bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification by site-directed mutagenesis of amino acids contributing to ligand-binding specificity or signal transduction properties of the human FP prostanoid receptor
Authors: NEUSCHÄFER-RUBE Frank , ENGEMAIER Eva , KOCH Sina , BÖER Ulrike , PÜSCHEL Gerhard P.
Published: April 2003

Prostanoid receptors belong to the class of heptahelical plasma membrane receptors. For the five prostanoids, eight receptor subtypes have been identified. They display an overall sequence similarity of roughly 30%. Based on sequence comparison, single amino acids in different subtypes of different species have previously been identified by site-directed mutagenesis or in hybrid receptors that appear to be essential for ligand binding or G-protein coupling. Based on this information, a series of mutants of the human FP receptor was generated and characterized in ligand-binding and second-messenger-formation studies. It was found that mutation of His-81 to Ala in transmembrane domain 2 and of Arg-291 to Leu in transmembrane domain 7, which are putative interaction partners for the prostanoid's carboxyl group, abolished ligand binding. Mutants in which Ser-263 in transmembrane domain 6 or Asp-300 in transmembrane domain 7 had been replaced by Ala or Gln, respectively, no longer discriminated between prostaglandins PGF2α and PGD2. Thus distortion of the topology of transmembrane domains 6 and 7 appears to interfere with the cyclopentane ring selectivity of the receptor. PGF2α-induced inositol formation was strongly reduced in the mutant Asp-300Gln, inferring a role for this residue in agonist-induced G-protein activation.
beta1 Integrin and alpha-dystroglycan binding sites are localized to different laminin-G-domain-like (LG) modules within the laminin alpha5 chain G domain
Authors: YU Hao , TALTS Jan F.
Published: April 2003

Laminins are a group of extracellular-matrix proteins important in development and disease. They are heterotrimers, and specific domains in the different chains have specialized functions. The G domain of the α5 chain has now been produced in transfected mammalian cells as single modules and two tandem arrays, α5LG1–3 and α5LG4–5 (LG is laminin G domain-like). Using these fragments we produced specific polyclonal antibodies functional in immunoblotting and immunofluorescence studies and in solid-phase assays. Both α5LG tandem arrays had physiologically relevant affinities for sulphated ligands such as heparin and sulphatides. Cells adhered to these fragments and acquired a spread morphology when plated on α5LG1–3. Binding of integrins α3β1 and α6β1 was localized to the α5LG1–3 modules, and α-dystroglycan binding was localized to the α5LG4–5 modules, thus locating these activities to different LG modules within the laminin α5 G domain. However, both these activities were of relatively low affinity, indicating that integrin-mediated cell adhesion to the laminin 10/11 α5G domain depends on contributions from the other chains of the heterotrimer and that high-affinity α-dystroglycan binding could be dependent on specific Ca2+-ion-co-ordinating amino acids absent from α5LG4–5.
The pro-urokinase plasminogen-activation system in the presence of serpin-type inhibitors and the urokinase receptor: rescue of activity through reciprocal pro-enzyme activation
Authors: BEHRENDT Niels , LIST Karin , ANDREASEN Peter A. , DANØ Keld
Published: April 2003

The reciprocal pro-enzyme activation system of plasmin, urokinase-type plasminogen activator (uPA) and their respective zymogens is a potent mechanism in the generation of extracellular proteolytic activity. Plasminogen activator inhibitor type 1 (PAI-1) acts as a negative regulator. This system is complicated by a poorly understood intrinsic reactivity of the uPA pro-enzyme (pro-uPA) before proteolytic activation, directed against both plasminogen and PAI-1. We have studied the integrated activation mechanism under the repression of PAI-1 in a purified system. A covalent reaction between pro-uPA and PAI-1 was positively demonstrated but the reaction of PAI-1 with two-chain uPA was found to be at least 1000-fold faster. However, in spite of this very fast inhibition, two-chain uPA still became the dominant plasminogen activator when plasminogen was incubated with pro-uPA and PAI-1. The activity pattern observed under these conditions revealed an initial lag phase, followed by a continuous generation of minute amounts of active two-chain uPA, this uPA having a short lifetime before inhibition but still succeeding to generate new plasmin activity, thus preventing a complete inactivation of the feedback system. This property of the activation system was retained even in the simultaneous presence of PAI-1 and α2-antiplasmin. Addition of soluble uPA receptor to the system did not change the role of pro-uPA and the same pattern was observed when pro-uPA was bound to the uPA receptor on U937 cells. The present mechanism maintains the system at standby level and may be triggered to increased activity without the need for an external initiating event.
3-Aminopropanal, formed during cerebral ischaemia, is a potent lysosomotropic neurotoxin
Authors: LI Wei , YUAN Xi-Ming , IVANOVA Svetlana , TRACEY Kevin J. , EATON John W. , BRUNK Ulf T.
Published: April 2003

Cytotoxic polyamine-derived amino aldehydes, formed during cerebral ischaemia, damage adjacent tissue (the so-called ‘penumbra') not subject to the initial ischaemic insult. One such product is 3-aminopropanal (3-AP), a potent cytotoxin that accumulates in ischaemic brain, although the precise mechanisms responsible for its formation are still unclear. More relevant to the present investigations, the mechanisms by which such a small aldehydic compound might be cytotoxic are also not known, but we hypothesized that 3-AP, having the structure of a weak lysosomotropic base, might concentrate within lysosomes, making these organelles a probable focus of initial toxicity. Indeed, 3-AP leads to lysosomal rupture of D384 glioma cells, a process which clearly precedes caspase activation and apoptotic cell death. Immunohistochemistry reveals that 3-AP concentrates in the lysosomal compartment and prevention of this accumulation by the lysosomotropic base ammonia, NH3, protects against 3-AP cytotoxicity by increasing lysosomal pH. A thiol compound, N-(2-mercaptopropionyl)glycine, reacts with and neutralizes 3-AP and significantly inhibits cytoxocity. Both amino and aldehyde functions of 3-AP are necessary for toxicity: the amino group confers lysosomotropism and the aldehyde is important for additional, presently unknown, reactions. We conclude that 3-AP exerts its toxic effects by accumulating intralysosomally, causing rupture of these organelles and releasing lysosomal enzymes which initiate caspase activation and apoptosis (or necrosis if the lysosomal rupture is extensive). These results may have implications for the development of new therapeutics designed to lessen secondary damage arising from focal cerebral ischaemia.
Tailoring tissue inhibitor of metalloproteinases-3 to overcome the weakening effects of the cysteine-rich domains of tumour necrosis factor-alpha converting enzyme
Authors: LEE Meng-Huee , DODDS Philippa , VERMA Vandana , MASKOS Klaus , KNÄUPER Vera , MURPHY Gillian
Published: April 2003

Tumour necrosis factor-α (TNF-α) converting enzyme (TACE) is a membrane-anchored, multiple-domain zinc metalloproteinase responsible for the release of the potent pro-inflammatory cytokine, TNF-α. The extracellular part of the active enzyme is composed of a catalytic domain and several cysteine-rich domains. Previously, we reported that these cysteine-rich domains significantly weakened the inhibitory potency of the N-terminal-domain form of tissue inhibitor of metalloproteinases-3 (N-TIMP-3). In the present paper, we describe a novel strategy developed to overcome this weakening effect. We have engineered a new generation of N-TIMP-3 mutants that are capable of withstanding the repulsion of the cysteine-rich domains by the formation of electrostatic bonds with the catalytic domain of the enzyme. These N-TIMP-3 mutants displayed markedly improved binding affinity with the soluble extracellular domain form of recombinant TACE. With Ki (app) values of <0.1nM, these mutants were dramatically better than the wild-type N-TIMP-3 [Ki (app) 1.7nM]. We accounted for this by proposing that Glu31, an acidic residue situated at the base of the AB-loop of N-TIMP-3, is drawn into contact with Lys315, a prominent basic residue adjacent to the TACE catalytic site. The mutagenesis strategy involved reorientation of the edge of N-TIMP-3; in particular, the β-strand A where Glu31 was located. Further expression of one of the mutants, Lys26/27/30/76→Glu, in a mammalian expression system confirmed that TIMP-3 associates with the extracellular matrix via its C-terminal domain.
Complement C1r and C1s genes are duplicated in the mouse: differential expression generates alternative isomorphs in the liver and in the male reproductive system
Authors: GARNIER Gérard , CIRCOLO Antonella , XU Yuanyuan , VOLANAKIS John E.
Published: April 2003

C1r and C1s are the serine proteases that form the catalytic unit of the C1 complex, the first component of complement. In the present study, we found that the genes encoding murine C1r and C1s are duplicated. One set of these genes, referred to as c1rA and c1sA, are primarily expressed in the liver and are therefore the homologues of the human C1r and C1s genes. The other two genes, termed c1rB and c1sB, are expressed exclusively in male reproductive tissues, specifically the coagulating gland and the prostate. The predicted C1rB and C1sB proteins share 96 and 93% amino acid identity with C1rA and C1sA respectively. Most of the substitutions are clustered in the serine protease domains, suggesting differences in catalytic efficiencies and/or substrate specificities or alternatively adaptation to different physiological environments. The high homology of C1rB and C1sB with C1rA and C1sA in the non-catalytic regions indicates that they are probably capable of assembling the C1 complex. The expression of alternative genes encoding isomorphs of activating components of complement in male reproductive tissues raises the possibility of new mechanisms of complement activation in the male genital tract or of novel functions for complement proteases in reproduction.
Adapter protein with a pleckstrin homology (PH) and an Src homology 2 (SH2) domain (APS) and SH2-B enhance insulin-receptor autophosphorylation, extracellular-signal-regulated kinase and phosphoinositide 3-kinase-dependent signalling
Authors: AHMED Zamal , PILLAY Tahir S.
Published: April 2003

Adapter protein with a pleckstrin homology (PH) and an Src homology 2 (SH2) domain (APS) and SH2-B are adapter proteins and substrates that interact with the activation loop of the insulin-receptor (IR) kinase. These proteins are homologous and share substantial sequence similarity. We previously showed [Ahmed, Smith and Pillay, FEBS Lett. 475, 31–34], for the first time, that insulin-stimulated phosphorylation of APS led to interaction with c-Cbl in 3T3-L1 adipocytes and in transfected Chinese-hamster ovary (CHO) cells. In the present study, we find that insulin stimulates the membrane translocation and phosphorylation of APS to a much greater extent than SH2-B, despite the structural similarity of these proteins. Expression of APS or SH2-B delays IR tyrosine and IR substrate (IRS) dephosphorylation. This enhancement of signalling is also observed downsteam of the receptor. In control cells that lack APS, following insulin stimulation, extracellular-signal-regulated kinase (ERK) and Akt kinase reach maximal activation and then decline to basal levels by 60min. In contrast, in APS- and SH2-B-expressing cells, ERK and Akt kinase activation remains at peak levels at 60min. These effects may occur because these proteins either stabilize the active conformation or prevent dephosphorylation of the IR. We therefore conclude that, despite the ability to couple to c-Cbl, APS functions as a positive regulator of IR signalling and, although SH2-B is a poor substrate for the IR, its association with the IR allows it to regulate pathways downstream of the receptor independently of its phosphorylation.
Catalytic properties and inhibition of proline-specific dipeptidyl peptidases II, IV and VII
Authors: LEITING Barbara , PRYOR KellyAnn D. , WU Joseph K. , MARSILIO Frank , PATEL Reshma A. , CRAIK Charles S. , ELLMAN Jonathan A. , CUMMINGS Richard T. , THORNBERRY Nancy A.
Published: April 2003

There is currently intense interest in the emerging group of proline-specific dipeptidases, and their roles in the regulation of biological processes. Dipeptidyl peptidase IV (DPP-IV) is involved in glucose metabolism by contributing to the regulation of glucagon family peptides and has emerged as a potential target for the treatment of metabolic diseases. Two other proline-specific dipeptidases, DPP-VII (also known as quiescent cell proline dipeptidase) and DPP-II, have unknown functions and have recently been suggested to be identical proteases based on a sequence comparison of human DPP-VII and rat DPP-II (78% identity) [Araki, Li, Yamamoto, Haneda, Nishi, Kikkawa and Ohkubo (2001) J. Biochem. 129, 279–288; Fukasawa, Fukasawa, Higaki, Shiina, Ohno, Ito, Otogoto and Ota (2001) Biochem. J. 353, 283–290]. To facilitate the identification of selective substrates and inhibitors for these enzymes, a complete biochemical profile of these enzymes was obtained. The pH profiles, substrate specificities as determined by positional scanning, Michaelis–Menten constants and inhibition profiles for DPP-VII and DPP-II were shown to be virtually identical, strongly supporting the hypothesis that they are the same protease. In addition, substrate specificities, catalytic constants and IC50 values were shown to be markedly different from those of DPP-IV. Selective DPP-IV and DPP-VII substrates were identified and they can be used to design selective inhibitors and probe further into the biology of these enzymes.
Physical interaction of tumour suppressor p53/p73 with CCAAT-binding transcription factor 2 (CTF2) and differential regulation of human high-mobility group 1 (HMG1) gene expression
Authors: URAMOTO Hidetaka , IZUMI Hiroto , NAGATANI Gunji , OHMORI Haruki , NAGASUE Naofumi , ISE Tomoko , YOSHIDA Takeshi , YASUMOTO Kosei , KOHNO Kimitoshi
Published: April 2003

The CCAAT-binding transcription factor (CTF)/nuclear factor I (NF-I) group of cellular DNA-binding proteins recognizes the sequence GCCAAT and is implicated in eukaryotic transcription, as well as DNA replication. Molecular analysis of human CTF/NF-I cDNA clones revealed multiple mRNA species that contain alternative coding regions, apparently as a result of differential splicing. Expression and functional analysis established that individual gene products can bind to GCCAAT recognition sites and serve as both promoter-selective transcriptional activators and initiation factors for DNA replication. The interaction between CTF2 and p53/p73 was shown to modulate their ability to regulate transcription of their respective target genes. In the present paper, we report that p53 down-regulates the activity of the high mobility group 1 (HMG1) gene promoter, whereas p73α up-regulates the activity of this promoter. Furthermore, CTF2 transactivates p53-induced p21 promoter activity, but inhibits p73α-induced p21 promoter activity. Using deletion mutants, we found that the DNA-binding domains of both p53 and p73α are required for physical interaction with CTF2 via the regions between amino acid residues 161 and 223, and 228 and 312 respectively. CTF2 enhances the DNA-binding activity of p53 and inhibits the DNA-binding activity of p73α. These results provide novel information on the functional interplay between CTF2 and p53/p73 as important determinants of their function in cell proliferation, apoptosis, DNA repair and cisplatin resistance.
Intersubunit interaction and catalytic activity of catechol 2,3-dioxygenases
Authors: OKUTA Akiko , OHNISHI Kouhei , YAGAME Sakiko , HARAYAMA Shigeaki
Published: April 2003

Catechol 2,3-dioxygenases (C23Os; EC 1.3.11.2) form a large protein family that is divided into several subgroups. Amino acid sequences of C23Os belonging to subgroup I.2.A and those belonging to I.2.B are found to be approx. 50% identical. When the central parts of the C23O sequences belonging to I.2.B were fused with the N-terminal and C-terminal sequences of I.2.A C23O, the hybrid enzymes were not active. To understand why these hybrid C23Os were inactive, hybrids between XylEP (C23O found in a Pseudomonas strain; subgroup I.2.A) and XylES (C23O found in a Sphingomonas strain; subgroup I.2.B) were constructed. HB3-C23O consisted mostly of the XylES sequence, except that its C-terminal end was derived from XylEP. While HB3-C23O was not active, HB4-C23O, carrying shorter C-terminal XylEP sequences than HB3-C23O, was active. This observation indicated that certain amino acid residues at the C-terminus were crucial for C23O activity in the hybrid forms of enzymes between XylEP and XylES. According to the crystal structure of XylEP, the C-terminal region is involved in the formation of a quaternary structure. Amino acid differences between HB3-C23O and HB4-C23O included the specific β-strand for oligomerization. Thus the quaternary structures of active C23Os, XylES, XylEP and HB4-C23O, as well as that of inactive HB3-C23O, were examined. Active enzymes XylES, XylEP and HB4-C23O were homotetrameric, while HB3-C23O existed only as a monomer. We concluded that hybrids of subgroups I.2.A and I.2.B were often inactive because of a defect in their oligomerization.
Cell-cycle arrest in Jurkat leukaemic cells: a possible role for docosahexaenoic acid
Authors: SIDDIQUI Rafat A. , JENSKI Laura J. , HARVEY Kevin A. , WIESEHAN Jacqueline D. , STILLWELL William , ZALOGA Gary P.
Published: April 2003

Docosahexaenoic acid (DHA) is known to have anti-cancer activities by mechanisms that are not well understood. In the present study, we test one possible pathway for DHA action in Jurkat leukaemic cells. Low doses of DHA (10μM) are shown to induce cell-cycle arrest, whereas higher doses are cytotoxic. However, when cells that were pre-treated with 10μM DHA are given an additional 10μM DHA dose, cell viability rapidly decreases. Immunoblotting reveals that repeated low doses of DHA results in activation of caspase 3, implying induction of apoptosis. DHA (10μM) is shown to increase ceramide levels after 6h of incubation and, after 24h, the cells appear to be arrested in S phase. With DHA, the amount of phosphorylated retinoblastoma protein (pRb) decreases significantly. Western blot analysis also shows that DHA greatly reduces the level of cyclin A, while increasing the level of p21 WAF1, a cellular inhibitor of cyclin A/cyclin-dependent kinase 2 (cdk2) activity. Furthermore, the observed DHA-induced doubling of the ratio of hypophosphorylated pRb (hypo-pRb) to total pRb is inhibited by tautomycin and phosphatidic acid (PA), known inhibitors of protein phosphatase 1 (PP1), and by the PP2 inhibitor okadaic acid. The present study demonstrates one possible connected pathway for DHA action. By this pathway, low doses of DHA increase ceramide levels, which leads to inhibition of cdk2 activity and stimulation of PP1 and PP2A. The net effect of cdk2 inhibition and protein phosphatase activation is an inhibition of pRb phosphorylation, consequently arresting Jurkat cell growth.
A proteomic approach to the identification of heterogeneous nuclear ribonucleoproteins as a new family of poly(ADP-ribose)-binding proteins
Authors: GAGNÉ Jean-Philippe , HUNTER Joanna M. , LABRECQUE Benoît , CHABOT Benoît , POIRIER Guy G.
Published: April 2003

A new class of poly(ADP-ribose) (pADPr)-binding proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), has been identified by a proteomic approach using matrix-assisted laser-desorption–ionization time-of-flight ('MALDI-TOF') MS. Liquid-phase isoelectric focusing with a Rotofor® cell (Bio-Rad) allowed pre-fractionation of proteins extracted from HeLa cells. Rotofor® protein fractions were further separated by SDS/PAGE and then transferred to a PVDF membrane. pADPr-binding proteins were analysed by autoradiography of the protein blot after incubation with 32P-labelled automodified pADPr polymerase-1 (PARP-1). Peptide mass fingerprinting of selected bands identified the most abundant pADPr-binding proteins as hnRNPs, a family of proteins that bind pre-mRNA into functional complexes involved in mRNA maturation and transport to the cytoplasm. Sequence homology database searching against a previously reported pADPr-binding sequence motif revealed that the hnRNPs contain a putative pADPr-binding sequence pattern [Pleschke, Kleczkowska, Strohm and Althaus (2000) J. Biol. Chem. 275, 40974–40980]. pADPr-binding assays performed with synthetic peptides by the dot-blot technique and with nitrocellulose-transferred recombinant hnRNPs confirmed the pADPr-binding protein identification and the specificity of the interaction. These results could establish a link between increased levels of pADPr in DNA damaged cells and the modified protein expression pattern resulting from altered mRNA trafficking.
Characterization of the Aspergillus nidulans transporters for the siderophores enterobactin and triacetylfusarinine C
Authors: HAAS Hubertus , SCHOESER Michelle , LESUISSE Emmanuel , ERNST Joachim F. , PARSON Walther , ABT Beate , WINKELMANN Günther , OBEREGGER Harald
Published: April 2003

The filamentous ascomyceteAspergillus nidulans produces three major siderophores: fusigen, triacetylfusarinine C, and ferricrocin. Biosynthesis and uptake of iron from these siderophores, as well as from various heterologous siderophores, is repressed by iron and this regulation is mediated in part by the transcriptional repressor SREA. Recently we have characterized a putative siderophore-transporter-encoding gene (mirA). Here we present the characterization of two further SREA- and iron-regulated paralogues (mirB and mirC), including the chromosomal localization and the complete exon/intron structure. Expression of mirA and mirB in a Saccharomyces cerevisiae strain, which lacks high affinity iron transport systems, showed that MIRA transports specifically the heterologous siderophore enterobactin and that MIRB transports exclusively the native siderophore triacetylfusarinine C. Construction and analysis of an A. nidulans mirA deletion mutant confirmed the substrate specificity of MIRA. Phylogenetic analysis of the available sequences suggests that the split of the species A. nidulans and S. cerevisiae predates the divergence of the paralogous Aspergillus siderophore transporters.
Aorsin, a novel serine proteinase with trypsin-like specificity at acidic pH
Authors: LEE Byung Rho , FURUKAWA Masato , YAMASHITA Koichiro , KANASUGI Yurie , KAWABATA Choko , HIRANO Kenichi , ANDO Kenichi , ICHISHIMA Eiji
Published: April 2003

A proteinase that hydrolyses clupeine and salmine at acidic pH, called aorsin, was found in the fungus Aspergillus oryzae. Purified aorsin also hydrolysed benzyloxycarbonyl-Arg-Arg-4-methylcoumaryl-7-amide optimally at pH4.0. The specificity of aorsin appeared to require a basic residue at the P1 position and to prefer paired basic residues. Aorsin activated plasminogen and converted trypsinogen to trypsin. The trypsin-like activity was inhibited strongly by antipain or leupeptin, but was not inhibited by any other standard inhibitors of peptidases. To identify the catalytic residues of aorsin, a gene was cloned and an expression system was established. The predicted mature protein of aorsin was 35% identical with the classical late-infantile neuronal ceroid lipofuscinosis protein CLN2p and was 24% identical with Pseudomonas serine-carboxyl proteinase, both of which are pepstatin-insensitive carboxyl proteinases. Several putative catalytic residues were mutated. The kcat/Km values of the mutant enzymes Glu86→Gln, Asp211→Asn and Ser354→Thr were 3–4 orders of magnitude lower and Asp90→Asn was 21-fold lower than that of wild-type aorsin, indicating that the positions are important for catalysis. Aorsin is another of the S53 family serine-carboxyl proteinases that are not inhibited by pepstatin.
Requirement for direct cross-talk between B1 and B2 kinin receptors for the proliferation of androgen-insensitive prostate cancer PC3 cells
Authors: BARKI-HARRINGTON Liza , BOOKOUT Angela L. , WANG Gaofeng , LAMB Maria E. , LEEB-LUNDBERG L.M. Fredrik , DAAKA Yehia
Published: April 2003

Stimulation of endogenous kinin receptors promotes growth of androgen-independent prostate cancer PC3 cells via activation of the mitogenic extracellular-signal-regulated kinase (ERK) pathway. In the present study, we show that kinin-mediated mitogenic signalling and prostate-cell growth involves two subtypes of bradykinin (BK) receptors, B1R and B2R. Specific stimulation of either B1R or B2R by their respective agonists des-Arg9-BK and Lys-BK promoted ERK activation and cell growth, whereas selective blockade with specific antagonists des-Arg9-[Leu8]BK and Hoe 140 respectively obliterated this effect, indicating the presence of both receptor subtypes. However, blockade of B1R also inhibited B2R-mediated ERK activation and cell growth, and, similarly, antagonism of B2R inhibited the B1R-mediated response. Furthermore, both B1R and B2R agonists promoted internalization of B1R, whereas both receptor antagonists blocked this effect. The B1R ligands des-Arg9-BK and des-Arg9-[Leu8]BK had no effect on the binding of BK to B2R, as demonstrated by radioligand competitive binding studies. However, blockade of either B1R or B2R impaired the ability of the reciprocal receptor to produce inositol phosphates, suggesting that the interaction between B1R and B2R is proximal to activation of phospholipase C. These results provide evidence for the existence of B1R–B2R complexes in prostate cancer PC3 cells and demonstrate that antagonism of one receptor interferes with the signalling ability of the other, possibly at the level of receptor–Gαq protein coupling. Selective inhibition of B1R, which is up-regulated in injured and cancerous tissue, may be beneficial for the treatment of advanced prostate cancer.
The Fn14 cytoplasmic tail binds tumour-necrosis-factor-receptor-associated factors 1, 2, 3 and 5 and mediates nuclear factor-kappaB activation
Authors: BROWN Sharron A.N. , RICHARDS Christine M. , HANSCOM Heather N. , FENG Sheau-Line Y. , WINKLES Jeffrey A.
Published: April 2003

Fn14 is a growth-factor-inducible immediate-early-response gene encoding a 102-amino-acid type I transmembrane protein. The human Fn14 protein was recently identified as a cell-surface receptor for the tumour necrosis factor (TNF) superfamily member named TWEAK (TNF-like weak inducer of apoptosis). In the present paper, we report that the human TWEAK extracellular domain can also bind the murine Fn14 protein. Furthermore, site-specific mutagenesis and directed yeast two-hybrid interaction assays revealed that the TNFR-associated factor (TRAF) 1, 2, 3 and 5 adaptor molecules bind the murine Fn14 cytoplasmic tail at an overlapping, but non-identical, amino acid sequence motif. We also found that TWEAK treatment of quiescent NIH 3T3 cells stimulates inhibitory κBα phosphorylation and transcriptional activation of a nuclear factor-κB (NF-κB) enhancer/luciferase reporter construct. Fn14 overexpression in transiently transfected NIH 3T3 cells also promotes NF-κB activation, and this cellular response requires an intact TRAF binding site. These results indicate that Fn14 is a functional TWEAK receptor that can associate with four distinct TRAF family members and stimulate the NF-κB transcription factor signalling pathway.
Haematopoietic lineage cell-specific protein 1 (HS1) promotes actin-related protein (Arp) 2/3 complex-mediated actin polymerization
Authors: URUNO Takehito , ZHANG Peijun , LIU Jiali , HAO Jian-Jiang , ZHAN Xi
Published: April 2003

HS1 (haematopoietic lineage cell-specific gene protein 1), a prominent substrate of intracellular protein tyrosine kinases in haematopoietic cells, is implicated in the immune response to extracellular stimuli and in cell differentiation induced by cytokines. Although HS1 contains a 37-amino acid tandem repeat motif and a C-terminal Src homology 3 domain and is closely related to the cortical-actin-associated protein cortactin, it lacks the fourth repeat that has been shown to be essential for cortactin binding to filamentous actin (F-actin). In this study, we examined the possible role of HS1 in the regulation of the actin cytoskeleton. Immunofluorescent staining demonstrated that HS1 co-localizes in the cytoplasm of cells with actin-related protein (Arp) 2/3 complex, the primary component of the cellular machinery responsible for de novo actin assembly. Furthermore, recombinant HS1 binds directly to Arp2/3 complex with an equilibrium dissociation constant (Kd) of 880nM. Although HS1 is a modest F-actin-binding protein with a Kd of 400nM, it increases the rate of the actin assembly mediated by Arp2/3 complex, and promotes the formation of branched actin filaments induced by Arp2/3 complex and a constitutively activated peptide of N-WASP (neural Wiskott–Aldrich syndrome protein). Our data suggest that HS1, like cortactin, plays an important role in the modulation of actin assembly.
Deglycosylation, processing and crystallization of human testis angiotensin-converting enzyme
Authors: GORDON Kerry , REDELINGHUYS Pierre , SCHWAGER Sylva L.U. , EHLERS Mario R.W. , PAPAGEORGIOU Anastassios C. , NATESH Ramanathan , ACHARYA K. Ravi , STURROCK Edward D.
Published: April 2003

Angiotensin I-converting enzyme (ACE) is a highly glycosylated type I integral membrane protein. A series of underglycosylated testicular ACE (tACE) glycoforms, lacking between one and five N-linked glycosylation sites, were used to assess the role of glycosylation in tACE processing, crystallization and enzyme activity. Whereas underglycosylated glycoforms showed differences in expression and processing, their kinetic parameters were similar to that of native tACE. N-glycosylation of Asn-72 or Asn-109 was necessary and sufficient for the production of enzymically active tACE but glycosylation of Asn-90 alone resulted in rapid intracellular degradation. All mutants showed similar levels of phorbol ester stimulation and were solubilized at the same juxtamembrane cleavage site as the native enzyme. Two mutants, tACEΔ36-g1234 and -g13, were successfully crystallized, diffracting to 2.8 and 3.0Å resolution respectively. Furthermore, a truncated, soluble tACE (tACEΔ36NJ), expressed in the presence of the glucosidase-I inhibitor N-butyldeoxynojirimycin, retained the activity of the native enzyme and yielded crystals belonging to the orthorhombic P212121 space group (cell dimensions, a = 56.47Å, b = 84.90Å, c = 133.99Å, α = 90°, β = 90° and γ = 90°). These crystals diffracted to 2.0Å resolution. Thus underglycosylated human tACE mutants, lacking O-linked oligosaccharides and most N-linked oligosaccharides or with only simple N-linked oligosaccharides attached throughout the molecule, are suitable for X-ray diffraction studies.
Alkaline transition of phytocyanins: a comparison of stellacyanin and umecyanin
Authors: DENNISON Christopher , HARRISON Mark D. , LAWLER Anne T.
Published: April 2003

The effect of pH on Cu(I) and Cu(II) forms of the isolated soluble domain of the stellacyanin from Rhus vernicifera (SCu), the Japanese lacquer tree, has been studied by electronic and NMR spectroscopy and using direct electrochemical measurements. A pKa value of 10.1–10.4 is observed for the alkaline transition in this oxidized phytocyanin and results in a slightly altered active-site structure, as indicated by changes in the visible and paramagnetic 1H NMR spectra. Electrochemical studies show that the pKa value for this transition in SCu(I) (reduced SCu) is 11.0. These results are compared with those recently obtained for other phytocyanins, and in particular umecyanin. In all cases, the alkaline transition is caused by the deprotonation of the surface lysine residue adjacent to the axial ligand. This lysine residue is completely conserved in known phytocyanin sequences. Also highlighted in these studies are the remarkable active-site similarities between stellacyanin and umecyanin.
A pheromone-binding protein from the cockroach Leucophaea maderae: cloning, expression and pheromone binding
Authors: RIVIÈRE Stéphane , LARTIGUE Audrey , QUENNEDEY Brigitte , CAMPANACCI Valérie , FARINE Jean-Pierre , TEGONI Mariella , CAMBILLAU Christian , BROSSUT Rémy
Published: April 2003

Odorant-binding proteins (OBPs) are thought to transport volatile compounds from air to their receptors through the sensillary lymph. In this protein family, the subgroup of pheromone-binding proteins (PBPs) is specifically tuned to the perception of the sexual pheromone. To date, the description of OBPs has been restricted to Endopterygota and Paraneoptera. Their expression in Orthopteroid has been hypothesized, but no evidence of OBP has been produced in this assemblage to date. In the present study, we describe the first OBP from a Dictyopteran insect that belongs to the cockroach Leucophaea maderae. The PBP of L. maderae (PBPLma) shares all the hallmarks of the OBP family and is expressed specifically in the female adult antennae, the sex that perceives the sexual pheromone. The affinity of the recombinant PBPLma produced in the Escherichia coli periplasm for the pheromonal compounds has been tested by displacement of a fluorophore, 8-anilino-1-naphtalenesulphonic acid (ANS). Our results suggest that two chemically close compounds of the pheromonal blend (3-hydroxy-butan-2-one and butane-2,3-diol) are capable of displacing ANS, whereas two other pheromone components (E-2-octenoic acid and senecioic acid) and other alkyl volatile compounds are not capable of displacing ANS, indicating a certain filtering of binding, which can be correlated with the putative function.
Killing tumours by ceramide-induced apoptosis: a critique of available drugs
Authors: RADIN Norman S.
Published: April 2003

Over 1000 research papers have described the production of programmed cell death (apoptosis) by interventions that elevate the cell content of ceramide (Cer). Other interventions, which lower cellular Cer, have been found to interfere with apoptosis induced by other agents. Some studies have shown that slowing the formation of proliferation-stimulating sphingolipids also induces apoptosis. These relationships are due to the two different aspects of Cer: Cer itself produces apoptosis, but metabolic conversion of Cer into either sphingosine 1-phosphate or glucosphingolipids leads to cell proliferation. The balance between these two aspects is missing in cancer cells, and yet intervention by stimulating or blocking only one or two of the pathways in Cer metabolism is very likely to fail. This results from two properties of cancer cells: their high mutation rate and the preferential survival of the most malignant cells. Tumours treated with only one or two drugs that elevate Cer can adjust the uncontrolled processes to either maintain or to ‘aggravate’ the excessive growth, angiogenesis and metastasis characteristics of tumours. These treatments might simply elevate the production of growth factors, receptors and other substances that reduce the effectiveness of Cer. Tumour cells that do not adapt in this way undergo apoptosis, leaving the adapted cells free to grow and, ultimately, to ‘subdue’ their host. Thus it is important to kill every type of cancer cell present in the tumour rapidly and simultaneously, using as many different agents to control as many pathways as possible. To aid this approach, this article catalogues many of the drugs that act on different aspects of Cer metabolism. The techniques described here may lead to the development of practical chemotherapy for cancer and other diseases of excess proliferation.
Steroid and bile acid conjugates are substrates of human multidrug-resistance protein (MRP) 4 (ATP-binding cassette C4)
Authors: ZELCER Noam , REID Glen , WIELINGA Peter , KUIL Annemieke , van der HEIJDEN Ingrid , SCHUETZ John D. , BORST Piet
Published: April 2003

Human multidrug-resistance protein (MRP) 4 transports cyclic nucleotides and when overproduced in mammalian cells mediates resistance to some nucleoside analogues. Recently, it has been shown that Mrp4 is induced in the livers of Fxr(-/-) mice, which have increased levels of serum bile acids. Since MRP4, like MRP1–3, also mediates transport of a model steroid conjugate substrate, oestradiol 17-β-d-glucuronide (E217βG), we tested whether MRP4 may be involved in the transport of steroid and bile acid conjugates. Bile salts, especially sulphated derivatives, and cholestatic oestrogens inhibited the MRP4-mediated transport of E217βG. Inhibition by oestradiol 3,17-disulphate and taurolithocholate 3-sulphate was competitive, suggesting that these compounds are MRP4 substrates. Furthermore, we found that MRP4 transports dehydroepiandrosterone 3-sulphate (DHEAS), the most abundant circulating steroid in humans, which is made in the adrenal gland. The ATP-dependent transport of DHEAS by MRP4 showed saturable kinetics with Km and Vmax values of 2μM and 45pmol/mg per min, respectively (at 27°C). We further studied the possible involvement of other members of the MRP family of transporters in the transport of DHEAS. We found that MRP1 transports DHEAS in a glutathione-dependent manner and exhibits Km and Vmax values of 5μM and 73pmol/mg per min, respectively (at 27°C). No transport of DHEAS was observed in membrane vesicles containing MRP2 or MRP3. Our findings suggest a physiological role for MRP1 and MRP4 in DHEAS transport and an involvement of MRP4 in transport of conjugated steroids and bile acids.
Nitrogen-source regulation of yeast gamma-glutamyl transpeptidase synthesis involves the regulatory network including the GATA zinc-finger factors Gln3, Nil1/Gat1 and Gzf3
Authors: SPRINGAEL Jean-Yves , PENNINCKX Michel J.
Published: April 2003

In Saccharomyces cerevisiae, the CIS2 gene encodes γ-glutamyl transpeptidase (γ-GT; EC 2.3.2.2), the main GSH-degrading enzyme. The promoter region of CIS2 contains one stress-response element (CCCCT) and eight GAT(T/A)A core sequences, probably involved in nitrogen-regulated transcription. We show in the present study that expression of CIS2 is indeed regulated according to the nature of the nitrogen source. Expression is highest in cells growing on a poor nitrogen source such as urea. Under these conditions, the GATA zinc-finger transcription factors Nil1 and Gln3 are both required for CIS2 expression, Nil1 appearing as the more important factor. We further show that Gzf3, another GATA zinc-finger protein, acts as a negative regulator in nitrogen-source control of CIS2 expression. During growth on a preferred nitrogen source like NH4+, CIS2 expression is repressed through a mechanism involving (at least) the Gln3-binding protein Ure2/GdhCR. Induction of CIS2 expression during nitrogen starvation is dependent on Gln3 and Nil1. Furthermore, rapamycin causes similar CIS2 activation, indicating that the target of rapamycin signalling pathway controls CIS2 expression via Gln3 and Nil1 in nitrogen-starved cells. Finally, our results show that CIS2 expression is induced mainly by nitrogen starvation but apparently not by other types of stress.
Functional characterization of the non-catalytic ectodomains of the nucleotide pyrophosphatase/phosphodiesterase NPP1
Authors: GIJSBERS Rik , CEULEMANS Hugo , BOLLEN Mathieu
Published: April 2003

The ubiquitous nucleotide pyrophosphatases/phosphodiesterases NPP1–3 consist of a short intracellular N-terminal domain, a single transmembrane domain and a large extracellular part, comprising two somatomedin-B-like domains, a catalytic domain and a poorly defined C-terminal domain. We show here that the C-terminal domain of NPP1–3 is structurally related to a family of DNA/RNA non-specific endonucleases. However, none of the residues that are essential for catalysis by the endonucleases are conserved in NPP1–NPP3, suggesting that the nuclease-like domain of NPP1–3 does not represent a second catalytic domain. Truncation analysis revealed that the nuclease-like domain of NPP1 is required for protein stability, for the targeting of NPP1 to the plasma membrane and for the expression of catalytic activity. We also demonstrate that 16 conserved cysteines in the somatomedin-B-like domains of NPP1, in concert with two flanking cysteines, mediate the dimerization of NPP1. The K173Q polymorphism of NPP1, which maps to the second somatomedin-B-like domain and has been associated with the aetiology of insulin resistance, did not affect the dimerization or catalytic activity of NPP1, and did not endow NPP1 with an affinity for the insulin receptor. Our data suggest that the non-catalytic ectodomains contribute to the subunit structure, stability and function of NPP1–3.
Cross-linking of cellular proteins by tissue transglutaminase during necrotic cell death: a mechanism for maintaining tissue integrity
Authors: NICHOLAS Ben , SMETHURST Peter , VERDERIO Elisabetta , JONES Richard , GRIFFIN Martin
Published: April 2003

Tissue transglutaminase (tTG) is a Ca2+-dependent enzyme which cross-links proteins via ε(γ-glutamyl)lysine bridges. There is increasing evidence that tTG is involved in wound repair and tissue stabilization, as well as in physiological mechanisms leading to cell death. To investigate the role of this enzyme in tissue wounding leading to loss of Ca2+ homoeostasis, we initially used a model involving electroporation to reproduce cell wounding under controlled conditions. Two cell models were used whereby tTG expression is regulated either by antisense silencing in ECV 304 cells or by using transfected Swiss 3T3 cells in which tTG expression is under the control of the tet regulatory system. Using these cells, loss of Ca2+ homoeostasis following electroporation led to a tTG-dependent formation of highly cross-linked proteinaceous shells from intracellular proteins. Formation of these structures is dependent on elevated intracellular Ca2+, but it is independent of intracellular proteases and is near maximal after only 20min post-wounding. Using labelled primary amines as an indicator of tTG activity within these ‘wounded cells’, we demonstrate that tTG modifies a wide range of proteins that are present in both the perinuclear and intranuclear spaces. The demonstration of entrapped DNA within these shell structures, which showed limited fragmentation, provides evidence that the high degree of transglutaminase cross-linking results in the prevention of DNA release, which may serve to dampen any subsequent inflammatory response. Comparable observations were shown when monolayers of cells were mechanically wounded by scratching. In this second model of cell wounding, redistribution of tTG activity to the extracellular matrix was also demonstrated, an effect which may serve to stabilize tissues post-trauma, and thus contribute to the maintenance of tissue integrity.
MIM-B, a putative metastasis suppressor protein, binds to actin and to protein tyrosine phosphatase delta
Authors: WOODINGS Jacquelyn A. , SHARP Stewart J. , MACHESKY Laura M.
Published: April 2003

We have found that MIM-B, a putative metastasis suppressor protein, is implicated in actin cytoskeletal control and interaction with a protein tyrosine phosphatase (PTP). MIM was originally described as a protein whose mRNA was Missing in Metastasis, as it was found not to be present in metastatic bladder carcinoma cell lines [Lee, Y.G., Macoska, J.A., Korenchuk, S. and Pienta, K.J. (2002) Neoplasia 4, 291–294]. We further characterized a variant of MIM, which we call MIM-B, and which we believe may be a link between tyrosine kinase signalling and the actin cytoskeleton. We have shown, using purified proteins and cell extracts, that MIM-B is an actin-binding protein, probably via a WASP (Wiskott–Aldrich syndrome protein)-homology 2 domain at its C-terminus. We have also found that MIM-B binds to the cytoplasmic domain of receptor PTPδ. Expression of full-length MIM-B induces actin-rich protrusions resembling microspikes and lamellipodia at the plasma membrane and promotes disassembly of actin stress fibres. The C-terminal portion of MIM-B is localized in the cytoplasm and does not affect the actin cytoskeleton when expressed, while the N-terminal portion localizes to internal vesicles and probably targets the protein to membranes. We postulate that MIM-B may be a regulator of actin assembly downstream of tyrosine kinase signalling and that this activity may explain the involvement of MIM in the metastasis of cancer cells.
Regulation of TG-interacting factor by transforming growth factor-beta
Authors: CHEN Feifei , OGAWA Kenji , NAGARAJAN Raman P. , ZHANG Meiyu , KUANG Chenzhong , CHEN Yan
Published: April 2003

TG-interacting factor (TGIF) is a transcriptional co-repressor that directly associates with Smad (Sma- and Mad-related protein) proteins and inhibits Smad-mediated transcriptional activation. By using Affymetrix (Santa Clara, CA, U.S.A.) oligonucleotide microarray analysis, we found that TGIF mRNA level was elevated by transforming-growth-factor-β (TGF-β) treatment in a human T-cell line, HuT78. Subsequent reverse-transcription PCR assays indicated that TGF-β1 and activin were able to induce a rapid and transient increase in the level of TGIF in both HuT78 and HepG2 hepatoma cells. To analyse whether or not the regulation of TGIF mRNA occurs at the transcriptional level, a 2.4kb human TGIF promoter was isolated. A primer extension assay was performed to localize the putative transcription initiation site of the promoter. When transiently expressed in HepG2 cells, this promoter was stimulated by TGF-β1 and activin treatment in a time-dependent manner. A series of deletion mutants of the TGIF promoter were also generated to further characterize the TGF-β responsive region of the promoter. In addition, expression of TGIF was able to cause a dose-dependent inhibition of TGF-β and activin signalling. Taken together, these experiments indicated that TGIF is a novel transcriptional target of TGF-β and activin signalling and is likely involved in a negative feedback loop to desensitize TGF-β/activin action.
Regulation of endocytic traffic by Rho GTPases
Authors: QUALMANN Britta , MELLOR Harry
Published: April 2003

The members of the Rho subfamily of small GTPases are key regulators of the actin cytoskeleton. However, recent studies have provided evidence for multiple additional roles for these signalling proteins in controlling endocytic traffic. Here we review our current understanding of Rho GTPase action within the endocytic pathway and examine the potential points of convergence with the more established, actin-based functions of these signalling proteins.
No Borders: The Case against Immigration Controls
Authors: Hayter Teresa
Published: April 2003

This article presents the case against immigration controls. Nation states, which are giving up controls on the movement of goods and capital, nevertheless still try to control the movement of people. Like controls under apartheid, immigration controls will eventually become untenable. They are also a relatively recent phenomenon. The actions of the governments of the rich countries, their international agencies and corporations increase both the opportunities and the need for migration. Together with arms sales and support for right-wing repressive regimes, they bear much responsibility for the wars and persecution from which people are forced to flee. The strongest reason for abolishing immigration controls is the increasingly harsh suffering they impose on refugees and migrants, largely to deter others. In the process, they undermine many human rights, including potentially those of existing residents.