bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the reaction mechanism for the XL-I form of bovine liver xenobiotic/medium-chain fatty acid:CoA ligase
Authors: VESSEY Donald A. , KELLEY Michael
Published: August 2015

The XL-I form of xenobiotic/medium-chain fatty acid:CoA ligase was purified to apparent homogeneity from bovine liver mitochondria and used to determine the reaction mechanism. A tersubstrate kinetic analysis was conducted by varying the concentrations of ATP, benzoate and CoA in turn. Both ATP and benzoate gave parallel double-reciprocal plots against CoA, which indicates a Ping Pong mechanism, with either pyrophosphate or AMP leaving before the binding of CoA. Addition of pyrophosphate to the assays changed the plots from parallel to intersecting; addition of AMP did not. This indicates that pyrophosphate is the product that leaves before binding of CoA. Based on end-product inhibition studies, it was concluded that the reaction follows a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding first, followed in order by benzoate binding, pyrophosphate release, CoA binding, benzoyl-CoA release and AMP release. A similar mechanism was obtained when the ligase was examined with butyrate as substrate. However, butyrate activation was characterized by a much higher affinity for CoA. This is attributed to steric factors resulting from the bulkier nature of the benzoate molecule. Also, with butyrate there is a bivalent cation activation distinct from that associated with binding to ATP. This activation by excess Mg2+ results in non-linear plots of 1/v against 1/[ATP] for butyrate unless the concentrations of Mg2+ and ATP are varied together.
Haem disorder in recombinant- and reticulocyte-derived haemoglobins: evidence for stereoselective haem insertion in eukaryotes
Authors: MATHEWS Antony J. , BRITTAIN Thomas
Published: August 2015

We have used NMR spectroscopy to measure haem disorder in adult human haemoglobin (HbA) obtained from mature erythrocyte cells and from yeast expressing recombinant HbA. Reticulocyte-derived HbA contained much higher levels of haem disorder (11% α- and 28% β-subunit disorder) than observed for HbA from mature erythrocytes (1.5% α- and 8% β-subunit disorder). Thus, unlike in vitro combination of haem and apoHb, biosynthetic haem insertion is not random with respect to orientation, but appears to show stereoselectivity. Recombinant HbA isolated from yeast showed 32% α- and 45% β-subunit haem disorder. These levels relaxed to their equilibrium positions after incubating the Hb in the ferric form. Recombinant embryonic human Hbs showed less haem disorder than recombinant HbA. The levels of haem disorder in embryonic Hbs ∊2∊2 and ∊2γ2 appear to have their equilibrium values. We propose that, in eukaryotes, in vivo haem insertion occurs via both co-translational mechanisms and insertion via semiHb-β.
Macrophage migration inhibitory factor of the parasitic nematode Trichinella spiralis
Authors: TAN Timothy H. P. , EDGERTON Steve A. V. , KUMARI Rashmi , McALISTER Mark S. B. , ROWE S. Mark , NAGL Sylvia , PEARL Laurence H. , SELKIRK Murray E. , BIANCO Albert E. , TOTTY Nicholas F. , ENGWERDA Chris , GRAY Carolyn A. , MEYER David J.
Published: August 2015

cDNAs were obtained for macrophage migration-inhibitory factor (MIF)/l-dopachrome methyl ester tautomerase homologues from the parasitic nematodes Trichinella spiralis (TsMIF) and Trichuris trichiura (TtMIF). The translated sequences, which were partly confirmed by sequencing of proteolytic fragments, show 42 and 44% identity respectively with human or mouse MIF, and are shorter by one C-terminal residue. Unlike vertebrate MIF and MIF homologues of filarial nematodes, neither TsMIF nor TtMIF contain cysteine residues. Soluble recombinant TsMIF, expressed in Escherichia coli showed secondary structure (by CD spectroscopy) and quaternary structure (by light-scattering and gel filtration) similar to that of the trimeric mammalian MIFs and d-dopachrome tautomerase. The catalytic specificity of recombinant TsMIF in the ketonization of phenylpyruvate (1.4×106M−1·s−1) was comparable with that of human MIF, while that of p-hydroxyphenylpyruvate (9.1×104M−1·s−1) was 71-fold lower. TsMIF showed high specificity in tautomerization of the methyl ester of l-dopachrome compared with non-esterified l-dopachrome (>87000-fold) and a high kcat (≈ 4×104s−1). The crystal structure, determined to 1.65 Å (1 Å = 0.1nm), was generally similar to that of human MIF, but differed in the boundaries of the putative active-site pocket, which can explain the low activity towards p-hydroxyphenylpyruvate. The central pore was blocked, but was continuous, with the three putative tautomerase sites. Recombinant TsMIF (5ng/ml–5pg/ml) inhibited migration of human peripheral-blood mononuclear cells in a manner similar to that shown by human MIF, but had no effect from 5 to 500ng/ml on anti-CD3-stimulated murine T-cell proliferation. TsMIF was detected in supernatants of T. spiralis larvae cultured in vitro at 6ng/ml (55ng/mg total secreted protein). In conclusion TsMIF has structural, catalytic and cell-migration-inhibitory properties which indicate that it is partially orthologous to mammalian MIF.
Site-directed removal of N-glycosylation sites in BST-1/CD157: effects on molecular and functional heterogeneity
Authors: YAMAMOTO-KATAYAMA Sumie , SATO Atsushi , ARIYOSHI Mariko , SUYAMA Mikita , ISHIHARA Katsuhiko , HIRANO Toshio , NAKAMURA Haruki , MORIKAWA Kosuke , JINGAMI Hisato
Published: August 2015

Cyclic ADP ribose (cADPR) is a novel second messenger that releases calcium from intracellular calcium stores, but works independently of inositol 1,4,5-trisphosphate. In mammals ADP-ribosyl cyclase function is found in two membrane proteins, CD38 and bone marrow stromal cell antigen 1 (BST-1)/CD157. These enzymes are exposed extracellularly and also possess cADPR hydrolase activity, but an intracellular soluble ADP-ribosyl cyclase has been reported in human T-cells. Previously, a soluble form of BST-1/CD157 (sBST-1), which lacked the glycosylphosphatidylinositol-anchored portion, was expressed by a baculovirus–insect-cell system. In this study, we have purified the sBST-1, and it migrated as two major bands by SDS/PAGE, suggesting that it is post-translationally modified. BST-1 contains four putative N-glycosylation sites. Tunicamycin treatment reduced sBST-1 expression in the culture medium, indicating that N-glycosylation is essential for secretion. Site-directed mutagenesis was performed to generate sBST-1 mutants (N1–N4), each preserving a single N-glycosylation site. N1, N3 and N4 were well secreted into the medium, and were each detected as a single band. Although N3 and N4 retained the ADP-ribosyl cyclase activity, the cADPR-hydrolase activity was retained only in N4. We conclude that N-glycosylation of sBST-1 facilitates the folding of the nascent polypeptide chain into a conformation that is conductive for intracellular transport and enzymic activity. Furthermore a crystal has been obtained using the N4 mutant, but not the wild-type sBST-1. Thus the artificial engineering of N-glycosylation sites could be an effective method to generate homogeneous material for structural studies.
Characterization of active-site residues in diadenosine tetraphosphate hydrolase from Lupinus angustifolius
Authors: MAKSEL Danuta , GOOLEY Paul R. , SWARBRICK James D. , GURANOWSKI Andrzej , GANGE Christine , BLACKBURN G. Michael , GAYLER Kenwyn R.
Published: August 2015

Site-directed mutagenesis has been used to characterize the functions of key amino acid residues in the catalytic site of the ‘nudix’ hydrolase, (asymmetrical) diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) hydrolase (EC 3.6.1.17) from Lupinus angustifolius, the three-dimensional solution structure of which has recently been solved. Residues within the nudix motif, Gly-(Xaa)5-Glu-(Xaa)7-Arg-Glu-Uaa-Xaa-(Glu)2-Xaa-Gly (where Xaa represents unspecified amino acids and Uaa represents the bulky aliphatic amino acids Ile, Leu or Val) conserved in ‘nudix enzymes’, and residues important for catalysis from elsewhere in the molecule, were mutated and the expressed proteins characterized. The results reveal a high degree of functional conservation between lupin asymmetric Ap4A hydrolase and the 8-oxo-dGTP hydrolase from Escherichia coli. Charged residues in positions equivalent to those that ligate an enzyme-bound metal ion in the E. coli 8-oxo-dGTP hydrolase [Harris, Wu, Massiah and Mildvan (2000) Biochemistry 39, 1655–1674] were shown to contribute to catalysis to similar extents in the lupin enzyme. Mutations E55Q, E59Q and E125Q all reduced kcat markedly, whereas mutations R54Q, E58Q and E122Q had smaller effects. None of the mutations produced a substantial change in the Km for Ap4A, but several extensively modified the pH-dependence and fluoride-sensitivities of the hydrolase. It was concluded that the precisely positioned glutamate residues Glu-55, Glu-59 and Glu-125 are conserved as functionally significant components of the hydrolytic mechanism in both of these members of the nudix family of hydrolases.
Temporal relationships between ceramide production, caspase activation and mitochondrial dysfunction in cell lines with varying sensitivity to anti-Fas-induced apoptosis
Authors: RODRIGUEZ-LAFRASSE Claire , ALPHONSE Gersende , BROQUET Pierre , ALOY Marie-Thérèse , LOUISOT Pierre , ROUSSON Robert
Published: August 2015

To clarify the chronology of events leading to anti-Fas-induced apoptosis, and the mechanisms of resistance to this death effector, we compared the response kinetics of three tumour cell lines that display varying sensitivity to anti-Fas (based on levels of apoptosis), in terms of ceramide release, mitochondrial function and the caspase-activation pathway. In the highly sensitive Jurkat cell line, early caspase-8 activation, observed from 2h after treatment, was chronologically associated with an acute depletion of glutathione and the cleavage of caspase-3 and poly-ADP ribosyl polymerase (PARP), followed by a progressive fall in the mitochondrial transmembrane potential (Δψm), between 4 and 48h after treatment. Ceramide levels began to increase 2h after the addition of anti-Fas (with no increase during the first hour), and increased continuously to 640% of control cells at 48h. In the moderately sensitive SCC61 adherent cells, comparable results were observed, though with lower levels of ceramide and a delay in the response kinetics, with apoptotic cells becoming flotant. Finally, despite early cleavage of caspase-8 at 2h, and a sustained level of activation until 48h, no apoptotic response was observed in anti-Fas-resistant SQ20B cells. This was confirmed by a lack of ceramide generation and mitochondrial changes, and by the absence of any detectable cleavage of caspase-3 or PARP. Inhibition of caspase processing, and amplification of endogenous ceramide signalling by pharmacological agents, allowed us to establish the order of cellular events, locating ceramide release after caspase-8 activation and before caspase-3 activation, and demonstrating a direct involvement for ceramide release in mitochondrial dysfunction. Furthermore, these experiments provide strong arguments for the role of endogenous ceramide as a key executor of apoptosis, rather than as a consequence of membrane alterations.
Cloning and characterization of the 5′-flanking region of the rat glutamate-cysteine ligase catalytic subunit
Authors: YANG Heping , WANG Jiaohong , HUANG Zong-Zhi , OU Xiaopeng , LU Shelly C.
Published: August 2015

Glutamate-cysteine ligase (GCL), the rate-limiting enzyme in glutathione synthesis, is made up of two subunits, a catalytic (heavy) subunit (GCLC) and a modifier (light) subunit (GCLM), which are differentially regulated. Increased hepatic GCLC expression occurs during rapid growth, oxidative stress and after ethanol treatment. To facilitate studies of GCLC transcriptional regulation, we have cloned and characterized a 1.8kb 5′-flanking region of the rat GCLC (GenBank accession number AF218362). A consensus TATA box and one transcriptional start site are located at 302 and 197 nucleotides upstream of the translational start site, respectively. The promoter contains consensus binding sites for many transcription factors including nuclear factor κB (NF-κB) and activator protein 1 (AP-1). The rat GCLC promoter was able to efficiently drive luciferase expression in H4IIE cells. Sequential deletion analysis revealed that three DNA regions, −595 to −111, −1108 to −705 and −705 to −595, are involved in positive (the first two regions) and negative (the latter region) gene regulation. Specific protein binding to these regions was confirmed by DNase I footprinting and electrophoretic mobility-shift assays (EMSAs). Ethanol-fed livers exhibit increased protein binding to region −416 to −336 on DNase I footprinting analysis, which was found to be NF-κB and AP-1 on EMSA and supershift analysis. Acetaldehyde treatment of H4IIE cells led to a time- and dose-dependent increase in GCLC mRNA levels, binding of NF-κB and AP-1 to the GCLC promoter, and luciferase activity driven by the GCLC promoter fragment containing these binding sites.
Amphibian DNases I are characterized by a C-terminal end with a unique, cysteine-rich stretch and by the insertion of a serine residue into the Ca2+-binding site
Authors: TAKESHITA Haruo , YASUDA Toshihiro , IIDA Reiko , NAKAJIMA Tamiko , MORI Shinjiro , MOGI Kouichi , KANEKO Yasushi , KISHI Koichiro
Published: August 2015

We purified four amphibian deoxyribonucleases I from the pancreases of one toad, two frog and one newt species, by using three different column chromatography methods in sequence. Each of the purified enzymes had a molecular mass of approx. 40kDa and an optimal pH for activity of approx. 8.0. These values were significantly greater than those for other vertebrate DNases I. The full-length cDNA encoding each amphibian DNase I was constructed from the total RNA of the pancreas by using rapid amplification of cDNA ends. Nucleotide sequence analyses revealed two structural characteristics unique to amphibian DNases I: a stretch of approx. 70 amino acids with a high cysteine content (approx. 15%) in the C-terminal region, and the insertion of a serine residue at position 205 (in a domain containing an essential Ca2+-binding site). Expression analysis of a series of mutant constructs indicated that both of these structures are essential in generating the active form of the enzyme. ‘DNase I signature sequences’, which are well conserved in other vertebrate DNases I, could not be found in any of the amphibian DNases I tested, whereas a ‘somatomedin B motif’ was identified in the Cys-rich stretches of all four. Although DNase I has so far been considered to be a secretory glycoprotein, amphibian DNase I seems to be non-glycosylated. These structural findings indicate strongly that amphibian DNases I are situated in a unique position on the phylogenetic tree of the DNase I family.
Comparison of the effects of dietary n−3 and n−6 polyunsaturated fatty acids on very-low-density lipoprotein secretion when delivered to hepatocytes in chylomicron remnants
Authors: ZHENG Xiaozhong , AVELLA Michael , BOTHAM Kathleen M.
Published: August 2015

The effects of chylomicron remnants enriched in n-3 or n-6 polyunsaturated fatty acids (derived from fish or corn oil respectively) on the secretion of very-low-density lipoprotein (VLDL) lipid and apolipoprotein B (apoB) by rat hepatocytes in culture was investigated. Remnants were prepared in vivo from chylomicrons obtained from rats given an oral dose of fish or corn oil and incubated with cultured hepatocytes for up to 16h. The medium was then removed and the secretion of cholesterol and triacylglycerol into the whole medium or the ρ < 1.050g/ml fraction during the following 7–24h was determined. After exposure of the cells to fish-oil as compared with corn-oil remnants, secretion of both cholesterol and triacylglycerol into the whole medium was decreased by 25–35%, and secretion into the ρ < 1.050g/ml fraction was decreased by 20–25%. In addition, the levels of apoB48 found in the ρ < 1.050g/ml fraction were significantly lower in cells treated with fish-oil rather than corn-oil remnants, although the levels of apoB100 remained unchanged. The expression of mRNA for apoB, as determined by reverse-transcriptase PCR, however, was not significantly changed after exposure of the cells to both types of remnants. These results demonstrate that the effects of dietary n-3 polyunsaturated fatty acids in depressing hepatic VLDL secretion occur directly when they are delivered to the liver from the intestine in chylomicron remnants, and that the secretion, but not the synthesis, of apoB is targeted.
Identification of a peroxisome-proliferator-activated-receptor response element in the apolipoprotein E gene control region
Authors: GALETTO Román , ALBAJAR Marta , POLANCO José I. , ZAKIN Mario M. , RODRÍGUEZ-REY José C.
Published: August 2015

Apolipoprotein E (apoE) is a protein involved in reverse cholesterol transport. Among other tissues, apoE is expressed in macrophages where its expression increases when macrophages develop into foam cells. It has been recently shown that peroxisome-proliferator-activated receptor gamma (PPARγ) is involved in this conversion. Northern-blot analysis was carried out in the macrophage cell line THP1 to determine whether apoE mRNA levels were regulated by ciglitazone, a PPARγ inducer. The results indicated that treatment with ciglitazone doubled the levels of apoE mRNA. To identify a possible PPARγ response element (PPRE), several portions of apoE gene control region were used to construct luciferase reporter plasmids. In U-87 MG cells, a 185bp fragment located in the apoE/apoCI intergenic region was sufficient to induce a 10-fold increase in the luciferase activity of the extract of cells co-transfected with a PPARγ expression plasmid. Subsequent analysis revealed the presence of a sequence with a high level of sequence similarity to the consensus PPRE. Mutations in this sequence resulted in a lack of functionality both in transient transfection and in electrophoretic-mobility-shift assays. These results demonstrated the presence of a functional PPRE in the apoE/apoCI intergenic region. These results have implications for the regulation of apoE gene expression and could be relevant for understanding the anti-atherogenic effect of thiazolidinediones.
Functional and conformational properties of the exclusive C-domain from the Arabidopsis copper chaperone (CCH)
Authors: MIRA Helena , VILAR Marçal , PÉREZ-PAYÁ Enrique , PEÑARRUBIA Lola
Published: August 2015

The Arabidopsis thaliana copper chaperone (CCH) is a small copper binding protein involved in copper trafficking. When compared to homologues from other eukaryotes, CCH has two different domains; the conserved N-domain and the plant-exclusive C-domain, a C-terminal extension with an unusual amino-acid composition. In order to characterize this extra C-domain, the CCH protein, the N-domain and the C-domain were all expressed separately in heterologous systems. While the N-domain retained the copper chaperone and antioxidant properties described for the yeast Atx1 and human HAH1 counterparts, the C-domain displayed particular structural properties that would be necessary to optimize copper homoeostasis in plant cells where it could be responsible for the metallochaperone plant-exclusive intercellular transport. The whole CCH protein and the C-domain, but not the N-domain, displayed altered SDS/PAGE mobilities. CD spectroscopy showed that the N-domain fold is representative of an α/β protein, while the C-domain adopts an extended conformation.
Inhibition of distant caspase homologues by natural caspase inhibitors
Authors: SNIPAS Scott J. , STENNICKE Henning R. , RIEDL Stefan , POTEMPA Jan , TRAVIS James , BARRETT Alan J. , SALVESEN Guy S.
Published: August 2015

Caspases play an important role in the ability of animal cells to kill themselves by apoptosis. Caspase activity is regulated in vivo by members of three distinct protease inhibitor families, two of which, baculovirus p35 and members of the inhibitor of apoptosis (IAP) family, are thought to be caspase specific. However, caspases are members of the clan of cysteine proteases designated CD, which also includes animal and plant legumains, and the bacterial proteases clostripain, gingipain-R and gingipain-K. Since these proteases have been proposed to have a common mechanism and evolutionary origin, we hypothesized that the caspase inhibitors may also regulate these other proteases. We tested this hypothesis by examining the effect of the natural caspase inhibitors on other members of protease clan CD. The IAP family proteins were found to have only a slight inhibitory effect on gingipain-R. The cowpox viral cytokine-response modifier A (CrmA) serpin had no effect on any of the proteases tested but a single point mutation of CrmA (Asp → Lys) resulted in strong inhibition of gingipain-K. More substantial, with respect to the hypothesis, was the strong inhibition of gingipain-K by wild-type p35. The site in p35, required for inhibition of gingipain-K, was mapped to Lys94, seven residues C-terminal to the caspase inhibitory site. Our data indicate that the virally encoded caspase inhibitors have adopted a mechanism that allows them to regulate disparate members of clan CD proteases.
Nitric oxide synthases: structure, function and inhibition
Authors: ALDERTON Wendy K. , COOPER Chris E. , KNOWLES Richard G.
Published: August 2015

This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, l-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated.
Isolation and characterization of two novel A20-like proteins
Authors: EVANS Paul C. , TAYLOR Ellen R. , COADWELL John , HEYNINCK Karen , BEYAERT Rudi , KILSHAW Peter J.
Published: August 2015

The transcription factor nuclear factor κB (NF-κB) plays a pivotal role in inflammatory processes through induction of adhesion molecules and chemokines. The zinc finger molecule A20 is an important negative regulator of NF-κB. The mechanism utilized by A20 is not fully understood, but A20 has been shown to bind to tumour-necrosis-factor-receptor-associated factor (TRAF) molecules, which are necessary for pro-inflammatory cytokine signalling. We report two novel genes, Cezanne (cellular zinc finger anti-NF-kB) and TRABID (TRAF-bnding domain), with sequence similarity to A20. Co-immunoprecipitation studies indicated that TRAF6 was able to interact with both Cezanne and TRABID. In contrast, reporter gene experiments revealed a specific ability of Cezanne to down-regulate NF-κB. It is likely, therefore, that Cezanne participates in the regulation of inflammatory processes.
Fingerprinting of polysaccharides attacked by hydroxyl radicals in vitro and in the cell walls of ripening pear fruit
Authors: FRY Stephen C. , DUMVILLE Jo C. , MILLER Janice G.
Published: August 2015

Hydroxyl radicals (•OH) may cause non-enzymic scission of polysaccharides in vivo, e.g. in plant cell walls and mammalian connective tissues. To provide a method for detecting the action of endogenous •OH in vivo, we investigated the products formed when polysaccharides were treated with •OH (generated in situ by ascorbate-H2O2-Cu2+ mixtures) followed by NaB3H4. Treatment with •OH increased the number of NaB3H4-reacting groups present in citrus pectin, homogalacturonan and tamarind xyloglucan. This increase is attributed partly to the formation of glycosulose and glycosulosuronic acid residues, which are then reduced back to the original (but radioactive) sugar residues and their epimers by NaB3H4. The glycosulose and glycosulosuronic acid residues were stable for > 16h at 20°C in ethanol or buffer (pH4.7), but were destroyed in alkali. Driselase-digestion of the radiolabelled polysaccharides yielded characteristic patterns of 3H-products, which included galactose and galacturonate from pectin, and isoprimeverose, galactose, glucose and arabinose from xyloglucan. Pectin yielded at least eight 3H-labelled anionic products, separable by electrophoresis at pH3.5. The patterns of radioactive products form useful ‘fingerprints’ by which •OH-attacked polysaccharides may be recognized. Applied to the cell walls of ripening pear (Pyrus communis) fruit, the method gave evidence for progressive •OH radical attack on polysaccharides during the softening process.
Phosphorylation-mimicking glutamate clusters in the proline-rich region are sufficient to simulate the functional deficiencies of hyperphosphorylated tau protein
Authors: EIDENMÜLLER Jochen , FATH Thomas , MAAS Thorsten , POOL Madeline , SONTAG Estelle , BRANDT Roland
Published: August 2015

The microtubule-associated tau proteins represent a family of closely related phosphoproteins that become enriched in the axons during brain development. In Alzheimer's disease (AD), tau aggregates somatodendritically in paired helical filaments in a hyperphosphorylated form. Most of the sites that are phosphorylated to a high extent in paired helical filament tau are clustered in the proline-rich region (P-region; residues 172–251) and the C-terminal tail region (C-region; residues 368–441) that flank tau's microtubule-binding repeats. This might point to a role of a region-specific phosphorylation cluster for the pathogenesis of AD. To determine the functional consequences of such modifications, mutated tau proteins were produced in which a P- or C-region-specific phosphorylation cluster was simulated by replacement of serine/threonine residues with glutamate. We show that a phosphorylation-mimicking glutamate cluster in the P-region is sufficient to block microtubule assembly and to inhibit tau's interaction with the dominant brain phosphatase protein phosphatase 2A isoform ABαC. P-region-specific mutations also decrease tau aggregation into filaments and decrease tau's process-inducing activity in a cellular transfection model. In contrast, a phosphorylation-mimicking glutamate cluster in the C-region is neutral with regard to these activities. A glutamate cluster in both the P- and C-regions induces the formation of SDS-resistant conformational domains in tau and suppresses tau's interaction with the neural membrane cortex. The results indicate that modifications in the proline-rich region are sufficient to induce the functional deficiencies of tau that have been observed in AD. They suggest that phosphorylation of the proline-rich region has a crucial role in mediating tau-related changes during disease.
Effect of ‘binary mitochondrial heteroplasmy’ on respiration and ATP synthesis: implications for mitochondrial diseases
Authors: KORZENIEWSKI Bernard , MALGAT Monique , LETELLIER Thierry , MAZAT Jean-Pierre
Published: August 2015

Respiratory-chain-complex subunits in mitochondria are encoded by nuclear or mitochondrial DNA. This property might have profound implications for the phenotypic expression of mutations affecting oxidative phosphorylation complexes. The aim of this paper is to study the importance of the origin of the mutation (nuclear or mitochondrial) on the expression of mitochondrial defects. We have therefore developed theoretical models illustrating three mechanisms of nuclear or mitochondrial DNA mutation giving rise to a deficiency in the respiratory-chain complex: (1) a partial deficiency, homogeneously distributed in all of the mitochondria; (2) a complete deficiency, only affecting some of the mitochondria (‘binary mitochondrial heteroplasmy’); and (3) a partial deficiency, affecting only some of the mitochondria. We show that mutations affecting oxidative phosphorylation complexes will be expressed in different ways depending on their origins. Although the expression of nuclear or mitochondrial mutations is evidence of a biochemical threshold, we demonstrate that the threshold value depends on the origin and distribution of the mutation (homogeneous or not) and also on the energy demand of the tissue. This last prediction has been confirmed in an experimental model using hexokinase for the simulation of the energy demand and a variation in mitochondrial concentration. We also emphasize the possible role of ‘binary mitochondrial heteroplasmy’ in the expression of mitochondrial DNA mutations and thus the importance of the origin of the deficit (mutation) for the diagnosis or therapy of mitochondrial diseases.
Ca2+/calmodulin-dependent protein kinase mediates the phosphorylation of CD44 required for cell migration on hyaluronan
Authors: LEWIS Charlotte A. , TOWNSEND Paul A. , ISACKE Clare M.
Published: August 2015

CD44 is the principal cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and binding to this ligand underlies CD44-mediated cell attachment and migration. As would be expected for a widely expressed adhesion receptor, CD44 is subject to complex regulatory events, and mis-regulation of the receptor has been associated with a number of disease pathologies, including chronic inflammatory conditions and the progression of metastatic tumours. In previous studies we have demonstrated that a key control point for this receptor is the phosphorylation of CD44 on a conserved cytoplasmic serine residue, Ser325. This modification is not required for efficient ligand binding, but is an essential component of CD44-dependent cell migration on a hyaluronan substratum. To understand better the mechanism regulating CD44 phosphorylation on Ser325, we have generated a monoclonal antibody that specifically recognizes CD44 phosphorylated on Ser325, and have developed assays to identify the Ser325 kinase. We demonstrate here that CD44 is phosphorylated to high stoichiometry in resting cells and that Ca2+/calmodulin-dependent protein kinase II is a CD44 Ser325 kinase.
A large family of endosome-localized proteins related to sorting nexin 1
Authors: TEASDALE Rohan D. , LOCI David , HOUGHTON Fiona , KARLSSON Lars , GLEESON Paul A.
Published: August 2015

Sorting nexin 1 (SNX1), a peripheral membrane protein, has previously been shown to regulate the cell-surface expression of the human epidermal growth factor receptor [Kurten, Cadena and Gill (1996) Science 272, 1008–1010]. Searches of human expressed sequence tag databases with SNX1 revealed eleven related human cDNA sequences, termed SNX2 to SNX12, eight of them novel. Analysis of SNX1-related sequences in the Saccharomyces cerevisiae genome clearly shows a greatly expanded SNX family in humans in comparison with yeast. On the basis of the predicted protein sequences, all members of this family of hydrophilic molecules contain a conserved 70–110-residue Phox homology (PX) domain, referred to as the SNX-PX domain. Within the SNX family, subgroups were identified on the basis of the sequence similarities of the SNX-PX domain and the overall domain structure of each protein. The members of one subgroup, which includes human SNX1, SNX2, SNX4, SNX5 and SNX6 and the yeast Vps5p and YJL036W, all contain coiled-coil regions within their large C-terminal domains and are found distributed in both membrane and cytosolic fractions, typical of hydrophilic peripheral membrane proteins. Localization of the human SNX1 subgroup members in HeLa cells transfected with the full-length cDNA species revealed a similar intracellular distribution that in all cases overlapped substantially with the early endosome marker, early endosome autoantigen 1. The intracellular localization of deletion mutants and fusions with green fluorescent protein showed that the C-terminal regions of SNX1 and SNX5 are responsible for their endosomal localization. On the basis of these results, the functions of these SNX molecules are likely to be unique to endosomes, mediated in part by interactions with SNX-specific C-terminal sequences and membrane-associated determinants.
Steady-state localization of a medial-Golgi glycosyltransferase involves transit through the trans-Golgi network
Authors: OPAT Andrew S. , HOUGHTON Fiona , GLEESON Paul A.
Published: August 2015

The steady-state localization of medial-Golgi enzymes is likely to involve retrograde transport pathways; however, the trafficking of these resident enzymes through the Golgi stack is unclear. To investigate if the medial-Golgi enzyme β-1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI) is transported to the late Golgi, a modified GlcNAc-TI bearing an N-glycan site on the C-terminus was constructed. The modified GlcNAc-TI was demonstrated to be functionally active in vivo, and was localized to the Golgi stack of transfected cells. In stable Chinese-hamster ovary (CHO) cell clones, the N-glycosylated GlcNAc-TI carried sialylated complex N-glycan chains. Pulse-chase studies showed that the majority of GlcNAc-TI was sialylated within 60min of synthesis. Treatment of transfected CHO cells with Brefeldin A resulted in the glycosylated GlcNAc-TI bearing endo-β-N-acetylglucosaminidase H resistant chains; however, the sialylation of glycosylated GlcNAc-TI was dramatically reduced. These data imply that, in CHO cells, newly synthesized GlcNAc-TI is transported rapidly through the Golgi stack to the trans-Golgi network, suggesting that GlcNAc-TI continuously recycles from the late Golgi. Furthermore, this data suggests that retrograde transport pathways play an important role in establishing the asymmetric distribution of GlcNAc-TI within the Golgi stack.
Multiple adaptive mechanisms affect asparagine synthetase substrate availability in asparaginase-resistant MOLT-4 human leukaemia cells
Authors: ASLANIAN Ara M. , KILBERG Michael S.
Published: August 2015

Childhood acute lymphoblastic leukaemia is treated by combination chemotherapy with a number of drugs, almost always including the enzyme l-asparaginase (ASNase). Although the initial remission rate is quite high, relapse and associated drug resistance remain a problem. In vitro studies have demonstrated an adaptive increase in asparagine synthetase (AS) expression in ASNase-resistant cells, which is believed to permit ASNase-resistant human leukaemia cells to survive in vivo. The present results, obtained with ASNase-sensitive and -resistant human MOLT-4 leukaemia cell lines, illustrate that several other adaptive processes occur to provide sufficient amounts of the AS substrates, aspartate and glutamine, required to support this increased enzymic activity. In both cell populations, aspartate is derived almost exclusively from intracellular sources, whereas the necessary glutamine arises from both intracellular and extracellular sources. Transport of glutamine into ASNase-resistant cells is significantly enhanced compared with the parental cells, whereas amino acid efflux (e.g. asparagine) is reduced. Most of the adaptive change for the amino acid transporters, Systems A, ASC and L, is rapidly (12h) reversed following ASNase removal. The enzymic activity of glutamine synthetase is also enhanced in ASNase-resistant cells by a post-transcriptional mechanism. The results demonstrate that there are several sites of metabolic adaptation in ASNase-treated leukaemia cells that serve to promote the replenishment of both glutamine and asparagine.
A maize insulin-like growth factor signals to a transduction pathway that regulates protein synthesis in maize
Authors: GARCÍA FLORES Cristina , AGUILAR Raúl , REYES DE LA CRUZ Homero , ALBORES Martha , SÁNCHEZ de JIMÉNEZ Estela
Published: August 2015

Insulin and insulin-like growth factors (IGFs) are well-characterized regulators in higher eukaryotic cells that control biological processes such as cell growth and survival, and selective translation of mRNAs. This research presents the purification of a 20kDa protein, isolated from maize tissue, with IGF activity. The protein was purified from 48h-germinated maize embryonic axes by G-50 Sephadex fractionation followed by affinity chromatography through a bovine insulin antibody–Sepharose column. This protein proved to significantly speed up maize germination and seedling growth. At the molecular level, Zea mays IGF (ZmIGF) enhanced phosphorylation of S6 ribosomal protein (rp) on the 40S ribosomal subunit, in a similar way as observed when bovine insulin is applied to maize axes during germination. Rapamycin, a specific inhibitor of the insulin-stimulated signal transduction pathway, prevented S6 rp phosphorylation in maize axes. Moreover, ZmIGF stimulated [35S]methionine incorporation into rps, above the level of overall cytoplasmic proteins. Either incubation with anti-insulin antibody, heat treatment (60°C) or trypsin digestion abolished this ZmIGF effect. It is proposed that ZmIGF is an endogenous maize growth factor that regulates the synthesis of specific proteins through a pathway similar to that of insulin or IGFs in animal tissues.
Functional and structural roles of the glutathione-binding residues in maize (Zea mays) glutathione S-transferase I
Authors: LABROU Nikolaos E. , MELLO Luciane V. , CLONIS Yannis D.
Published: August 2015

The isoenzyme glutathione S-transferase (GST) I from maize (Zea mays) was cloned and expressed in Escherichia coli, and its catalytic mechanism was investigated by site-directed mutagenesis and dynamic studies. The results showed that the enzyme promotes proton dissociation from the GSH thiol and creates a thiolate anion with high nucleophilic reactivity by lowering the pKa of the thiol from 8.7 to 6.2. Steady-state kinetics fit well to a rapid equilibrium, random sequential Bi Bi mechanism, with intrasubunit modulation between the GSH binding site (G-site) and the electrophile binding site (H-site). The rate-limiting step of the reaction is viscosity-dependent, and thermodynamic data suggest that product release is rate-limiting. Five residues of GST I (Ser11, His40, Lys41, Gln53 and Ser67), which are located in the G-site, were individually replaced with alanine and their structural and functional roles in the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction were investigated. On the basis of steady-state kinetics, difference spectroscopy and limited proteolysis studies it is concluded that these residues: (1) contribute to the affinity of the G-site for GSH, as they are involved in side-chain interaction with GSH; (2) influence GSH thiol ionization, and thus its reactivity; (3) participate in kcat regulation by affecting the rate-limiting step of the reaction; and (4) in the cases of His40, Lys41 and Gln53 play an important role in the structural integrity of, and probably in the flexibility of, the highly mobile short 310-helical segment of α-helix 2 (residues 35–46), as shown by limited proteolysis experiments. These structural perturbations are probably transmitted to the H-site through changes in Phe35 conformation. This accounts for the modulation of Kcdnbm by His40, Lys41 and Gln53, and also for the intrasubunit communication between the G- and H-sites. Computer simulations using CONCOORD were applied to maize GST I monomer and dimer structures, each with bound lactoylglutathione, and the results were analysed by the essential dynamics technique. Differences in dynamics were found between the monomer and the dimer simulations showing the importance of using the whole structure in dynamic analysis. The results obtained confirm that the short 310-helical segment of α-helix 2 (residues 35–46) undergoes the most significant structural rearrangements. These rearrangements are discussed in terms of enzyme catalytic mechanism.
Elongated dermatan sulphate in post-inflammatory healing skin distributes among collagen fibrils separated by enlarged interfibrillar gaps
Authors: KUWABA Kumiko , KOBAYASHI Miya , NOMURA Yoshihiro , IRIE Shinkichi , KOYAMA Yoh-ichi
Published: August 2015

It has been reported that the disaccharide composition of dermatan sulphate shows transient changes after epicutaneous application of the hapten 2,4-dinitrofluorobenzene to mouse skin, and that these changes are most conspicuous in healing skin on day 15 after chemical insult [Kuwaba, Nomura, Irie and Koyama (1999) J. Dermatol. Sci. 19, 23–30]. In the present study it was found that the molecular size of dermatan sulphate was increased on day 15 after hapten application. The molecular size of decorin increased in healing skin, whereas the size of dermatan-sulphate-depleted core protein did not increase. The length and localization of decorin dermatan sulphate were investigated by electron microscopy. Dermatan sulphate filaments oriented orthogonally to collagen fibrils were longer in healing skin than in control skin. In control skin, dermatan sulphate filaments were found among tightly packed collagen fibrils. In contrast, the interfibrillar gaps between each collagen fibril were enlarged in healing skin; elongated dermatan sulphate filaments extended from the surface of collagen fibrils across the enlarged gap. These results suggest that the increase in molecular size of decorin dermatan sulphate is important in organizing collagen fibrils separated by enlarged interfibrillar gaps in healing skin.
Structure and expression of human mitochondrial adenylate kinase targeted to the mitochondrial matrix
Authors: NOMA Takafumi , FUJISAWA Koichi , YAMASHIRO Yasuhiro , SHINOHARA Miho , NAKAZAWA Atsushi , GONDO Toshikazu , ISHIHARA Tokuhiro , YOSHINOBU Kumiko
Published: August 2015

The previously isolated cDNA encoding human adenylate kinase (AK) isozyme 3 was recently renamed AK4. Consequently, human AK3 cDNA remains to be identified and we have little information about the functional relationship between human AK3 and AK4. In pursuit of the physiological roles of both the AK3 and AK4 proteins, we first isolated an authentic human AK3 cDNA and compared their expression. Nucleotide sequencing revealed that the cDNA encoded a 227-amino-acid protein, with a deduced molecular mass of 25.6kDa, that shares greater homology with the AK3 cDNAs isolated from bovine and rat than that from human. We named the isolated cDNA AK3. Northern-blot analysis revealed that AK3 mRNA was present in all tissues examined, and was highly expressed in heart, skeletal muscle and liver, moderately expressed in pancreas and kidney, and weakly expressed in placenta, brain and lung. On the other hand, we found that human AK4 mRNA was highly expressed in kidney, moderately expressed in heart and liver and weakly expressed in brain. Western-blot analysis demonstrated expression profiles of AK3 and AK4 that were similar to their mRNA expression patterns in each tissue. Over expression of AK3, but not AK4, in both Escherichia coli CV2, a temperature-sensitive AK mutant, and a human embryonic kidney-derived cell line, HEK-293, not only produced significant GTP:AMP phosphotransferase (AK3) activity, but also complemented the CV2 cells at 42°C. Subcellular and submitochondrial fractionation analysis demonstrated that both AK3 and AK4 are localized in the mitochondrial matrix.
Palmitoylethanolamide inhibits the expression of fatty acid amide hydrolase and enhances the anti-proliferative effect of anandamide in human breast cancer cells
Authors: Di MARZO Vincenzo , MELCK Dominique , ORLANDO Pierangelo , BISOGNO Tiziana , ZAGOORY Orna , BIFULCO Maurizio , VOGEL Zvi , de PETROCELLIS Luciano
Published: August 2015

Palmitoylethanolamide (PEA) has been shown to act in synergy with anandamide (arachidonoylethanolamide; AEA), an endogenous agonist of cannabinoid receptor type 1 (CB1). This synergistic effect was reduced by the CB2 cannabinoid receptor antagonist SR144528, although PEA does not activate either CB1 or CB2 receptors. Here we show that PEA potently enhances the anti-proliferative effects of AEA on human breast cancer cells (HBCCs), in part by inhibiting the expression of fatty acid amide hydrolase (FAAH), the major enzyme catalysing AEA degradation. PEA (1–10μM) enhanced in a dose-related manner the inhibitory effect of AEA on both basal and nerve growth factor (NGF)-induced HBCC proliferation, without inducing any cytostatic effect by itself. PEA (5μM) decreased the IC50 values for AEA inhibitory effects by 3–6-fold. This effect was not blocked by the CB2 receptor antagonist SR144528, and was not mimicked by a selective agonist of CB2 receptors. PEA enhanced AEA-evoked inhibition of the expression of NGF Trk receptors, which underlies the anti-proliferative effect of the endocannabinoid on NGF-stimulated MCF-7 cells. The effect of PEA was due in part to inhibition of AEA degradation, since treatment of MCF-7 cells with 5μM PEA caused a ∼ 30–40% down-regulation of FAAH expression and activity. However, PEA also enhanced the cytostatic effect of the cannabinoid receptor agonist HU-210, although less potently than with AEA. PEA did not modify the affinity of ligands for CB1 or CB2 receptors, and neither did it alter the CB1/CB2-mediated inhibitory effect of AEA on adenylate cyclase type V, nor the expression of CB1 and CB2 receptors in MCF-7 cells. We suggest that long-term PEA treatment of cells may positively affect the pharmacological activity of AEA, in part by inhibiting FAAH expression.
Purification and characterization of a glutathione S-transferase Omega in pig: evidence for two distinct organ-specific transcripts
Authors: ROUIMI Patrick , ANGLADE Patricia , BENZEKRI Anne , COSTET Philippe , DEBRAUWER Laurent , PINEAU Thierry , TULLIEZ Jacques
Published: August 2015

A cytosolic glutathione S-transferase (GST, EC 2.5.1.18) from the recently characterized Omega class [GSTO; Board et al. 2000, J. Biol. Chem. 275, 24798–24806] has been identified in pig organs. It was found widely distributed in the different tissues investigated and especially abundant in liver and muscle. The hepatic enzyme has been purified to homogeneity by using its selective affinity for S-hexylglutathione over GSH, thus providing a simple method to isolate mammalian GSTO. The dimeric protein has a subunit molecular mass of 27328Da as measured by electrospray ionization MS. Internal peptide sequencing and complete cDNA sequencing revealed strong similarities with its human recombinant orthologue and two rodent GST-like proteins with the ability to catalyse the GSH-dependent reduction of dehydroascorbate. Additional similarities, including the presence of a specific N-terminal extension and of immunological cross-reactivity, support the results. Moreover, this gene encoding GSTO generates two organ-specific transcripts, suggesting transcriptional mechanisms with a significance that is as yet uncharacterized.