bluuebunny/crossref_metadata_2025 huggingface dataset:
The multidrug/oligosaccharidyl‐lipid/polysaccharide (MOP) exporter superfamily
Authors: Hvorup Rikki N. , Winnen Brit , Chang Abraham B. , Jiang Yong , Zhou Xiao‐Feng , Saier Milton H.
Published: March 2003

The multidrug/oligosaccharidyl‐lipid/polysaccharide (MOP) exporter superfamily (TC #2.A.66) consists of four previously recognized families: (a) the ubiquitous multi‐drug and toxin extrusion (MATE) family; (b) the prokaryotic polysaccharide transporter (PST) family; (c) the eukaryotic oligosaccharidyl‐lipid flippase (OLF) family and (d) the bacterial mouse virulence factor family (MVF). Of these four families, only members of the MATE family have been shown to function mechanistically as secondary carriers, and no member of the MVF family has been shown to function as a transporter. Establishment of a common origin for the MATE, PST, OLF and MVF families suggests a common mechanism of action as secondary carriers catalyzing substrate/cation antiport. Most protein members of these four families exhibit 12 putative transmembrane α‐helical segments (TMSs), and several have been shown to have arisen by an internal gene duplication event; topological variation is observed for some members of the superfamily. The PST family is more closely related to the MATE, OLF and MVF families than any of these latter three families are related to each other. This fact leads to the suggestion that primordial proteins most closely related to the PST family were the evolutionary precursors of all members of the MOP superfamily. Here, phylogenetic trees and average hydropathy, similarity and amphipathicity plots for members of the four families are derived and provide detailed evolutionary and structural information about these proteins. We show that each family exhibits unique characteristics. For example, the MATE and PST families are characterized by numerous paralogues within a single organism (58 paralogues of the MATE family are present in Arabidopsis thaliana), while the OLF family consists exclusively of orthologues, and the MVF family consists primarily of orthologues. Only in the PST family has extensive lateral transfer of the encoding genes occurred, and in this family as well as the MVF family, topological variation is a characteristic feature. The results serve to define a large superfamily of transporters that we predict function to export substrates using a monovalent cation antiport mechanism.
β3‐Adrenoceptor knockout in C57BL/6J mice depresses the occurrence of brown adipocytes in white fat
Authors: Jimenez Maria , Barbatelli Giorgio , Allevi Roberta , Cinti Saverio , Seydoux Josiane , Giacobino Jean‐Paul , Muzzin Patrick , Preitner Frédéric
Published: March 2003

White and brown adipocytes are usually located in distinct depots; however, in response to cold, brown adipocytes appear in white fat. This response is mediated by β‐adrenoceptors but there is a controversy about the subtype(s) involved. In the present study, we exposed to cold β3‐adrenoceptor knockout mice (β3KO) on a C57BL/6J genetic background and measured in white adipose tissue the density of multilocular cells and the expression of the brown adipocyte marker uncoupling protein‐1 (UCP1). In brown fat of β3KO mice, UCP1 expression levels were normal at 24 °C as well as after a 10‐day cold exposure. Strikingly, under both conditions, in the white fat of β3KO mice the levels of UCP1 mRNA and protein as well as the density of multilocular cells were decreased. These results indicate that β3‐adrenoceptors play a major role in the appearance of brown adipocytes in white fat and suggest that the brown adipocytes present in white fat differ from those in brown fat.
The relationship between thermal stability and pH optimum studied with wild‐type and mutant Trichoderma reesei cellobiohydrolase Cel7A
Authors: Boer Harry , Koivula Anu
Published: March 2003

The major cellulase secreted by the filamentous fungus Trichoderma reesei is cellobiohydrolase Cel7A. Its three‐dimensional structure has been solved and various mutant enzymes produced. In order to study the potential use of T. reesei Cel7A in the alkaline pH range, the thermal stability of Cel7A was studied as a function of pH with the wild‐type and two mutant enzymes using different spectroscopic methods. Tryptophan fluorescence and CD measurements of the wild‐type enzyme show an optimal thermostability between pH 3.5–5.6 (Tm, 62 ± 2 °C), at which the highest enzymatic activity is also observed, and a gradual decrease in the stability at more alkaline pH values. A soluble substrate, cellotetraose, was shown to stabilize the protein fold both at optimal and alkaline pH. In addition, unfolding of the Cel7A enzyme and the release of the substrate seem to coincide at both acidic and alkaline pH, demonstrated by a change in the fluorescence emission maximum. CD measurements were used to show that the five point mutations (E223S/A224H/L225V/T226A/D262G) that together result in a more alkaline pH optimum [Becker, D., Braet, C., Brumer, H., III, Claeyssens, M., Divne, C., Fagerström, R.B., Harris, M., Jones, T.A., Kleywegt, G.J., Koivula, A., et al. (2001) Biochem. J.356, 19–30], destabilize the protein fold both at acidic and alkaline pH when compared with the wild‐type enzyme. In addition, an interesting time‐dependent fluorescence change, which was not observed by CD, was detected for the pH mutant. Our data show that in order to engineer more alkaline pH cellulases, a combination of mutations should be found, which both shift the pH optimum and at the same time improve the thermal stability at alkaline pH range.
Site‐directed mutagenesis of a loop at the active site of E1 (α2β2) of the pyruvate dehydrogenase complex
Authors: Fries Markus , Chauhan Hitesh J. , Domingo Gonzalo J. , Jung Hyo‐Il , Perham Richard N.
Published: March 2003

Limited proteolysis of the pyruvate decarboxylase (E1, α2β2) component of the pyruvate dehydrogenase (PDH) multienzyme complex of Bacillus stearothermophilus has indicated the importance for catalysis of a site (Tyr281‐Arg282) in the E1α subunit (Chauhan, H.J., Domingo, G.J., Jung, H.‐I. & Perham, R.N. (2000) Eur. J. Biochem. 267, 7158–7169). This site appears to be conserved in the α‐subunit of heterotetrameric E1s and multiple sequence alignments suggest that there are additional conserved amino‐acid residues in this region, part of a common pattern with the consensus sequence ‐YR‐H‐D‐YR‐DE‐. This region lies about 50 amino acids on the C‐terminal side of a 30‐residue motif previously recognized as involved in binding thiamin diphosphate (ThDP) in all ThDP‐dependent enzymes. The role of individual residues in this set of conserved amino acids in the E1α chain was investigated by means of site‐directed mutagenesis. We propose that particular residues are involved in: (a) binding the 2‐oxo acid substrate, (b) decarboxylation of the 2‐oxo acid and reductive acetylation of the tethered lipoyl domain in the PDH complex, (c) an ‘open–close’ mechanism of the active site, and (d) phosphorylation by the E1‐specific kinase (in eukaryotic PDH and branched chain 2‐oxo acid dehydrogenase complexes).
Modulation of DNA‐protein interactions in the P1 and P2 c‐myc promoters by two intercalating drugs
Authors: Vaquero Alejandro , Portugal José
Published: March 2003

Regulation of transcription from the oncogene c‐myc has an important role in the genesis of various tumors. Therefore, c‐myc is a potential target for chemotherapy by drugs which are able to modify its activity directly. In this article, we identify the binding sites in the P1 and P2 promoter regions of c‐myc for the intercalating antibiotics actinomycin D and elsamicin A. Gel retardation experiments indicate that actinomycin D or elsamicin A binding can inhibit the formation of several DNA‐protein complexes. However, relatively low concentrations of elsamicin A, but not actinomycin D, appear to increase the level of binding to the P1 promoter of a protein factor. Using pure Sp1 transcription factor and an oligonucleotide containing the Sp1 putative binding site, we determined that the binding enhancement induced by small amounts of elsamicin was on the Sp1‐DNA complex. Run‐off transcription experiments in vitro showed that the effect of elsamicin A on Sp1 binding is followed by the maintenance or a relative rise in transcription levels from the P1 promoter of c‐myc, while actinomycin D always inhibited the transcription from the P1 c‐myc promoter in a concentration‐dependent manner. Higher concentrations of elsamicin acted as an inhibitor of the transcription from the P1 start site but not from the P2.
The urokinase‐type‐plasminogen‐activator receptor (CD87) is a pleiotropic molecule
Authors: Dear Anthony E. , Medcalf Robert L.
Published: March 2003

Since its discovery over a decade ago, evidence has accumulated implicating the cell‐surface urokinase receptor (u‐PAR), in numerous biological processes. Most notable has been the identification of a critical role for u‐PAR in the regulation of cell‐surface plasminogen activation in physiological and pathological conditions. Recent evidence suggests that u‐PAR, a glycosylphosphatidylinositol‐linked receptor, lacking transmembrane and cytoplasmic domains, is also involved in processes not related to plasminogen activation, including cellular adhesion and the transmission of extracellular signals across the plasma membrane. Involvement of activated u‐PAR in these events identifies previously unsuspected roles for this molecule and defines a new field of research in u‐PAR biology. We discuss the molecular biology of u‐PAR together with the underlying mechanisms responsible for the novel functional roles recently ascribed to this pleiotropic molecule.
Purification, properties and in situ localization of the amphibolic enzymes	D‐ribulose 5‐phosphate 3‐epimerase and transketolase from spinach chloroplasts
Authors: Teige Markus , Melzer Michael , Süss Karl‐Heinz
Published: March 2003

The amphibolic enzymes D‐ribulose 5‐phosphate 3‐epimerase and transketolase have been purified from stroma extracts of spinach chloroplasts using ammonium sulfate fractionation and FPLC. For the native enzymes, a molecular mass of 180 kDa for epimerase and 160 kDa for transketolase was found and the molecular masses of the subunits was determined to be 23 kDa for epimerase and 74 kDa for transketolase. Protein sequencing of the purified chloroplast enzymes revealed the NH2‐terminal amino acid sequences of mature epimerase (NH2‐TSRVDKFSKSDIIVSP) and transketolase (NH2‐AAVEALESTDTDQLVEG). The enzymic properties of both enzymes such as Km values or pH optima, were found to be very similar to those for epimerases and transketolases from other sources, including yeast and animal cells. In contrast to the light‐activated enzymes of the Calvin cycle, the activity of these amphibolic enzymes was not redox‐dependent. Immunogold electron microscopy on spinach leaf thin sections revealed that about 90 % of the total epimerase and transketolase, and 96 % of the total chloroplast H+‐ATP synthase portion CF1 are associated with thylakoid membranes in situ. Ribulose‐1,5‐bisphosphate carboxylase/oxygenase, in contrast, was evenly distributed throughout chloroplasts. These and other results indicate that minor chloroplast enzymes are arranged in a thin layer on thylakoid membrane surfaces in vivo.
Functional analysis of the guanylate kinase‐like domain in the synapse‐associated protein SAP97
Authors: Kuhlendahl Sven , Spangenberg Oliver , Konrad Manfred , Kim Eunjoon , Garner Craig C.
Published: March 2003

SAP97 is a membrane cytoskeletal protein localized at the presynaptic nerve terminals of type 1 asymmetric synapses. It has been implicated in the assembly of synapses and in particular in the localization and clustering of ion channels. The C‐terminal GK domain of SAP97 shares a high degree of sequence similarity with low‐molecular‐mass guanylate kinases. These enzymes are involved in the guanine nucleotide metabolic cycle and in the maintenance of GTP/GDP pools required for example in Ras‐mediated cell signaling. It has therefore been hypothesized that SAP97 plays an essential role in cellular signaling by regulating the guanine nucleotide pools at synaptic junctions. Here, we test this hypothesis by assessing whether the GK domain in SAP97 encodes an authentic guanylate kinase. We show that the GK domain in and of itself does not encode an active guanylate kinase, that it cannot be activated by its binding partner GKAP and that flanking regions are not acting as inhibitory regulators for enzymatic activity. Thus, it would appear that the GK domain of SAP97 is not involved in the metabolism of guanine nucleotides required for signaling events.
Occurrence of two plastidic ATP/ADP transporters in Arabidopsis thaliana L
Authors: Möhlmann Torsten , Tjaden Joachim , Schwöppe Christian , Winkler Herbert H. , Kampfenkel Karlheinz , Neuhaus H. Ekkehard
Published: March 2003

Recently, we sequenced a cDNA clone from Arabidopsis thaliana L. encoding an ATP/ADP transporter protein (AATP1) located in the plastid envelope membrane. The deduced amino acid sequence of AATP1 exhibits a high degree of similarity (> 66 %) to the ATP/ADP transporter from the obligate intracellular gram‐negative bacterium Rickettsia prowazekii. Here we report a second plastidic ATP/ADP carrier from A. thaliana (AATP2). As deduced from the amino acid sequence, AATP2 exhibits 77.6 % identity to AATP1 and 36 % to the rickettsial protein. Hydropathy analysis indicates that all three translocators are highly hydrophobic membrane proteins, which exhibit marked similarities and differences. The AATP1 translocator lacks the sixth transmembrane domain that is present in AATP2 and the bacterial transporter in R. prowazekii. In contrast to AATP1 and the bacterial transport protein, only AATP2 exhibits a truncated C‐terminal end. To compare the general biochemical properties of AATP2 with the known transport properties of AATP1 we cloned the entire AATP2 cDNA into plasmid pJT118, leading to the presence of an additional N‐terminal histidine tag of 10 amino acids. For heterologous expression of His10‐AATP2 we chose the Escherichia coli strain C43, which was reported recently to allow overproduction of eucaryotic membrane transport proteins. After transformation and subsequent induction by isopropylthio‐2‐
D‐galactopyranoside intact E. coli cells harbouring plasmid pJT118 showed import of radioactively labelled ATP and ADP. As deduced from a Lineweaver‐Burk analysis His10‐AATP2 exhibited apparent Km values for ATP and ADP of 22 μM and 20 μM, respectively. Import of ADP into His10‐AATP2−expressing E. coli cells occurred at a rate of 24 nmol mg protein−1 h−1, which was about threefold faster than import of ATP. These biochemical characteristics are similar to transport properties of the heterologously expressed His10‐AATP1 protein.
Effect of reversible reactions on isotope label redistribution
Authors: Follstad Brian D. , Stephanopoulos Gregory
Published: March 2003

The pentose phosphate pathway plays several key roles in metabolism including supply of biosynthetic carbon skeletons and reducing power. Previous research has focused on determining the fluxes through the reactions of this pathway using carbon‐labeled substrates and models that make certain assumptions about the reversibility of the transketolase and transaldolase reactions in the nonoxidative pathway. These assumptions, however, have resulted in inconsistencies between the predicted carbon label distributions using these models and those determined experimentally. A general metabolic reaction network model developed in this paper and applied to the pentose phosphate pathway not only incorporates reaction reversibility but also accounts for the effect of individually varying extents of reaction reversibility on labeled carbon fractional enrichment values for intermediate metabolites. In addition, an algorithm is presented that can be used to calculate the three individual transaldolase and transketolase extents of reversibility. The results of this method show that varying extents of reaction reversibility have an observable effect on the metabolite carbon label distributions which can in turn affect flux calculation for other parts of the metabolic network such as the tricarboxylic acid cycle. In addition, the observability of reversibility extent and accuracy of flux calculations depend on the particular choice of metabolite carbon enrichments measured. In particular, [6‐13C]hexose 6‐phosphate and [4‐13C]erythrose 4‐phosphate carbon enrichment values resulting from [1‐13C]glucose feeding contained more information as compared to those from ribose 5‐phosphate. This analysis was applied to literature data of metabolite carbon labeling that resulted from supplying either 13C‐ or 14C‐enriched substrates to several cell types growing under various conditions. The specific activities of metabolite carbon atoms taken from rat epididymal adipose tissue, goosefish islet cells, Corynebacterium glutamicum, and Escherichia coli supplied with either [2‐14C]glucose or [1‐13C]glucose demonstrate how reversibility is present in the pentose phosphate pathway and the extents of reversibility can be estimated from labeled carbon data sets.
Galectin‐1 from ovine placenta
Authors: Iglesias M. Mercedes , Rabinovich Gabriel A. , Ivanovic Valeria , Sotomayor Claudia , Wolfenstein‐Todel Carlota
Published: March 2003

In the present study we report the amino‐acid sequence, carbohydrate specificity and overall biochemical and physicochemical properties of galectin‐1, a β‐galactoside‐binding lectin from ovine placenta. The complete amino‐acid sequence, obtained by tryptic and chymotryptic digestion, revealed that this carbohydrate‐binding protein shares all the absolutely preserved and critical residues found in other members of the mammalian galectin‐1 subfamily. Moreover, conformational changes induced by protein interaction with its specific disaccharide were investigated by fourth‐derivative spectral analysis, intrinsic tryptophan fluorescence measurements and circular dichroism. The first two methods indicated changes in the environment of aromatic residues, in agreement with the role of Trp in carbohydrate binding. The quenching of the fluorescence emission upon addition of lactose, allowed us to calculate the Kd for its interaction with the galectin, which was 0.157 ± 0.02 mM. The far‐ultraviolet CD spectra is consistent with the large extent of β‐sheet structure described for other galectins. Addition of lactose produced no significant changes, suggesting that it causes no modifications in the secondary structure of the lectin. In addition, we explored its potential cell‐growth inhibitory activity and implications in T‐cell death. Finally, we also provide evidence showing that antagonic properties of galectins‐1 and ‐3 are reciprocally neutralized in a natural mixture of both proteins, suggesting that they could play an important role in the regulation of cell proliferation and death, according to physiological requirements at particular developmental stages of the placenta, thus allowing successful pregnancy to occur.
Localization of carbohydrate chains of pig sperm ligand in the glycoprotein ZPB of egg zona pellucida
Authors: Kudo Katsuyasu , Yonezawa Naoto , Katsumata Toshiyuki , Aoki Hironori , Nakano Minoru
Published: March 2003

The three glycoproteins of pig zona pellucida (ZPA, ZPB and ZPC) can be separated into ZPA and a mixture of ZPB/ZPC by gel‐filtration HPLC. We have shown previously that the neutral complex‐type N‐linked carbohydrate chains obtained from ZPB/ZPC possess sperm‐binding activity. Intact ZPB and ZPC cannot be separated from each other unless acidic N‐acetyllactosamine regions of their carbohydrate chains are removed by endo‐β‐galactosidase digestion. The endo‐β‐galactosidase−digested ZPB retains the sperm‐binding activity. Recently, we have reported that N‐linked carbohydrate chains of N‐terminal fragment (residues 137−247) obtained from endo‐β‐galactosidase−digested ZPB are involved mainly in sperm binding [Yonezawa, N., Mitsui, S., Kudo, K. & Nakano, M. (1997) Eur. J. Biochem. 248, 86−92]. In this study, we separated the intact neutral N‐linked chains from the ZPB/ZPC mixture into diantennary chains and triantennary and tetraantennary chains by affinity chromatography on Concanavalia ensiformis agglutinin. An in vitro competition assay revealed that triantennary and tetraantennary chains possess a sperm‐binding activity stronger than that of diantennary chains. Three glycopeptides, having one Asn residue to which the carbohydrate chain is linked, were obtained by lysyl endopeptidase digestion of the heat‐solubilized zonae containing intact ZPB and lysyl endopeptidase and chymotrypsin A digestion of endo‐β‐galactosidase−digested ZPB. From sugar‐mapping analysis of the carbohydrate chains from these glycopeptides and comparison with the carbohydrate structures of the main intact neutral N‐linked chains of ZPB/ZPC, the triantennary and tetraantennary chains were shown to be localized mainly at Asn220 of ZPB, and diantennary chains were present on all the three potential residues (Asn203, Asn220 and Asn333). These results suggest that the carbohydrate chains linked to Asn220 of ZPB participate predominantly in sperm‐egg binding.
Selective reduction of 6‐O‐sulfation in heparan sulfate from transformed mammary epithelial cells
Authors: Safaiyan Fariba , Lindahl Ulf , Salmivirta Markku
Published: March 2003

Heparan sulfate at cell surfaces and in the extracellular matrix regulates cell proliferation and adhesion by binding to growth factors and matrix proteins via structurally specific oligosaccharide domains. We have used the hormonally regulated mouse mammary carcinoma cell line S115 as a model to elucidate the effect of malignant transformation on the structure of heparan sulfate. When cultured in the presence of testosterone, S115 cells form tumor cell colonies in soft agar and exhibit fibroblast‐like morphology; withdrawal of testosterone results in a loss of the tumorigenic capacity and a switch towards epithelial morphology. Metabolically 35SO4‐labeled heparan sulfate was isolated from testosterone‐treated and non‐treated S115 cells and subjected to structural analysis. We found that the testosterone‐dependent malignant transformation was associated with reduced sulfation of heparan sulfate due to a approximately 40 % decrease in the amount of GlcN6S units. By contrast, no significant differences were observed in the amounts of 2‐O‐sulfate or N‐sulfate groups. The reduced 6‐O‐sulfation of GlcN units in heparan sulfate from transformed S115 cells led to a marked decrease in the amount of trisulfated IdoA2S‐GlcNS6S units (IdoA,
L‐iduronic acid), implicated in many heparan sulfate‐protein interactions.
Enzymatic activity of two caspases related to interleukin‐1β‐converting enzyme
Authors: Fassy Florence , Krebs Odile , Rey Hélène , Komara Bentou , Gillard Corinne , Capdevila Cécile , Yea Christopher , Faucheu Chi , Blanchet Anne‐Marie , Miossec Christine , Diu‐Hercend Anita
Published: March 2003

Interleukin‐1β‐converting enzyme is a member of a family of human cysteine proteases with specificity for aspartic acid, which have been named caspases. Within this family of enzymes, transcript X (TX) and transcript Y (TY) (caspases 4 and 5, respectively) are very similar to ICE (caspase 1) and form the ICE subfamily. Given the high degree of conservation in the sequences of these proteases (more than 50 % amino acid identity in the mature enzymes), it was of interest to examine whether they shared similar substrate specificities. The three enzymes, ICE, TX and TY, were therefore expressed in baculovirus‐infected insect cells, as 30‐kDa proteins lacking the propeptide. Automaturation into p20 and p10 subunits occured within the cells. Active ICE, TX and TY were collected in the cell culture supernatants. In addition, their production induced the activation of an endogenous 32‐kDa putative cysteine protease (CPP32) like caspase. T7‐tagged ICE, TX and TY were purified by immunoaffinity and tested for their catalytic efficiency on YVAD‐containing synthetic substrates and on the ICE natural substrate, pro‐interleukin‐1β. TX cleaved the same synthetic substrates as ICE (Km of 90 μM and kcat of 0.4 s−1 for Suc‐YVAD‐NH‐Mec, where Suc represents succinyl and NH‐Mec represents amino‐4‐methylcoumarin) and could cleave pro‐interleukin‐1β into the same peptides as ICE but less efficiently. On the other hand, TY showed very little efficacy on the different ICE substrates (Km of 860 μM for Suc‐YVAD‐NH‐Mec). These results show that the ICE/TX/TY subfamily has functional heterogeneity and that ICE remains the preferred enzyme for pro‐interleukin‐1β cleavage.
Effect of single mutations on the structural dynamics of a DNA repair enzyme, the Escherichia coli formamidopyrimidine‐DNA glycosylase
Authors: Kuznetsov Serguei V. , Sidorkina Olga M. , Jurado Juan , Bazin Marc , Tauc Patrick , Brochon Jean‐Claude , Laval Jacques , Santus René
Published: March 2003

The effects on the structure dynamics of the Escherichia coli wild‐type formamidopyrimidine‐DNA glycosylase (Fpg) protein of the single mutations Lys57←Gly (FpgK57G), Pro2←Gly (FpgP2G) and Pro2←Glu (FpgP2E) were studied by fluorescence techniques, namely : lifetime measurements and acrylamide quenching of the fluorescence of Trp residues. The fluorescence decays of Fpg and its mutant forms were analysed by the maximum‐entropy method and lifetime distributions in the range 200 ps to 9 ns were obtained. The lifetime distribution profiles of FpgK57G, FpgP2G and FpgP2E are different from that of wild‐type Fpg. Both dynamic and static quenching by acrylamide were observed for all the proteins. At 20 +C, the bimolecular collisional quenching rate constant of the FpgP2E fluorescence by acrylamide was only 0.8 M−1 s−1 as compared to about 1.4 M−1 s−1 for the three other proteins. At 6 +C, all the spectroscopic properties of these four proteins are about the same. The analysis of experimental data demonstrates that all three mutations induce a structural reorganization of the Fpg protein. However, only the P2E mutation lead to a reduced accessibility of some Trp residues to acrylamide quenching. It is concluded that the single P2E replacement induces a conformational change leading to a more rigid globular structure as opposed to the wild type and K57G and P2G mutations. The influence of the single mutations on the enzyme activities of the Fpg protein is discussed.
Processing and release of tumor necrosis factor α
Authors: Watanabe Naoko , Nakada Kumiko , Kobayashi Yoshiro
Published: March 2003

Tumor necrosis factor α (TNFα) is synthesized as a transmembrane precursor form that is proteolytically processed and released as the soluble mature form. In human monocytes and monocytic cell lines, the production, processing, and release of TNFα are co‐induced by certain activators, such as lipopolysaccharide. To investigate the mechanism of TNFα processing and release, we established a cell line which constitutively produced TNFα, by transfecting the TNFα precursor form cDNA into NIH/3T3 cells. In these cells, the processing and release of TNFα were augmented by phorbol 12‐myristate 13‐acetate (PMA), mediated through a protein kinase C (PKC) signalling pathway. Various protease inhibitors were tested and it was found that matrix metalloproteinase (MMP) inhibitors blocked the processing and release of TNFα both in the absence and presence of PMA. This result is compatible with the recent reports that MMP are involved in the processing and release of TNFα. In contrast, 3,4‐dichloroisocoumarin, Nα‐p‐tosyl‐
L‐phenylalanine chloromethane, iodoacetamide, and o‐phenanthroline inhibited the processing and release of TNFα only in the presence of PMA, suggesting that serine proteases requiring SH for their activity, a combination of serine proteases and cysteine proteases, or MMP, may be involved in the PKC‐mediated induction of TNFα processing and release.
Glucocorticoids induce the expression of the leptin gene through a non‐classical mechanism of transcriptional activation
Authors: De Vos Piet , Lefebvre Anne‐Marie , Shrivo Iris , Fruchart Jean‐Charles , Auwerx Johan
Published: March 2003

Leptin is a hormone which is produced in adipose tissue and which plays a role in the regulation of energy homeostasis. The expression of the ob gene, encoding leptin, is under multi‐hormonal control. We have shown previously that high doses of glucocorticoids are positive regulators of leptin expression in rats and that they concomitantly reduce food intake and body mass gain in these animals. In the present report we analyse the molecular mechanism of this glucocorticoid regulation of leptin expression. In cultured rat adipocytes dexamethasone induces leptin mRNA levels, an effect not inhibited by the protein synthesis inhibitor cycloheximide. In addition, our data indicate that the induction of the expression of the leptin gene by dexamethasone is at least in part due to a transcriptional activation that is mediated by the glucocorticoid receptor. Deletion mapping of the human leptin promoter shows that cis‐elements involved in the glucocorticoid effect are located between −55 and +31 relative to the transcription initiation site. Since this region does not contain a binding site for the glucocorticoid receptor, the effect does not rely on the classical molecular mechanism of glucocorticoid receptor action. A role of C/EBP and Sp‐1 in mediating this glucocorticoid effect was furthermore excluded. Multiple nuclear factors from 3T3‐L1 preadipocytes interact with this promoter region of the human leptin gene and may be potential mediators of the induction by glucocorticoids.
Kinetic mechanism of vanillyl‐alcohol oxidase with short‐chain 4‐alkylphenols
Authors: Fraaije Marco W. , van den Heuvel Robert H. H. , Roelofs Jules C. A. A. , van Berkel Willem J. H.
Published: March 2003

The kinetic mechanism of vanillyl‐alcohol oxidase with 4‐methylphenol, 4‐ethylphenol, 4‐propylphenol and their Cα‐deuterated analogs has been studied at pH 7.5 and 25 +C. Conversion of 4‐methylphenol is extremely slow (0.005 s−1) while the enzyme is largely in the reduced form during turnover. 4‐Ethylphenol and 4‐propylphenol are readily converted while the enzyme is mainly in the oxidized form during turnover. The deuterium kinetic isotope effect for overall catalysis ranges between 7−10 whereas the intrinsic deuterium kinetic isotope effect for flavin reduction ranges over 9−10. With all three 4‐alkylphenols, flavin reduction appeared to be a reversible process with the rate of reduction being in the same range as the rate for the reverse reaction. During the reductive half‐reaction of vanillyl‐alcohol oxidase with 4‐ethylphenol and 4‐propylphenol, a transient intermediate is formed with an absorbance maximum at 330 nm. This intermediate has been tentatively identified as the p‐quinone methide of the aromatic substrate in complex with reduced enzyme. It is concluded that vanillyl‐alcohol oxidase catalysis with 4ethylphenol and 4‐propylphenol favors an ordered sequential binding mechanism in which the rate of flavin reduction determines the turnover rate while the reduced enzyme−p‐quinone methide binary complex rapidly reacts with dioxygen. During the reaction of vanillyl‐alcohol oxidase with 4‐methylphenol, a fluorescent enzyme species is stabilized. Based on its spectal characteristics and crystallographic data [Mattevi, A., Fraaije, M. W., Mozzarelli, A., Olivi, L., Coda, A. & van Berkel, W. J. H. (1997) Structure 5, 907−920], it is proposed that this species represents a covalent 5‐(4′‐hydroxybenzyl)‐FAD adduct. With 4‐ethylphenol and 4‐propylphenol, similar N5 flavin adducts may be formed but their rate of formation is too slow to be of catalytic relevance.
The 58‐kDa microspherule protein (MSP58), a nucleolar protein, interacts with nucleolar protein p120
Authors: Ren Yong , Busch Rose K. , Perlaky Laszlo , Busch Harris
Published: March 2003

Proteinp120 is a proliferation‐related nucleolar protein which is detectable early in the G1 phase of the cell cycle and peaks early in the S phase. Most human malignant tumors contain much higher levels of protein p120 than normal resting cells. To identify p120‐associated protein(s), a yeast two‐hybrid screen was carried out using protein p120 as the bait. Two positive clones encoded portions of a novel protein, designated microspherule protein 58 kDa (MSP58). MSP58 mRNA is 1.9 kb and encodes an approximately 58‐kDa polypeptide of 462 amino acids as shown by Western blotting of HeLa nucleolar proteins. The mouse MSP58 homolog has 97 % amino acid similarity to human MSP58, but no MSP58 homolog was found in the yeast genome. The MSP58 N‐terminal region contains serine‐rich clusters and its C‐terminal region has a coiled‐coil domain. In insect Sf9 cells, recombinant p120 and MSP58 proteins associated with each other, confirming the results of the yeast two‐hybrid assay. Deletion mutations revealed that the binding of MSP58 to p120 required a previously unrecognized coiled‐coil domain within the N‐terminal region of p120 and the C‐terminal region of MSP58 protein. Immunofluorescence indicated that the MSP58 protein is localized in microspherules in the nucleolus. Anti‐MSP58 Ig labeled nucleolar ’caps' when HeLa cells were treated with actinomycin D. When the MSP58 protein was overexpressed in COS‐7 cells, the nucleolus became irregularly enlarged, which suggests that MSP58 may affect the size and shape of the nucleolus.
Oxygen‐regulated erythropoietin gene expression is dependent on a CpG methylation‐free hypoxia‐inducible factor‐1 DNA‐binding site
Authors: Wenger Roland H. , Kvietikova Ivica , Rolfs Andreas , Camenisch Gieri , Gassmann Max
Published: March 2003

The hypoxia‐inducible factor‐1 (HIF‐1) is a transcriptional activator involved in the expression of oxygen‐regulated genes such as that for erythropoietin. Following exposure to low oxygen partial pressure (hypoxia), HIF‐1 binds to an hypoxia‐response element located 3′ to the erythropoietin gene and confers activation of erythropoietin expression. The conserved core HIF‐1 binding site (HBS) of the erythropoietin 3′ enhancer (CGTG) contains a CpG dinucleotide known to be a potential target of cytosine methylation. We found that methylation of the HBS abolishes HIF‐1 DNA binding as well as hypoxic reporter gene activation, suggesting that a methylation‐free HBS is mandatory for HIF‐1 function. The in vivo methylation pattern of the erythropoietin 3′ HBS in various human cell lines and mouse organs was assessed by genomic Southern blotting using a methylation‐sensitive restriction enzyme. Whereas this site was essentially methylation‐free in the erythropoietin‐producing cell line Hep3B, a direct correlation between erythropoietin protein expression and the degree of erythropoietin 3′ HBS methylation was found in different HepG2 sublines. However, the finding that this site is partially methylation‐free in human cell lines and mouse tissues that do not express erythropoietin suggests that there might be a general selective pressure to keep this site methylation‐free, independent of erythropoietin expression.
Structural basis of the chiral selectivity of Pseudomonas cepacia lipase
Authors: Lang Dietmar A. , Mannesse Maurice L. M. , De Haas Gerard H. , Verheij Hubertus M. , Dijkstra Bauke W.
Published: March 2003

To investigate the enantioselectivity of Pseudomonas cepacia lipase, inhibition studies were performed with SC‐ and RC‐(RP ,SP)‐1,2‐dialkylcarbamoylglycero‐3‐O‐p‐nitrophenyl alkylphosphonates of different alkyl chain lengths. P. cepacia lipase was most rapidly inactivated by RC‐(RP ,SP)‐1,2‐dioctylcarbamoylglycero‐3‐O‐p‐nitrophenyl octylphosphonate (RC‐trioctyl) with an inactivation half‐time of 75 min, while that for the SC‐(RP ,SP)‐1,2‐dioctylcarbamoylglycero‐3‐O‐p‐nitrophenyl octyl‐phosphonate (SC‐trioctyl) compound was 530 min. X‐ray structures were obtained of P. cepacia lipase after reaction with RC‐trioctyl to 0.29‐nm resolution at pH 4 and covalently modified with RC‐(RP ,SP)‐1,2‐dibutylcarbamoylglycero‐3‐O‐p‐nitrophenyl butyl‐phosphonate (RC‐tributyl) to 0.175‐nm resolution at pH 8.5. The three‐dimensional structures reveal that both triacylglycerol analogues had reacted with the active‐site Ser87, forming a covalent complex. The bound phosphorus atom shows the same chirality (SP) in both complexes despite the use of a racemic (RP ,SP) mixture at the phosphorus atom of the triacylglycerol analogues. In the structure of RC‐tributyl‐complexed P. cepacia lipase, the diacylglycerol moiety has been lost due to an aging reaction, and only the butyl phosphonate remains visible in the electron density. In the RC‐trioctyl complex the complete inhibitor is clearly defined; it adopts a bent tuning fork conformation. Unambiguously, four binding pockets for the triacylglycerol could be detected : an oxyanion hole and three pockets which accommodate the sn‐1, sn‐2, and sn‐3 fatty acid chains. Van der Waals' interactions are the main forces that keep the radyl groups of the triacylglycerol analogue in position and, in addition, a hydrogen bond to the carbonyl oxygen of the sn‐2 chain contributes to fixing the position of the inhibitor.
The ubiquitin‐protein ligase E6‐associated protein (E6‐AP) serves as its own substrate
Authors: Nuber Ulrike , Schwarz Sylvia E. , Scheffner Martin
Published: March 2003

Recognition of substrate proteins by the ubiquitin‐conjugation system is a highly specific and regulated event and involves the action of ubiquitin‐conjugating enzymes (E2) and ubiquitin‐protein ligases (E3). However, the E2 and E3 involved in the recognition of particular substrates have been identified in only a few cases. The ubiquitin‐protein ligase E6‐associated protein (E6‐AP) was originally identified as a protein involved in the human papillomavirus E6‐oncoprotein‐induced degradation of p53. The substrate proteins of E6‐AP in the absence of the E6 oncoprotein, however, have not been identified. We show here that E6‐AP can target itself for ubiquitination in vitro and provide evidence that, under conditions of overexpression, E6‐AP efficiently promotes its own degradation in vivo. Autoubiquitination of E6‐AP is mediated mainly by intermolecular transfer of ubiquitin. In addition, highly ubiquitinated forms of E6‐AP cannot bind to p53 in the presence of the E6 oncoprotein and, conversely, binding of E6‐AP to p53 interferes with ubiquitination of E6‐AP. These results suggest that autoubiquitination and subsequent degradation of E6‐AP represents a mechanism to control intracellular E6‐AP levels by inactivating E6‐AP molecules that are not bound to substrate proteins.
Characterization of a partially unfolded structure of cytochrome c induced by sodium dodecyl sulphate and the kinetics of its refolding
Authors: Das Tapan Kanti , Mazumdar Shyamalava , Mitra Samaresh
Published: March 2003

The mechanism of unfolding of ferricytochrome c induced by the surfactant sodium dodecyl sulfate has been studied by heme absorption, tryptophan fluorescence, circular dichroism, resonance Raman scattering, stopped‐flow and time‐resolved resonance energy transfer to obtain a comprehensive view of the whole process. Unfolding occurred at an almost specific molecular ratio of SDS/cytochrome c in the concentration range (20−50 μM) studied here. However there appears to be a point at ≈0.6 mM SDS where unfolding begins to occur for lower cytochrome c concentrations. The kinetics of unfolding revealed only a single transition with a rate constant of 33 s−1 (at 298 K, [SDS] = 8.7 mM) and activation energy barrier of ≈ 16 kJ/mol, indicating that other associated steps, if any, are too fast to be significantly populated. The free energy change (ΔG+) involved with the unfolding transition was estimated to be about 16.8 kJ/mol. The CD spectrum at 220 nm of SDS‐unfolded cytochrome c shows only a partial decrease (25 %), indicating that a significant amount of helical structure remains folded in contrast to a complete loss of helical structure in GdnHCl‐denatured cytochrome c. The heme structure in SDS‐unfolded cytochrome c, as deduced from heme absorption and resonance Raman spectra, shows a major population (≈95 %) of mis‐ligated histidine to the heme which acts as a kinetic trap in the folding process. The structural changes associated with cytochrome c unfolding were also monitored by time‐resolved resonance energy transfer which shows a drastic increase in tryptophan fluorescence lifetime from 12 ps in the native protein to 0.63 ns in the unfolded one, associated with a movement of Trp59 by 10 Å away from heme. The maximum entropy method analysis of fluorescence decay indicated the growth of various conformational substates in SDS‐unfolded cytochrome c in contrast to narrowly distributed conformations in the native protein. The refolding was comprised of three kinetic steps; the first was significantly fast (≈8 ms) and was assigned to the dissociation of His26 that paves the protein towards correct folding pathway. The other two slower steps probably arise from chain misorganization and prolyl isomerization. The absence of a burst‐phase amplitude supports the idea that the burst phase observed in the folding from completely unfolded cytochrome c corresponds to a molecular collapse that produces significant secondary structure. The partially unfolded state represents a unique intermediate state in the folding pathway.
The thermosome from Methanopyrus kandleri possesses an NH.es+.rb.ei4.rb‐dependent ATPase activity
Authors: Andrä Stefan , Frey Gerhard , Jaenicke Rainer , Stetter Karl O.
Published: March 2003

The ATPase activity of the thermosome from a methanogen, Methanopyrus kandleri, was characterized in detail. In contrast to all other known chaperonins, enzymatic ATP hydrolysis was found to be strictly dependent on high levels of ammonium salts in vitro. The ths gene encoding the thermosome subunit from the hyperthermophilic M. kandleri was functionally expressed in Escherichia coli and the overproduced polypeptide was assembled into intact thermosome complexes in the mesophilic host. The recombinant particles could be purified by a simple two‐step procedure including only one chromatographic step. Structural and biochemical properties of the recombinant protein were closely similar to those of the natural complex. Western blot analysis with an antiserum against the M. kandleri thermosome indicated the presence of at least two subfamilies of archaeal chaperonins.
Mutational analysis of residues forming hydrogen bonds in the Rieske [2Fe‐2S] cluster of the cytochrome bc1 complex in Paracoccus denitrificans
Authors: Schröter Thomas , Hatzfeld Oliver M. , Gemeinhardt Sabine , Korn Marcus , Friedrich Thorsten , Ludwig Bernd , Link Thomas A.
Published: March 2003

Two mutations (S157A and Y159F) in the Rieske iron‐sulfur subunit of the ubihydroquinone‐cytochrome c oxidoreductase from Paracoccus denitrificans have been characterized with respect to the protein and [2Fe‐2S] cluster stability, the enzyme activity and the redox potential of the [2Fe‐2S] cluster.

In the structure of the water‐soluble fragment of the Rieske iron‐sulfur protein of the bovine heart mitochondrial bc1 complex, both residues (S163 and Y165 in the bovine sequence) form the following hydrogen bonds to sulfur atoms in the [2Fe‐2S] cluster : Ser163 Oγ‐S‐1 and Tyr165 Oη‐Cys139 Sγ [Iwata, S., Saynovits, M., Link, T. A. & Michel, H. (1996) Structure 4, 567−579]. They are conserved in all known Rieske iron‐sulfur proteins from bc1 complexes including that from P. denitrificans.

Both amino acid exchanges, introduced as separate point mutations on plasmids containing the fbc operon, lead to a fully assembled three‐subunit complex in P. denitrificans, with a metal and heme content as well as subunit composition indistinguishable from the parental strain.

The purified complexes show decreased turnover numbers and redox potentials of the Rieske cluster. Whereas the turnover number of the bc1 complex isolated from the parental strain was 145 s−1, the turnover numbers for the mutants S157A and Y159F were 10 s−1 and 52 s−1, respectively, corresponding to 7 % and 36 % activity, respectively. The midpoint potential Em of the Rieske cluster at pH 6 and 5 +C was +360 mV for the bc1 complex from the parental strain; the values in the mutant complexes were +316 mV (Y159F) and +265 mV (S157A). Shifts of the g values in the electron paramagnetic resonance spectra indicate an altered electron distribution in the mutants compared to in the wild‐type protein.
Dehydroascorbic acid prevents apoptosis induced by oxidized low‐density lipoprotein in human monocyte‐derived macrophages
Authors: Asmis Reto , Wintergerst Eva S.
Published: March 2003

Human low‐density lipoprotein (LDL) oxidized with Cu2+ or the radical generator 2,2′‐azobis(2‐methyl‐propionamidine) hydrochloride (AAPH) induces apoptosis in mature human monocyte‐derived macrophages as assessed by staining with fluorescein‐isothiocyanate‐labeled annexin V, by terminal deoxynucleotidyltransferase‐mediated dUTP nick end labeling, and by staining of the 7A6 mitochondrial antigen. Oxidized LDL‐induced apoptosis was dose and time dependent and clearly distinct from apoptosis induced by serum deprivation. Human autologous serum and lipoprotein‐deficient human serum prevented apoptosis induced by oxidized LDL. Supplementation of serum‐free culture medium with 25 μM ascorbic or isoascorbic acid only partially protected macrophages from apoptosis, whereas dehydroascorbic acid (DHAA) completely inhibited apoptosis induced by either Cu2+‐ or AAPH‐oxidized LDL. Apoptosis was also inhibited by the structural analogue alloxan. Both cyclic multiketones dose‐dependently inhibited oxidized LDL‐induced apoptosis with IC50 in the submicromolar range. Prior loading of macrophages with ascorbic acid did not prevent the induction of apoptosis. Apoptosis was reduced by more than 90 % after treatment of oxidized LDL with DHAA, whereas after incubation with either ascorbic or isoascorbic acid there was no such reduction. Removal of free DHAA by gel filtration did not reverse the inactivation. Parameters of LDL oxidation such as electrophoretic mobility, α‐tocopherol content, thiobarbituric‐acid‐reactive subtances and lipid peroxide levels did not correlate to apoptotic activity. Also, binding and uptake of Texas‐red‐labeled oxidized LDL was not prevented by DHAA. Dithiothreitol‐treatment of oxidized LDL, however, reduced the apoptotic activity by 76 %. Our results suggest that oxidized thiols on apoB may be essential for the induction of apoptosis by oxidized LDL in human macrophages.
Alkaline unfolding and salt‐induced folding of bovine liver catalase at high pH
Authors: Prajapati Shashi , Bhakuni Vinod , Babu Kodali Ravindra , Jain Swatantra Kumar
Published: March 2003

We have studied the alkaline unfolding of bovine liver catalase and its dependence on ionic strength by enzymic activity measurements and a combination of optical methods like circular dichroism, fluorescence and absorption spectroscopies. Under conditions of high pH (11.5) and low ionic strength, the native tetrameric enzyme dissociates into monomers with complete loss of enzymic activity and a significant loss of α‐helical content. Increase in ionic strength by addition of salts like potassium chloride and sodium sulphate resulted in folding of alkaline‐unfolded enzyme by association of monomers to tetramer but with significantly different structural properties compared to native enzyme. The salt‐induced tetrameric intermediate is characterized by a significant exposure of the buried hydrophobic clusters and significantly reduced α‐helical content compared to the native enzyme. The refolding/reconstitution studies showed that the salt‐induced partially folded tetrameric intermediate shows significantly higher efficiency of refolding/reconstitution as compared to alkaline‐denatured catalase in the absence of salts. These studies suggest that folding of multimeric enzymes proceeds probably through the hydrophobic collapse of partially folded multimeric intermediate with exposed hydrophobic clusters.