bluuebunny/crossref_metadata_2025 huggingface dataset:
Human MUC5AC mucin dimerizes in the rough endoplasmic reticulum, similarly to the MUC2 mucin
Authors: ASKER Noomi , AXELSSON Magnus A. B. , OLOFSSON Sven-Olof , HANSSON Gunnar C.
Published: August 2015

Biosynthetic studies on the human MUC5AC mucin were performed by immunoprecipitations with antisera recognizing only the non-O-glycosylated apomucin in the colon adenocarcinoma cell line LS 174T. Pulse–chase studies and subcellular fractionations showed that MUC5AC formed dimers in the rough endoplasmic reticulum within 15 min of the initiation of biosynthesis. No non-O-glycosylated species larger than dimers were identified. The dimerization was N-glycosylation-dependent, because tunicamycin treatment significantly lowered the rate of dimerization. When the biosynthesis of MUC5AC apomucin was compared with that of MUC2 apomucin, also produced in the LS 174T cell line, both apomucins were assembled in similar ways with respect to their rates of dimerization with and without inhibition of N-glycosylation. No heterodimerization was observed between the human MUC5AC and the MUC2 apomucins despite the extensive sequence similarities in the positions of the cysteine residues in the C-termini proposed to be involved in mucin dimerization.
Atypical protein kinase Cλ binds and regulates p70 S6 kinase
Authors: AKIMOTO Kazunori , NAKAYA Masaaki , YAMANAKA Tomoyuki , TANAKA Junpei , MATSUDA Shin-ichi , WENG Qing-Ping , AVRUCH Joseph , OHNO Shigeo
Published: August 2015

p70 S6 kinase (p70 S6K) has been implicated in the regulation of cell cycle progression. However, the mechanism of its activation is not fully understood. In the present work, evidence is provided that an atypical protein kinase C (PKC) isotype, PKCλ, is indispensable, but not sufficient, for the activation of p70 S6K. Both the regulatory and kinase domains of PKCλ associate directly with p70 S6K. Overexpression of the kinase domain without kinase activity or the regulatory domain of PKCλ results in the suppression of the serum-induced activation of p70 S6K. In addition, two types of dominant-negative mutants of PKCλ, as well as a kinase-deficient mutant of p70 S6K, suppress serum-induced DNA synthesis and E2F activation. The overexpresion of the active form of PKCλ, however, fails to activate p70 S6K. These results suggest that PKCλ is a mediator in the regulation of p70 S6K activity and plays an important role in cell cycle progression.
Involvement of a local Fenton reaction in the reciprocal modulation by O2 of the glucagon-dependent activation of the phosphoenolpyruvate carboxykinase gene and the insulin-dependent activation of the glucokinase gene in rat hepatocytes
Authors: KIETZMANN Thomas , PORWOL Torsten , ZIEROLD Karl , JUNGERMANN Kurt , ACKER Helmut
Published: August 2015

H2O2 mimicked the action of periportal pO2 in the modulation by O2 of the glucagon-dependent activation of the phosphoenolpyruvate carboxykinase (PCK) gene and the insulin-dependent activation of the glucokinase (GK) gene. H2O2 can be converted in the presence of Fe2+ in a Fenton reaction into hydroxyl anions and hydroxyl radicals (•OH). The hydroxyl radicals are highly reactive and might interfere locally with transcription factors. It was the aim of the present study to investigate the role of and to localize such a Fenton reaction. Hepatocytes cultured for 24 h were treated under conditions mimicking periportal or perivenous pO2 with glucagon or insulin plus the iron chelator desferrioxamine (DSF) or the hydroxyl radical scavenger dimethylthiourea (DMTU) to inhibit the Fenton reaction. PCK mRNA was induced by glucagon maximally under conditions of periportal pO2 and half-maximally under venous pO2. GK mRNA was induced by insulin with reciprocal modulation by O2. DSF and DMTU reduced the induction of PCK mRNA to about half-maximal and increased the induction of GK mRNA to maximal under both O2 tensions. Hydroxyl radical formation was maximal under arterial pO2. Perivenous pO2, DSF and DMTU each decreased the formation of •OH to about 70% of control. The Fenton reaction could be localized in a perinuclear space by confocal laser microscopy and three-dimensional reconstruction techniques. In the same compartment, iron could be detected by electron-probe X-ray microanalysis. Thus a local Fenton reaction is involved in the O2 signalling, which modulated the glucagon- and insulin-dependent PCK gene and GK gene activation.
Specificity of interaction between adaptor-complex medium chains and the tyrosine-based sorting motifs of TGN38 and lgp120
Authors: STEPHENS David J. , BANTING George
Published: August 2015

Multiple sorting steps within eukaryotic cells are mediated by tyrosine-based sorting motifs. Motifs conforming to the consensus -YXX∅- (where ∅ indicates a bulky hydrophobic residue) have been shown to specify high-efficiency internalization from the plasma membrane, targeting from the plasma membrane to the trans-Golgi network and targeting to lysosomal compartments as well as being involved in basolateral sorting in polarized cells. These motifs are recognized by the medium-chain subunits of heterotetrameric adaptor complexes. Whereas these motifs have been shown to be sufficient to mediate interaction with the µ-chains, we and others have shown that their context is important in determining the affinity of interaction. In this study we have investigated the interaction between the tyrosine motifs of the type-1 integral membrane proteins TGN38 and lgp120 with medium-chain subunits using the yeast two-hybrid system. Whereas the wild-type version of the cytosolic domain of TGN38 interacts with highest affinity with µ2, we show that the cytosolic domain of lgp120 interacts almost exclusively with µ3A. The specificity of binding of tyrosine-based sorting motifs to µ-chains is shown to be highly sensitive to the context in which the motif lies. For example, the -YQTI- motif of lgp120 is effectively non-functional with regard to µ-chain binding when placed in the context of the TGN38 cytosolic domain. Deletion of four amino acids (NLKL) at the extreme C-terminus of TGN38, leaving the YXX∅ motif as the C-terminus, greatly enhances the affinity of interaction with µ2. Furthermore, addition of these same residues to the extreme C-terminus of lgp120 effectively abolishes the interaction of the cytosolic domain of lgp120 with µ-chains. We also show that the newly identified µ-adaptin-related protein 2 (µ4) only interacts weakly with tyrosine-based sorting motifs.
Structural and functional properties of a yeast xylitol dehydrogenase, a Zn2+-containing metalloenzyme similar to medium-chain sorbitol dehydrogenases
Authors: LUNZER Regina , MAMNUN Yasmine , HALTRICH Dietmar , KULBE Klaus D. , NIDETZKY Bernd
Published: August 2015

The NAD+-dependent xylitol dehydrogenase from the xylose-assimilating yeast Galactocandida mastotermitis has been purified in high yield (80%) and characterized. Xylitol dehydrogenase is a heteronuclear multimetal protein that forms homotetramers and contains 1 mol of Zn2+ ions and 6 mol of Mg2+ ions per mol of 37.4 kDa protomer. Treatment with chelating agents such as EDTA results in the removal of the Zn2+ ions with a concomitant loss of enzyme activity. The Mg2+ ions are not essential for activity and are removed by chelation or extensive dialysis without affecting the stability of the enzyme. Results of initial velocity studies at steady state for d-sorbitol oxidation and d-fructose reduction together with the characteristic patterns of product inhibition point to a compulsorily ordered Theorell–Chance mechanism of xylitol dehydrogenase in which coenzyme binds first and leaves last. At pH 7.5, the binding of NADH (Ki ≈ 10 µM) is approx. 80-fold tighter than that of NAD+. Polyhydroxyalcohols require at least five carbon atoms to be good substrates of xylitol dehydrogenase, and the C-2 (S), C-3 (R) and C-4 (R) configuration is preferred. Therefore xylitol dehydrogenase shares structural and functional properties with medium-chain sorbitol dehydrogenases.
Iron in cytosolic ferritin can be recycled through lysosomal degradation in human fibroblasts
Authors: RADISKY Derek C. , KAPLAN Jerry
Published: August 2015

Examination of the mechanism of intracellular iron recovery from lysosomally-degraded ferritin in vivo has been complicated by the continuous flux of cellular iron through ferritin molecules. Here we incubated human fibroblasts with cationic ferritin, a derivative of horse spleen ferritin, as a technique for delivering immunologically distinct ferritin molecules directly to lysosomes. Using this method, we found increased endogenous ferritin levels after the cellular degradation of cationic ferritin, demonstrating that cells can utilize lysosomal ferritin to produce increased cytosolic ferritin levels. Further, using an in vitro assay, we showed that isolated lysosomes degrade endogenous ferritin in a time- and temperature-dependent manner. These results are consistent with a model in which cytosolic ferritin is taken into the lysosomes and degraded. The solubilized iron from the ferric core could then be transported across the lysosomal membrane back into the cytosol.
Effect of mutation of lysine-128 of the large subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans
Authors: BAINBRIDGE Graeme , ANRALOJC P. John , MADGWICK Pippa J. , PITTS Jim E. , PARRY Martin A. J.
Published: August 2015

The contribution of lysine-128 within the active site of Anacystis nidulansd-ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was investigated by the characterization of mutants in which lysine-128 was replaced with arginine, glycine, glutamine, histidine or aspartic acid. Mutated genes encoding the Rubisco large subunit were expressed in Escherichia coliand the resultant polypeptides assembled into active complexes. All of the mutant enzymes had a lower affinity for ribulose 1,5-bisphosphate (RuBP) and lower rates of carboxylation. Substitution of lysine-128 with glutamine, histidine or aspartic acid decreased the specificity factor and led to the production of an additional monophosphate reaction product. We show that this product results from the loss of the phosphate from C-1 of RuBP, most probably by β-elimination from the 2,3-enediolate derivative of RuBP. The results confirm that lysine-128 is important in determining the position of the essential ε-amino group of lysine-334 within the active site and in loop dynamics. This further demonstrates that residues remote from the active site can be manipulated to modify catalytic function.
Lysine-691 of the anion exchanger from human erythrocytes is located on its cytoplasmic surface
Authors: ERICKSON Hans K. , KYTE Jack
Published: August 2015

A combination of vectorial modification and site-directed immunochemistry has been used to determine the disposition, with respect to the membrane, of Lys-691 of the anion exchanger from human erythrocytes. Intact erythrocytes and inside-out vesicles were vectorially modified in the same container with pyridoxal phosphate and sodium [3H]borohydride. The modified inside-out vesicles were separated from erythrocytes by differential centrifugation and the vesicles and erythrocyte membranes were treated with alkali and digested with trypsin and thermolysin to liberate the peptides IVSKPER and IVSK{Nε-[4´-(5´-phospho-[4-3H]pyridoxyl)]}PER. These peptides, containing the unmodified and modified versions of Lys-691, were retrieved from the digests by site-directed immunochemistry and were identified by HPLC and liquid scintillation spectroscopy. Both the inside-out vesicles and the intact erythrocytes contained the peptide IVSKPER, however, the 3H-label from the phosphopyridoxylated peptide could be detected only in the inside-out vesicles. The incorporation of 3H into Lys-691 of the anion exchanger from inside-out vesicles was at least 30-fold greater than the incorporation into Lys-691 of the anion exchanger from intact erythrocytes. It follows that Lys-691 of the anion exchanger is located on the cytoplasmic surface of the plasma membrane.
S-phase-dependent action of cycloheximide in relieving chromatin-mediated general transcriptional repression
Authors: CESARI Maya , HÉLIOT Laurent , MEPLAN Catherine , PABION Michel , KHOCHBIN Saadi
Published: August 2015

Chromatin plays a major role in the tight regulation of gene expression and in constraining inappropriate gene activity. Replication-coupled chromatin assembly ensures maintenance of these functions of chromatin during S phase of the cell cycle. Thus treatment of cells with an inhibitor of translation, such as cycloheximide (CX), would be expected to have a dramatic effect on chromatin structure and function, essentially in S phase of the cell cycle, due to uncoupled DNA replication and chromatin assembly. In this work, we confirm this hypothesis and show that CX can induce a dramatic S-phase-dependent alteration in chromatin structure that is associated with general RNA polymerase II-dependent transcriptional activation. Using two specific RNA polymerase II-transcribed genes, we confirm the above conclusion and show that CX-mediated transcriptional activation is enhanced during the DNA replication phase of the cell cycle. Moreover, we show co-operation between an inhibitor of histone deacetylase and CX in inducing gene expression, which is again S-phase-dependent. The modest effect of CX in inducing the activity of a transiently transfected promoter shows that the presence of the promoter in an endogenous chromatin context is necessary in order to observe transcriptional activation. We therefore suggest that the uncoupled DNA replication and histone synthesis that occur after CX treatment induces a general modification of chromatin structure, and propose that this general disorganization of chromatin structure is responsible for a widespread activation of RNA polymerase II-mediated gene transcription.
Cholesterol relieves the inhibitory effect of sphingomyelin on type II secretory phospholipase A2
Authors: KOUMANOV Kamen S. , QUINN Peter J. , BÉRÉZIAT Gilbert , WOLF Claude
Published: August 2015

Secretory type II phospholipase A2 (sPLA2) is inhibited by sphingomyelin (SPH); cholesterol either mixed with the model glycerophospholipid substrate or added to the assay medium as separated liposomes counteracts this inhibition efficiently. The inhibition of fatty acid release assayed by quantitative gas chromatography–MS is observed when SPH is added to erythrocyte membranes as the substrate instead of a readily hydrolysable phosphatidylethanolamine/phosphatidylserine model mixture. Hydrolysis of SPH by Staphylococcus aureus sphingomyelinase suppresses its inhibitory potency. The addition of cholesterol to SPH liposomes with a 1:1 stoichiometry relieves completely the inhibition of sPLA2 exerted by SPH. The mechanism of inhibition suggested by the binding assay is that sPLA2 binds with affinity to the SPH interface, after either phase segregation at the assay temperature or on the pure SPH liposomes added to the incubation medium. Cholesterol is shown to suppress the binding affinity of the enzyme for the SPH interface. A model for inhibition is suggested in which binding of the sphingosine moiety is competitive for sPLA2 (inhibition) or for cholesterol (release of the enzyme).
Design of a transferrin–proteinase inhibitor conjugate to probe for active cysteine proteinases in endosomes
Authors: XING Ruye , MASON Robert W.
Published: August 2015

A new technique has been developed to identify active proteinases in endosomes that does not require prior isolation of organelles and extraction of the active enzymes. [125I]Iodotyrosylalanyldiazomethane was reversibly conjugated to transferrin to selectively deliver it to endosomes. The protein was conjugated to the inhibitor via a disulphide bond using N-succinimidyl 3-(2-pyridyldithio)propionate. The inhibitor portion of the conjugate bound irreversibly to active cathepsins B and L, and subsequently the reacted enzymes were separated from the transferrin after SDS/PAGE under reducing conditions. Uptake of the protein–inhibitor conjugate and incorporation of inhibitor into cathepsins was blocked at 4 °C, demonstrating that the conjugate enters cells by receptor-mediated endocytosis. Furthermore, endocytosed transferrin–inhibitor conjugate could be recycled back to the extracellular medium and binding to the transferrin receptor could be blocked by native transferrin. Labelling of the enzymes was not blocked by incubating cells at 16 °C, consistent with the majority of the reagent being targeted to endosomes. The inhibited enzymes remained conjugated to transferrin, showing that the disulphide bond between the transferrin and inhibitor was not reduced in the endosome. Results from these studies show that endosomes contain both intermediate and late biosynthetic forms of active cathepsin B, which are indistinguishable from those found in mature lysosomes. These results indicate that the active enzymes in endosomes are not early biosynthetic forms in transit to lysosomes but most probably enter the endosome via retrograde traffic from the lysosome.
Attenuation of drug-stimulated topoisomerase II–DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium buffer is correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase IIα
Authors: AOYAMA Masako , GRABOWSKI Dale R. , DUBYAK George R. , CONSTANTINOU Andreas I. , RYBICKI Lisa A. , BUKOWSKI Ronald M. , GANAPATHI Mahrukh K. , HICKSON Ian D. , GANAPATHI Ram
Published: August 2015

Topoisomerase II (topo II), an essential enzyme for cell viability, is also the target for clinically important anti-neoplastic agents that stimulate topo II-mediated DNA scission. The role of alterations in topo IIα phosphorylation and its effect on drug-induced DNA damage and cytotoxicity were investigated. Following loading of HL-60 cells with the calcium buffer 1,2-bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid tetra(acetoxymethyl) ester (BAPTA-AM), which abrogates intracellular Ca2+ transients, a significant decrease in etoposide (VP-16)- or amsacrine (m-AMSA)-stabilized topo II–DNA cleavable complex formation and a corresponding decrease in cytotoxicity was observed. In a cell-free system, nuclear extracts from BAPTA-AM-treated cells exhibited markedly less activity when assayed for VP-16-stabilized topo II–DNA complex formation, but not decatenation of kinetoplast DNA. In contrast, the loading of HL-60 cells with N,N,N´,N´-tetrakis-(2-pyridyl)ethylenediamine (TPEN), which binds heavy metals without disturbing calcium or magnesium concentrations, did not significantly affect VP-16-stimulated topo II–DNA cleavable complex formation or cytotoxicity. In HL-60 cells the accumulation of BAPTA, but not TPEN, also led to the hypophosphorylation of topo IIα. Tryptic phosphopeptide mapping of topo IIα protein from HL-60 cells revealed: (a) eight major phosphorylation sites in untreated cells; (b) hypophosphorylation of two out of eight sites in BAPTA-AM-treated cells; and (c) hypophosphorylation of between two and four out of eight sites in topo II-poison-resistant HL-60 cells. The two hypophosphorylated sites present following BAPTA-AM treatment of wild-type cells were identical with the hypophosphorylated sites in the resistant cells, but were not the same as the sites that are substrates for casein kinase II [Wells, Addison, Fry, Ganapathi and Hickson (1994) J. Biol. Chem. 269, 29746–29751]. In summary, changes in intracellular Ca2+ transients that lead to the site-specific hypophosphorylation of topo IIα are possibly involved in regulating the DNA damage caused by and the cytotoxic potential of topo II poisons.
Role of plasma membrane transporters in muscle metabolism
Authors: ZORZANO Antonio , FANDOS César , PALACÍN Manuel
Published: August 2015

Muscle plays a major role in metabolism. Thus it is a major glucose-utilizing tissue in the absorptive state, and changes in muscle insulin-stimulated glucose uptake alter whole-body glucose disposal. In some conditions, muscle preferentially uses lipid substrates, such as fatty acids or ketone bodies. Furthermore, muscle is the main reservoir of amino acids and protein. The activity of many different plasma membrane transporters, such as glucose carriers and transporters of carnitine, creatine and amino acids, play a crucial role in muscle metabolism by catalysing the influx or the efflux of substrates across the cell surface. In some cases, the membrane transport process is subjected to intense regulatory control and may become a potential pharmacological target, as is the case with the glucose transporter GLUT4. The goal of this review is the molecular characterization of muscle membrane transporter proteins, as well as the analysis of their possible regulatory role.
14-3-3 proteins are required for the inhibition of Ras by exoenzyme S
Authors: HENRIKSSON Maria Lena , TROLLÉR Ulrika , HALLBERG Bengt
Published: August 2015

14-3-3 proteins play a regulatory role and participate in both signal transduction and checkpoint control pathways. 14-3-3 proteins bind phosphoserine ligands, such as Raf-1 kinase and Bad, by recognizing the phosphorylated consensus motif, Arg-Ser-Xaa-pSer-Xaa-Pro (where ‘Xaa’ represents ‘any residue’, and ‘pSer’ is ‘phosphoserine’) . However, 14-3-3 proteins must bind unphosphorylated ligands, such as glycoprotein Ibα and Pseudomonas aeruginosa exoenzyme S (ExoS), since it has been suggested that specific residues of 14-3-3 proteins are required for activation of ExoS. Furthermore, an unphosphorylated peptide derived from a phage display library inhibited the binding of both ExoS and Raf-1 to 14-3-3, and bound within the same conserved amphipathic groove on the surface of 14-3-3 as the Raf-derived phosphopeptide (pS-Raf-259). In the present study we identify the interaction site on ExoS for 14-3-3, and show that ExoS and 14-3-3 do indeed interact in vivo. In addition, we show that this interaction is critical for the ADP-ribosylation of Ras by ExoS, both in vitro and in vivo. Loss of the 14-3-3 binding site on ExoS results in an ExoS molecule that is unable to efficiently inactivate Ras, and displays reduced killing activity.
Involvement of the membrane lipid bilayer in sorting prohormone convertase 2 into the regulated secretory pathway
Authors: BLÁZQUEZ Mercedes , THIELE Christoph , HUTTNER Wieland B. , DOCHERTY Kevin , SHENNAN Kathleen I. J.
Published: August 2015

Prohormone convertase 2 (PC2) is a neuroendocrine-specific protease involved in the intracellular maturation of prohormones and proneuropeptides. PC2 is synthesised as a proprotein (proPC2) that undergoes proteolysis, aggregation and membrane association during its transit through the regulated secretory pathway. We have previously shown that the pro region of proPC2 plays a key role in its aggregation and membrane association. To investigate this further, we determined the binding properties of a peptide containing amino acids 45–84 of proPC2 (proPC245–84) to trans-Golgi network/granule-enriched membranes from the AtT20 cell line. Removal of peripheral membrane proteins or hydrolysis of integral membrane proteins did not affect the binding properties of proPC245–84. Rather, proPC245–84 was shown to bind to protein-free liposomes in a pH- and Ca2+-dependent manner. To identify the component of the lipid bilayer involved in this membrane association, we used chromaffin-granule membranes and studied the binding properties of the endogenous PC2. Treatment of the membranes with saponin, a cholesterol-depleting detergent, failed to extract PC2 from the membranes, whereas chromogranin A (CgA) was removed. Treatment of the membranes with Triton X-100 yielded a low-density detergent-insoluble fraction enriched in PC2, but not CgA. The detergent-insoluble fraction also contained glycoprotein III, known to be part of the lipid rafts (membrane microdomains rich in sphingolipids). Finally, sphingolipid depletion of AtT20 cells resulted in the mis-sorting of PC2, suggestive of a link between the association of PC2 with lipid rafts in the membrane and its sorting into the regulated secretory pathway.
Crystal structure of the NADP+-dependent aldehyde dehydrogenase from Vibrio harveyi: structural implications for cofactor specificity and affinity
Authors: AHVAZI Bijan , COULOMBE Rene , DELARGE Marc , VEDADI Masoud , ZHANG Lei , MEIGHEN Edward , VRIELINK Alice
Published: August 2015

Aldehyde dehydrogenase from the bioluminescent bacterium, Vibrio harveyi, catalyses the oxidation of long-chain aliphatic aldehydes to acids. The enzyme is unique compared with other forms of aldehyde dehydrogenase in that it exhibits a very high specificity and affinity for the cofactor NADP+. Structural studies of this enzyme and comparisons with other forms of aldehyde dehydrogenase provide the basis for understanding the molecular features that dictate these unique properties and will enhance our understanding of the mechanism of catalysis for this class of enzyme. The X-ray structure of aldehyde dehydrogenase from V. harveyi has been solved to 2.5-Å resolution as a partial complex with the cofactor NADP+ and to 2.1-Å resolution as a fully bound ‘holo’complex. The cofactor preference exhibited by different forms of the enzyme is predominantly determined by the electrostatic environment surrounding the 2´-hydroxy or the 2´-phosphate groups of the adenosine ribose moiety of NAD+ or NADP+, respectively. In the NADP+-dependent structures the presence of a threonine and a lysine contribute to the cofactor specificity. In the V. harveyi enzyme an arginine residue (Arg-210) contributes to the high cofactor affinity through a pi stacking interaction with the adenine ring system of the cofactor. Further differences between the V. harveyi enzyme and other aldehyde dehydrogenases are seen in the active site, in particular a histidine residue which is structurally conserved with phosphorylating glyceraldehyde-3-phosphate dehydrogenase. This may suggest an alternative mechanism for activation of the reactive cysteine residue for nucleophilic attack.
Tissue-specific activity of lipoprotein lipase in skeletal muscle regulates the expression of uncoupling protein 3 in transgenic mouse models
Authors: KRATKY Dagmar , STRAUSS Juliane G. , ZECHNER Rudolf
Published: August 2015

Uncoupling protein (UCP)-2 and UCP-3 are two recently discovered proteins similar to UCP-1, which regulates thermogenesis in brown adipose tissue (BAT). Whereas UCP-1 expression is restricted to BAT, UCP-2 is widely expressed. UCP-3 is found mainly in skeletal muscle and BAT. A large body of evidence exists that the expression of UCP-2 and UCP-3 in skeletal muscle of mice is regulated by feeding/fasting, and some studies have suggested that this effect might be caused by the changing concentration of plasma non-esterified fatty acids (NEFAs). In an attempt to determine whether the increased import of triacylglycerol-derived NEFAs can also affect UCP expression, we determined the mRNA levels of UCP-1, UCP-2 and UCP-3 in BAT and muscle of induced mutant mouse lines that overexpressed or lacked lipoprotein lipase (LPL) in these tissues. The expression levels of UCP-1 and UCP-2 in BAT and in skeletal and cardiac muscle respectively were not affected by variations in tissue LPL activities. In contrast, UCP-3 mRNA levels were induced 3.4-fold in mice with high levels of LPL in skeletal muscle, and down-regulated in mice that lacked LPL in skeletal muscle. The presence or absence of LPL in BAT had no effect on UCP-3 expression levels. The response of UCP-3 mRNA expression to variations in LPL activity in skeletal muscle was independent of the feeding status or of plasma NEFA concentrations. These findings indicated that NEFAs as lipolytic products of LPL-mediated triacylglycerol hydrolysis markedly affect UCP-3 expression and that increased LPL activities occurring during fasting in skeletal muscle contribute to the induction of UCP-3 expression by promoting the increased uptake of NEFAs. In addition, our results demonstrate that UCP-2 and UCP-3 are differentially regulated in response to LPL-mediated NEFA uptake in skeletal muscle of mice.
Cytoplasmic interactions between phospholamban residues 1–20 and the calcium-activated ATPase of the sarcoplasmic reticulum
Authors: SHARMA Parveen , PATCHELL Valerie B. , GAO Yuan , EVANS James S. , LEVINE Barry A.
Published: August 2015

Phospholamban regulates the activity of the calcium-activated ATPase (CaATPase) of cardiac sarcoplasmic reticulum. Equilibrium fluorescence studies have shown that the N-terminal cytoplasmic region of phospholamban (residues 1–20, domain 1) causes a decrease in the intrinsic tryptophan fluorescence of the CaATPase. The interaction of phospholamban residues 1–20 with the CaATPase also results in spectral changes for the extrinsic chromophore FITC covalently attached to the cytoplasmic region of the calcium pump. The fluorescence changes for both reporter groups correlate with a dissociation constant of ≈ 40µM for the complex between phospholamban residues 1–20 and the CaATPase. Complex formation is notably weaker when phospholamban 1–20 is titrated into the CaATPase in the presence of calcium, with altered conformational effects resulting from binding. The interaction of domain 1 of phospholamban with the CaATPase is also reduced upon phosphorylation of phospholamban 1–20 at Ser-16. This region of phospholamban 1–20 is shown by isotope-edited NMR study to be involved in interaction with the CaATPase. Binding of the phosphorylated peptide is not abolished, however, indicating that phospholamban 1–20 remains associated with the CaATPase even after phosphorylation. The data provide direct evidence for the interaction between the cytoplasmic regions of phospholamban and the pump, and are discussed in the context of the mechanism for inhibition of cardiac CaATPase activity by phospholamban.
The same rat Δ6-desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis
Authors: D'ANDREA Sabine , GUILLOU Hervé , JAN Sophie , CATHELINE Daniel , THIBAULT Jean-Noël , BOURIEL Monique , RIOUX Vincent , LEGRAND Philippe
Published: August 2015

The recently cloned Δ6-desaturase is known to catalyse the first step in very-long-chain polyunsaturated fatty acid biosynthesis, i.e. the desaturation of linoleic and α-linolenic acids. The hypothesis that this enzyme could also catalyse the terminal desaturation step, i.e. the desaturation of 24-carbon highly unsaturated fatty acids, has never been elucidated. To test this hypothesis, the activity of rat Δ6-desaturase expressed in COS-7 cells was investigated. Recombinant Δ6-desaturase expression was analysed by Western blot, revealing a single band at 45kDa. The putative involvement of this enzyme in the Δ6-desaturation of C24:5n-3 to C24:6n-3 was measured by incubating transfected cells with C22:5n-3. Whereas both transfected and non-transfected COS-7 cells were able to synthesize C24:5n-3 by elongation of C22:5n-3, only cells expressing Δ6-desaturase were also able to produce C24:6n-3. In addition, Δ6-desaturation of [1-14C]C24:5n-3 was assayed invitro in homogenates from COS-7 cells expressing Δ6-desaturase or not, showing that Δ6-desaturase catalyses the conversion of C24:5n-3 to C24:6n-3. Evidence is therefore presented that the same rat Δ6-desaturase catalyses not only the conversion of C18:3n-3 to C18:4n-3, but also the conversion of C24:5n-3 to C24:6n-3. A similar mechanism in the n-6 series is strongly suggested.
Mining proteases in the genome databases
Authors: Coates David
Published: August 2015

Protease data mining can take advantage both of the many specialist, Web-available databases that cover the genetic, protein and nucleic acid sequence information that is specific to a variety of organisms, and of a flexible, but defined, classification system. However, precomputed data, such as gene predictions, should be used with care. Unless there is definitive supporting information, ideally sequencing of a cDNA to show that the predictions are accurate, followed by expression and biochemical characterization of the predicted protein, the predicted gene and its product remains a possibility, rather than a certainty.
Oncogenes as regulators of apoptosis
Authors: Labazi Mohamed , Phillips Andrew C
Published: August 2015

Although cancer is a disease that will afflict one out of three people in the Western world, when considered at a cellular level, it is a rare clonal event. Long-lived organisms, such as humans, have evolved strategies to restrict the development of potentially malignant cells, and one such mechanism is the coupling of proliferative and apoptotic pathways. Multiple oncogenes have the ability to trigger apoptosis when expressed in an inappropriate fashion, and this is thought to restrict tumour formation by eliminating potentially malignant cells that have acquired a mutation stimulating proliferation. Hence for a tumour to arise, in addition to mutations that drive proliferation, mutations that prevent apoptosis are also a prerequisite.
Apoptosis in disease: about shortage and excess
Authors: Brunner Thomas , Mueller Christoph
Published: August 2015

The death of cells by apoptosis is a fundamental event in development and the maintenance of cell homoeostasis. The other side of the coin, however, is that excessive cell death by apoptosis or the lack of apoptosis is often the driving force of many diseases. Whereas reduced apoptosis sensitivity is a basic characteristic of many tumour cells, accelerated tissue cell death and loss of tissue functions is the underlying cause of many auto-immune and inflammatory diseases.
Sex, drugs and gene expression: signalling by members of the nuclear receptor superfamily
Authors: McEwan Iain J
Published: August 2015

It is almost 20 years since the first steroid receptor cDNAs were cloned, a development that led to the concept of a superfamily of ligand-activated transcription factors: the nuclear receptors. Natural ligands for nuclear receptors are generally lipophilic in nature and include steroid hormones, bile acids, fatty acids, thyroid hormones, certain vitamins and prostaglandins. Nuclear receptors act principally to directly control patterns of gene expression and play vital roles during development and in the regulation of metabolic and reproductive functions in the adult organism. Since the original cloning experiments, considerable progress has been made in our understanding of the structure, mechanisms of action and biology of this important family of proteins.
Plant polyphenols: free radical scavengers or chain-breaking antioxidants?
Authors: Rice-Evans Catherine
Published: August 2015

There is increasing interest in the biological effects of tea- and wine-derived polyphenols and many studies in vitro and in vivo are demonstrating their antioxidant properties. Tea is a major source of dietary polyphenols and an even richer source of the flavanols, the catechins and catechin/gallate esters. Although there are limited studies on the bioavailability of the polyphenols, the absorption of flavanols in humans has been shown. The studies described in this chapter discuss the relative antioxidant potentials of the polyphenolic flavonoids in vitro against radicals generated in the aqueous phase in comparison with their relative effectiveness as antioxidants against propagating lipid peroxyl radicals, and how their activity influences that of α-tocopherol in low-density lipoproteins exposed to oxidative stress.
Tamoxifen as an antioxidant and cardioprotectant
Authors: Wiseman Helen
Published: August 2015

Tamoxifen is widely used in the treatment of breast cancer and has been proposed as a prophylactic agent in this disease. Tamoxifen is an effective antioxidant and protects membranes and low-density lipoprotein (LDL) particles against oxidative damage. This antioxidant action is shared by endogenous and synthetic oestrogens. The ability of tamoxifen to protect LDL particles against the oxidative damage implicated in atherosclerosis may be an important factor in the reported cardioprotective action of tamoxifen in women being treated for breast cancer. In addition, tamoxifen has been found to act in a similar manner to oestrogens to lower plasma cholesterol levels. The cardioprotective action of tamoxifen may be a key factor in predicting the likely risk/benefit ratio for prophylactic tamoxifen treatment in otherwise healthy women, who have been calculated to be genetically predisposed to developing breast cancer. In the future, predisposition to breast cancer may be determined by genetic screening.
Free radicals and food irradiation
Authors: Dodd "N.J.F.
Published: August 2015

Ionizing radiation can be used to control insect and microbial infestation of foodstuffs, inhibit sprouting, delay ripening and reduce the dangers from food-poisoning bacteria. Irradiation produces free radicals, most of which decay rapidly, although some are more persistent. These latter radicals can be detected and characterized by electron spin resonance (ESR). In bone and other calcified tissues, the radiation-induced radicals are distinguishable from naturally occurring radicals, and their stability makes them ideal for radiation dosimetry. The radicals induced in plant material, such as seeds and dried spices, are generally indistinguishable from the endogenous radicals and decay over a period of days or weeks. However, in many of these materials, a radiation-specific radical can be detected at low concentration, thereby permitting identification of irradiated samples, although precluding accurate dosimetry. ESR, although not universally applicable, currently provides the most specific method for the detection of irradiated food.
Excitotoxicity and mitochondria
Authors: Nicholls D.G. , Budd S.L. , Ward M.W. , Castilho R.F.
Published: August 2015

Excitotoxicity is the process whereby a massive glutamate release in the central nervous system in response to ischaemia or related trauma leads to the delayed, predominantly necrotic death of neurons. Excitotoxicity is also implicated in a variety of slow neurodegenerative disorders. Mitochondria accumulate much of the post-ischaemic calcium entering the neurons via the chronically activated N-methyl-d-aspartate receptor. This calcium accumulation plays a key role in the subsequent death of the neuron. Cultured cerebellar granule cells demonstrate delayed calcium de-regulation (DCD) followed by necrosis upon exposure to glutamate. DCD is unaffected by the ATP synthase inhibitor oligomycin but is inhibited by the further addition of a respiratory chain inhibitor to depolarize the mitochondria and inhibit mitochondrial calcium accumulation without depleting ATP [Budd and Nicholls (1996) J. Neurochem. 67, 2282-2291]. Mitochondrial depolarization paradoxically decreases the cytoplasmic calcium elevation following glutamate addition, probably due to an enhanced calcium efflux from the cell. Cells undergo immediate calcium de-regulation in the presence of glutamate if the respiratory chain is inhibited; this is due to ATP depletion following ATP synthase reversal and can be reversed by oligomycin. In contrast, DCD is irreversible. Elevated cytoplasmic calcium is not excitotoxic as long as mitochondria are depolarized; alternative substrates do not rescue cells about to undergo DCD, suggesting that glycolytic failure is not involved. Mitochondria in situ remain sufficiently polarized during granule cell glutamate exposure to continue to generate ATP and show a classic mitochondrial state 3-state 4 hyperpolarization on inhibiting ATP synthesis; mitochondrial depolarization follows, and may be a consequence of rather than a cause of DCD. In addition, our studies show no evidence of the mitochondrial permeability transition prior to DCD. The mitochondrial generation of superoxide anions is enhanced during glutamate exposure and a working hypothesis is that DCD may be caused by oxidative damage to calcium extrusion pathways at the plasma membrane.
Apolipoprotein E and Alzheimer's disease: signal transduction mechanisms
Authors: Strittmatter Warren J.
Published: August 2015

The three common apolipoprotein E (ApoE) alleles differentially contribute to the risk of Alzheimer's disease (AD). While the APOE genotype alters susceptibility to disease expression, individuals with APOE ϵ4 alleles have the highest risk of developing AD; the APOE ϵ4 allele is neither essential nor sufficient on its own to cause AD. Since the discovery, in 1992, of the involvement of APOE in AD, many scientists have explored the role of the ApoE isoforms in the central nervous system in an effort to elucidate their roles in the pathophysiological mechanism of this disease. While many hypotheses have been proposed, none has been proven. ApoE was discovered through investigations into cholesterol metabolism. In serum and in cerebrospinal fluid ApoE binds lipoprotein particles, which contain cholesterol esters, and is critical in the shuttling of cholesterol from cell to cell. Trafficking of ApoE is mediated by specific interactions with cell-surface receptors. As described later, several families of ApoE receptors with diverse functions have been discovered. The roles of these receptors are proving increasingly complex since additional interactions with other ligands and with other intracellular proteins are rapidly being identified. It was once thought that these receptors only shuttle ApoE-containing phospholipid particles from the extracellular environment into the cell, but they also transduce a number of additional intracellular signals and interactions. Molecular signalling cascades initiated by the various ApoE receptors modulate a number of critical cellular processes. To date, two functional classes of ApoE receptors have been identified. The first is the low-density lipoprotein receptor family and the second the scavenger receptor families.
Modelling Alzheimer's disease in multiple transgenic mice
Authors: Dewachter Ilse , Moechars Dieder , van Dorpe Jo , Tesseur Ina , Van den Haute Chris , Spittaels Kurt , Van Leuven Fred
Published: August 2015

We have reported transgenic mice with neuronal overexpression of the clinical mutant ϐ-amyloid precursor protein (APP) known as London, which develop an AD-related phenotype [Moechers, Dewachter, Lorent, Reversé, Baekelandt, Nadiu, Tesseur, Spittaels, Van den Haute, Checler, et al. (1999) J. Biol. Chem. 274, 6483-6492]. Characterized early symptoms (3-9 months) include disturbed behaviour, neophobia, aggression, hypersensitivity to kainic acid, hyposensitivity to N-methyl-D-aspartate, defective cognition and memory, and decreased long-term potentiation. Late in life, at 12-15 months, amyloid plaques develop in the brain and correlate with increased levels of ϐ-amyloid (Aϐ)40/42 (the 40- and 42-amino-acid forms of Aϐ). The formation of amyloid plaques is dissociated in time from and not involved in the early phenotype. Hyperphosphorylated protein tau is present but no tangle pathology is observed. In double-transgenic mice, i.e. APP/London x Presenilin 1, the increased production of Aϐ results in amyloid plaques developing by the age of 6 months. Transgenic mice with overexpression of either human apolipoprotein E4 (ApoE4) or human protein tau in central neurons develop severe axonopathy in the brain and spinal cord. Progressive degeneration of nerves and muscles is demonstrated by motor problems, wasting and premature death. Tau is hyperphosphorylated but there is no formation of filaments or neurofibrillary tangles. The tangle aspect of AD pathology is still missing from all current transgenic amyloid models. Its implementation will require insight into the cellular signalling pathways which regulate the microtubule-stabilizing function by phosphorylation of neuronal tau.
Defining the structure of the substrate-free state of the DnaK molecular chaperone
Authors: Swain Joanna F. , Sivendran Renuka , Gierasch Lila M.
Published: August 2015

Members of the Hsp70 (heat-shock protein of 70 kDa) family of molecular chaperones bind to exposed hydrophobic stretches on substrate proteins in order to dissociate molecular complexes and prevent aggregation in the cell. Substrate affinity for the C-terminal domain of the Hsp70 is regulated by ATP binding to the N-terminal domain utilizing an allosteric mechanism. Our multi-dimensional NMR studies of a substrate-binding domain fragment (amino acids 387-552) from an Escherichia coli Hsp70, DnaK(387-552), have uncovered a pH-dependent conformational change, which we propose to be relevant for the full-length protein also. At pH 7, the C-terminus of DnaK(387-552) mimics substrate by binding to its own substrate-binding site, as has been observed previously for truncated Hsp70 constructs. At pH 5, the C-terminus is released from the binding site, such that DnaK is in the substrate-free state 10-20% of the time. We propose that the mechanism for the release of the tail is a loss of affinity for substrate at low pH. The pH-dependent fluorescence changes at a tryptophan residue near the substrate-binding pocket in full-length DnaK lead us to extend these conclusions to the full-length DnaK as well. In the context of the DnaK substrate-binding domain fragment, the release of the C-terminus from the substrate-binding site provides our first glimpse of the empty conformation of an Hsp70 substrate-binding domain containing a portion of the helical subdomain.
Lectins and protein traffic early in the secretory pathway
Authors: Hauri Hans-Peter , Nufer Oliver , Breuza Lionel , Tekaya Houchaima Ben , Liang Lu
Published: August 2015

Lectins of the early secretory pathway are involved in selective transport of newly synthesized glycoproteins from the endoplasmic reticulum (ER) to the ER–Golgi intermediate compartment (ERGIC). The most prominent cycling lectin is the mannose-binding type I membrane protein ERGIC-53 (ERGIC protein of 53 kDa), a marker for the ERGIC, which functions as a cargo receptor to facilitate export of an increasing number of glycoproteins with different characteristics from the ER. Two ERGIC-53-related proteins, VIP36 (vesicular integral membrane protein 36) and a novel ERGIC-53-like protein, ERGL, are also found in the early secretory pathway. ERGL may act as a regulator of ERGIC-53. Studies of ERGIC-53 continue to provide new insights into the organization and dynamics of the early secretory pathway. Analysis of the cycling of ERGIC-53 uncovered a complex interplay of trafficking signals and revealed novel cytoplasmic ER-export motifs that interact with COP-II coat proteins. These motifs are common to type I and polytopic membrane proteins including presenilin 1 and presenilin 2. The results support the notion that protein export from the ER is selective.
Structural basis of matrix metalloproteinase function
Authors: Bode Wolfram
Published: August 2015

The matrix metalloproteinases (MMPs) constitute a family of multidomain zinc endopeptidases which contain a catalytic domain with a common metzincin-like topology. The MMPs are involved not only in extracellular matrix degradation, but also in a number of other biological processes. Normally, their proteolytic activity is regulated precisely by their main endogenous protein inhibitors, in particular the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases, such as arthritis, tumour growth and metastasis, rendering the MMPs attractive targets for inhibition therapy. Knowledge of their tertiary structures is crucial for a full understanding of their functional properties. Since the first publication of atomic MMP structures in 1994, much more structural information has become available on details of the catalytic domain, on its interaction with synthetic and protein inhibitors, on domain organization and on the formation of complexes with other proteins. This review will outline our current knowledge of MMP structure.