bluuebunny/crossref_metadata_2025 huggingface dataset:
Yeast cell-free system that catalyses joint-molecule formation in a Rad51p- and Rad52p-dependent fashion
Authors: NAGARAJ Vijayalakshmi , NORRIS David
Published: August 2015

One of the central reactions of homologous recombination is the invasion of a single strand of DNA into a homologous duplex to form a joint molecule. Here we describe the isolation of a cell-free system from meiotic yeast cells that catalyses joint-molecule formation in vitro. The active components in the system required ATP and homologous DNA and operated in both 0.5 and 13 mM MgCl2. When the cell-free system was prepared from rad51/rad51 and rad52/rad52 mutants and joint-molecule formation was assayed at 0.5 mM MgCl2, the specific activity decreased to 6% and 13.8% respectively of the wild-type level. However, when the same mutant extracts were premixed, joint-molecule formation increased 4-8-fold, i.e. the mutant extracts exhibited complementation in vitro. These results demonstrated that Rad51p and Rad52p were required for optimal joint-molecule formation at 0.5 mM MgCl2. Intriguingly, however, Rad51p and Rad52p seemed to be more dispensable at higher concentrations of MgCl2 (13 mM). Further purification of the responsible activity has proven problematical, but it did flow through a sizing column as a single peak (molecular mass 1.2 MDa) that was co-eluted with Rad51p and RFA, the eukaryotic single-stranded DNA-binding protein. All of these characteristics are consistent with the known properties of the reaction in vivo and suggest that the new cell-free system will be suitable for purifying enzymes involved in homologous recombination.
Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans
Authors: CHEN Sharon C. A. , WRIGHT Lesley C. , GOLDING John C , SORRELL Tania C.
Published: August 2015

Infection caused by the fungus Cryptococcus neoformans is potentially fatal. A highly active extracellular phospholipase, demonstrating phospholipase B (PLB), lysophospholipase (LPL) and lysophospholipase/transacylase (LPTA) activities, was purified to homogeneity from C. neoformans using (NH4)2SO4 fractionation, and hydrophobic-interaction, anion-exchange and gel-filtration chromatography. All three enzyme activities co-purified as a single protein with an apparent molecular mass of 70-90 kDa by SDS/PAGE and 160-180 kDa by gel filtration. The ratio of the three activities remained constant after each purification step. The amino acid composition, as well as the sequences of the N-terminus and of five internal peptide fragments were novel. The protein was an acidic glycoprotein containing N-linked carbohydrate moieties, with pI values of 5.5 and 3.5. The apparent Vmax values for PLB and LPL activities were 12.3 and 870 μmol/min per mg of protein respectively; the corresponding Km values were approx. 185.3 and 92.2 μM. The enzyme was active only at acidic pH (pH optimum of 4.0 for PLB and 4.0-5.0 for LPL and LPTA). Enzyme activity did not require added cations, but was inhibited by Fe3+. LPL and LPTA activities were decreased by 0.1% (v/v) Triton X-100 to 50% of the control value. Palmitoylcarnitine (0.5 mM) inhibited PLB (97% inhibition) and LPL and LPTA activities (35% inhibition) competitively. All phospholipids except phosphatidic acid were degraded by PLB, but dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylcholine were the preferred substrates. This is the first complete description of the purification and properties of a phospholipase, which may be involved in virulence, from a pathogenic fungus.
Regional evolution of venom-gland phospholipase A2 isoenzymes of Trimeresurus flavoviridis snakes in the southwestern islands of Japan
Authors: CHIJIWA Takahito , DESHIMARU Masanobu , NOBUHISA Ikuo , NAKAI Makoto , OGAWA Tomohisa , ODA Naoko , NAKASHIMA Kin-ichi , FUKUMAKI Yasuyuki , SHIMOHIGASHI Yasuyuki , HATTORI Shosaku , OHNO Motonori
Published: August 2015

Conventional chromatographic analysis showed that phospholipase A2 (PLA2) isoenzymes of the venom of Trimeresurus flavoviridis (Habu snake) of Okinawa island are profoundly different in composition from those of T. flavoviridis of Amami-Oshima and Tokunoshima islands. The most striking feature was that myotoxic [Lys49]PLA2 isoenzymes, called BPI and BPII, which are expressed abundantly in the venoms of Amami-Oshima and Tokunoshima T. flavoviridis, are missing from the venom of Okinawa T. flavoviridis. Northern blot analysis of Okinawa T. flavoviridis venom-gland mRNA species showed the absence of BPI and BPII mRNA species. Analysis by single-stranded conformational polymorphism-PCR of venom-gland mRNA species of T. flavoviridis from three islands, with reference to five DNA species each encoding different PLA2 isoenzymes from Tokunoshima T. flavoviridis venom gland, also suggested that BPI and BPII mRNA species are not expressed in Okinawa T. flavoviridis venom gland. In contrast, genomic Southern blot analysis with a variety of probes showed that only the bands corresponding to the upstream and downstream regions of the genes for BPI and/or BPII can be detected in Okinawa T. flavoviridis. These results suggested that the genes for BPI and BPII in Okinawa T. flavoviridis genome had been inactivated to form pseudogenes. Differently from Amami-Oshima and Tokunoshima T. flavovirdis genomic DNAs, PCR amplification of the segments of BPI and BPII genes between the 5ʹ moiety of second exon and the middle portion of second intron failed for Okinawa T. flavoviridis genomic DNAs. In sequence analysis of the two segments involving polymorphism between BPI and BPII genes, which are located in first exon and third exon, respectively, only one base was detected at the polymorphic positions for pseudogene in Okinawa T. flavoviridis genome. Based on these facts, it became evident for pseudogene that the upstream region of BPI gene down to the 5ʹ moiety of second exon and the downstream region of BPII gene starting from the middle portion of second intron are in a linked form with a possible insertion. Such observations suggest that venom-gland genes for PLA2 isoenzymes in T. flavoviridis snakes isolated for one to two million years have evolved independently. Their evolution is regional and seems, from several lines of consideration and observation, to be adaptive to the environment.
Regulation of expression of the human β-1,2-N-acetylglucosaminyltransferase II gene (MGAT2) by Ets transcription factors
Authors: ZHANG Wenli , REVERS Leigh , PIERCE Michael , SCHACHTER Harry
Published: August 2015

Oncogenic transformation of fibroblasts by the src oncogene has long been known to cause an increase in the size of cell-surface protein-bound oligosaccharides, owing primarily to increased N-glycan branching mediated by increased β-1,6-N-acetylglucosaminyltransferase V (GnT V) activity. The src-responsive element of the GnT V promoter was localized to Ets-binding sites and the promoter was transcriptionally stimulated by both ets-1 and ets-2 expression [Buckhaults, Chen, Fregien and Pierce (1997) J. Biol. Chem. 272, 19575-19581; Kang, Saito, Ihara, Miyoshi, Koyama, Sheng and Taniguchi (1996) J. Biol. Chem. 271, 26706-26712]. Because GnT V action requires the prior action of β-1,2-N-acetylglucosaminyltransferase II (GnT II) and the human GnT II promoter contains four putative Ets-binding sites [Chen, Zhou, Tan and Schachter (1998) Glycoconj. J. 15, 301-308], GnT II might also be under oncogenic control via Ets transcription factors. We now report that co-transfection into HepG2 or COS-1 cells of either ets-1 or ets-2 expression plasmids together with chimaeric GnT II promoter-chloramphenicol acetyltransferase plasmids results in a 2-4-fold stimulation of promoter activity. Mobility-shift assays and South-Western blots localized the functional Ets-binding site to one of the four putative sites on the GnT II promoter. The GnT II promoter, unlike the GnT V promoter, is not activated by either src or neu. Therefore although both promoters are stimulated by a member of the Ets family of transcription factors, the functional role of this Ets transcriptional control seems to be different for the two genes.
Prominent expression of the selenoprotein thioredoxin reductase in the medullary rays of the rat kidney and thioredoxin reductase mRNA variants differing at the 5′ untranslated region
Authors: RUNDLÖF Anna-Klara , CARLSTEN Mattias , GIACOBINI MaiBritt M. J. , ARNÉR Elias S. J.
Published: August 2015

The mammalian selenoprotein thioredoxin reductase is a central enzyme in protection against oxidative damage or the redox control of cell function. Previously a neuroblastoma-cell-derived 2193 bp cDNA for rat thioredoxin reductase 1 (TrxR1) was characterized [Zhong, Arnér, Ljung, Åslund and Holmgren (1998) J. Biol. Chem. 273, 8581-8591]. Here, the major rat TrxR1 mRNA was determined as 3.5 kb by Northern blotting. A corresponding full-length 3360 bp liver-derived cDNA was cloned and sequenced, being extended in the 3ʹ untranslated region (3ʹ UTR) compared with the previous clone. Among tissues examined, lowest TrxR1 mRNA levels were found in spleen and testis and highest in liver and kidney. High expression in kidney was unexpected and in situ hybridization of adult rat kidney was performed. This revealed a highly structured expression pattern with the mRNA being prominently synthesized in the proximal tubules of the medullary rays. Analysing rat kidney cDNA, a 5ʹ UTR domain of TrxR1 was found that was different from that in liver- or neuroblastoma-derived cDNA clones. The kidney-derived 5ʹ UTR mRNA domain was instead highly similar to kidney-derived cDNA variants of murine apolipoprotein E. By sequence determination of the rat genomic sequence upstream of the open reading frame for TrxR1, an exon was encountered that encoded a third alternative 5ʹ UTR domain that could also be expressed, as confirmed by its presence in a mouse skin TrxR1 cDNA clone. It can therefore be concluded that TrxR1 mRNA is expressed in at least three different variants that differ at their 5ʹ UTRs.
Serine protease inhibitors suppress cytochrome c-mediated caspase-9 activation and apoptosis during hypoxia–reoxygenation
Authors: DONG Zheng , SAIKUMAR Pothana , PATEL Yogendra , WEINBERG Joel M. , VENKATACHALAM Manjeri A.
Published: August 2015

We have shown that reoxygenation of hypoxic rat kidney proximal tubule cells leads to apoptosis. This is mediated by translocation of Bax from the cytosol to mitochondria, accompanied by release of mitochondrial cytochrome c (cyt.c). The present study has examined the proteolytic mechanisms responsible for apoptosis during hypoxia-reoxygenation. Caspases were activated during hypoxia, as shown by cleavage of fluorogenic peptide substrates. By 5 h caspase-3-like activity to cleave carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin was increased approx. 30-fold. This was accompanied by specific processing of pro-caspase-3, -8 and -9 into active forms. Caspase activation during hypoxia was blocked by carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone and overexpression of Bcl-2. Of particular interest, caspase activation was also suppressed by the chymotryptic inhibitors N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and Ala-Pro-Phe chloromethyl ketone (APF), and the general serine protease inhibitor 4-(2-aminoethyl)benzenesulphonyl fluoride. Inhibition of caspase activation by these compounds resulted in arrest of apoptosis. On the other hand, the serine protease inhibitors did not prevent release of mitochondrial cyt.c during hypoxia, suggesting that these compounds blocked a critical step in post-mitochondrial caspase activation. Further studies using an in vitro reconstitution model showed that cyt.c/dATP stimulated caspase-9 processing and downstream caspase activation were significantly suppressed in the presence of TPCK and APF. Based on these results, we speculate that serine proteases may be involved in post-mitochondrial apoptotic events that lead to activation of the initiator, caspase-9.
Purification and characterization of a Baeyer–Villiger mono-oxygenase from Rhodococcus erythropolis DCL14 involved in three different monocyclic monoterpene degradation pathways
Authors: VAN DER WERF Mariët J.
Published: August 2015

A Baeyer-Villiger mono-oxygenase (BVMO), catalysing the NADPH- and oxygen-dependent oxidation of the monocyclic monoterpene ketones 1-hydroxy-2-oxolimonene, dihydrocarvone and menthone, was purified to homogeneity from Rhodococcus erythropolis DCL14. Monocyclic monoterpene ketone mono-oxygenase (MMKMO) is a monomeric enzyme of molecular mass 60 kDa. It contains 1 mol of FAD/monomer as the prosthetic group. The N-terminal amino acid sequence showed homology with many other NADPH-dependent and FAD-containing (Type 1) BVMOs. Maximal enzyme activity was measured at pH 9 and 35 °C. MMKMO has a broad substrate specificity, catalysing the lactonization of a large number of monocyclic monoterpene ketones and substituted cyclohexanones. The natural substrates 1-hydroxy-2-oxolimonene, dihydrocarvone and menthone were converted stoichiometrically into 3-isopropenyl-6-oxoheptanoate (the spontaneous rearrangement product of the lactone formed by MMKMO), 4-isopropenyl-7-methyl-2-oxo-oxepanone and 7-isopropyl-4-methyl-2-oxo-oxepanone respectively. The MMKMO-catalysed conversion of iso-dihydrocarvone showed an opposite regioselectivity to that of dihydrocarvone; in this case, 6-isopropenyl-3-methyl-2-oxo-oxepanone was formed as the product. MMKMO converted all enantiomers of the natural substrates with almost equal efficiency. MMKMO is involved in the conversion of the monocyclic monoterpene ketone intermediates formed in the degradation pathways of all stereoisomers of three different monocyclic monoterpenes, i.e. limonene, (dihydro)carveol and menthol.
Shedding of somatic angiotensin-converting enzyme (ACE) is inefficient compared with testis ACE despite cleavage at identical stalk sites
Authors: WOODMAN Zenda L. , OPPONG Sylvester Y. , COOK Sarah , HOOPER Nigel M. , SCHWAGER Sylva L. U. , BRANDT Wolf F. , EHLERS Mario R. W. , STURROCK Edward D.
Published: August 2015

The somatic and testis isoforms of angiotensin-converting enzyme (ACE) are both C-terminally anchored ectoproteins that are shed by an unidentified secretase. Although testis and somatic ACE both share the same stalk and membrane domains the latter was reported to be shed inefficiently compared with testis ACE, and this was ascribed to cleavage at an alternative site [Beldent, Michaud, Bonnefoy, Chauvet and Corvol (1995) J. Biol. Chem. 270, 28962-28969]. These differences constitute a useful model system of the regulation and substrate preferences of the ACE secretase, and hence we investigated this further. In transfected Chinese hamster ovary cells, human somatic ACE (hsACE) was indeed shed less efficiently than human testis ACE, and shedding of somatic ACE responded poorly to phorbol ester activation. However, using several analytical techniques, we found no evidence that the somatic ACE cleavage site differed from that characterized in testis ACE. First, anti-peptide antibodies raised to specific sequences on either side of the reported cleavage site (Arg1137/Leu1138) clearly recognized soluble porcine somatic ACE, indicating that cleavage was C-terminal to Arg1137. Second, a competitive ELISA gave superimposable curves for porcine plasma ACE, secretase-cleaved porcine somatic ACE (eACE), and trypsin-cleaved ACE, suggesting similar C-terminal sequences. Third, mass-spectral analyses of digests of released soluble hsACE or of eACE enabled precise assignments of the C-termini, in each case to Arg1203. These data indicated that soluble human and porcine somatic ACE, whether generated in vivo or in vitro, have C-termini consistent with cleavage at a single site, the Arg1203/Ser1204 bond, identical with the Arg627/Ser628 site in testis ACE. In conclusion, the inefficient release of somatic ACE is not due to cleavage at an alternative stalk site, but instead supports the hypothesis that the testis ACE ectodomain contains a motif that activates shedding, which is occluded by the additional domain found in somatic ACE.
Mutational analysis of the N-linked glycosylation sites of the human insulin receptor
Authors: ELLEMAN Thomas C. , FRENKEL Maurice J. , HOYNE Peter A. , MCKERN Neil M. , COSGROVE Leah , HEWISH Dean R. , JACHNO Kim M. , BENTLEY John D. , SANKOVICH Sonia E. , WARD Colin W.
Published: August 2015

Site-directed mutagenesis has been used to remove 15 of the 18 potential N-linked glycosylation sites, in 16 combinations, from the human exon 11-minus receptor isoform. The three glycosylation sites not mutated were asparagine residues 25, 397 and 894, which are known to be important in receptor biosynthesis or function. The effects of these mutations on proreceptor processing into α and β subunits, cell-surface expression, insulin binding and receptor autophosphorylation were assessed in Chinese hamster ovary cells. The double mutants 16+78, 16+111, 16+215, 16+255, 337+418, the triple mutants 295+337+418, 295+418+514, 337+418+514 and 730+743+881 and the quadruple mutants 606+730+743+881 and 671+730+743+881 seemed normal by all criteria examined. The triple mutant 16+215+255 showed only low levels of correctly processed receptor on the cell surface, this processed receptor being autophosphorylated in response to insulin. The quadruple mutant 624+730+743+881 showed normal processing and ligand binding but exhibited a constitutively active tyrosine kinase as judged by autophosphorylation. Three higher-order mutants were constructed, two of which, 16+337+418+730+743+881 (∆6) and 16+295+337+418+730+743+881 (∆7a), seemed normal. The third construct, 16+337+418+514+730+743+881 (∆7b), was expressed at high levels on the cell surface, essentially as uncleaved proreceptor with only the small proportion of ∆7b that was correctly processed showing insulin-stimulated autophosphorylation. The mutations of ∆6 and ∆7a were incorporated into soluble ectodomains, which had affinities for insulin that were 4-fold that of wild-type ectodomain. The ∆6 ectodomain expressed in Lec8 cells was produced in quantity in a bioreactor for subsequent structural analysis.
Protein kinase C activation by acidic proteins including 14-3-3
Authors: VAN DER HOEVEN Paulus C. J. , VAN DER WAL José C. M. , RUURS Paula , VAN BLITTERSWIJK Wim J.
Published: August 2015

14-3-3 proteins may function as adapter or scaffold proteins in signal transduction pathways. We reported previously that several 14-3-3 isotypes bind to protein kinase C (PKC)-ζ and facilitate coupling of PKC-ζ to Raf-1 [van der Hoeven, van der Wal, Ruurs, van Dijk and van Blitterswijk (2000) Biochem. J. 345, 297-306], an event that boosts the mitogen-activated protein kinase (ERK) pathway in Rat-1 fibroblasts. The present work investigated whether bound 14-3-3 would affect PKC-ζ activity. Using recombinant 14-3-3 proteins and purified PKC-ζ in a convenient, newly developed in vitro kinase assay, we found that 14-3-3 proteins stimulated PKC-ζ activity in a dose-dependent fashion up to approx. 2.5-fold. Activation of PKC-ζ by 14-3-3 isotypes was unrelated to their mutual affinity, estimated by co-immunoprecipitation from COS cell lysates. Accordingly, PKC-ζ with a defective (point-mutated) 14-3-3-binding site, showed the same 14-3-3-stimulated activity as wild-type PKC-ζ. As 14-13-3 proteins are acidic, we tested several other acidic proteins, which turned out to stimulate PKC-ζ activity in a similar fashion, whereas neutral or basic proteins did not. These effects were not restricted to the atypical PKC-ζ, but were also found for classical PKC. Together, the results suggest that the stimulation of PKC activity by 14-3-3 proteins is non-specific and solely due to the acidic nature of these proteins, quite similar to that known for acidic lipids.
Intragenic and intergenic suppression of the Escherichia coli ATP synthase subunit a mutation of Gly-213 to Asn: functional interactions between residues in the proton transport site
Authors: KUO Phillip H. , NAKAMOTO Robert K.
Published: August 2015

Subunit a of the ATP synthase Fo sector contains a transmembrane helix that interacts with subunit c and is critical for H+ transport activity. From a cysteine scan in the region around the essential subunit a residue, Arg-210, we found that the replacement of aGly-213 greatly attenuated ATP hydrolysis, ATP-dependent proton pumping and ∆μH+-dependent ATP synthesis. Various amino acid substitutions caused similar effects, suggesting that functional perturbations were caused by altering the environment or conformation of aArg-210. aG213N, which was particularly severe in effect, was suppressed by two second-site mutations, aL251V and cD61E. These mutations restored efficient coupling; the latter also increased ATP-dependent proton transport rates. These results were consistent with the proposed functional interaction between aArg-210 and cAsp-61, the likely carrier of the transported proton. From Arrhenius analysis of steady-state ATP hydrolytic activity, the transport mutants had large increases in the transition-state enthalpic and entropic parameters. Linear isokinetic relationships demonstrate that the transport mechanism is coupled to the rate-limiting catalytic transition-state step, which we have previously shown to involve the rotation of the γ subunit in multi-site, co-operative catalysis.
Epoxyalkyl glycosides of d-xylose and xylo-oligosaccharides are active-site markers of xylanases from glycoside hydrolase family 11, not from family 10
Authors: NTARIMA Patricia , NERINCKX Wim , KLARSKOV Klaus , DEVREESE Bart , BHAT Mahalingeshwara K. , BEEUMEN Jozef VAN , CLAEYSSENS Marc
Published: August 2015

A series of Ω-epoxyalkyl glycosides of D-xylopyranose, xylobiose and xylotriose were tested as potential active-site-directed inhibitors of xylanases from glycoside hydrolase families 10 and 11. Whereas family-10 enzymes (Thermoascus aurantiacus Xyn and Clostridium thermocellum Xyn Z) are resistant to electrophilic attack of active-site carboxyl residues, glycoside hydrolases of family 11 (Thermomyces lanuginosus Xyn and Trichoderma reesei Xyn II) are irreversibly inhibited. The apparent inactivation and association constants (ki, 1/Ki) are one order of magnitude higher for the xylobiose and xylotriose derivatives. The effects of the aglycone chain length can clearly be described. Xylobiose and n-alkyl β-D-xylopyranosides are competitive ligands and provide protection against inactivation. MS measurements showed 1:1 stoichiometries in most labelling experiments. Electrospray ionization MS/MS analysis revealed the nucleophile Glu86 as the modified residue in the T. lanuginosus xylanase when 2,3-epoxypropyl β-D-xylopyranoside was used, whereas the acid/base catalyst Glu178 was modified by the 3,4-epoxybutyl derivative. The active-site residues Glu86 and Glu177 in T. reesei Xyn II are similarly modified, confirming earlier X-ray crystallographic data [Havukainen, Törrönen, Laitinen and Rouvinen (1996) Biochemistry 35, 9617-9624]. The inability of the Ω-epoxyalkyl xylo(oligo)saccharide derivatives to inactivate family-10 enzymes is discussed in terms of different ligand-subsite interactions.
Roles of individual EF-hands in the activation of m-calpain by calcium
Authors: DUTT Previn , ARTHUR J. C. Simon , GROCHULSKI Pawel , CYGLER Miroslaw , ELCE John S.
Published: August 2015

m-Calpain is a heterodimeric, cytosolic, thiol protease, which is activated by Ca2+-binding to EF-hands in the C-terminal domains of both subunits. There are four potential Ca2+-binding EF-hands in each subunit, but their relative affinities for Ca2+ are not known. In the present study mutations were made in both subunits to reduce the Ca2+-binding affinity at one or more EF-hands in one or both subunits. X-ray crystallography of some of the mutated small subunits showed that Ca2+ did not bind to the mutated EF-hands, but that its binding at other sites was not affected. The structures of the mutant small subunits in the presence of Ca2+ were otherwise identical to that of the Ca2+-bound wild-type small subunit. In the whole enzyme the wild-type macroscopic Ca2+ requirement (Kd) was approx. 350 μM. The mutations did not affect the maximum specific activity of the enzyme, but caused increases in Kd, which were characteristic of each site. All the EF-hands could be mutated in various combinations without loss of activity, but preservation of at least one wild-type EF-hand 3 sequence was required to maintain Kd values lower than 1 mM. The results suggest that all the EF-hands can contribute co-operatively to calpain activation, but that EF-hand 3, in both subunits, has the highest intrinsic affinity for Ca2+ and provides the major driving force for conformational change.
Sphingosine 1-phosphate stimulates proliferation and migration of human endothelial cells possibly through the lipid receptors, Edg-1 and Edg-3
Authors: KIMURA Takao , WATANABE Tomoko , SATO Koichi , KON Junko , TOMURA Hideaki , TAMAMA Ken-ichi , KUWABARA Atsushi , KANDA Tsugiyasu , KOBAYASHI Isao , OHTA Hideo , UI Michio , OKAJIMA Fumikazu
Published: August 2015

Sphingosine 1-phosphate (S1P) stimulates thymidine incorporation (DNA synthesis), cell growth and cell migration in human aortic endothelial cells (HAECs). The extent of the S1P-induced responses are comparable to those stimulated by vascular endothelial growth factor, one of the most potent stimulators of angiogenesis. These responses to S1P were mimicked by dihydrosphingosine 1-phosphate, an S1P receptor agonist, and inhibited by pertussis toxin (PTX), an inactivator of Gi/Go-proteins. S1P also induced activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAP kinase). The activation of these enzymes was inhibited again by PTX and also by suramin, a non-selective receptor antagonist. S1P-induced DNA synthesis and ERK activation were inhibited by PD98059, an ERK kinase inhibitor, but not by SB203580, a p38 MAP kinase inhibitor. In contrast, cell migration and p38 MAP kinase activation, in response to S1P, were inhibited by SB203580 but not by PD98059. In HAECs, high-affinity S1P binding activity and expression of Edg-1 and Edg-3 mRNA were detected. These results suggest that S1P might be a novel angiogenesis factor and that the lipid-induced proliferation and migration of endothelial cells are possibly mediated through cell-surface S1P receptors, Edg-1 and Edg-3, which are linked to signalling pathways.
Chronic insulin effects on insulin signalling and GLUT4 endocytosis are reversed by metformin
Authors: PRYOR Paul R. , LIU Simon C. H. , CLARK Avril E. , YANG Jing , HOLMAN Geoffrey D. , TOSH David
Published: August 2015

Decreases in insulin-responsive glucose transport and associated levels of cell surface GLUT4 occur in rat adipocytes maintained in culture for 20 h under hyperinsulinaemic and hyperglycaemic conditions. We have investigated whether this defect is due to reduced signalling from the insulin receptor, GLUT4 expression or impaired GLUT4 trafficking. The effects of chronic insulin treatment on glucose transport and GLUT4 trafficking were ameliorated by inclusion of metformin in the culture medium. In comparison with the acute effects of insulin, chronic insulin treatment attenuated changes in signalling processes leading to glucose transport. These included insulin receptor tyrosine phosphorylation, phosphoinositide 3-kinase activity and Akt activity, which were all reduced by 60-70%. Inclusion of metformin in the culture medium prevented the effects of the chronic insulin treatment on these signalling processes. In comparison with cells maintained in culture without insulin, the total expression of GLUT4 protein was not significantly altered by chronic insulin treatment, although the level of GLUT1 expression was increased. Trafficking rate constants for wortmannin-induced cell-surface loss of GLUT4 and GLUT1 were assessed by 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-yloxy)-2-propylamine (ATB-BMPA) photolabelling. In comparison with cells acutely treated with insulin, chronic insulin treatment resulted in a doubling of the rate constants for GLUT4 endocytosis. These results suggest that the GLUT4 endocytosis process is very sensitive to the perturbations in signalling that occur under hyperinsulinaemic and hyperglycaemic conditions, and that the resulting elevation of endocytosis accounts for the reduced levels of net GLUT4 translocation observed.
Identification of amino acids imparting acceptor substrate selectivity to human arylamine acetyltransferases NAT1 and NAT2
Authors: GOODFELLOW Geoffrey H. , DUPRET Jean-Marie , GRANT Denis M.
Published: August 2015

The human arylamine N-acetyltransferases NAT1 and NAT2 catalyse the acetyl-CoA-dependent N- and O-acetylation of primary arylamine and hydrazine xenobiotics and their N-hydroxylated metabolites. We previously used a panel of recombinant NAT1/NAT2 chimaeric proteins to identify linear amino acid segments that have roles in imparting the distinct catalytic specificities to these proteins [Dupret, Goodfellow, Janezic and Grant (1994) J. Biol. Chem. 269, 26830-26835]. These studies indicated that a conserved central region (residues 112-210) distinct from that containing the active-site cysteine residue Cys68 was important in determining NAT substrate selectivity. In the present study we have refined our analysis through further chimaera generation of this conserved region and by subsequent site-directed mutagenesis of individual amino acids. Enzyme-kinetic analysis of these mutant proteins with the NAT1-selective and NAT2-selective substrates p-aminosalicylic acid (PAS) and sulphamethazine (SMZ) respectively suggests that residues 125, 127 and 129 are important determinants of NAT1-type and NAT2-type substrate selectivity. Modification of Arg127 had the greatest effect on specificity for PAS, whereas changing Phe125 had the greatest effect on specificity for SMZ. Selected NAT mutants exhibited Km values for acetyl-CoA that were comparable with those of the wild-type NATs, implying that the mutations affected acceptor substrate specificity rather than cofactor binding affinity. Taken together with previous observations, these results suggest that residues 125, 127 and 129 might contribute to the formation of the active-site pocket surrounding Cys68 and function as important determinants of NAT substrate selectivity.
Designing transthyretin mutants affecting tetrameric structure: implications in amyloidogenicity
Authors: REDONDO Clara , DAMAS Ana M. , SARAIVA Maria João M.
Published: August 2015

The molecular mechanisms that convert soluble transthyretin (TTR) tetramers into insoluble amyloid fibrils are still unknown; dissociation of the TTR tetramer is a pre-requisite for amyloid formation in vitro and involvement of monomers and/or dimers in fibril formation has been suggested by structural studies. We have designed four mutated proteins with the purpose of stabilizing [Ser117 → Cys (S117C) and Glu92 → Cys (E92C)] or destabilizing [Asp18 → Asn (D18N) and Leu110 → Ala (D110A)] the dimer/tetramer interactions in TTR, aiming at elucidating structural determinants in amyloidogenesis. The resistance of the mutated proteins to dissociation was analysed by HPLC studies of diluted TTR preparations. Both ‘stabilized’ mutants migrated as tetramers and, upon dilution, no other TTR species was observed, confirming the increased resistance to dissociation. For the ‘destabilized’ mutants, a mixture of tetrameric and monomeric forms co-existed at low dilution and the latter increased upon 10-fold dilution. Both of the destabilizing mutants formed amyloid in vitro when acidified. This result indicated that both the AB loop of TTR, destabilized in D18N, and the hydrophobic interactions affecting the dimer-dimer interfaces in L110A are implicated in the stability of the tetrameric structure. The stabilized mutants, which were dimeric in nature through disulphide bonding, were unable to polymerize into amyloid, even at pH 3.2. When the amyloid formation assay was repeated in the presence of 2-mercaptoethanol, upon disruption of the S-S bridges of these stable dimers, amyloid fibril formation was observed. This experimental evidence suggests that monomers, rather than dimers, are the repeating structural subunit comprising the amyloid fibrils.
Structure and function of Humicola insolens family 6 cellulases: structure of the endoglucanase, Cel6B, at 1.6 Å resolution
Authors: DAVIES Gideon J. , BRZOZOWSKI A. Marek , DAUTER Miroslawa , VARROT Annabelle , SCHÜLEIN Martin
Published: August 2015

Cellulases are traditionally classified as either endoglucanases or cellobiohydrolases on the basis of their respective catalytic activities on crystalline cellulose, which is generally hydrolysed more efficiently only by the cellobiohydrolases. On the basis of the Trichoderma reesei cellobiohydrolase II structure, it was proposed that the active-site tunnel of cellobiohydrolases permitted the processive hydrolysis of cellulose, whereas the corresponding endoglucanases would display open active-site clefts [Rouvinen, Bergfors, Teeri, Knowles and Jones (1990) Science 249, 380-386]. Glycoside hydrolase family 6 contains both cellobiohydrolases and endoglucanases. The structure of the catalytic core of the family 6 endoglucanase Cel6B from Humicola insolens has been solved by molecular replacement with the known T. reesei cellobiohydrolase II as the search model. Strangely, at the sequence level, this enzyme exhibits the highest sequence similarity to family 6 cellobiohydrolases and displays just one of the loop deletions traditionally associated with endoglucanases in this family. However, this enzyme shows no activity on crystalline substrates but a high activity on soluble substrates, which is typical of an endoglucanase. The three-dimensional structure reveals that the deletion of just a single loop of the active site, coupled with the resultant conformational change in a second ‘cellobiohydrolase-specific’ loop, peels open the active-site tunnel to reveal a substrate-binding groove.
Rho GTPases and their effector proteins
Authors: BISHOP Anne L. , HALL Alan
Published: August 2015

Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, gene transcription, cell-cycle progression and cell adhesion. About 30 potential effector proteins have been identified that interact with members of the Rho family, but it is still unclear which of these are responsible for the diverse biological effects of Rho GTPases. This review will discuss how Rho GTPases physically interact with, and regulate the activity of, multiple effector proteins and how specific effector proteins contribute to cellular responses. To date most progress has been made in the cytoskeleton field, and several biochemical links have now been established between GTPases and the assembly of filamentous actin. The main focus of this review will be Rho, Rac and Cdc42, the three best characterized mammalian Rho GTPases, though the genetic analysis of Rho GTPases in lower eukaryotes is making increasingly important contributions to this field.
Tyrosine phosphorylation of the vascular endothelial-growth-factor receptor-2 (VEGFR-2) is modulated by Rho proteins
Authors: GINGRAS Denis , LAMY Sylvie , BÉLIVEAU Richard
Published: August 2015

The effects of Rho-specific modifying toxins on the tyrosine phosphorylation of endothelial cell proteins were investigated. Incubation of the cells with the Rho-activating toxin cytotoxic necrotizing factor 1 (CNF1) induced a marked increase in the tyrosine phosphorylation of a number of signalling intermediates of the vascular endothelial growth factor (VEGF)-mediated cascade, including focal adhesion kinase, paxillin, phospholipase Cγ1 and a Shc-associated protein of 195 kDa. Both CNF1- and VEGF-dependent tyrosine phosphorylation of these proteins were significantly reduced by prior incubation with C3 transferase, a known inhibitor of RhoA function, suggesting a Rho-dependent mechanism. The stimulation of endothelial cells with CNF1 resulted in a marked increase in the tyrosine phosphorylation of the VEGF receptor (VEGFR)-2, which was correlated with a stimulation of its kinase activity and with its association with downstream tyrosine phosphorylated proteins. The stimulatory effect of CNF1 was specific for VEGFR-2 since the phosphotyrosine content of VEGFR-1 was not affected by the toxin. Transient overexpression of a dominant-active RhoA mutant also induced an increase in the tyrosine phosphorylation of the VEGFR-2, whereas overexpression of a dominant-inactive form of the protein was without effect. Taken together, these results indicate that Rho proteins may play an important role in angiogenesis by modulating the tyrosine phosphorylation levels of VEGFR-2.
Cholesterol decreases secretion of the secreted form of amyloid precursor protein by interfering with glycosylation in the protein secretory pathway
Authors: GALBETE Josè Luis , RODRIGUEZ-MARTIN Teresa , PERESSINI Elisa , MODENA Piergiorgio , BIANCHI Roberto , FORLONI Gianluigi
Published: August 2015

Cerebral deposits of β-amyloid (βA) are a major feature in Alzheimer's disease. βA is derived from amyloid precursor protein (APP). APP is subject to N- and O-glycosylation and undergoes a series of proteolytic cleavages that lead to the release of βA or of a non-amyloidogenic secreted form of APP (APPs). We used primary neuronal and glial cultures to investigate how cholesterol affects the production and secretion of APPs. Exposure to cholesterol for 2 h did not change the neuronal release of APPs; after 6 h APPs release was slightly lower, whereas 24 h of exposure decreased APPs in the medium by approx. 60%. The time courses were similar in astrocytes and microglia preparations. To verify whether the effect of cholesterol was a consequence of membrane rigidification we tested the activity of ganglioside GM1 and prion protein fragment PrP 106-126, which affect membrane fluidity similarly to cholesterol, on APPs secretion. Neither altered the production of APPs. APP mRNA and the total amount of APP in the cells were slightly decreased by cholesterol after 2 and 24 h respectively. Immunoblot analysis of APP associated with neuronal cells and astrocytes indicated that cholesterol progressively decreased the glycosylated forms of the protein; a similar tendency was noted in cells treated with brefeldin A and monensin, two substances that interfere with protein glycosylation. The cell-surface biotinylation method showed that in cholesterol-treated cells APP reached the plasma membrane. Our results indicate that cholesterol decreases the secretion of APPs by interfering with APP maturation and inhibiting glycosylation of the protein; although APP is inserted in the membrane it is not cleaved by α-secretase.
Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells
Authors: LEE Hsin-Chen , YIN Pen-Hui , LU Ching-You , CHI Chin-Wen , WEI Yau-Huei
Published: August 2015

Mitochondrial respiratory function is impaired in the target tissues of patients with mitochondrial diseases and declines with age in various human tissues. It is generally accepted that respiratory-chain defects result in enhanced production of reactive oxygen species and free radicals in mitochondria. Recently, we have demonstrated that the copy number of mitochondrial DNA (mtDNA) is increased in the lung tissues of elderly human subjects. The mtDNA copy number was suggested to be increased by a feedback mechanism that compensates for defects in mitochondria harbouring mutated mtDNA and a defective respiratory system. However, the detailed mechanism remains unclear. In this study, we treated a human lung fibroblast cell line, MRC-5, with H2O2 at concentrations of 90-360 μM. After the treatment for 24-72 h, we found that cells were arrested at G0 and G1 phases but that mitochondrial mass and mtDNA content were significantly increased in a concentration- and time-dependent manner. Moreover, the oxidative stress induced by buthionine sulphoximine was also found to cause an increase in mitochondrial mass of the treated cells. Increased uptake of a vital mitochondrial dye Rhodamine 123 and enhanced tetrazolium [MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] reduction revealed that the mitochondria increased by H2O2 treatment were functional. In addition, the increase in the mitochondrial mass was also observed in cell-cycle-arrested cells induced by mimosine, lovastatin and genistein. Taken together, these findings suggest that the increase in mitochondrial mass and mtDNA content are the early molecular events of human cells in response to endogenous or exogenous oxidative stress through cell-cycle arrest.
Retinoid-binding proteins: mediators of retinoid action
Authors: NOY Noa
Published: August 2015

Active vitamin A metabolites, known as retinoids, are essential for multiple physiological processes, ranging from vision to embryonic development. These small hydrophobic compounds associate in vivo with soluble proteins that are present in a variety of cells and in particular extracellular compartments, and which bind different types of retinoids with high selectivity and affinity. Traditionally, retinoid-binding proteins were viewed as transport proteins that act by solubilizing and protecting their labile ligands in aqueous spaces. It is becoming increasingly clear, however, that, in addition to this general role, retinoid-binding proteins have diverse and specific functions in regulating the disposition, metabolism and activities of retinoids. Some retinoid-binding proteins appear to act by sequestering their ligands, thereby generating concentration gradients that allow cells to take up retinoids from extracellular pools and metabolic steps to proceed in energetically unfavourable directions. Other retinoid-binding proteins regulate the metabolic fates of their ligands by protecting them from some enzymes while allowing metabolism by others. In these cases, delivery of a bound retinoid from the binding protein to the ‘correct’ enzyme is likely to be mediated by direct and specific interactions between the two proteins. One retinoid-binding protein was reported to enhance the ability of its ligand to regulate gene transcription by directly delivering this retinoid to the transcription factor that is activated by it. ‘Channelling’ of retinoids between their corresponding binding protein and a particular protein target thus seems to be a general theme through which some retinoid-binding proteins exert their effects.
The molecular basis of phosphatidylcholine preference of human group-V phospholipase A2
Authors: KIM Kwang Pyo , HAN Sang Kyou , HONG Mihae , CHO Wonhwa
Published: August 2015

Human group-V phospholipase A2 (hVPLA2) is a secretory phospholipase A2 (PLA2) that is involved in eicosanoid formation in such inflammatory cells as macrophages and mast cells. We showed that hVPLA2 can bind phosphatidylcholine membranes and hydrolyse phosphatidylcholine molecules much more efficiently than human group-IIa PLA2, which accounts for its high activity on the outer plasma membrane of mammalian cells. To understand the molecular basis of the high phosphatidylcholine specificity of hVPLA2, we mutated several residues (Gly-53, Glu-56 and Glu-57) that might be involved in interaction with an active-site-bound phospholipid molecule. Phospholipid head-group specificities of mutants determined using polymerized mixed-liposome substrates indicate that a small glycine residue in position 53 is important for accommodating a bulky choline head group. Also, results indicated that two anionic residues, Glu-56 and Glu-57, favourably interact with cationic head groups of phosphatidylcholine and phosphatidylethanolamine. Together, these steric and electrostatic properties of the active site of hVPLA2 allow for effective binding and hydrolysis of a bulky cationic choline head group of phosphatidylcholine, which is unique among mammalian secretory PLA2s.
DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts
Authors: MURAKAMI Hiroshi , NURSE Paul
Published: August 2015

The cell cycle checkpoint mechanisms ensure the order of cell cycle events to preserve genomic integrity. Among these, the DNA-replication and DNA-damage checkpoints prevent chromosome segregation when DNA replication is inhibited or DNA is damaged. Recent studies have identified an outline of the regulatory networks for both of these controls, which apparently operate in all eukaryotes. In addition, it appears that these checkpoints have two arrest points, one is just before entry into mitosis and the other is prior to chromosome separation. The former point requires the central cell-cycle regulator Cdc2 kinase, whereas the latter involves several key regulators and substrates of the ubiquitin ligase called the anaphase promoting complex. Linkages between these cell-cycle regulators and several key checkpoint proteins are beginning to emerge. Recent findings on post-translational modifications and protein-protein interactions of the checkpoint proteins provide new insights into the checkpoint responses, although the functional significance of these biochemical properties often remains unclear. We have reviewed the molecular mechanisms acting at the DNA-replication and DNA-damage checkpoints in the fission yeast Schizosaccharomyces pombe, and the modifications of these controls during the meiotic cell cycle. We have made comparisons with the controls in fission yeast and other organisms, mainly the distantly related budding yeast.
Regulation of sterol regulatory-element binding protein 1 gene expression in liver: role of insulin and protein kinase B/cAkt
Authors: FLEISCHMANN Mark , IYNEDJIAN Patrick B.
Published: August 2015

Insulin stimulates the transcription of the sterol regulatory- element binding protein (SREBP) 1/ADD1 gene in liver. Hepatocytes in primary culture were used to delineate the insulin signalling pathway for induction of SREBP1 gene expression. The inhibitors of phosphoinositide 3-kinase (PI 3-kinase), wortmannin and LY 294002, abolished the insulin-dependent increase in SREBP1 mRNA, whereas the inhibitor of the mitogen- activated protein kinase cascade, PD 98059, was without effect. To investigate the role of protein kinase B (PKB)/cAkt downstream of PI 3-kinase, hepatocytes were transduced with an adenovirus encoding a PKB-oestrogen receptor fusion protein. The PKB activity of this recombinant protein was rapidly activated in hepatocytes challenged with 4-hydroxytamoxifen (OHT), as was endogenous PKB in hepatocytes challenged with insulin. The addition of OHT to transduced hepatocytes resulted in accumulation of SREBP1 mRNA, with a time-course and magnitude similar to the effect of insulin in non-transduced cells. The level of SREBP1 mRNA was not increased by OHT in hepatocytes expressing a mutant form of the recombinant protein whose PKB activity was not activated by OHT. Thus acute activation of PKB is sufficient to induce SREBP1 mRNA accumulation in primary hepatocytes, and might be the major signalling event by which insulin induces SREBP1 gene expression in the liver.