bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutations of the serine phosphorylated in the protein phosphatase-1-binding motif in the skeletal muscle glycogen-targeting subunit
Authors: LIU Jun , WU Jun , OLIVER Carey , SHENOLIKAR Shirish , BRAUTIGAN David L.
Published: August 2015

Cellular functions of protein phosphatase-1 (PP1) are determined by regulatory subunits that contain the consensus PP1-binding motif, RVXF. This motif was first identified as the site of phosphorylation by cAMP-dependent protein kinase (PKA) in a skeletal muscle glycogen-targeting subunit (GM). We reported previously that a recombinant fusion protein of glutathione S-transferase (GST) and the N-terminal domain of GM [GST-GM-(1-240)] bound PP1 in a pull down assay, and phosphorylation by PKA prevented PP1 binding. Here we report that substitution of either Ala or Val for Ser-67 in the RVS67F motif in GST-GM-(1-240) essentially eliminated PP1 binding. This was unexpected because other glycogen-targeting subunits have a Val residue at the position corresponding to Ser-67. In contrast, a mutation of Ser-67 to Thr (S67T) in GST-GM(1-240) gave a protein that bound PP1 the same as wild type and was unaffected by PKA phosphorylation. Full length GM tagged with the epitope sequence DYKDDDDK (FLAG) expressed in COS7 cells bound PP1 that was recovered by co-immunoprecipitation, but this association was prevented by treatment of the cells with forskolin. By comparison, PP1 binding with FLAG-GM(S67T) was not disrupted by forskolin treatment. Neither FLAG-GM(S67A) nor FLAG-GM(S67V) formed stable complexes with PP1 in COS7 cells. These results emphasise the unique contribution of Ser-67 in PP1 binding to GM. The constitutive PP1-binding activity shown by GM(S67T) opens the way for studying the role of GM multisite phosphorylation in hormonal control of glycogen metabolism.
Interactions between β-enolase and creatine kinase in the cytosol of skeletal muscle cells
Authors: FOUCAULT Georges , VACHER Monique , CRIBIER Sophie , ARRIO-DUPONT Martine
Published: August 2015

We studied interactions in vivo between the cytosolic muscle isoform of creatine kinase (M-CK) and the muscle isoform of 2-phospho-D-glycerate hydrolyase (β-enolase) in muscle sarcoplasm by incubating glycerol-skinned fibres with FITC-labelled β-enolase in the presence or absence of free CK. A small amount of bound β-enolase was observed in the presence of large concentrations of CK. The mobility of enolase was measured in cultured satellite cells by modulated-fringe-pattern photobleaching. FITC-labelled β-enolase was totally mobile in both the presence and the absence of CK but its diffusion coefficient was slightly lower in the presence of CK. This suggests a weak interaction in vivo between enolase and CK.
Purification and inhibitory profile of phospholipase A2 inhibitors from Australian elapid sera
Authors: HAINS Peter G. , BROADY Kevin W.
Published: August 2015

Although the resistance of snakes to their own venom is well known, until now no investigators have examined the serum of Australian snakes. Here we describe the identification and purification of a range of phospholipase A2 (PLA2) inhibitors from the serum of Australian elapids. All PLA2 inhibitors were composed of two protein chains, an α-chain and a β-chain. The α-chains were approx. 22.5 kDa in size and variably glycosylated, whereas the β-chains were approx. 19.8 kDa in size and not glycosylated. Identification of isoforms of the two subunit chains was significant because three of the six sera examined were from single snake specimens. In addition, the glycosylation patterns of the α-chains were thoroughly investigated in these unpooled sera. The functional and structural properties of the purified inhibitors were studied. Uniquely, a snake PLA2 inhibitor was found to inhibit human type II PLA2 enzyme, which has implications for the treatment of the many diseases in which PLA2 enzymes have been implicated. Further, we demonstrate that the inhibitor forms a non-covalent association with a purified PLA2 enzyme. Finally, the purified PLA2 inhibitor was shown to protect in vivo against the lethal affects of a homologous PLA2 enzyme, suggesting a role for PLA2 inhibitors in the treatment of snake bite victims.
Cloning and characterization of mouse extracellular-signal-regulated protein kinase 3 as a unique gene product of 100 kDa
Authors: TURGEON Benjamin , SABA-EL-LEIL Marc K. , MELOCHE Sylvain
Published: August 2015

MAP (mitogen-activated protein) kinases are a family of serine/threonine kinases that have a pivotal role in signal transduction. Here we report the cloning and characterization of a mouse homologue of extracellular-signal-regulated protein kinase (ERK)3. The mouse Erk3 cDNA encodes a predicted protein of 720 residues, which displays 94% identity with human ERK3. Transcription and translation of this cDNA in vitro generates a 100 kDa protein similar to the human gene product ERK3. Immunoblot analysis with an antibody raised against a unique sequence of ERK3 also recognizes a 100 kDa protein in mouse tissues. A single transcript of Erk3 was detected in every adult mouse tissue examined, with the highest expression being found in the brain. Interestingly, expression of Erk3 mRNA is acutely regulated during mouse development, with a peak of expression observed at embryonic day 11. The mouse Erk3 gene was mapped to a single locus on central mouse chromosome 9, adjacent to the dilute mutation locus and in a region syntenic to human chromosome 15q21. Finally, we provide several lines of evidence to support the existence of a unique Erk3 gene product of 100 kDa in mammalian cells.
Crystal structures of human pancreatic α-amylase in complex with carbohydrate and proteinaceous inhibitors
Authors: NAHOUM Virginie , ROUX Geneviève , ANTON Véronique , ROUGÉ Pierre , PUIGSERVER Antoine , BISCHOFF Hilmar , HENRISSAT Bernard , PAYAN Françoise
Published: August 2015

Crystal structures of human pancreatic α-amylase (HPA) in complex with naturally occurring inhibitors have been solved. The tetrasaccharide acarbose and a pseudo-pentasaccharide of the trestatin family produced identical continuous electron densities corresponding to a pentasaccharide species, spanning the -3 to +2 subsites of the enzyme, presumably resulting from transglycosylation. Binding of the acarviosine core linked to a glucose residue at subsites -1 to +2 appears to be a critical part of the interaction process between α-amylases and trestatin-derived inhibitors. Two crystal forms, obtained at different values of pH, for the complex of HPA with the protein inhibitor from Phaseolus vulgaris (α-amylase inhibitor) have been solved. The flexible loop typical of the mammalian α-amylases was shown to exist in two different conformations, suggesting that loop closure is pH-sensitive. Structural information is provided for the important inhibitor residue, Arg-74, which has not been observed previously in structural analyses.
Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli
Authors: GRIEßLER Richard , D'AURIA Sabato , SCHINZEL Reinhard , TANFANI Fabio , NIDETZKY Bernd
Published: August 2015

Maltodextrin phosphorylase from Escherichia coli (MalP) is a dimeric protein in which each ≈ 90-kDa subunit contains active-site pyridoxal 5ʹ-phosphate. To unravel factors contributing to the stability of MalP, thermal denaturations of wild-type MalP and a thermostable active-site mutant (Asn-133 → Ala) were compared by monitoring enzyme activity, cofactor dissociation, secondary structure content and aggregation. Small structural transitions of MalP are shown by Fourier-transform infrared spectroscopy to take place at ≈ 45 °C. They are manifested by slight increases in unordered structure and 1H/2H exchange, and reflect reversible inactivation of MalP. Aggregation of the MalP dimer is triggered by these conformational changes and starts at ≈ 45 °C without prior release into solution of pyridoxal 5ʹ-phosphate. It is driven by electrostatic rather than hydrophobic interactions between MalP dimers, and leads to irreversible inactivation of the enzyme. Aggregation is inhibited efficiently and specifically by oxyanions such as phosphate, and AMP which therefore, stabilize MalP against the irreversible denaturation step at 45 °C. Melting of the secondary structure in soluble and aggregated MalP takes place at much higher temperatures of approx. 58 and 67 °C, respectively. Replacement of Asn-133 by Ala does not change the mechanism of thermal denaturation, but leads to a shift of the entire pathway to a ≈ 15 °C higher value on the temperature scale. Apart from greater stability, the Asn-133 → Ala mutant shows a 2-fold smaller turnover number and a 4.6-fold smaller energy of activation than wild-type MalP, probably indicating that the site-specific replacement of Asn-133 brings about a greater rigidity of the active-site environment of the enzyme. A structure-based model is proposed which explains the stabilizing interaction between MalP and oxyanions, or AMP.
Pancreatic eukaryotic initiation factor-2α kinase (PEK) homologues in humans, Drosophila melanogaster and Caenorhabditis elegans that mediate translational control in response to endoplasmic reticulum stress
Authors: SOOD Ruchira , PORTER Amy C. , MA Kun , QUILLIAM Lawrence A. , WEK Ronald C.
Published: August 2015

In response to different cellular stresses, a family of protein kinases regulates translation by phosphorylation of the α subunit of eukaryotic initiation factor-2 (eIF-2α). Recently, we identified a new family member, pancreatic eIF-2α kinase (PEK) from rat pancreas. PEK, also referred to as RNA-dependent protein kinase (PKR)-like endoplasmic reticulum (ER) kinase (PERK) is a transmembrane protein implicated in translational control in response to stresses that impair protein folding in the ER. In this study, we identified and characterized PEK homologues from humans, Drosophila melanogaster and Caenorhabditis elegans. Expression of human PEK mRNA was found in over 50 different tissues examined, with highest levels in secretory tissues. In mammalian cells subjected to ER stress, we found that elevated eIF-2α phosphorylation was coincident with increased PEK autophosphorylation and eIF-2α kinase activity. Activation of PEK was abolished by deletion of PEK N-terminal sequences located in the ER lumen. To address the role of C. elegans PEK in translational control, we expressed this kinase in yeast and found that it inhibits growth by hyperphosphorylation of eIF-2α and inhibition of eIF-2B. Furthermore, we found that vaccinia virus K3L protein, an inhibitor of the eIF-2α kinase PKR involved in an anti-viral defence pathway, also reduced PEK activity. These results suggest that decreased translation initiation by PEK during ER stress may provide the cell with an opportunity to remedy the folding problem prior to introducing newly synthesized proteins into the secretory pathway.
HIV-2 protease is inactivated after oxidation at the dimer interface and activity can be partly restored with methionine sulphoxide reductase
Authors: DAVIS David A. , NEWCOMB Fonda M. , MOSKOVITZ Jackob , WINGFIELD Paul T. , STAHL Stephen J. , KAUFMAN Joshua , FALES Henry M. , LEVINE Rodney L. , YARCHOAN Robert
Published: August 2015

Human immunodeficiency viruses encode a homodimeric protease that is essential for the production of infectious virus. Previous studies have shown that HIV-1 protease is susceptible to oxidative inactivation at the dimer interface at Cys-95, a process that can be reversed both chemically and enzymically. Here we demonstrate a related yet distinct mechanism of reversible inactivation of the HIV-2 protease. Exposure of the HIV-2 protease to H2O2 resulted in conversion of the two methionine residues (Met-76 and Met-95) to methionine sulphoxide as determined by amino acid analysis and mass spectrometry. This oxidation completely inactivated protease activity. However, the activity could be restored (up to 40%) after exposure of the oxidized protease to methionine sulphoxide reductase. This treatment resulted in the reduction of methionine sulphoxide 95 but not methionine sulphoxide 76 to methionine, as determined by peptide mapping/mass spectrometry. We also found that exposure of immature HIV-2 particles to H2O2 led to the inhibition of polyprotein processing in maturing virus particles comparable to that demonstrated for HIV-1 particles. Thus oxidative inactivation of the HIV protease in vitro and in maturing viral particles is not restricted to the type 1 proteases. These studies indicate that two distinct retroviral proteases are susceptible to inactivation after a very minor modification at residue 95 of the dimer interface and suggest that the dimer interface might be a viable target for the development of novel protease inhibitors.
Zinc-regulated ubiquitin conjugation signals endocytosis of the yeast ZRT1 zinc transporter
Authors: GITAN Raad S. , EIDE David J.
Published: August 2015

The yeast ZRT1 zinc transporter is regulated by zinc at both transcriptional and post-translational levels. At the post-translational level, zinc inactivates ZRT1 by inducing the removal of the protein from the plasma membrane by endocytosis. The zinc transporter is subsequently degraded in the vacuole. This regulatory system allows for the rapid shut off of zinc uptake activity in cells exposed to high zinc concentrations, thereby preventing overaccumulation of this potentially toxic metal. In this report, we examine the role of ubiquitin conjugation in this process. First, we show that ZRT1 is ubiquitinated shortly after zinc treatment and before endocytosis. Secondly, mutations in various components of the ubiquitin conjugation pathway, specifically the RSP5 ubiquitin-protein ligase and the UBC4 and UBC5 ubiquitin conjugating enzymes, inhibit both ubiquitination and endocytosis. Finally, mutation of a specific lysine residue in ZRT1 blocks both ubiquitination and endocytosis. This critical lysine, Lys-195, is located in a cytoplasmic loop region of the protein and may be the residue to which ubiquitin is attached. These results demonstrate that ubiquitin conjugation is a critical step in the signal transduction pathway that controls the rate of ZRT1 endocytosis in response to zinc.
Stress-induced generation of N-acylethanolamines in mouse epidermal JB6 P+ cells
Authors: BERDYSHEV Evgueni V. , SCHMID Patricia C. , DONG Zigang , SCHMID Harald H. O.
Published: August 2015

It has long been known that N-acylethanolamine phospholipids [N-acylphosphatidylethanolamine (N-acyl PE)] and N-acylethanolamines (NAEs) accumulate in mammalian tissues undergoing degenerative membrane changes associated with necrosis. Here we studied the effects of stress factors (UVB irradiation and serum deprivation) on the endogenous levels of N-acyl PE and NAE in mouse epidermal JB6 P+ cells. We found that 16:0, 18:0, 18:1,n-9 and 18:1,n-7 are the predominant amide-linked fatty acids in both N-acyl PE and NAE in these cells. UVB irradiation and serum deprivation resulted in significantly increased levels of N-acyl PE and NAE, especially 18:1,n-9 N-acyl PE and NAE. UVB challenge increased the cellular content of anandamide (20:4,n-6 NAE), but this increase was the lowest of all NAEs measured. Serum deprivation resulted in a decreased cellular anandamide level, as well as a decrease in 20:4,n-6 N-acyl PE. Interestingly, the replacement of serum-free medium with medium containing 5% (v/v) fetal calf serum after 36 h of serum deprivation restored N-acyl PE and NAE levels almost completely within 4-8 h. These data suggest the involvement of N-acyl PE and NAE in cellular responses to stress.
Ferredoxin III of Desulfovibrio africanus: sequencing of the native gene and characterization of a histidine-tagged form
Authors: BUSCH Johanneke L. H. , BRETON Jacques L. , DAVY Sharon L. , JAMES Richard , MOORE Geoffrey R. , ARMSTRONG Fraser A. , THOMSON Andrew J.
Published: August 2015

Desulfovibrio africanus ferredoxin III (Da FdIII) contains one [4Fe-4S]2+/1+ cluster and one [3Fe-4S]1+/0 cluster, bound by seven Cys residues, in which the [3Fe-4S] cluster is co-ordinated by the unusual sequence, Cys11-Xaa-Xaa-Asp14-Xaa-Xaa-Cys17-Xaan-Cys51-Glu. The [3Fe-4S] core of this ferredoxin is so far unique in showing rapid bi-directional [3Fe-4S] ↔ [4Fe-4S] cluster interconversion with a wide range of metal ions. In order to obtain protein for mutagenesis studies Da FdIII has been cloned, sequenced, and expressed as a hexa-histidine tagged (ht) polypeptide in Escherichia coli strain BL21(DE3) pLysS. Expression of ht Da FdIII, whether translated from a synthetic gene (pJB10) or from the native nucleotide sequence (pJB11), occurred at similar levels (approx. 6 mg·l-1), but without incorporation of metal clusters. The nucleotide sequence confirms the protein sequence reported previously [Bovier-Lapierre, Bruschi, Bonicel and Hatchikian (1987) Biochim. Biophys. Acta 913, 20-26]. Cluster incorporation was achieved using FeCl3 together with cysteine sulphur transferase, NifS, plus cysteine to generate low levels of sulphide ions. Absorption and EPR spectroscopy show that both [3Fe-4S] and [4Fe-4S] clusters are correctly inserted. Thin-film electrochemistry provides evidence that the [3Fe-4S] cluster undergoes reversible cluster transformation in the presence of Fe(II) and Zn(II) ions with properties identical to the native protein. Nevertheless the protein has lower stability than native Da FdIII during chromatography. The one-dimensional 600 MHz NMR spectrum of the apoprotein indicates an unstructured protein with random coil chemical shifts whereas spectra of the reconstituted ht protein show secondary structural elements and 18 peaks shifted downfield of 9.6 p.p.m. The spectra are unique but have similarities with the shift patterns seen with 7Fe Desulfurolobus ambivalens Fd. The ht does not affect iron-sulphur cluster incorporation, but NMR evidence suggests that excess Fe binds to the tag. This may account for the lower stability of the ht compared with the native protein.
Investigation of the mechanism by which glucose analogues cause translocation of glucokinase in hepatocytes: evidence for two glucose binding sites
Authors: AGIUS Loranne , STUBBS Mark
Published: August 2015

Glucokinase translocates between the cytoplasm and nucleus of hepatocytes where it is bound to a 68 kDa protein. The mechanism by which glucose induces translocation of glucokinase from the nucleus was investigated using glucose analogues that are not phosphorylated by glucokinase. There was strong synergism on glucokinase translocation between effects of glucose analogues (glucosamine, 5-thioglucose, mannoheptulose) and sorbitol, a precursor of fructose 1-phosphate. In the absence of glucose or glucose analogues, sorbitol had a smaller effect than glucose on translocation. However, sorbitol potentiated the effects of glucose analogues. In the absence of sorbitol the effect of glucose on glucokinase translocation is sigmoidal with a Hill coefficient of 1.9 suggesting involvement of two glucose-binding sites. The effects of glucosamine and 5-thioglucose were also sigmoidal but with lower Hill Coefficients. In the presence of sorbitol, the effects of glucose, glucosamine and 5-thioglucose were hyperbolic. Mannoheptulose, unlike the other glucose analogues, had a hyperbolic effect on glucokinase translocation in the absence of sorbitol suggesting interaction with one site and was synergistic rather than competitive with glucose. The results favour a two-site model for glucokinase translocation involving either two glucose-binding sites or one binding-site for glucose and one for fructose 1-phosphate. The glucose analogues differed in their effects on the kinetics of purified glucokinase. Mannoheptulose caused the greatest decrease in co-operativity of glucokinase for glucose whereas N-acetylglucosamine had the smallest effect. The anomalous effects of mannoheptulose on glucokinase translocation and on the kinetics of purified glucokinase could be explained by a second glucose-binding site on glucokinase.
Identification and expression of an allergen Asp f 13 from Aspergillus fumigatus and epitope mapping using human IgE antibodies and rabbit polyclonal antibodies
Authors: CHOW Lu-Ping , LIU Shu-Ling , YU Chia-Jung , LIAO Hsin-Kai , TSAI Jaw-Ji , TANG Tswen-Kei
Published: August 2015

The Aspergillus genus of fungi is known to be one of the most prevalent aeroallergens. On two-dimensional immunoblotting using patients' sera containing IgE specific for Asp f 13, an allergen with a molecular mass of 33 kDa and a pI of 6.2 was identified. This allergen was also present in A. fumigatus culture filtrates. Furthermore, the sequence of the Asp f 13 cDNA was identical to that for alkaline protease isolated from A. fumigatus and showed 42-49% identity of amino acids with two proteases from P. cyclopium and T. album and with the Pen c 1 allergen from P. citrinum. Asp f 13 coding sequences were expressed in Escherichia coli as a [His]6-tagged fusion protein which was purified by Ni2+-chelate affinity chromatography. Recombinant Asp f 13 was recognized by rabbit polyclonal antibodies against Asp f 13 and by IgE antibodies from subject allergic to A. fumigatus. To identify and characterize the linear epitopes of this allergen, a combination of chemical and enzymatic cleavage and immunoblotting techniques, with subsequent N-terminal sequencing and mass spectrometry, were performed. At least 13 different linear epitopes reacting with the rabbit anti-Asp f 13 antiserum were identified, located throughout the entire molecule. In contrast, IgE from A. fumigatus-sensitive patients bound to three immunodominant epitopes at the C-terminal of the protein.
Expression of heparan sulphate l-iduronyl 2-O-sulphotransferase in human kidney 293 cells results in increased d-glucuronyl 2-O-sulphation
Authors: RONG Jianhui , HABUCHI Hiroko , KIMATA Koji , LINDAHL Ulf , KUSCHE-GULLBERG Marion
Published: August 2015

Functionally important interactions between heparan sulphate and a variety of proteins depend on the precise location of O-sulphate groups. Such residues occur at C-2 of L-iduronic (IdoA) and D-glucuronic acid (GlcA) units, and at C-3 and C-6 of D-glucosamine (GlcN) units. Stable transfection of human embryonic kidney 293 cells with a cDNA encoding mouse mastocytoma IdoA 2-O-sulphotransferase resulted in an approx. 6-fold increase in O-sulphotransferase activity, compared with control cells, as determined using O-desulphated heparin as an acceptor. Structural analysis of endogenous heparan sulphate in the transfected cells, following metabolic labelling with either [3H]GlcN or [35S]sulphate, showed appreciable formation of -GlcA(2-OSO3)-GlcNSO3- disaccharide units (6% of total disaccharide units; 17% of total O-sulphated disaccharide units) that were essentially absent from heparan sulphate from control cells. The increase in GlcA 2-O-sulphation was accompanied by a decrease in the amount of IdoA formed, whereas overall 2-O-sulphation or 6-O-sulphation remained largely unaffected. These findings indicate that 2-O-sulphation of IdoA and GlcA residues is catalysed by the same enzyme in heparan sulphate biosynthesis.
Tandem mass spectrometric analysis of Aspergillus niger pectin methylesterase: mode of action on fully methyl-esterified oligogalacturonates
Authors: KESTER Harry C. M. , BENEN Jacques A. E. , VISSER Jaap , WARREN Maria Esteban , ORLANDO Ron , BERGMANN Carl , MAGAUD Didier , ANKER Daniel , DOUTHEAU Alain
Published: August 2015

The substrate specificity and the mode of action of Aspergillus niger pectin methylesterase (PME) was determined using both fully methyl-esterified oligogalacturonates with degrees of polymerization (DP) 2-6 and chemically synthesized monomethyl trigalacturonates. The enzymic activity on the different substrates and a preliminary characterization of the reaction products were performed by using high-performance anion-exchange chromatography at neutral pH. Electrospray ionization tandem MS (ESI-MS/MS) was used to localize the methyl esters on the 18O-labelled reaction products during the course of the enzymic reaction. A. niger PME is able to hydrolyse the methyl esters of fully methyl-esterified oligogalacturonates with DP 2, and preferentially hydrolyses the methyl esters located on the internal galacturonate residues, followed by hydrolysis of the methyl esters towards the reducing end. This PME is unable to hydrolyse the methyl ester of the galacturonate moiety at the non-reducing end.
Cloning and functional characterization of the 5′-flanking region of human methionine adenosyltransferase 1A gene
Authors: ZENG Zhaohui , HUANG Zong-Zhi , CHEN Changjin , YANG Heping , MAO Zebin , LU Shelly C.
Published: August 2015

Methionine adenosyltransferase (MAT) is an essential cellular enzyme which catalyses the formation of S-adenosylmethionine, the principal methyl donor and precursor for polyamines. In mammals, two different genes, MAT1A and MAT2A, encode for liver-specific and non-liver-specific MAT respectively. We previously described a switch in the MAT expression from MAT1A to MAT2A in human liver cancer, which offered the cancerous cell a growth advantage. Loss of MAT1A expression was due to lack of gene transcription. To study regulation of the MAT1A gene, we have cloned and characterized a 1.9 kb 5ʹ-flanking region of the human MAT1A gene. One transcriptional start site, located 25 nt downstream from a consensus TATA box, was identified by primer extension and RNase protection assays. The promoter contains several consensus binding sites for CAAT enhancer binding protein (C/EBP) and hepatocyte-enriched nuclear factor (HNF), transcriptional factors important in liver-specific gene expression. The human MAT1A promoter was able to efficiently drive luciferase expression in Chang cells, a human liver cell line, but not in HeLa cells. Sequential deletion analysis of the promoter revealed two DNA regions upstream of the translational start site, -705 to -839 bp and -1111 to -1483 bp, which are involved in positive and negative gene regulation, respectively. Specific protein binding to these regions was confirmed by electrophoretic-mobility-shift and DNase I footprinting assays. Similar to the situation with the rat MAT1A, glucocorticoid treatment also increased human MAT1A expression and promoter activity in a dose- and time-dependent manner.
The N-terminal 34 residues of the 55 kDa regulatory subunits of phosphoinositide 3-kinase interact with tubulin
Authors: INUKAI Kouichi , FUNAKI Makoto , NAWANO Masao , KATAGIRI Hideki , OGIHARA Takehide , ANAI Motonobu , ONISHI Yukiko , SAKODA Hideyuki , ONO Hiraku , FUKUSHIMA Yasushi , KIKUCHI Masatoshi , OKA Yoshitomo , ASANO Tomoichiro
Published: August 2015

There are five regulatory subunit isoforms of phosphoinositide 3-kinase (PI 3-kinase), which are classified into three groups: proteins of 85 kDa (p85α and p85β), 55 kDa (p55α and p55γ) and 50 kDa (p50α). Structural differences between the three groups reside in the N-terminus. To elucidate the unique functional role of the 55 kDa regulatory subunits, GST (glutathione S-transferase) fusion proteins containing a unique N-terminal portion consisting of a 34-amino-acid sequence of p55α or p55γ (GST-p55α/γN1-34) were used as affinity matrices to screen rat brain cell extracts for proteins to which this portion binds specifically. A protein that bound was identified as β-tubulin by protein sequencing. In addition, not only the β isoform of tubulin, but also the α and γ isoforms, were detected in the protein absorbed from cell lysates with GST-p55γN1-34 and GST-p55αN1-34 by immunoblotting. Indeed, the only regulatory subunit present in the purified microtubule assembly from rat brain was the 55 kDa isoform; neither 85 kDa nor 50 kDa subunits were detected. These results indicate endogenous binding of 55 kDa regulatory subunits of PI 3-kinase to tubulin in the brain. Finally, we measured tubulin-associated PI 3-kinase activity in CHO/IR cells overexpressing each of the five regulatory subunit isoforms. Only in cells expressing p55α or p55γ was there a significant elevation of tubulin-associated PI 3-kinase activity in response to insulin. These results suggest that the p55α and p55γ regulatory subunits have important roles in regulating PI 3-kinase activity, particularly for microtubules at the cell periphery.
Mutations in the reduced-folate carrier affect protein localization and stability
Authors: SADLISH Heather , MURRAY Richard C. , WILLIAMS Frederick M. R. , FLINTOFF Wayne F.
Published: August 2015

The reduced-folate-carrier (rfc) gene has been shown to be functionally important for reduced-folate transport in mammalian cells. In the present paper we describe the identification of alterations in both alleles of the rfc gene in a mutant Chinese-hamster ovary cell line deficient in methotrexate transport. One allele of the rfc gene contains a point mutation resulting in a Gly345 → Arg substitution in the predicted amino acid sequence. In this case, a protein of similar size to the wild-type protein is produced, although it remains as an immature, core-glycosylated, form. The second allele contains a point mutation in the last base of intron 5 that results in the utilization of a cryptic splice site leading to a seven-base deletion in the mRNA. The use of an alternate splice site changes the reading frame to yield a truncated protein with 68 different C-terminal amino acids as compared with the wild-type. Both of these altered gene products were monitored by fusion with green fluorescent protein and found to be non-functional with an increased rate of turnover. The protein with the point mutation is trapped in the endoplasmic reticulum with subsequent degradation, whereas the product of the splice mutation is not membrane-associated and is partially degraded. Thus mutations in both alleles of the rfc gene in this resistant cell line account for the loss of reduced-folate transport. The observations made regarding the degradation of these mutant gene products also provide support for putative checkpoints in the endoplasmic reticulum.
Interaction between the homeodomain proteins Cdx2 and HNF1α mediates expression of the lactase-phlorizin hydrolase gene
Authors: MITCHELMORE Cathy , TROELSEN Jesper T. , SPODSBERG Nikolaj , SJÖSTRÖM Hans , NORÉN Ove
Published: August 2015

Lactase-phlorizin hydrolase is a brush-border enzyme which is specifically expressed in the small intestine where it hydrolyses lactose, the main carbohydrate found in milk. We have previously demonstrated in transgenic mice that the tissue-specific and developmental expression of lactase is controlled by a 1 kb upstream region of the pig lactase gene. Two homeodomain transcription factors, caudal-related homeodomain protein (Cdx2) and hepatic nuclear factor 1α (HNF1α), are known to bind to regulatory cis elements in the promoters for several intestine-specific genes, including lactase, and are present in mammalian intestinal epithelia from an early stage in development. In the present study, we examined whether Cdx2 and HNF1α physically interact and co-operatively activate transcription from the lactase-phlorizin hydrolase promoter. We show that the presence of both factors leads to a much higher level of transcription than the sum of the activation by either factor alone. The N-terminal activation domain of Cdx2 is required for maximal synergy with HNF1α. With the use of pull-down assays, we demonstrate a direct protein-protein interaction between Cdx2 and HNF1α. The interaction domain includes the homeodomain region of both proteins. This is the first demonstration of a functional interaction between two transcription factors involved in the activation of a number of intestine-specific genes. Synergistic interaction between tissue-restricted factors is likely to be an important mechanism for reinforcing developmental and tissue-specific gene expression within the intestine.
α-Synuclein overexpression promotes aggregation of mutant huntingtin
Authors: FURLONG Robert A. , NARAIN Yolanda , RANKIN Julia , WYTTENBACH Andreas , RUBINSZTEIN David C.
Published: August 2015

Protein aggregates are a neuropathological feature of Huntington's disease and Parkinson's disease. Mutant huntingtin exon 1 with 72 CAG repeats fused to enhanced green fluorescent protein (EGFP) forms hyperfluorescent inclusions in PC12 cells. Inclusion formation is enhanced in cells co-transfected with EGFP-huntingtin-(CAG)72 and α-synuclein, a major component of Parkinson's disease aggregates. However, α-synuclein does not form aggregates by itself, nor does it appear in huntingtin inclusions in vitro.
Hepatic expression, synthesis and secretion of a novel fibrinogen/angiopoietin-related protein that prevents endothelial-cell apoptosis
Authors: KIM Injune , KIM Hwan-Gyu , KIM Hyun , KIM Hong-Hee , PARK Sung Kwang , UHM Chang-Sub , LEE Zang Hee , KOH Gou Young
Published: August 2015

Using degenerate PCR we isolated a cDNA encoding a novel 406- and 410-amino acid protein from human and mouse embryonic cDNAs and have designated it ‘hepatic fibrinogen/angiopoietin-related protein’ (HFARP). The N-terminal and C-terminal portions of HFARP contain the characteristic coiled-coil domains and fibrinogen-like domains that are conserved in angiopoietins. In human and mouse tissues, HFARP mRNA is specifically expressed in the liver. HFARP mRNA and protein are mainly present in the hepatocytes. HFARP has a highly hydrophobic region at the N-terminus that is typical of a secretory signal sequence and one consensus glycosylation site. Recombinant HFARP expressed in COS-7 cells is secreted and glycosylated. HFARP protein is present not only in the hepatocytes, but also in the circulating blood. Recombinant HFARP acts as an apoptosis survival factor for vascular endothelial cells, but does not bind to Tie1 or Tie2 (endothelial-cell tyrosine kinase receptors). These results suggest that HFARP may exert a protective function on endothelial cells through an endocrine action.
Histidine to aspartate phosphotransferase activity of nm23 proteins: phosphorylation of aldolase C on Asp-319
Authors: WAGNER Paul D. , VU Ngoc-Diep
Published: August 2015

nm23 genes have been implicated in the suppression of tumour metastasis and cell motility; however, the biochemical mechanisms for these suppressions are not known. We have previously described the transfer of phosphate from the catalytic histidine residues of nm23 proteins to an aspartic or a glutamic residue on one or more 43 kDa proteins in detergent extracts of bovine brain membranes. To gain a better understanding of this transferase activity, we partly purified this 43 kDa protein and identified aldolases A and C as the major 43 kDa proteins present in the preparation. Aldolase was purified from brain cytosol; its phosphorylation by rat liver nm23 proteins and by recombinant human nm23-H1 was examined. The site of phosphorylation was identified as Asp-319 on aldolase C. The equivalent residue on aldolase A, a glutamic residue, was not phosphorylated. Aldolase C was rapidly phosphorylated by wild-type nm23-H1 but was not phosphorylated, or was phosphorylated very slowly, by either nm23-H1P96S or nm23-H1S120G, mutants of nm23-H1 that do not suppress cell motility. This is the first identification of a protein that is phosphorylated on an aspartic residue by nm23 proteins. The sequence around Asp-319 of aldolase C has some similarities to those around the histidine residues on ATP-citrate lyase and succinic thiokinase that are phosphorylated by nm23 proteins.
Evidence for regulation of NF-κB by poly(ADP-ribose) polymerase
Authors: KAMEOKA Masanori , OTA Katsuya , TETSUKA Toshifumi , TANAKA Yasuharu , ITAYA Asako , OKAMOTO Takashi , YOSHIHARA Koichiro
Published: August 2015

The DNA-binding activity of NF-ĸB in nuclear extracts of poly(ADP-ribose) polymerase (PARP)-defective mutant L1210 cell clones was markedly increased and was inversely correlated with the PARP content in these cells. The DNA-binding activity of NF-ĸB in a clone with the lowest PARP content (Cl-3527, contained 6% of PARP of wild type cells) was about 35-fold of that of the wild-type cells, whereas the change in the DNA-binding activity of AP-1 and SP-1 in the mutant was relatively small or not so significant. Transfection of a PARP-expressing plasmid to the mutant cells decreased the abnormally high levels of NF-ĸB complexes, especially p50/p65(Rel A) complex, to near the normal level. Moreover, poly(ADP-ribosyl)ation of nuclear extracts in vitro suppressed the ability of NF-ĸB to form a complex with its specific DNA probe by approx. 80%. Further analysis with purified recombinant NF-ĸB proteins revealed that both rp50 and rMBP-p65 (Rel A) proteins, but not rGST-IĸB, could be poly(ADP-ribosyl)ated in vitro and that the modification resulted in a marked decrease in the DNA-binding activity of rMBP-p65, whereas a slight activation was observed in rp50. Poly(ADP-ribosyl)ated p65/NF-ĸB was detected in the cytosol of wild type L1210 cells by immunoblotting with anti-poly(ADP-ribose) and anti-p65 antibodies. Taken together, these results strongly suggest that PARP is involved in the regulation of NF-ĸB through the protein modification.
Mechanism of an antibody-catalysed allylic isomerization
Authors: GONÇALVES Olivier , DINTINGER Thierry , LEBRETON Jacques , BLANCHARD Dominique , TELLIER Charles
Published: August 2015

The catalytic antibody 4B2, which was generated against a substituted amidine 1, catalyses the allylic isomerization of β,γ-unsaturated ketones with an acceleration factor (kcat/kuncat) of 1.5×103. On the basis of the ‘bait and switch’ strategy, it was reasoned that the positively charged hapten could elicit, by charge complementarity, an acidic residue (Asp or Glu) in the antibody-binding site in the right position to catalyse this proton transfer reaction. The pH dependence curve of kcat/Km shows a bell-shaped feature with an optimum at approx. pH 4.5. By cloning and sequencing the light and heavy chains of the 4B2 antibody, we confirmed the presence of several Asp and Glu residues in the complementarity-determining region loops. The antibody catalyses the α-proton exchange on the same substrates, demonstrating the involvement of a dienol intermediate in the reaction mechanism. Kinetic studies with 2H-NMR provide evidence that α-proton abstraction is stereospecific. Whether the process involves one or two acid/base residues in this simple proton transfer or whether it is a concerted mechanism is discussed.
Identification of copper/zinc superoxide dismutase as a nitric oxide-regulated gene in human (HaCaT) keratinocytes: implications for keratinocyte proliferation
Authors: FRANK Stefan , KÄMPFER Heiko , PODDA Maurizio , KAUFMANN Roland , PFEILSCHIFTER Josef
Published: August 2015

Recent studies have demonstrated an induction of expression of inducible nitric oxide synthase that is associated with several inflammatory diseases of the skin. To define the mechanisms of action of nitric oxide (NO) in the skin, we attempted to identify genes that are regulated by NO in keratinocytes. Using the human keratinocyte cell line HaCaT as a model system, we identified a Cu/Zn superoxide dismutase (SOD) that was strongly induced by high concentrations (500 μM) of NO-donating agents {S-nitrosoglutathione, sodium nitroprusside and (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO)}, but not by serum or by single recombinant growth factors and inflammatory cytokines or by treatment with superoxide anions. Furthermore, endogenously produced NO increased the expression of Cu/Zn SOD mRNA in keratinocytes. Moreover, treatment of HaCaT cells with NO was associated with a biphasic effect on cell proliferation, because low doses (100 μM) of different NO donors (S-nitrosoglutathione and DETA-NO) mediated a proliferative signal to the cells, whereas high concentrations (500 μM) were cytostatic. To determine a possible correlation between the close regulation of Cu/Zn SOD expression and proliferation by NO in keratinocytes, we established a cell line (psp1CZ1N) carrying a human Cu/Zn SOD cDNA under the control of a ponasterone-inducible promoter construct. Ponasterone-induced overexpression of Cu/Zn SOD caused a cytostatic effect in proliferating psp1CZ1N cells. We therefore suggest that the up-regulation of Cu/Zn SOD expression by NO establishes an inhibitory mechanism on keratinocyte proliferation.
Deuterium-labelled isotopomers of 2-C-methyl-d-erythritol as tools for the elucidation of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis
Authors: CHARON Lionel , HOEFFLER Jean-François , PALE-GROSDEMANGE Catherine , LOIS Luisa-Maria , CAMPOS Narciso , BORONAT Albert , ROHMER Michel
Published: August 2015

Escherichia coli synthesizes its isoprenoids via the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The MC4100dxs::CAT strain, defective in deoxyxylulose-5-phosphate synthase, which is the first enzyme in this metabolic route, exclusively synthesizes its isoprenoids from exogenous 2-C-methyl-D-erythritol (ME) added to the culture medium. The fate of the hydrogen atoms in the MEP pathway was followed by the incorporation of [1,1-2H2]ME and [3,5,5,5-2H4]ME. The two C-1 hydrogen atoms of ME were found without any loss in the prenyl chain of menaquinone and/or ubiquinone on the carbon atoms derived from C-4 of isopentenyl diphosphate (IPP) and on the E-methyl group of dimethylallyl diphosphate (DMAPP), the C-5 hydrogen atoms on the methyl groups derived from IPP C-5 methyl group and the Z-methyl group of DMAPP. This showed that no changes in the oxidation state of these carbon atoms occurred in the reaction sequence between MEP and IPP. Furthermore, no deuterium scrambling was observed between the carbon atoms derived from C-4 and C-5 of IPP or DMAPP, suggesting a completely stereoselective IPP isomerase or no significant activity of this enzyme. The C-3 deuterium atom of [3,5,5,5-2H4]ME was preserved only in the DMAPP starter unit and was completely missing from all those derived from IPP. This finding, aided by the non-essential role of the IPP isomerase gene, suggests the presence in E. coli of two different routes towards IPP and DMAPP, starting from a common intermediate derived from MEP.
Involvement of lipoxygenase in lysophosphatidic acid-stimulated hydrogen peroxide release in human HaCaT keratinocytes
Authors: SEKHARAM Madhavi , CUNNICK Jess M. , WU Jie
Published: August 2015

Although it is now recognized that low levels of reactive oxygen species (ROS) are required for the mitogenic response, mitogen-induced signalling pathways that regulate ROS generation in non-phagocytic cells remain largely uncharacterized. Using a real-time assay for measuring hydrogen peroxide (H2O2) formation, we analysed H2O2 release in human HaCaT keratinocytes in response to lysophosphatidic acid (LPA), a mitogen for keratinocytes. LPA rapidly increased H2O2 release in HaCaT cells. Unlike LPA-induced mitogen-activated protein (MAP) kinase activation, LPA-stimulated H2O2 release was independent of the tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Calcium chelators, phospholipase A2 inhibitors, and lipoxygenase inhibitors effectively blocked LPA-stimulated H2O2 release, whereas cyclooxygenase inhibitors were without effect. Addition of 5-lipoxygenase products 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene B4, but not 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene C4, restored LPA-stimulated H2O2 release in cells treated with the lipoxygenase inhibitors nordihydroguaiaretic acid and Zileuton. These results suggest that the lipoxygenase products 5-HPETE and leukotriene B4 are required for LPA-stimulated H2O2 release in HaCaT cells.
Protective effect of thioredoxin upon NO-mediated cell injury in THP1 monocytic human cells
Authors: FERRET P. J. , SOUM E. , NEGRE O. , WOLLMAN E. E. , FRADELIZI D.
Published: August 2015

Although NO has been postulated to play important roles in host defences, it is potentially damaging for exposed cells, including for the macrophages producing the NO. Thus a network of radical acceptors and enzymes is thought to play an important redox-buffering role to protect cells against NO-mediated injury. We examined the properties of the redox systems superoxide dismutase (SOD)/catalase, glutathione (GSH) and thioredoxin (Trx), in regulating the viability of two human monocytic cell lines (THP1 and U937) exposed to the NO-generating compound diethylene triamine-nitric oxide (DETA-NO). We observed that NO-induced cytotoxic effects were time- and dose-dependent towards the two cell lines. After vitamin-induced differentiation in vitro with retinoic acid (RA) and 1,25-dihydroxy vitamin D3 (VD), termed RA/VD, we observed that THP1 RA/VD cells became more resistant to NO-mediated cytotoxicity whereas the susceptibility of U937 cells was not modified. Using Western blotting and reverse-transcriptase PCR methods, we observed that gene transcription and protein expression of Trx and thioredoxin reductase were significantly increased upon RA/VD treatment and differentiation in THP1 cells. By contrast, SOD/catalase and GSH redox state remained unmodified. Finally, a stable transfectant THP1 line overexpressing Trx was found to be more resistant than THP1 control cells that were untransfected or transfected with an empty plasmid, when exposed to DETA-NO in vitro. In conclusion, we observed an inverse correlation between cell susceptibility to NO damaging effects and Trx expression, suggesting that the Trx system may have important preventative capacities towards NO-mediated cellular injury in monocytic macrophage cells.
Altered toxicity of the prion protein peptide PrP106–126 carrying the Ala117→Val mutation
Authors: BROWN David R.
Published: August 2015

The inherited prion diseases such as Gerstmann-Sträussler-Scheinker syndrome (GSS) are linked to point mutations in the gene coding for the cellular isoform of the prion protein (PrPC). One particular point mutation A117V (Ala117 → Val) is linked to a variable pathology that usually includes deposition of neurofibrillary tangles. A prion protein peptide carrying this point mutation [PrP106-126(117V)] was generated and compared with a peptide based on the normal human sequence [PrP106-126(117A)]. The inclusion of this point mutation increased the toxicity of PrP106-126 which could be linked to an increased β-sheet content. An assay of microtubule formation in the presence of tau indicated that PrP106-126 decreased the rate of microtubule formation that could be related to the displacement of tau. PrP106-126 carrying the 117 mutation was more efficient at inhibiting microtubule formation. These results suggest a possible mechanism of toxicity for protein carrying this mutation via destabilization of the cytoskeleton and deposition of tau in filaments, as observed in GSS.