bluuebunny/crossref_metadata_2025 huggingface dataset:
The oxidative effect of bacterial lipopolysaccharide on native and cross‐linked human hemoglobin as a function of the structure of the lipopolysaccharide
Authors: Currell Douglas L. , Levin Jack
Published: March 2003

The binding of lipopolysaccharide (LPS, also known as bacterial endotoxin) to human hemoglobin is known to result in oxidation of hemoglobin to methemoglobin and hemichrome. We have investigated the effects of the LPSs from smooth and rough Escherichia coli and Salmonella minnesota on the rate of oxidation of native oxyhemoglobin A0 and hemoglobin cross‐linked between the α‐99 lysines. For cross‐linked hemoglobin, both smooth LPSs produced a rate of oxidation faster than the corresponding rough LPSs, indicating the importance of the binding of LPS to the hemoglobin. The effect of the LPS appeared to be largely on the initial fast phase of the oxidation reaction, suggestingmodification of the heme pocket of the α chains. For hemoglobin A0, the rates of oxidation produced by rough and smooth LPSs were very similar, suggesting the possibility that the effect of the LPSs was to cause dissociation of hemoglobin into dimers. The participation of cupric ion in the oxidation process was demonstrated in most cases. In contrast, the rate of oxidation of cross‐linked hemoglobin by the LPSs of both the rough and smooth E. coli was not affected by the presence of chelators, suggesting that cupric ion had previously bound to these LPSs. Overall, these data suggest that the physiological effectiveness of hemoglobin solutions now being developed for clinical use may be decreased by the presence of lipopolysaccharide in the circulation of recipients.
A nonphosphorylated 14‐3‐3 binding motif on exoenzyme S that is functional in vivo
Authors: Henriksson Maria L. , Francis Matthew S. , Peden Alex , Aili Margareta , Stefansson Kristina , Palmer Ruth , Aitken Alastair , Hallberg Bengt
Published: March 2003

14‐3‐3 proteins play an important role in a multitude of signalling pathways. The interactions between 14‐3‐3 and other signalling proteins, such as Raf and KSR (kinase suppressor of Ras), occur in a phospho‐specific manner. Recently, a phosphorylation‐independent interaction has been reported to occur between 14‐3‐3 and several proteins, for example 5‐phosphatase, p75NTR‐associated cell death executor (NADE) and the bacterial toxin Exoenzyme S (ExoS), an ADP‐ribosyltransferase from Pseudomonas aeruginosa. In this study we have identified the amino acid residues on ExoS, which are responsible for its specific interaction with 14‐3‐3. Furthermore, we show that a peptide derived from ExoS, containing the 14‐3‐3 interaction site, effectively competes out the interaction between ExoS and 14‐3‐3. In addition, competition with this peptide blocks ExoS modification of Ras in our Ras modification assay. We show that the ExoS protein interacts with all isoforms of the 14‐3‐3 family tested. Moreover, in vivo an ExoS protein lacking the 14‐3‐3 binding site has a reduced capacity to ADP ribosylate cytoplasmic proteins, e.g. Ras, and shows a reduced capacity to change the morphology of infected cells.
Probing the role of glutamic acid 139 of Anabaena ferredoxin‐NADP+ reductase in the interaction with substrates
Authors: Faro Merche , Frago Susana , Mayoral Tomas , Hermoso Juan A. , Sanz‐Aparicio Julia , Gómez‐Moreno Carlos , Medina Milagros
Published: March 2003

The role of the negative charge of the E139 side‐chain of Anabaena Ferredoxin‐NADP+ reductase (FNR) in steering appropriate docking with its substrates ferredoxin, flavodoxin and NADP+/H, that leads to efficient electron transfer (ET) is analysed by characterization of several E139 FNR mutants. Replacement of E139 affects the interaction with the different FNR substrates in very different ways. Thus, while E139 does not appear to be involved in the processes of binding and ET between FNR and NADP+/H, the nature and the conformation of the residue at position 139 of Anabaena FNR modulates the precise enzyme interaction with the protein carriers ferredoxin (Fd) and flavodoxin (Fld). Introduction of the shorter aspartic acid side‐chain at position 139 produces an enzyme that interacts more weakly with both ET proteins. Moreover, the removal of the charge, as in the E139Q mutant, or the charge‐reversal mutation, as in E139K FNR, apparently enhances additional interaction modes of the enzyme with Fd, and reduces the possible orientations with Fld to more productive and stronger ones. Hence, removal of the negative charge at position 139 of Anabaena FNR produces a deleterious effect in its ET reactions with Fd whereas it appears to enhance the ET processes with Fld. Significantly, a large structural variation is observed for the E139 side‐chain conformer in different FNR structures, including the E139K mutant. In this case, a positive potential region replaces a negative one in the wild‐type enzyme. Our observations further confirm the contribution of both attractive and repulsive interactions in achieving the optimal orientation for efficient ET between FNR and its protein carriers.
Soluble silk‐like organic matrix in the nacreous layer of the bivalve Pinctada maxima
Authors: Pereira‐Mouriès Lucilia , Almeida Maria‐José , Ribeiro Cristina , Peduzzi Jean , Barthélemy Michel , Milet Christian , Lopez Evelyne
Published: March 2003

Nacre organic matrix has been conventionally classified as both ‘water‐soluble’ and ‘water‐insoluble’, based on its solubility in aqueous solutions after decalcification with acid or EDTA. Some characteristics (aspartic acid‐rich, silk‐fibroin‐like content) were specifically attributed to either one or the other. The comparative study on the technique of extraction (extraction with water alone vs. demineralization with EDTA) presented here, seems to reveal that this generally accepted classification may need to be reconsidered. Actually, the nondecalcified soluble organic matrix, extracted in ultra‐pure water, displays many of the characteristics of what until now has been called ‘insoluble matrix’. We present the results obtained on this extract and on a conventional EDTA‐soluble matrix, with various characterization methods: fractionation by size‐exclusion and anion‐exchange HPLC, amino acid analysis, glycosaminoglycan and calcium quantification, SDS/PAGE and FTIR spectroscopy. We propose that the model for the interlamellar matrix sheets of nacre given by Nakahara [In: Biomineralization and Biological Metal Accumulation, Westbroek, P. & deJong, E.W., eds, (1983) pp. 225–230. Reidel, Dordrecht, Holland] and Weiner and Traub [Phil. Trans. R. Soc. Lond. B (1984) 304, 425–434] may no longer be valid. The most recent model, proposed by Levi‐Kalisman et al. [J. Struct. Biol. (2001) 135, 8–17], seemed to be more in accordance with our findings.
Spectroscopic characterization and ligand‐binding properties of chlorite dismutase from the chlorate respiring bacterial strain GR‐1
Authors: Hagedoorn Peter L. , de Geus Daniel C. , Hagen Wilfred R.
Published: March 2003

Chlorite dismutase (EC 1.13.11.49), an enzyme capable of reducing chlorite to chloride while producing molecular oxygen, has been characterized using EPR and optical spectroscopy. The EPR spectrum of GR‐1 chlorite dismutase shows two different high‐spin ferric heme species, which we have designated ‘narrow’ (gx,y,z = 6.24, 5.42, 2.00) and ‘broad’ (gz,y,x = 6.70, 5.02, 2.00). Spectroscopic evidence is presented for a proximal histidine co‐ordinating the heme iron center of the enzyme. The UV/visible spectrum of the ferrous enzyme and EPR spectra of the ferric hydroxide and imidazole adducts are characteristic of a heme protein with an axial histidine co‐ordinating the iron. Furthermore, the substrate analogs nitrite and hydrogen peroxide have been found to bind to ferric chlorite dismutase. EPR spectroscopy of the hydrogen peroxide adduct shows the loss of both high‐spin and low‐spin ferric signals and the appearance of a sharp radical signal. The NO adduct of the ferrous enzyme exhibits a low‐spin EPR signal typical of a five‐co‐ordinate heme iron nitrosyl adduct. It seems that the bond between the proximal histidine and the iron is weak and can be broken upon binding of NO. The midpoint potential, Em(Fe3+/2+) = −23 mV, of chlorite dismutase is higher than for most heme enzymes. The spectroscopic features and redox properties of chlorite dismutase are more similar to the gas‐sensing hemoproteins, such as guanylate cyclase and the globins, than to the heme enzymes.
Probing intermolecular protein–protein interactions in the calcium‐sensing receptor homodimer using bioluminescence resonance energy transfer (BRET)
Authors: Jensen Anders A. , Hansen Jakob L. , Sheikh Søren P. , Bräuner‐Osborne Hans
Published: March 2003

The calcium‐sensing receptor (CaR) belongs to family C of the G‐protein coupled receptor superfamily. The receptor is believed to exist as a homodimer due to covalent and non‐covalent interactions between the two amino terminal domains (ATDs). It is well established that agonist binding to family C receptors takes place at the ATD and that this causes the ATD dimer to twist. However, very little is known about the translation of the ATD dimer twist into G‐protein coupling to the 7 transmembrane moieties (7TMs) of these receptor dimers.

In this study we have attempted to delineate the agonist‐induced intermolecular movements in the CaR homodimer using the new bioluminescence resonance energy transfer technique, BRET2, which is based on the transference of energy from Renilla luciferase (Rluc) to the green fluorescent protein mutant GFP2. We tagged CaR with Rluc and GFP2 at different intracellular locations. Stable and highly receptor‐specific BRET signals were obtained in tsA cells transfected with Rluc‐ and GFP2‐tagged CaRs under basal conditions, indicating that CaR is constitutively dimerized. However, the signals were not enhanced by the presence of agonist. These results could indicate that at least parts of the two 7TMs of the CaR homodimer are in close proximity in the inactivated state of the receptor and do not move much relative to one another upon agonist activation. However, we cannot exclude the possibility that the BRET technology is unable to register putative conformational changes in the CaR homodimer induced by agonist binding because of the bulk sizes of the Rluc and GFP2 molecules.
Purification, crystallization, NMR spectroscopy and biochemical analyses of α‐phycoerythrocyanin peptides
Authors: Wiegand Georg , Parbel Axel , Seifert Markus H. J. , Holak Tad A. , Reuter Wolfgang
Published: March 2003

The α‐phycoerythrocyanin subunits of the different phycoerythrocyanin complexes of the phycobilisomes from the cyanobacteriumMastigocladus laminosusperform a remarkable photochemistry. Similar to phytochromes – the photoreceptors of higher plants – the spectral properties of the molecule reversibly change according to the irradiation wavelength. To enable extensive analyses, the protein has been produced at high yield by improving purification protocols. As a result, several comparative studies on theZ‐ andE‐configurations of the intact α‐subunit, and also on photoactive peptides originating from nonspecific degradations of the chromoprotein, were possible. The analyses comprise absorbance, fluorescence and CD spectroscopy, crystallization, preliminary X‐ray measurements, mass spectrometry, N‐terminal amino acid sequencing and 1D NMR spectroscopy. Intact α‐phycoerythrocyanin aggregates significantly, due to hydrophobic interactions between the two N‐terminal helices. Removal of these helices reduces the aggregation but also destabilizes the protein fold. The complete subunit could be crystallized in itsE‐configuration, but the X‐ray measurement conditions must be improved. Nevertheless, NMR spectroscopy on a soluble photoactive peptide presents the first insight into the complex chromophore protein interactions that are dependent on the light induced state. The chromophore environment in theZ‐configuration is rigid whereas other regions of the protein are more flexible. In contrast, theE‐configuration has a mobile chromophore, especially the pyrrole ring D, while other regions of the protein rigidified compared to theZ‐configuration.
Distinct parts of minichromosome maintenance protein 2 associate with histone H3/H4 and RNA polymerase II holoenzyme
Authors: Holland Linda , Downey Michael , Song Xiaomin , Gauthier Laura , Bell‐Rogers Patricia , Yankulov Krassimir
Published: March 2003

Minichromosome maintenance (MCM) proteins are part of the replication licensing factor (RLF‐M), which limits the initiation of DNA replication to once per cell cycle. We have previously reported that higher order complexes of mammalian pol II and general pol II transcription factors, referred to as pol II holoenzyme, also contain MCM proteins. In the present study we have analyzed in detail the interaction between MCM2 and pol II holoenzyme. N‐ and C‐ terminal deletions were introduced into epitope‐tagged MCM2 and the truncated proteins were transiently expressed in 293 cells. Affinity chromatography was used to purify RNA pol II holoenzyme and histone binding MCM complexes. We found that amino acids 168–230 of MCM2 are required for its binding to pol II holoenzyme in vivo. We also showed that bacterially expressed amino acids 169–212 of MCM2 associate with pol II and several general transcription factors in vitro. Point mutations within the 169–212 domain of MCM2 disrupted its interaction with pol II holoenzyme both in vitro and in vivo. This region is distinct from the previously characterized histone H3 binding domain of MCM2.
Association of human tumor necrosis factor‐related apoptosis inducing ligand with membrane upon acidification
Authors: Nam Gyu Hyun , Choi Kwan Yong
Published: March 2003

Tumor necrosis factor (TNF)‐related apoptosis inducing ligand (TRAIL) has been known to induce tumor‐specific apoptosis and to share the structural and functional characteristics with the proteins of TNF family. Recently, the crystal structure of human TRAIL showed that TRAIL is a homotrimeric protein whose subunits contain mainly β‐sheets. We characterized the structural changes of recombinant human TRAIL induced by acidification and the biological implication of the structural characteristics at acidic pH in the interaction with the lipid bilayer. At acidic pH below pH 4.5, TRAIL resulted in substantial structural changes to a molten globule (MG)‐like state. Far‐UV CD spectrum of TRAIL indicated that the acidification induced α‐helices that are absent in the native state. TRAIL at acidic pH exhibited significant change of tertiary structures as reflected in the near‐UV CD spectrum. Thermal transition curve indicated that there was less cooperation at acidic pH than at neutral pH in the thermal denaturation of TRAIL. Moreover, TRAIL at the MG‐like state not only enhanced the binding ability to liposomes, but also increased the release rate of a fluorescent dye, calcein, encapsulated in liposomes. The binding assay with anilinonaphthalene‐8‐sulfonic acid revealed that the surface hydrophobicity of TRAIL was increased while tryptophan residues became more exposed to solvent as judged by blue shift of the maximum fluorescence wavelength. Taken together, our results demonstrate that the acidification of human TRAIL induces the MG‐like state in vitro and makes the membrane permeable through the favorable interaction of TRAIL with the membrane, implicating that general intrinsic properties such as TRAIL, TNF‐α and lymphotoxin are shared by TNF family members.
Functional analysis of DM64, an antimyotoxic protein with immunoglobulin‐like structure from Didelphis marsupialis serum
Authors: Rocha Surza L. G. , Lomonte Bruno , Neves‐Ferreira Ana G. C. , Trugilho Monique R. O. , Junqueira‐de‐Azevedo Inácio de L. M. , Ho Paulo L. , Domont Gilberto B. , Gutiérrez José M. , Perales Jonas
Published: March 2003

Bothrops snake venoms are known to induce local tissue damage such as hemorrhage and myonecrosis. The opossum Didelphis marsupialis is resistant to these snake venoms and has natural venom inhibitors in its plasma. The aim of this work was to clone and study the chemical, physicochemical and biological properties of DM64, an antimyotoxic protein from opossum serum. DM64 is an acidic protein showing 15% glycosylation and with a molecular mass of 63 659 Da when analysed by MALDI‐TOF MS. It was cloned and the amino acid sequence was found to be homologous to DM43, a metalloproteinase inhibitor from D. marsupialis serum, and to human α1B‐glycoprotein, indicating the presence of five immunoglobulin‐like domains. DM64 neutralized both the in vivo myotoxicity and the in vitro cytotoxicity of myotoxins I (mt‐I/Asp49) and II (mt‐II/Lys49) from Bothrops asper venom. The inhibitor formed noncovalent complexes with both toxins, but did not inhibit the PLA2 activity of mt‐I. Accordingly, DM64 did not neutralize the anticoagulant effect of mt‐I nor its intracerebroventricular lethality, effects that depend on its enzymatic activity, and which demonstrate the dissociation between the catalytic and toxic activities of this Asp49 myotoxic PLA2. Furthermore, despite its similarity with metalloproteinase inhibitors, DM64 presented no antihemorrhagic activity against Bothrops jararaca or Bothrops asper crude venoms, and did not inhibit the fibrinogenolytic activity of jararhagin or bothrolysin. This is the first report of a myotoxin inhibitor with an immunoglobulin‐like structure isolated and characterized from animal blood.
Differences in the binding capacity of human apolipoprotein E3 and E4 to size‐fractionated lipid emulsions
Authors: Perugini Matthew A. , Schuck Peter , Howlett Geoffrey J.
Published: March 2003

We describe sensitive new approaches for detecting and quantitating protein–lipid interactions using analytical ultracentrifugation and continuous size‐distribution analysis [Schuck (2000) Biophys. J.78, 1606–1619]. The new methods were developed to investigate the binding of human apolipoprotein E (apoE) isoforms to size‐fractionated lipid emulsions, and demonstrate that apoE3 binds preferentially to small lipid emulsions, whereas apoE4 exhibits a preference for large lipid particles. Although the apparent binding affinity for large emulsions is similar (Kd ≈ 0.5 µm), the maximum binding capacity for apoE4 is significantly higher than for apoE3 (3.0 and 1.8 amino acids per phospholipid, respectively). This indicates that apoE4 has a smaller binding footprint at saturation. We propose that apoE isoforms differentiate between lipid surfaces on the basis of size, and that these differences in lipid binding are due to a greater propensity of apoE4 to adopt a more compact closed conformation. Implications for the role of apoE4 in blood lipid transport and disease are discussed.
Complexation of ytterbium to human transferrin and its uptake by K562 cells
Authors: Du Xiu‐lian , Zhang Tian‐lan , Yuan Lan , Zhao Yong‐yuan , Li Rong‐chang , Wang Kui , Yan Siu Cheong , Zhang Li , Sun Hongzhe , Qian Zhong‐ming
Published: March 2003

There is an increasing interest in the use of lanthanides in medicine. However, the mechanism of their accumulation in cells is not well understood. Lanthanide cations are similar to ferric ions with regard to transferrin binding, suggesting transferrin‐receptor mediated transport is possible; however, this has not yet been confirmed. In order to clarify this mechanism, we investigated the binding of Yb3+ to apotransferrin by UV‐Vis spectroscopy and stopped‐flow spectrophotometry, and found that Yb3+ binds to apotransferrin at the specific iron sites in the presence of bicarbonate. The apparent binding constants of these sites showed that the affinity of Yb3+ is lower than that of Fe3+and binding of Yb3+ in the N‐lobe is kinetically favored while the C‐lobe is thermodynamically favored. The first Yb3+ bound to the C‐lobe quantitatively with a Yb/apotransferrin molar ratio of < 1, whereas the binding to the other site is weaker and approaches completeness by a higher molar ratio only. As demonstrated by 1H NMR spectra, Yb3+ binding disturbed the conformation of apotransferrin in a manner similar to Fe3+. Flow cytometric studies on the uptake of fluorescein isothiocyanate labeled Yb3+‐bound transferrin species by K562 cells showed that they bind to the cell receptors. Laser scanning confocal microscopic studies with fluorescein isothiocyanate labeled Yb3+‐bound transferrin and propidium iodide labeled DNA and RNA in cells indicated that the Yb3+ entered the cells. The Yb3+‐transferrin complex inhibited the uptake of the fluorescein labeled ferric‐saturated transferrin (Fe2‐transferrin) complex into K562 cells. The results demonstrate that the complex of Yb3+‐transferrin complex was recognized by the transferrin receptor and that the transferrin‐receptor‐mediated mechanism is a possible pathway for Yb3+ accumulation in cells.
Oxidation of propionate to pyruvate in Escherichia coli
Authors: Brock Matthias , Maerker Claudia , Schütz Alexandra , Völker Uwe , Buckel Wolfgang
Published: March 2003

The pathway of the oxidation of propionate to pyruvate in Escherichia coli involves five enzymes, only two of which, methylcitrate synthase and 2‐methylisocitrate lyase, have been thoroughly characterized. Here we report that the isomerization of (2S,3S)‐methylcitrate to (2R,3S)‐2‐methylisocitrate requires a novel enzyme, methylcitrate dehydratase (PrpD), and the well‐known enzyme, aconitase (AcnB), of the tricarboxylic acid cycle. AcnB was purified as 2‐methylaconitate hydratase from E. coli cells grown on propionate and identified by its N‐terminus. The enzyme has an apparent Km of 210 µm for (2R,3S)‐2‐methylisocitrate but shows no activity with (2S,3S)‐methylcitrate. On the other hand, PrpD is specific for (2S,3S)‐methylcitrate (Km = 440 µm) and catalyses in addition only the hydration of cis‐aconitate at a rate that is five times lower. The product of the dehydration of enzymatically synthesized (2S,3S)‐methylcitrate was designated cis‐2‐methylaconitate because of its ability to form a cyclic anhydride at low pH. Hence, PrpD catalyses an unusual syn elimination, whereas the addition of water to cis‐2‐methylaconitate occurs in the usual anti manner. The different stereochemistries of the elimination and addition of water may be the reason for the requirement for the novel methylcitrate dehydratase (PrpD), the sequence of which seems not to be related to any other enzyme of known function. Northern‐blot experiments showed expression of acnB under all conditions tested, whereas the RNA of enzymes of the prp operon (PrpE, a propionyl‐CoA synthetase, and PrpD) was exclusively present during growth on propionate. 2D gel electrophoresis showed the production of all proteins encoded by the prp operon during growth on propionate as sole carbon and energy source, except PrpE, which seems to be replaced by acetyl‐CoA synthetase. This is in good agreement with investigations on Salmonella enterica LT2, in which disruption of the prpE gene showed no visible phenotype.
RE‐1 silencing transcription factor (REST) regulates human synaptophysin gene transcription through an intronic sequence‐specific DNA‐binding site
Authors: Lietz Michael , Hohl Mathias , Thiel Gerald
Published: March 2003

Synaptophysin, one of the major proteins on synaptic vesicles, is ubiquitously expressed throughout the brain. Synaptophysin and synapsin I, another synaptic vesicle protein, are also expressed by retinoic acid‐induced neuronally differentiated P19 teratocarcinoma cells. Here, we show that inhibition of histone deacetylase activity in P19 cells is sufficient to activate transcription of the synaptophysin and synapsin I genes, indicating that neuronal differentiation and impairment of histone deacetylases results in a similar gene expression pattern. The transcription factor REST, a repressor of neuronal genes in non‐neuronal tissues, has been shown to function via recruitment of histone deacetylases to the transcription unit, indicating that modulation of the chromatin structure via histone deacetylation is of major importance for REST function and neuron‐specific gene transcription. Furthermore, REST has been shown to be the major regulator of neuronal expression of synapsin I. Here, we have identified a functional binding site for REST in the first intron of the human synaptophysin gene indicating that REST blocks human synaptophysin gene transcription through an intronic neuron‐specific silencer element. The synaptophysin promoter is, however, devoid of neuron‐specific genetic elements and directs transcription in both neuronal and non‐neuronal cells. Using a dominant‐negative approach we have identified the transcription factor Sp1 as one of the regulators responsible for constitutive transcription of the human synaptophysin gene.
The −148 to −124 region of c‐jun interacts with a positive regulatory factor in rat liver and enhances transcription
Authors: Sharma Dipali , Ohri Sujata , Dixit Aparna
Published: March 2003

The c‐jun gene encodes the protein Jun, a component of the essential transcription factor, AP1. Jun/AP‐1 occupies a central position in signal transduction pathways as it is responsible for the induction of a number of genes in response to growth promoters. However, the exact mechanisms leading to an enhanced expression of the c‐jun gene itself during proliferation, differentiation, cell growth and development are not fully understood. Cell culture studies have given some insight in the mechanisms involved in the up‐regulation of c‐jun expression by UV irradiation and phorbol esters. However, it is well known that transformed cells do not accurately reflect the biology of a normal cell. We now report the identification of a positive regulatory factor from normal rat liver that activates transcription from the c‐jun promoter by binding to the −148 to −124 region of c‐jun. Preincubation of fractionated rat liver nuclear extract with an oligonucleotide encompassing this region of the gene significantly reduced transcription from cloned c‐jun promoter. In vitro transfection studies using green fluorescent protein as a reporter gene under the control of the c‐jun promoter with (−148 to +53) and without (−123 to +53) this region further confirmed its role in transcription. A DNA‐binding protein factor, interacting with this region of c‐jun was identified from rat liver by using electrophoretic mobility shift assays. This factor binds to its recognition sequence only in the phosphorylated form and exhibits high affinity and specificity. UV cross‐linking studies, South‐Western analysis and affinity purification collectively indicated the factor to be ≈40 kDa and to bind to its recognition sequence as a dimer.
A hydrophobic segment within the C‐terminal domain is essential for both client‐binding and dimer formation of the HSP90‐family molecular chaperone
Authors: Yamada Shin‐ichi , Ono Toshio , Mizuno Akio , Nemoto Takayuki K
Published: March 2003

The α isoform of human 90‐kDa heat shock protein (HSP90α) is composed of three domains: the N‐terminal (residues 1–400); middle (residues 401–615) and C‐terminal (residues 621–732). The middle domain is simultaneously associated with the N‐ and C‐terminal domains, and the interaction with the latter mediates the dimeric configuration of HSP90. Besides one in the N‐terminal domain, an additional client‐binding site exists in the C‐terminal domain of HSP90. The aim of the present study is to elucidate the regions within the C‐terminal domain responsible for the bindings to the middle domain and to a client protein, and to define the relationship between the two functions. A bacterial two‐hybrid system revealed that residues 650–697 of HSP90α were essential for the binding to the middle domain. An almost identical region (residues 657–720) was required for the suppression of heat‐induced aggregation of citrate synthase, a model client protein. Replacement of either Leu665‐Leu666 or Leu671‐Leu672 to Ser‐Ser within the hydrophobic segment (residues 662–678) of the C‐terminal domain caused the loss of bindings to both the middle domain and the client protein. The interaction between the middle and C‐terminal domains was also found in human 94‐kDa glucose‐regulated protein. Moreover, Escherichia coli HtpG, a bacterial HSP90 homologue, formed heterodimeric complexes with HSP90α and the 94‐kDa glucose‐regulated protein through their middle‐C‐terminal domains. Taken together, it is concluded that the identical region including the hydrophobic segment of the C‐terminal domain is essential for both the client binding and dimer formation of the HSP90‐family molecular chaperone and that the dimeric configuration appears to be similar in the HSP90‐family proteins.
Kininogen‐derived peptides for investigating the putative vasoactive properties of human cathepsins K and L
Authors: Desmazes Claire , Galineau Laurent , Gauthier Francis , Brömme Dieter , Lalmanach Gilles
Published: March 2003

Macrophages at an inflammatory site release massive amounts of proteolytic enzymes, including lysosomal cysteine proteases, which colocalize with their circulating, tight‐binding inhibitors (cystatins, kininogens), so modifying the protease/antiprotease equilibrium in favor of enhanced proteolysis. We have explored the ability of human cathepsins B, K and L to participate in the production of kinins, using kininogens and synthetic peptides that mimic the insertion sites of bradykinin on human kininogens. Although both cathepsins processed high‐molecular weight kininogen under stoichiometric conditions, only cathepsin L generated significant amounts of immunoreactive kinins. Cathepsin L exhibited higher specificity constants (kcat/Km) than tissue kallikrein (hK1), and similar Michaelis constants towards kininogen‐derived synthetic substrates. A 20‐mer peptide, whose sequence encompassed kininogen residues Ile376 to Ile393, released bradykinin (BK; 80%) and Lys‐bradykinin (20%) when incubated with cathepsin L. By contrast, cathepsin K did not release any kinin, but a truncated kinin metabolite BK(5–9) [FSPFR(385–389)]. Accordingly cathepsin K rapidly produced BK(5–9) from bradykinin and Lys‐bradykinin, and BK(5–8) from des‐Arg9‐bradykinin, by cleaving the Gly384‐Phe385 bond. Data suggest that extracellular cysteine proteases may participate in the regulation of kinin levels at inflammatory sites, and clearly support that cathepsin K may act as a potent kininase.
Endotoxic activity and chemical structure of lipopolysaccharides from Chlamydia trachomatis serotypes E and L2 and Chlamydophila psittaci 6BC
Authors: Heine Holger , Müller‐Loennies Sven , Brade Lore , Lindner Buko , Brade Helmut
Published: March 2003

The lipopolysaccharide (LPS) of Chlamydia trachomatis serotype E was isolated from tissue culture‐grown elementary bodies and analyzed structurally by mass spectrometry and 1H, 13C and 31P nuclear magnetic resonance. The LPS is composed of the same pentasaccharide bisphosphate αKdo‐(2–8)‐αKdo‐(2–4)‐αKdo‐(2–6)‐βGlcN‐4P‐(1–6)‐αGlcN‐1P (Kdo is 3‐deoxy‐α‐d‐manno‐oct‐2‐ulosonic acid) as reported for C. trachomatis serotype L2[Rund, S., Lindner, B., Brade, H. and Holst, O. (1999) J. Biol. Chem. 274, 16819–16824]. The glucosamine disaccharide backbone is substituted with a complex mixture of fatty acids with ester or amide linkage whereby no ester‐linked hydroxy fatty acids were found. The LPS was purified carefully (with contaminations by protein or nucleic acids below 0.3%) and tested for its ability to induce proinflammatory cytokines in several readout systems in comparison to LPS from C. trachomatis serotype L2 and Chlamydophila psittaci strain 6BC as well as enterobacterial smooth and rough LPS and synthetic hexaacyl lipid A. The chlamydial LPS were at least 10 times less active than typical endotoxins; specificity of the activities was confirmed by inhibition with the LPS antagonist, B1233, or with monoclonal antibodies against chlamydial LPS. Like other LPS, the chlamydial LPS used toll‐like receptor TLR4 for signalling, but unlike other LPS activation was strictly CD14‐dependent.
Upstream and intronic regulatory sequences interact in the activation of the glutamine synthetase promoter
Authors: Garcia de Veas Lovillo Rocio M. , Ruijter Jan M. , Labruyère Wil T. , Hakvoort Theodorus B. M. , Lamers Wouter H.
Published: March 2003

Glutamine synthetase (GS) is expressed at high levels in subsets of cells in some tissues and at low levels in all cells of other tissues, suggesting that the GS gene is surrounded by multiple regulatory elements. We searched for such elements in the 2.5‐kb upstream region and in the 2.6‐kb first intron of the GS gene, using FTO‐2B hepatoma and C2/7 muscle cells as representatives of both cell types and transient transfection assays as our tools. In addition to the entire upstream region and entire intron, an upstream enhancer module at −2.5 kb, and 5′, middle and 3′ modules of the first intron were tested. The main effects of the respective modules and their combinatorial interactions were quantified using the analysis of variance (anova) technique. The upstream enhancer was strongly stimulatory, the middle intron module strongly inhibitory, and the 3′‐intron module weakly stimulatory in both hepatoma and muscle cells. The 5′‐intron module was strongly stimulatory in muscle cells only. The major new finding was that in both cell types, the upstream enhancer and 5′‐intron module needed to be present simultaneously to fully realize their transactivational potencies. This interaction was responsible for a pronounced inhibitory effect of the 5′‐intron module in the absence of the upstream enhancer in hepatoma cells, and for a strong synergistic effect of these two modules, when present simultaneously in muscle cells. The main difference between hepatoma and muscle cells therefore appeared to reside in tissue‐specific differences in activity of the respective regulatory elements due to interactions rather than in the existence of tissue‐specific regulatory elements.
Identification and functional expression of a second human β‐galactoside α2,6‐sialyltransferase, ST6Gal II
Authors: Krzewinski‐Recchi Marie‐Ange , Julien Sylvain , Juliant Sylvie , Teintenier‐Lelièvre Mélanie , Samyn‐Petit Bénédicte , Montiel Maria‐Dolores , Mir Anne‐Marie , Cerutti Martine , Harduin‐Lepers Anne , Delannoy Philippe
Published: March 2003

blast analysis of the human and mouse genome sequence databases using the sequence of the human CMP‐sialic acid:β‐galactoside α‐2,6‐sialyltransferase cDNA (hST6Gal I, EC2.4.99.1) as a probe allowed us to identify a putative sialyltransferase gene on chromosome 2. The sequence of the corresponding cDNA was also found as an expressed sequence tag of human brain. This gene contained a 1590 bp open reading frame divided in five exons and the deduced amino‐acid sequence didn't correspond to any sialyltransferase already known in other species. Multiple sequence alignment and subsequent phylogenic analysis showed that this new enzyme belonged to the ST6Gal subfamily and shared 48% identity with hST6Gal‐I. Consequently, we named this new sialyltransferase ST6Gal II. A construction in pFlag vector transfected in COS‐7 cells gave raise to a soluble active form of ST6Gal II. Enzymatic assays indicate that the best acceptor substrate of ST6Gal II was the free disaccharide Galβ1–4GlcNAc structure whereas ST6Gal I preferred Galβ1–4GlcNAc‐R disaccharide sequence linked to a protein. The α2,6‐linkage was confirmed by the increase of Sambucus nigra agglutinin‐lectin binding to the cell surface of CHO transfected with the cDNA encoding ST6Gal II and by specific sialidases treatment. In addition, the ST6Gal II gene showed a very tissue specific pattern of expression because it was found essentially in brain whereas ST6Gal I gene is ubiquitously expressed.
Nerve growth factor promotes activation of the α, β and γ isoforms of protein kinase B in PC12 pheochromocytoma cells
Authors: Andjelković Mirjana , Suidan Hana S. , Meier Roger , Frech Matthias , Alessi Dario R. , Hemmings Brian A.
Published: March 2003

The activation of phosphatidylinositol (PtdIns) 3‐kinase is considered to be a key event occurring after stimulation of cells with growth factors. The proto‐oncogenic protein kinase B (PKB; also known as RAC protein kinase or Akt) has recently been shown to be a downstream target of PtdIns 3‐kinase and may be involved in cell survival. We therefore asked whether stimulation of neuronal cells with nerve growth factor (NGF), on which certain types of neurons are dependent for survival, causes activation of PKB. Stimulation of serum‐starved PC12 rat pheochromocytoma cells with NGF caused an increase of up to 14‐fold in PKB activity. This activation was detected within 1 min of stimulation and occurred at NGF concentrations that are consistent with TrkA‐mediated signaling. PKB activation was accompanied by a decrease in electrophoretic mobility of the kinase, which is characteristic of phosphorylation. Both PKB activation and mobility changes were prevented by wortmannin, indicating the upstream involvement of PtdIns 3‐kinase in these events. Analyses employing isoform‐specific antibodies for immunoprecipitation suggested that all three isoforms of PKB (α, β and γ) are activated in response to NGF. G‐protein‐coupled‐receptor agonists, lysophosphatidic acid (lyso‐PtdH) and thrombin, which induce rapid neurite retraction, neither stimulated PKB activity, nor affected NGF‐induced or insulin‐induced kinase activation. Wortmannin treatment did not prevent neurite retraction induced by lyso‐PtdH or thrombin. These data suggest that PtdIns 3‐kinase and PKB are not involved in cytoskeletal changes mediated by the small GTPase Rho.
Demonstration of leukotriene‐C4 synthase in platelets and species distribution of the enzyme activity
Authors: Tornhamre Susanne , Sjölinder Mikael , Lindberg Åsa , Ericsson Inger , Näsman‐Glaser Barbro , Griffiths William J. , Lindgren Jan Åke
Published: March 2003

Human platelets have been demonstrated to possess leukotriene (LT)‐C4 synthase activity and may thus be involved in transcellular 5(S)‐hydroxy‐6(R)‐S‐glutathionyl‐7,9‐trans‐11,14‐cis‐eicosatetraenoic acid (LTC4) synthesis. In this study, platelets from seven different species were screened for LTC4 synthase activity. Very high enzyme activity was observed in suspensions of bovine platelets, with approximately 70 % conversion of 5(S)‐trans‐5,6‐oxido‐7,9‐trans‐11,14‐cis‐eicosatetraenoic acid (LTA4) to LTC4. The capacity of equine platelets to produce LTC4 was similar to that of human platelets. In addition, ovine, rabbit, and rat platelets also produced LTC4 after incubation with LTA4. The results demonstrate that LTC4 synthase activity is a common feature among platelets from various species. In contrast, porcine platelets failed to transform LTA4 to LTC4. Instead, these cells produced 5(S),12(R)‐dihydroxy‐6,14‐cis‐8,10‐trans‐eicosatetraenoic acid (LTB4), indicating the presence of LTA4 hydrolase in porcine platelets.

A protein with a molecular mass of approximately 18 kDa and LTC4 synthase activity was solubilised from lyophilised bovine platelet concentrates and purified to near homogeneity by affinity chromatography and gel filtration. The N‐terminal amino acid sequence of this protein was analysed and found to be almost identical to the corresponding sequence of human LTC4 synthase (17 of 18 amino acid residues identical). Kinetic analysis of partially purified bovine platelet LTC4 synthase revealed Km (for LTA4) and Vmax values of 3.3 μM and 521 nmol mg protein−1 min−1, respectively. In addition, the presence of a mRNA transcript encoding LTC4 synthase was demonstrated in equine platelets by reverse transcription (RT) PCR using primers derived from the human LTC4 synthase cDNA sequence. Cloning and sequencing of the PCR fragment corresponding to a region near the N‐terminus demonstrated very high identity between equine and human leukotriene‐C4 synthase in this region.

In summary, the present study establishes that platelets contain LTC4 synthase and indicates that this enzyme is widely distributed among platelets from various species.
Aggregates of an amphiphilic synthetic peptide bind and deliver all‐trans retinol and all‐trans retinoic acid into fibroblast cells
Authors: Pellegrin Pierre , Chaloin Laurent , Roustan Claude , Méry Jean , Bennes René
Published: March 2003

The structure and conformational behaviour of a vector peptide, designed by association of a fusion peptide and a nuclear localization sequence, are described. A β‐sheet domain is observed in which fluorescence measurements show that ten peptide molecules bind one all‐trans retinol or all‐trans retinoic acid molecule with a strong affinity (.cf2.K.cf2..cf1..es.cf1.′.rb.eid.rb = 40 nM). Stoichiometry and affinity of the binding can be compared with those of cellular retinoid binding proteins, the structure of which is an anti‐parallel β barrel. Analogy between the system under study and cellular retinoid‐binding proteins is discussed. Peptide‐helped internalization and subsequent perinuclear localization of retinol in human fibroblast cells confirm this analogy. Also, this last result shows that the peptide is an efficient carrier for insoluble substances like retinoids.
Chitotriosidase, a chitinase, and the 39‐kDa human cartilage glycoprotein, a chitin‐binding lectin, are homologues of family 18 glycosyl hydrolases secreted by human macrophages
Authors: Renkema G. Herma , Boot Rolf G. , Au Fung Lin , Donker‐Koopman Wilma E. , Strijland Anneke , Muijsers Anton O. , Hrebicek Martin , Aerts Johannes M. F. G.
Published: March 2003

In various mammals, enzymatically active and inactive members of family 18 glycosyl hydrolases, containing chitinases, have been identified. In man, chitotriosidase is the functional chitinolytic enzyme, whilst the homologous human cartilage 39‐kDa glycoprotein (HC gp‐39) does not exhibit chitinase activity and its function is unknown. This study establishes that HC gp‐39 is a chitin‐specific lectin. It is experimentally demonstrated that a single amino acid substitution in the catalytic centre of the 39‐kDa isoform of chitotriosidase, which generates a similar sequence to that in HC gp‐39, results in a loss of hydrolytic activity and creates the capacity to bind to chitin. The possible implication of the finding for chitinolytic and chitin‐binding proteins that are produced in high quantities by activated macrophages are discussed.
Changes in the proton potential and the cellular energetics of Escherichia coli during growth by aerobic and anaerobic respiration or by fermentation
Authors: Tran Quang Hon , Unden Gottfried
Published: March 2003

The energetic parameters of Escherichia coli were analyzed for the aerobic/anaerobic transition. The electrochemical proton potential (Δ p) across the cytoplasmic membrane was determined in the steady state of respiration with O2, nitrate, fumarate, dimethylsulfoxide (Me2SO), and for fermentation. With O2, a proton potential of −160 mV was obtained. For anaerobic respiration with nitrate, fumarate or Me2SO, Δ p decreased only slightly by about 20 mV in contrast to earlier assumptions, whereas Δ p dropped by approximately 40 mV during fermentation. Under all conditions, the membrane potential (Δ @KY) contributed the major portion to Δ p. The cellular ATP levels were highest for aerobic growth (about 13 μmol/g dry cells) and decreased to 3−6 μmol/g in anaerobic metabolism. Δ .cf2.G.cf2..cf1..es.cf1.′.cf5..rb.eiPhos.rb, however, was constant due to equivalent changes of the ADP contents. Transition to the stationary growth phase caused a massive drop in the ATP content. It is concluded that, during anaerobic respiration, the energetic situation for the bacteria is very similar to that for aerobic growth with respect to Δ .cf2.G.cf2..cf1..es.cf1.′.cf5..rb.eiPhos.rb and Δ p whereas, for fermentation, a significant decrease in Δ p was observed. The consequences for the cellular energetics and for the regulation of the aerobic/anaerobic transition are discussed.
Characterization of recombinant epidermal growth factor (EGF)‐like modules from vitamin‐K‐dependent protein S expressed in Spodoptera cells
Authors: Stenberg Yvonne , Drakenberg Torbjörn , Dahlbäck Björn , Stenflo Johan
Published: March 2003

Epidermal growth factor (EGF)‐like modules in protein S, a physiological anticoagulant protein that functions as a cofactor to activated protein C, have been expressed in Spodoptera cells using baculovirus. EGF modules 1‐3, 1‐4, 2‐3 and 2‐4 were produced on a preparative scale. The isolated modules were more than 95 % homogenous, as judged by sequence determination. 45Ca2+‐ligand blotting experiments indicated that recombinant proteins that contained the fourth EGF module, i.e. EGF 1‐4 and 2‐4, bound Ca2+ with high affinity. The 45Ca2+‐ligand blotting results, together with results of competitive binding experiments using monoclonal antibodies as structural probes, indicated that the recombinant proteins had been folded to a native conformation. EGF modules 1‐3 and 1‐4 inhibited the interaction between activated protein C and protein S, whereas modules 2‐3 and 2‐4 had no effect on this interaction. It is thus apparent that EGF module 1 is crucial for the interaction between protein S and activated protein C. Moreover, EGF modules 1‐4 were approximately 10‐fold more effective in inhibiting the interaction than modules 1‐3, suggesting a very weak interaction between module 4 and activated protein C or that this module is important to keep module 1 in a conformation that is optimal for interaction with activated protein C.
Cooperation of the DnaK and GroE chaperone systems in the folding pathway of plant ferredoxin‐NADP+ reductase expressed in Escherichia coli
Authors: Dionisi Hebe M. , Checa Susana K. , Krapp Adriana R. , Arakaki Adrián K. , Ceccarelli Eduardo A. , Carrillo Néstor , Viale Alejandro M.
Published: March 2003

The DnaK system is required for the productive folding of pea chloroplast ferredoxin‐NADP+ reductase (FNR) expressed in Escherichia coli. The formation of a mature active enzyme was severely impaired in E. coli dnaK, dnaJ or grpE mutants expressing either the cytosolic precursor of the reductase (preFNR) or the mature apoenzyme, and these forms aggregated extensively in these cells. Coexpression of dnaK from a multicopy plasmid in the dnaK‐null mutants restored preFNR processing and folding of FNR, rendering a mature‐sized active enzyme. Overexpression of GroESL chaperonins failed to prevent preFNR aggregation, but it restored productive folding of FNR in dnaK‐null mutants expressing the mature enzyme. Expression of preFNR in OmpT‐protease‐deficient E. coli cells resulted in the accumulation of the unprocessed precursor in the soluble fraction of the cells. The interaction of this soluble preFNR, but not the mature reductase, with DnaK and GroEL was evidenced by immunoprecipitation studies. We conclude that, in addition to the GroE chaperonins [Carrillo, N., Ceccarelli, E. A., Krapp, A. R., Boggio, S., Ferreyra, R. G. & Viale, A. M. (1992) J. Biol. Chem. 267, 15 537−15 541], the DnaK chaperone system plays a crucial role in the folding pathway of FNR.