bluuebunny/crossref_metadata_2025 huggingface dataset:
Ara12 subtilisin-like protease from Arabidopsis thaliana: purification, substrate specificity and tissue localization
Authors: HAMILTON John M.U. , SIMPSON David J. , HYMAN Stefan C. , NDIMBA Bongani K. , SLABAS Antoni R.
Published: February 2003

A C-terminal portion of Ara12 subtilisin-like protease (residues 542—757) was expressed in Escherichia coli cells as a fusion protein bound to maltose binding protein. Polyclonal antisera raised against the expressed protein were used to examine the tissue specificity and subcellular localization of Ara12. The protease was found predominantly in the silique and stem of plants, but was hardly detectable in leaf and not seen in root tissue. The distribution observed using immunological techniques is different from that seen by an RNA analysis study, which demonstrated similar mRNA abundance in the stem and leaves. Using immunogold labelling, Ara12 was shown to have an extracellular localization and was found in the intercellular spaces in stem tissue. Ara12 protease was purified to homogeneity from Arabidopsis thaliana cell suspension cultures by anion exchange and hydrophobic interaction chromatography. Proteolytic activity of Ara12 was inhibited by a number of serine protease inhibitors, but was almost unaffected by inhibitors of other catalytic classes of proteases. Optimal proteolytic activity was displayed under acidic conditions (pH5.0). Ara12 activity was relatively thermostable and was stimulated in the presence of Ca2+ ions. Substrate specificity studies were conducted using a series of internally quenched fluorogenic peptide substrates. At the P1 position of substrates, hydrophobic residues, such as Phe and Ala, were preferred to Arg, whilst at the P1′ position, Asp, Leu and Ala were most favoured. Possible functions of Ara12 are discussed in the light of the involvement of a number of plant subtilisin-like proteases in morphogenesis.
Cloning, expression and characterization of a mammalian Nudix hydrolase-like enzyme that cleaves the pyrophosphate bond of UDP-glucose
Authors: YAGI Toshihiro , BAROJA-FERNÁNDEZ Edurne , YAMAMOTO Ryuji , MUÑOZ Francisco José , AKAZAWA Takashi , HONG Kyoung Su , POZUETA-ROMERO Javier
Published: February 2003

A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney (Sus scrofa). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna, Baroja-Fernández, Muñoz, Bastarrica-Berasategui, Zandueta-Criado, Rodríguez-López, Lasa, Akazawa and Pozueta-Romero (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8128—8132], UGPPase appears to be a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated Nudix hydrolases. A complete cDNA of the UGPPase-encoding gene, designated UGPP, was isolated from a human thyroid cDNA library and expressed in E. coli. The resulting cells accumulated a protein that showed kinetic properties identical to those of pig UGPPase.
Identification of amino acid residues, essential for maintaining the tetrameric structure of sheep liver cytosolic serine hydroxymethyltransferase, by targeted mutagenesis
Authors: JALA Venkatakrishna Rao , RAO Naropantul APPAJI , SAVITHRI Handanahal Subbarao
Published: February 2003

Serine hydroxymethyltransferase (SHMT), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyses the transfer of the hydroxymethyl group from serine to tetrahydrofolate to yield glycine and N5,N10-methylenetetrahydrofolate. An analysis of the known SHMT sequences indicated that several amino acid residues were conserved. In this paper, we report the identification of the amino acid residues essential for maintaining the oligomeric structure of sheep liver cytosolic recombinant SHMT (scSHMT) through intra- and inter-subunit interactions and by stabilizing the binding of PLP at the active site. The mutation of Lys-71, Arg-80 and Asp-89, the residues involved in intra-subunit ionic interactions, disturbed the oligomeric structure and caused a loss of catalytic activity. Mutation of Trp-110 to Phe was without effect, while its mutation to Ala resulted in the enzyme being present in the insoluble fraction. These results suggested that Trp-110 located in a cluster of hydrophobic residues was essential for proper folding of the enzyme. Arg-98 and His-304, residues involved in the inter-subunit interactions, were essential for maintaining the tetrameric structure. Mutation of Tyr-72, Asp-227 and His-356 at the active site which interact with PLP resulted in the loss of PLP, and hence loss of tetrameric structure. Mutation of Cys-203, located away from the active site, weakened PLP binding indirectly. The results demonstrate that in addition to residues involved in inter-subunit interactions, those involved in PLP binding and intra-subunit interactions also affect the oligomeric structure of scSHMT.
The transition between active and de-activated forms of NADH:ubiquinone oxidoreductase (Complex I) in the mitochondrial membrane of Neurospora crassa
Authors: GRIVENNIKOVA Vera G. , SEREBRYANAYA Darya V. , ISAKOVA Elena P. , BELOZERSKAYA Tatyana A. , VINOGRADOV Andrei D.
Published: February 2003

The mammalian mitochondrial NADH:ubiquinone oxidoreductase (Complex I) has been shown to exist in two kinetically and structurally distinct slowly interconvertible forms, active (A) and de-activated (D) [Vinogradov and Grivennikova (2001) IUBMB Life 52, 129—134]. This work was undertaken to investigate the putative Complex I A—D transition in the mitochondrial membrane of the lower eukaryote Neurospora crassa and in plasma membrane of the prokaryote Paracoccus denitrificans, organisms that are eligible for molecular genetic manipulations. The potential interconversion between A and D forms was assessed by examination of the initial and steady-state rates of NADH oxidation catalysed by inside-out submitochondrial (N. crassa) and sub-bacterial (P. denitrificans) particles and their sensitivities to N-ethylmaleimide and Mg2+. All diagnostic tests provide evidence that slow temperature- and turnover-dependent A—D transition is an explicit feature of eukaryotic N. crassa Complex I, whereas the phenomenon is not seen in the membranes of the prokaryote P. denitrificans. Significantly lower activation energy for A-to-D transition characterizes the N. crassa enzyme compared with that determined previously for the mammalian Complex I. Either a lag or a burst in the onset of the NADH oxidase assayed in the presence of Mg2+ is seen when the reaction is initiated by the thermally de-activated or NADH-activated particles, whereas the delayed final activities of both preparations are the same. We conclude that continuous slow cycling between A and D forms occurs during the steady-state operation of Complex I in N. crassa mitochondria.
Upstream stimulatory factor activates the vasopressin promoter via multiple motifs, including a non-canonical E-box
Authors: COULSON Judy M. , EDGSON Jodie L. , MARSHALL-JONES Zoe V. , MULGREW Robert , QUINN John P. , WOLL Penella J.
Published: February 2003

We have described previously a complex E-box enhancer (-147) of the vasopressin promoter in small-cell lung cancer (SCLC) extracts [Coulson, Fiskerstrand, Woll and Quinn, (1999) Biochem. J. 344, 961—970]. Upstream stimulatory factor (USF) heterodimers were one of the complexes binding to this site in vitro. We now report that USF overexpression in non-SCLC (NSCLC) cells can functionally activate vasopressin promoter-driven reporters that are otherwise inactive in this type of lung cancer cell. Site-directed mutagenesis and electrophoretic mobility-shift analysis demonstrate that although the −147 E-box contributes, none of the previously predicted E-boxes (-147, −135, −34) wholly account for this USF-mediated activation in NSCLC. 5′ Deletion showed the key promoter region as −52 to +42; however, USF-2 binding was not reliant on the −34 E-box, but on a novel adjacent CACGGG non-canonical E-box at −42 (motif E). This mediated USF binding in both SCLC and USF-2-transfected NSCLC cells. Mutation of motif E or the non-canonical TATA box abolished activity, implying both are required for transcriptional initiation on overexpression of USF-2. Co-transfected dominant negative USF confirmed that binding was required through motif E for function, but that the classical activation domain of USF was not essential. USF-2 bound motif E with 10-fold lower affinity than the −147 E-box. In NSCLC, endogenous USF-2 expression is low, and this basal level appears to be insufficient to activate transcription of arginine vasopressin (AVP). In summary, we have demonstrated a novel mechanism for USF activation, which contributes to differential vasopressin expression in lung cancer.
Fgr but not Syk tyrosine kinase is a target for beta2 integrin-induced c-Cbl-mediated ubiquitination in adherent human neutrophils
Authors: MELANDER Fredrik , ANDERSSON Tommy , DIB Karim
Published: February 2003

An early and critical event in β2 integrin signalling during neutrophil adhesion is activation of Src tyrosine kinases and Syk. In the present study, we report Src kinase-dependent β2 integrin-induced tyrosine phosphorylation of Cbl occurring in parallel with increased Cbl-associated tyrosine kinase activity. These events concurred with activation of Fgr and, surprisingly, also with dissociation of this Src tyrosine kinase from Cbl. Moreover, the presence of the Src kinase inhibitor PP1 in an in vitro assay had only a limited effect on the Cbl-associated kinase activity. These results suggest that an additional active Src-dependent tyrosine kinase associates with Cbl. The following observations imply that Syk is such a kinase: (i) β2 integrins activated Syk in a Src-dependent manner, (ii) Syk was associated with Cbl much longer than Fgr was, and (iii) the Syk inhibitor piceatannol (3,4,3′,5′-tetrahydroxy-trans-stilbene) abolished the Cbl-associated kinase activity in an in vitro assay. Effects of the mentioned interactions between these two kinases and Cbl may be related to the finding that Cbl is a ubiquitin E3 ligase. Indeed, we detected β2 integrin-induced ubiquitination of Fgr that, similar to the phosphorylation of Cbl, was abolished in cells pretreated with PP1. However, the ubiquitination of Fgr did not cause any apparent degradation of the protein. In contrast with Fgr, Syk was not modified by the E3 ligase. Thus Cbl appears to be essential in β2 integrin signalling, first by serving as a matrix for a subsequent agonist-induced signalling interaction between Fgr and Syk, and then by mediating ubiquitination of Fgr which possibly affects its interaction with Cbl.
A novel splice variant of mouse interleukin-1-receptor-associated kinase-1 (IRAK-1) activates nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK)
Authors: YANAGISAWA Ken , TAGO Kenji , HAYAKAWA Morisada , OHKI Motomichi , IWAHANA Hiroyuki , TOMINAGA Shin-ichi
Published: February 2003

Interleukin-1 (IL-1)-receptor-associated kinase (IRAK) is an indispensable signalling molecule for host-defence responses initiated by a variety of ligands that bind to members of the Toll/IL-1 receptor family. Here we report a novel splice variant of mouse IRAK-1, IRAK-1-S, which is generated by utilizing a new splicing acceptor site within exon 12. IRAK-1-S cDNA is shorter than the originally reported IRAK-1 (IRAK-1-W) cDNA by 271 nucleotides, and the subsequent frameshift causes a premature termination of translation after 23 amino acids, which are unique to the IRAK-1-S protein. To elucidate the physiological function of IRAK-1-S, we overexpressed it in 293T cells and studied the effects on the IL-1 signalling cascade. As it lacks the C-terminal region of IRAK-1-W that has been reported to contain the TRAF6 (tumour necrosis factor receptor-associated factor 6) binding domain, IRAK-1-S was unable to bind TRAF6 protein, which is a proposed downstream signalling molecule. However, IRAK-1-S overexpressed in 293T cells induced constitutive activation of nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) independent of stimulation by IL-1, as did IRAK-1-W. To clarify the mechanism of NF-κB activation by IRAK-1-S in the absence of binding to TRAF6, we demonstrated that IRAK-1-S binds to IRAK-1-W through its death domain; the findings suggested that overexpressed IRAK-1-S may bind endogenous IRAK-1-W and activate TRAF6 through IRAK-1-W. These results also indicate that this novel variant may play roles in the activation of NF-κB and JNK by IL-1 and other ligands whose signal transduction is dependent on IRAK-1 under physiological conditions.
Hepatocyte nuclear factor-6 stimulates transcription of the alpha-fetoprotein gene and synergizes with the retinoic-acid-receptor-related orphan receptor alpha-4
Authors: NACER-CHERIF Habib , BOIS-JOYEUX Brigitte , ROUSSEAU Guy G. , LEMAIGRE Frédéric P. , DANAN Jean-Louis
Published: February 2003

The rat α-fetoprotein (afp) gene is controlled by three enhancers whose function depends on their interaction with liver-enriched transcription factors. The afp enhancer III, located at −6kb, is composed of three regions that act in synergy. Two of these regions, called s1 and s2, contain a putative binding site for hepatocyte nuclear factor-6 (HNF-6). This factor is the prototype of the ONECUT family of cut-homoeodomain proteins and is a known regulator of liver gene expression in adults and during development. We show here that the two splicing isoforms of HNF-6 bind to a site in the s1 region and in the s2 region. The core sequence of the s1 site corresponds to none of the known HNF-6 binding sites. Nevertheless, the binding properties of the s1 site are identical with those of the s2 site and of previously characterized HNF-6 binding sequences. The HNF-6 consensus should therefore be rewritten as DRRTCVATND. Binding of HNF-6 to the s1 and s2 sites requires both the cut and the homoeo domains, is co-operative and induces DNA bending. HNF-6 strongly stimulates the activity of the afp enhancer III in transient transfection experiments. This effect requires the stereo-specific alignment of the two HNF-6 sites. Moreover, HNF-6 stimulates the enhancer in synergy with the retinoic-acid-receptor-related orphan receptor α (RORα), which binds to a neighbouring site in the s1 region. Thus expression of the afp gene requires functional interactions between HNF-6 molecules and between HNF-6 and RORα.
Identification of HTF (HER2 transcription factor) as an AP-2 (activator protein-2) transcription factor and contribution of the HTF binding site to ERBB2 gene overexpression
Authors: VERNIMMEN Douglas , BEGON Dominique , SALVADOR Christophe , GOFFLOT Stéphanie , GROOTECLAES Madeleine , WINKLER Rosita
Published: February 2003

The ERBB2 gene is overexpressed in 30% of human breast cancers and this is correlated with poor prognosis. Overexpression of the ERBB2 gene is due to increased transcription and gene amplification. Our previous studies have identified a new cis element in the ERBB2 promoter which is involved in the gene's overexpression. This cis element, located 501bp upstream from the main ERBB2 transcription initiation site, binds a transcription factor called HTF (HER2 transcription factor). We report here the identification of HTF as an AP-2 (activator protein-2) transcription factor. The new cis element is bound by AP-2 with high affinity, compared with a previously described AP-2 binding site located 284bp downstream. Co-transfection of an AP-2α expression vector with a reporter vector containing the newly identified AP-2 binding site in front of a minimal ERBB2 promoter induced a dose-dependent increase in transcriptional activity. We examined the contribution of the new AP-2 binding site to ERBB2 overexpression. For this purpose we abolished the new and/or the previously described AP-2 binding sequence by site-directed mutagenesis. The results show that the two functional AP-2 sites in the first 700bp of the ERBB2 promoter co-operate to achieve maximal transcriptional activity.
Interactions of the designed antimicrobial peptide MB21 and truncated dermaseptin S3 with lipid bilayers: molecular-dynamics simulations
Authors: SHEPHERD Craig M. , VOGEL Hans J. , TIELEMAN D. Peter
Published: February 2003

Molecular-dynamics simulations covering 30ns of both a natural and a synthetic antimicrobial peptide in the presence of a zwitterionic lipid bilayer were performed. In both simulations, copies of the peptides were placed in an α-helical conformation on either side of the bilayer about 10Å (1Å = 0.1nm) from the interface, with either the hydrophobic or the positively charged face of the helix directed toward the bilayer surface. The degree of peptide—lipid interaction was dependent on the starting configuration: surface binding and subsequent penetration of the bilayer was observed for the hydrophobically oriented peptides, while the charge-oriented peptides demonstrated at most partial surface binding. Aromatic residues near the N-termini of the peptides appear to play an important role in driving peptide—lipid interactions. A correlation between the extent of peptide—lipid interactions and helical stability was observed in the simulations. Insertion of the peptides into the bilayer caused a dramatic increase in the lateral area per lipid and decrease in the bilayer thickness, resulting in substantial disordering of the lipid chains. Results from the simulations are consistent with early stages of proposed mechanisms for the lytic activity of antimicrobial peptides. In addition to these ‘free’ simulations, 25ns simulations were carried out with the peptides constrained at three different distances relative to the bilayer interface. The constraint forces are in agreement with the extent of peptide—bilayer insertion observed in the free simulations.
Control of glucose phosphorylation in L6 myotubes by compartmentalization, hexokinase, and glucose transport
Authors: WHITESELL Richard R. , ARDEHALI Hossein , PRINTZ Richard L. , BEECHEM Joseph M. , KNOBEL Susan M. , PISTON David W. , GRANNER Daryl K. , MEER Wieb Van Der , PERRIOTT Laureta M. , MAY James M.
Published: February 2003

In muscle, insulin enhances influx of glucose and its conversion to glucose 6-phosphate (G6P) by hexokinase (HK). While effects of insulin on glucose transport have been demonstrated, its effect on the activity of HK of cells has not. In L6 myotubes treated for 24h with insulin there was increased expression of the HK isoform, HKII, and increased glucose phosphorylation without a concomitant increase in glucose transport, indirectly suggesting that phosphorylation of glucose was a target of insulin action [Osawa, Printz, Whitesell and Granner (1995) Diabetes 44, 1426—1432]. In the present work the same treatment led to a 2-fold rise in G6P, suggesting that transport and/or HK were important targets of insulin action. We used a method to identify the site of rate control involving the specificity of phosphorylation towards 2-deoxy-[1-14C]glucose and d-[2-3H]glucose. Glucose transport does not greatly discriminate between these two tracers while HK shows increased specificity for glucose. Specificity of the glucose phosphorylation of the cells increased with addition of insulin and when extracellular glucose was raised. Specificity was reduced at low glucose concentrations or when the inhibitor of transport, cytochalasin B, was added. We conclude that transport and HK share nearly equal control over glucose phosphorylation in these cells. A computer program was used to test models for compatibility with the different types of experiments. The predicted intracellular glucose and transport rates associated with phosphorylation activity were lower than their measured values for the whole cell. In the most likely model, 15±4% of the glucose transporters serve a proportionate volume of the cytoplasm. Insulin activation of glucose phosphorylation might then result from stimulation of these transporters together with HK recruitment or relief from inhibition by G6P.
Folding of thyroglobulin in the calnexin/calreticulin pathway and its alteration by loss of Ca2+ from the endoplasmic reticulum
Authors: JESO Bruno DI , ULIANICH Luca , PACIFICO Francesco , LEONARDI Antonio , VITO Pasquale , CONSIGLIO Eduardo , FORMISANO Silvestro , ARVAN Peter
Published: February 2003

During its initial folding in the endoplasmic reticulum (ER), newly synthesized thyroglobulin (Tg) is known to interact with calnexin and other ER molecular chaperones, but its interaction with calreticulin has not been examined previously. In the present study, we have investigated the interactions of endogenous Tg with calreticulin and with several other ER chaperones. We find that, in FRTL-5 and PC-Cl3 cells, calnexin and calreticulin interact with newly synthesized Tg in a carbohydrate-dependent manner, with largely overlapping kinetics that are concomitant with the maturation of Tg intrachain disulphide bonds, preceding Tg dimerization and exit from the ER. Calreticulin co-precipitates more newly synthesized Tg than does calnexin; however, using two different experimental approaches, calnexin and calreticulin were found in ternary complexes with Tg, making this the first endogenous protein reported in ternary complexes with calnexin and calreticulin in the ER of live cells. Depletion of Ca2+ from the ER elicited by thapsigargin (a specific inhibitor of ER Ca2+-ATPases) results in retention of Tg in this organelle. Interestingly, thapsigargin treatment induces the premature exit of Tg from the calnexin/calreticulin cycle, while stabilizing and prolonging interactions of Tg with BiP (immunoglobulin heavy chain binding protein) and GRP94 (glucose-regulated protein 94), two chaperones whose binding is not carbohydrate-dependent. Our results suggest that calnexin and calreticulin, acting in ternary complexes with a large glycoprotein substrate such as Tg, might be engaged in the folding of distinct domains, and indicate that lumenal Ca2+ strongly influences the folding of exportable glycoproteins, in part by regulating the balance of substrate binding to different molecular chaperone systems within the ER.
The liver isoform of carnitine palmitoyltransferase 1 is not targeted to the endoplasmic reticulum
Authors: BROADWAY Neil M. , PEASE Richard J. , BIRDSEY Graeme , SHAYEGHI Majid , TURNER Nigel A. , SAGGERSON E. DAVID
Published: February 2003

Liver microsomal fractions contain a malonyl-CoA-inhibitable carnitine acyltransferase (CAT) activity. It has been proposed [Fraser, Corstorphine, Price and Zammit (1999) FEBS Lett. 446, 69—74] that this microsomal CAT activity is due to the liver form of carnitine palmitoyltransferase 1 (L-CPT1) being targeted to the endoplasmic reticulum (ER) membrane as well as to mitochondria, possibly by an N-terminal signal sequence [Cohen, Guillerault, Girard and Prip-Buus (2001) J. Biol. Chem. 276, 5403—5411]. COS-1 cells were transiently transfected to express a fusion protein in which enhanced green fluorescent protein was fused to the C-terminus of L-CPT1. Confocal microscopy showed that this fusion protein was localized to mitochondria, and possibly to peroxisomes, but not to the ER. cDNAs corresponding to truncated (amino acids 1—328) or full-length L-CPT1 were transcribed and translated in the presence of canine pancreatic microsomes. However, there was no evidence of authentic insertion of CPT1 into the ER membrane. Rat liver microsomal fractions purified by sucrose-density-gradient centrifugation contained an 88kDa protein (p88) which was recognized by an anti-L-CPT1 antibody and by 2,4-dinitrophenol-etomoxiryl-CoA, a covalent inhibitor of L-CPT1. Abundance of p88 and malonyl-CoA-inhibitable CAT activity were increased approx. 3-fold by starvation for 24h. Deoxycholate solubilized p88 and malonyl-CoA-inhibitable CAT activity from microsomes to approximately the same extent. The microsomal fraction contained porin, which, relative to total protein, was as abundant as in crude mitochondrial outer membranes fractions. It is concluded that L-CPT1 is not targeted to the ER membrane and that malonyl-CoA CAT in microsomal fractions is L-CPT1 that is derived from mitochondria, possibly from membrane contact sites.
Leptin activates the promoter of the interleukin-1 receptor antagonist through p42/44 mitogen-activated protein kinase and a composite nuclear factor kappaB/PU.1 binding site
Authors: DREYER Magali G. , JUGE-AUBRY Cristiana E. , GABAY Cem , LANG Ursula , ROHNER-JEANRENAUD Françoise , DAYER Jean-Michel , MEIER Christoph A.
Published: February 2003

We have recently shown that leptin strongly induces the expression and secretion of the interleukin-1 receptor antagonist (IL-1Ra) [Gabay, Dreyer, Pellegrinelli, Chicheportiche and Meier (2001) J. Clin. Endocrinol. Metab. 86, 783—791] in monocytes. However, the intracellular signalling mechanisms involved remained unknown. We now demonstrate that the activation of the IL-1Ra promoter by leptin is strictly dependent on the presence of the long form of the leptin receptor (OB-Rb), and that it also requires the activation of the p42/44 mitogen-activated protein kinases (MAPKs) as well as the presence of a nuclear factor κB (NF-κB)/PU.1 composite site at position −80 of the IL-1Ra promoter. Although leptin is capable of activating a NF-κB reporter element in transient transfection experiments, the protein complex binding to the NF-κB/PU.1 site of the IL-1Ra promoter is not composed of the p65/p50 subunits of NF-κB, as is evident in electrophoretic gel mobility-shift experiments. In contrast, a protein complex which does not contain PU.1 binds to this composite element in a leptin-dependent manner. In summary, we characterize the signalling pathway for leptin and OB-Rb involved in the induction of IL-1Ra, involving p42/44 MAPK, and a yet uncharacterized complex of transcription factor(s) binding to a NF-κB/PU.1 composite element of the IL-1Ra promoter.
Detection of bovine spongiform encephalopathy, ovine scrapie prion-related protein (PrPSc) and normal PrPc by monoclonal antibodies raised to copper-refolded prion protein
Authors: THACKRAY Alana M. , MADEC Jean-Yves , WONG Edmond , MORGAN-WARREN Robert , BROWN David R. , BARON Thierry , BUJDOSO Raymond
Published: February 2003

Prion-related protein (PrP) is a glycosylphosphatidylinositol-linked cell-surface protein expressed by a wide variety of cells, including those of the nervous system and the immune system. Several functions of normal cellular PrP (PrPc) have been proposed that may be associated with the capacity of this protein to bind copper. In the present study, we describe the generation of a panel of monoclonal antibodies raised to copper-refolded PrP, which may be used to analyse the normal and disease-associated forms of this protein. The anti-PrP monoclonal antibodies were reactive by Western blot and ELISA with recombinant murine PrPc refolded in the presence or absence of either copper or manganese, and with the disease-susceptible allelic form V136R154Q171 ('VRQ'; where single-letter amino-acid notation has been used) and disease-resistant allelic form A136R154R171 ('ARR') of recombinant ovine PrPc. FACS analysis of lymphoid cells using these monoclonal antibodies showed that wild-type non-activated mouse lymphocytes expressed little, if any, PrPc. These monoclonal antibodies were shown to react with the unglycosylated and monoglycosylated forms of PrPSc (abnormal disease-specific conformation of PrP) in prion-infected tissue samples from all of the different species tested by Western blot. In addition, this analysis allowed one to make a distinction between bovine spongiform encephalopathy ('BSE') and scrapie PrPSc isolates from experimentally infected sheep on the basis of their different electrophoretic mobilities.
Cloning, expression and biochemical characterization of one Epsilon-class (GST-3) and ten Delta-class (GST-1) glutathione S-transferases from Drosophila melanogaster, and identification of additional nine members of the Epsilon class
Authors: SAWICKI Rafał , SINGH Sharda P. , MONDAL Ashis K. , BENEŠ Helen , ZIMNIAK Piotr
Published: February 2003

From the fruitfly, Drosophila melanogaster, ten members of the cluster of Delta-class glutathione S-transferases (GSTs; formerly denoted as Class I GSTs) and one member of the Epsilon-class cluster (formerly GST-3) have been cloned, expressed in Escherichia coli, and their catalytic properties have been determined. In addition, nine more members of the Epsilon cluster have been identified through bioinformatic analysis but not further characterized. Of the 11 expressed enzymes, seven accepted the lipid peroxidation product 4-hydroxynonenal as substrate, and nine were active in glutathione conjugation of 1-chloro-2,4-dinitrobenzene. Since the enzymically active proteins included the gene products of DmGSTD3 and DmGSTD7 which were previously deemed to be pseudogenes, we investigated them further and determined that both genes are transcribed in Drosophila. Thus our present results indicate that DmGSTD3 and DmGSTD7 are probably functional genes. The existence and multiplicity of insect GSTs capable of conjugating 4-hydroxynonenal, in some cases with catalytic efficiencies approaching those of mammalian GSTs highly specialized for this function, indicates that metabolism of products of lipid peroxidation is a highly conserved biochemical pathway with probable detoxification as well as regulatory functions.
Kinetic model of the inositol trisphosphate receptor that shows both steady-state and quantal patterns of Ca2+ release from intracellular stores
Authors: DAWSON Alan P. , LEA Edward J.A. , IRVINE Robin F.
Published: February 2003

The release of Ca2+ from intracellular stores via InsP3 receptors shows anomalous kinetics. Successive additions of low concentrations of InsP3 cause successive rapid transients of Ca2+ release. These quantal responses have been ascribed to all-or-none release from stores with differing sensitivities to InsP3 or, alternatively, to a steady-state mechanism where complex kinetic properties of the InsP3 receptor allow partial emptying of all the stores. We present here an adaptive model of the InsP3 receptor that can show either pattern, depending on the imposed experimental conditions. The model proposes two interconvertible conformational states of the receptor: one state binds InsP3 rapidly, but with low affinity, whereas the other state binds slowly, but with high affinity. The model shows repetitive increments of Ca2+ release in the absence of a Ca2+ gradient, but more pronounced incremental behaviour when released Ca2+ builds up at the mouth of the channel. The sensitivity to InsP3 is critically dependent on the density of InsP3 receptors, so that different stores can respond to different concentration ranges of InsP3. Since the model generates very high Hill coefficients (h7), it allows all-or-none release of Ca2+ from stores of differing receptor density, but questions the validity of the use of h values as a guide to the number of InsP3 molecules needed to open the channel. The model presents a mechanism for terminating Ca2+ release in the presence of positive feedback from released Ca2+, thereby providing an explanation of why elementary Ca2+ signals ('blips’ and ‘puffs') do not inevitably turn into regenerative waves.
Ellman's-reagent-mediated regeneration of trypanothione in situ: substrate-economical microplate and time-dependent inhibition assays for trypanothione reductase
Authors: HAMILTON Chris J. , SARAVANAMUTHU Ahilan , EGGLESTON Ian M. , FAIRLAMB Alan H.
Published: February 2003

Trypanothione reductase (TryR) is a key enzyme involved in the oxidative stress management of the Trypanosoma and Leishmania parasites, which helps to maintain an intracellular reducing environment by reduction of the small-molecular-mass disulphide trypanothione (T[S]2) to its di-thiol derivative dihydrotrypanothione (T[SH]2). TryR inhibition studies are currently impaired by the prohibitive costs of the native enzyme substrate T[S]2. Such costs are particularly notable in time-dependent and high-throughput inhibition assays. In the present study we report a protocol that greatly decreases the substrate quantities needed for such assays. This is achieved by coupling the assay with the chemical oxidant 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), which can rapidly re-oxidize the T[SH]2 product back into the disulphide substrate T[S]2, thereby maintaining constant substrate concentrations and avoiding deviations from rate linearity due to substrate depletion. This has enabled the development of a continuous microplate assay for both classical and time-dependent TryR inhibition in which linear reaction rates can be maintained for 60min or more using minimal substrate concentrations (<1μM, compared with a substrate Km value of 30μM) that would normally be completely consumed within seconds. In this manner, substrate requirements are decreased by orders of magnitude. The characterization of a novel time-dependent inhibitor, cis-3-oxo-8,9b-bis-(N1-acrylamidospermidyl)-1,2,3,4,4a,9b-hexahydrobenzofuran (PK43), is also described using these procedures.
Down-regulation of alphav/beta3 integrin via misrouting to lysosomes by overexpression of a beta3Lamp1 fusion protein
Authors: CONESA Magali , PRAT Annik , MORT John S. , MARVALDI Jacques , LISSITZKY Jean-Claude , SEIDAH Nabil G.
Published: February 2003

We present a general strategy for the dominant negative reduction in the levels of type-1 membrane-bound heterodimeric proteins within the secretory pathway through fusion of the soluble ectodomain of one of the partners to the transmembrane-cytosolic tail of the lysosomal protein Lamp1. Thus, in human embryonic kidney (HEK)-293 cells, overexpression of an integrin β3Lamp1 chimera resulted in a drastic reduction of its endogenous partner, the integrin αv subunit. The mechanism involves the formation in the endoplasmic reticulum of a αv/β3Lamp1 complex that is subsequently sorted towards a lysosomal/endosomal degradation pathway. The specificity of this approach is afforded by the invariance in the levels of the endogenous integrins α5 and β1 as compared with control cells. Conversely overexpression of integrin β3 in HEK-293 cells led to an increased level of αvβ3 at the cell surface. Functionally β3Lamp1 and β3 overexpressors exhibit decreased and increased adhesion to vitronectin, respectively, as well as diminished cellular aggregation. The application of this technology should enable the analysis of the functional importance of homodimers or heterodimers in the cell types of choice and the identification of novel partner proteins by proteomic approaches.
Effects of nitrogen monoxide and carbon monoxide on molecular and cellular iron metabolism: mirror-image effector molecules that target iron
Authors: WATTS Ralph N. , PONKA Prem , RICHARDSON Des R.
Published: February 2003

Many effector functions of nitrogen monoxide (NO) and carbon monoxide (CO) are mediated through their high-affinity for iron (Fe). In this review, the roles of NO and CO are examined in terms of their effects on the molecular and cellular mechanisms involved in Fe metabolism. Both NO and CO avidly form complexes with a plethora of Fe-containing molecules. The generation of NO and CO is mediated by the nitric oxide synthase and haem oxygenase (HO) families of enzymes respectively. The effects of NO on Fe metabolism have been well characterized, whereas knowledge of the effects of CO remains within its infancy. In terms of the role of NO in Fe metabolism, one of the best characterized interactions includes its effect on the iron regulatory proteins. These molecules are mRNA-binding proteins that control the expression of the transferrin receptor 1 and ferritin, molecules that are involved in Fe uptake and storage respectively. Apart from this, activated macrophages impart their cytotoxic activity by generating NO, which results in marked Fe mobilization from tumour-cell targets. This deprives the cell of the Fe that is required for DNA synthesis and energy production. Considering that HO degrades haem, resulting in the release of CO, Fe(II) and biliverdin, it is suggested that a CO—Fe complex will form. This may account for the rapid Fe mobilization observed from macrophages after haemoglobin catabolism. Intriguingly, overexpression of HO results in cellular Fe mobilization, suggesting that CO has a similar effect to NO on Fe trafficking. Preliminary evidence suggests that, like NO, CO plays important roles in Fe metabolism.
Distance of sequons to the C-terminus influences the cellular N-glycosylation of the prion protein
Authors: WALMSLEY Adrian R. , HOOPER Nigel M.
Published: February 2003

Cell-specific differences in the utilization of the two N-glycosylation sequons (Asn180-Ile-Thr and Asn196-Phe-Thr) of the prion protein (PrP) have been proposed to influence the aetiology of the neurodegenerative prion diseases. As the N-glycosylation of PrP is ablated by deletion of the C-terminal glycosyl-phosphatidylinositol (GPI) anchor signal sequence, we have investigated the determinants for PrP sequon utilization in human neuronal cells using the novel approach of restoring N-glycosylation to secreted forms of PrP lacking a GPI anchor. N-glycosylation was restored to an efficiency comparable with that of GPI anchored PrP when the distance of the sequon to the C-terminus was increased so that it was sufficient to reach the active site of oligosaccharyltransferase before chain termination. Our findings indicate that sequon utilization in PrP is a co-translational process that precedes GPI anchor addition and, as such, will be greatly influenced by the dynamics of the translocon—oligosaccharyltransferase complex.
D53 is a novel endosomal SNARE-binding protein that enhances interaction of syntaxin 1 with the synaptobrevin 2 complex in vitro
Authors: PROUX-GILLARDEAUX Véronique , GALLI Thierry , CALLEBAUT Isabelle , MIKHAILIK Anatoly , CALOTHY Georges , MARX Maria
Published: February 2003

Synaptobrevin 2 (Sb2), syntaxin1 (Stx1), and synaptosomal-associated protein of 25kDa (SNAP-25) are the main components of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex involved in fusion of synaptic vesicles with the presynaptic plasma membrane. We report the characterization of D53, a novel SNARE-binding protein preferentially expressed in neural and neuro-endocrine cells. Its two-dimensional organization, established by the hydrophobic cluster analysis, is reminiscent of SNARE proteins. D53 contains two putative helical regions, one of which includes a large coiled-coil domain involved in the interaction with Sb2 in vitro. Following subcellular fractionation, endogenous D53 was specifically detected in the membrane-containing fraction of PC12 cells, where it co-immunoprecipitated with Sb2. Analysis by confocal microscopy showed that, in these cells, endogenous D53 co-localized partially with the transferrin receptor in early endosomes. In vitro assays revealed that binding properties of D53 to Stx1 and Sb2 are comparable with those of SNAP-25. Furthermore, D53 forms Sb2/Stx1/D53 complexes in vitro in a manner similar to SNAP-25. We propose that D53 could be involved in the assembly or disassembly of endosomal SNARE complexes by regulating Sb2/Stx interaction.
Cloning and kinetic characterization of Arabidopsis thaliana solanesyl diphosphate synthase
Authors: HIROOKA Kazutake , BAMBA Takeshi , FUKUSAKI Ei-ichiro , KOBAYASHI Akio
Published: February 2003

trans-Long-chain prenyl diphosphate synthases catalyse the sequential condensation of isopentenyl diphosphate (C5) units with allylic diphosphate to produce the C30—C50 prenyl diphosphates, which are precursors of the side chains of prenylquinones. Based on the relationship between product specificity and the region around the first aspartate-rich motif in trans-prenyl diphosphate synthases characterized so far, we have isolated the cDNA for a member of trans-long-chain prenyl diphosphate synthases from Arabidopsis thaliana. The cDNA was heterologously expressed in Escherichia coli, and the recombinant His6-tagged protein was purified and characterized. Product analysis revealed that the cDNA encodes solanesyl diphosphate (C45) synthase (At-SPS). At-SPS utilized farnesyl diphosphate (FPP; C15) and geranylgeranyl diphosphate (GGPP; C20), but did not accept either the C5 or the C10 allylic diphosphate as a primer substrate. The Michaelis constants for FPP and GGPP were 5.73μM and 1.61μM respectively. We also performed an analysis of the side chains of prenylquinones extracted from the A. thaliana plant, and showed that its major prenylquinones, i.e. plastoquinone and ubiquinone, contain the C45 prenyl moiety. This suggests that At-SPS might be devoted to the biosynthesis of either or both of the prenylquinone side chains. This is the first established trans-long-chain prenyl diphosphate synthase from a multicellular organism.
Degradation of human thymidine kinase is dependent on serine-13 phosphorylation: involvement of the SCF-mediated pathway
Authors: KE Po-Yuan , YANG Che-Chuan , TSAI I-Chun , CHANG Zee-Fen
Published: February 2003

The expression level of human thymidine kinase (hTK) is regulated in a cell-cycle-dependent manner. One of the mechanisms responsible for the fluctuation of TK expression in the cell cycle can be attributed to protein degradation during mitosis. Given the facts that cell-cycle-dependent proteolysis is highly conserved in all eukaryotes and yeast cells are an excellent model system for protein-degradation study, here we report on the use of Saccharomyces cerevisiae and Schizosaccharomyces pombe to investigate the degradation signal and mechanism required for hTK degradation. We found that the stability of hTK is significantly reduced in mitotic yeasts. Previously, we have observed that Ser-13 is the site of mitotic phosphorylation of hTK in HeLa cells [Chang, Huang and Chi (1998) J. Biol. Chem. 273, 12095—12100]. Here, we further provide evidence that the replacement of Ser-13 by Ala (S13A) renders hTK stable in S. pombe and S. cerevisiae. Most interestingly, we demonstrated that degradation of hTK is impaired in S. cerevisiae carrying a temperature-sensitive mutation in the proteasomal gene pre1-1 or the Skp1-Cullin-1/CDC53-F-box (SCF) complex gene cdc34 or cdc53, suggesting the contribution of the SCF-mediated pathway in hTK degradation. As phosphorylation is a prerequisite signal for SCF recognition, our results implied that phosphorylation of Ser-13 probably contributes to the degradation signal for hTK via the SCF-mediated proteolytic pathway.
Vitamin E (alpha-tocopherol) attenuates cyclo-oxygenase 2 transcription and synthesis in immortalized murine BV-2 microglia
Authors: EGGER Tamara , SCHULIGOI Rufina , WINTERSPERGER Andrea , AMANN Rainer , MALLE Ernst , SATTLER Wolfgang
Published: February 2003

One of the immediate early microglial genes that are up-regulated in response to proinflammatory stimuli is cyclo-oxygenase 2 (COX-2). In the present study, we have investigated the effects of α-tocopherol (αTocH), an essential constituent of the nervous system, on the activation of COX-2 in lipopolysaccharide (LPS)-stimulated mouse BV-2 microglia. In unstimulated BV-2 cells, COX-2 mRNA and protein were almost undetectable but were strongly up-regulated in response to LPS. Activation of COX-2 protein synthesis in LPS-stimulated BV-2 cells involved activation of the extracellular-signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathway and was sensitive to the protein kinase C (PKC) inhibitors staurosporine and chelerythrine, and the MAP kinase/ERK kinase 1/2 inhibitors PD98059 and U0126. Supplementation of BV-2 cells with αTocH before LPS stimulation resulted in pronounced up-regulation of protein phosphatase 2A (PP2A) activity, down-regulation of PKC activity, ERK1/2 phosphorylation and nuclear factor κB (NFκB) activation. As a result, COX-2 protein levels and prostaglandin E2 production were significantly lower in αTocH-supplemented cells. The effects of αTocH on PKC activity could be reverted by calyculin A and okadaic acid, two PP inhibitors. In summary, our results suggest that αTocH activates microglial PP2A activity and thereby silences an LPS-activated PKC/ERK/NFκB signalling cascade resulting in significantly attenuated COX-2 protein synthesis. These in vitro results imply that αTocH could induce quiescence to pathways that are associated with acute or chronic inflammatory conditions in the central nervous system.
Glutaminase isoform expression in cell lines derived from human colorectal adenomas and carcinomas
Authors: TURNER Abigail , McGIVAN John D.
Published: February 2003

This paper describes some properties of glutamine oxidation and glutaminase isoform expression in cell lines derived from human colorectal adenomas and carcinomas. The slow-growing adenoma-derived cell line AA/C1, and the rapidly proliferating carcinoma cell line HT29, both required glutamine for growth. The rate of 14CO2 production from [U-14C]glutamine was faster in AA/C1 cells than in HT29 cells. Conversely HT29 cells showed faster rates of glucose oxidation and lactate production. The activity of glutaminase was 3 times higher in AA/C1 cell extracts than in extracts of HT29 cells. Glutaminase activity in the two cell lines had similar Km values for glutamine, but the activity in AA/C1 cells had a higher K0.5 for activation by phosphate. Glutaminase activity in extracts of both cells was inhibited by glutamate. Western blotting showed the presence, in both cell lines, of isoform(s) of glutaminase with an molecular mass of 63kDa, intermediate between that of kidney glutaminase and liver glutaminase. PCR-based analysis showed that an mRNA species identical to the kidney-type isoform glutaminase C was present in both cell types as was an additional mRNA species identical to the liver-type glutaminase isoform from human breast tumour cells. Northern blotting using isoform-specific cDNA probes demonstrated that mRNA for both glutaminase isoforms was expressed at significant levels in both cell types. Similar results to those in AA/C1 cells and HT29 cells were obtained in two further adenoma and carcinoma cell lines respectively. These results contrast with those reported previously in hepatocyte/hepatoma model systems with respect to fuel selection, glutaminase activity and isoform expression. They also constitute the first demonstration of simultaneous expression of two glutaminase isoforms in a single cell type.
SDP1 is a peroxisome-proliferator-activated receptor gamma2 co-activator that binds through its SCAN domain
Authors: BABB Robert , BOWEN Benjamin R.
Published: February 2003

Peroxisome-proliferator-activated receptors (PPARs), members of the nuclear hormone receptor superfamily, play an important role in the regulation of lipid metabolism and energy homoeostasis. In a yeast two-hybrid experiment using the zinc-finger transcription factor ZNF202 as bait, we previously identified the SCAN-domain-containing protein SDP1. SDP1 shares a high degree of amino acid sequence identity with PGC-2, a previously identified PPARγ2 co-activator from the mouse. Here we show that SDP1 and PGC-2 interact with PPARγ2 through their SCAN domains, even though PPARγ2 does not contain a SCAN domain. Similar to PGC-2, SDP1 enhanced PPARγ2-dependent gene transcription in transiently transfected cells but did not alter the affinity of PPARγ2 for agonists. Although the SCAN domain was necessary for binding to PPARγ2, it was not sufficient for co-activation in cells, suggesting that other features of SDP1 are responsible for transcriptional co-activation. The ability of SDP1 to interact with two different transcription factors that regulate genes involved in lipid metabolism, ZNF202 and PPARγ2, suggests that SDP1 may be an important co-regulator of such genes.