatively superior. If the same metal–sulfur connectivities reported in the literature from 1H‐109Ag heteronuclear multiple quantum coherence spectroscopy are assumed to hold for the present copper derivative, a peptide structure is obtained which is again similar, but still not identical, within indetermination, to that available. The structure of the copper polymetallic center may well be different from that proposed for the silver derivative, and indeed a number of different arrangements of the seven copper ions are consistent with the present highly refined structure of the protein part.