bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxidation of ubiquinol by peroxynitrite: implications for protection of mitochondria against nitrosative damage
Authors: SCHÖPFER Francisco , RIOBÓ Natalia , CARRERAS María Cecilia , ALVAREZ Beatriz , RADI Rafael , BOVERIS Alberto , CADENAS Enrique , PODEROSO Juan José
Published: August 2015

A major pathway of nitric oxide utilization in mitochondria is its conversion to peroxynitrite, a species involved in biomolecule damage via oxidation, hydroxylation and nitration reactions. In the present study the potential role of mitochondrial ubiquinol in protecting against peroxynitrite-mediated damage is examined and the requirements of the mitochondrial redox status that support this function of ubiquinol are established. (1) Absorption and EPR spectroscopy studies revealed that the reactions involved in the ubiquinol/peroxynitrite interaction were first-order in peroxynitrite and zero-order in ubiquinol, in agreement with the rate-limiting formation of a reactive intermediate formed during the isomerization of peroxynitrite to nitrate. Ubiquinol oxidation occurred in one-electron transfer steps as indicated by the formation of ubisemiquinone. (2) Peroxynitrite promoted, in a concentration-dependent manner, the formation of superoxide anion by mitochondrial membranes. (3) Ubiquinol protected against peroxynitrite-mediated nitration of tyrosine residues in albumin and mitochondrial membranes, as suggested by experimental models, entailing either addition of ubiquinol or expansion of the mitochondrial ubiquinol pool caused by selective inhibitors of complexes III and IV. (4) Increase in membrane-bound ubiquinol partially prevented the loss of mitochondrial respiratory function induced by peroxynitrite. These findings are analysed in terms of the redox transitions of ubiquinone linked to both nitrogen-centred radical scavenging and oxygen-centred radical production. It may be concluded that the reaction of mitochondrial ubiquinol with peroxynitrite is part of a complex regulatory mechanism with implications for mitochondrial function and integrity.
Activation of phosphatidylinositol 3-kinase is required for transcriptional activity of F-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: assessment of the role of protein kinase B and p70 S6 kinase
Authors: FERNÁNDEZ DE MATTOS Silvia , DE LOS PINOS Elisabet , JOAQUIN Manel , TAULER Albert
Published: August 2015

Previous studies have demonstrated that the F isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase(6PF2K/Fru-2,6-BPase) is transcriptionally regulated by growth factors. The aim of this study was to investigate the importance of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway in the regulation of 6PF2K/Fru-2,6-BPase gene expression. We have completed studies using chemical inhibitors and expression vectors for the proteins involved in this signalling cascade. Treatment of cells with LY 294002, an inhibitor of PI 3-kinase, blocked the epidermal growth factor (EGF)-dependent stimulation of 6PF2K/Fru-2,6-BPase gene transcription. Transient transfection of a constitutively active PI 3-kinase was sufficient to activate transcription from the F-type 6PF2K/Fru-2,6-BPase promoter. In contrast, co-transfection with a dominant-negative form of PI 3-kinase completely abrogated the stimulation by EGF, and down-regulated the basal promoter activity. In an attempt to determine downstream proteins that lie between PI 3-kinase and 6PF2K/Fru-2,6-BPase gene expression, the overexpression of a constitutively active form of protein kinase B (PKB) was sufficient to activate 6PF2K/Fru-2,6-BPase gene expression, even in the presence of either a dominant-negative form of PI 3-kinase or LY 294002. The over-expression of p70/p85 ribosomal S6 kinase or the treatment with its inhibitor rapamycin did not affect 6PF2K/Fru-2,6-BPase transcription. We conclude that PI 3-kinase is necessary for the transcriptional activity of F-type 6PF2K/Fru-2,6-BPase, and that PKB is a downstream effector of PI 3-kinase directly involved in the regulation of 6PF2K/Fru-2,6-BPase gene expression.
Expression, purification and characterization of recombinant human choline acetyltransferase: phosphorylation of the enzyme regulates catalytic activity
Authors: DOBRANSKY Tomas , DAVIS Wanda L. , XIAO Gong-Hua , RYLETT R. Jane
Published: August 2015

Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons and, in humans, may be produced in 82- and 69-kDa forms. In this study, recombinant choline acetyltransferase from baculovirus and bacterial expression systems was used to identify protein isoforms by two-dimensional SDS/PAGE and as substrate for protein kinases. Whereas hexa-histidine-tagged 82- and 69-kDa enzymes did not resolve as individual isoforms on two-dimensional gels, separation of wild-type choline acetyltransferase expressed in insect cells revealed at least nine isoforms for the 69-kDa enzyme and at least six isoforms for the 82-kDa enzyme. Non-phosphorylated wild-type choline acetyltransferase expressed in Escherichia coli yielded six (69 kDa) and four isoforms (82 kDa) respectively. Immunofluorescent labelling of insect cells expressing enzyme showed differential subcellular localization with the 69-kDa enzyme localized adjacent to plasma membrane and the 82-kDa enzyme being cytoplasmic at 24 h. By 64 h, the 69-kDa form was in cytoplasm and the 82-kDa form was only present in nucleus. Studies in vitro showed that recombinant 69-kDa enzyme was a substrate for protein kinase C (PKC), casein kinase II (CK2) and α-calcium/calmodulin-dependent protein kinase II (α-CaM kinase), but not for cAMP-dependent protein kinase (PKA); phosphorylation by PKC and CK2 enhanced enzyme activity. The 82-kDa enzyme was a substrate for PKC and CK2 but not for PKA or α-CaM kinase, with only PKC yielding increased enzyme activity. Dephosphorylation of both forms of enzyme by alkaline phosphatase decreased enzymic activity. These studies are of functional significance as they report for the first time that phosphorylation enhances choline acetyltransferase catalytic activity.
Unifying mechanism for Aplysia ADP-ribosyl cyclase and CD38/NAD+ glycohydrolases
Authors: CAKIR-KIEFER Céline , MULLER-STEFFNER Hélène , SCHUBER Francis
Published: August 2015

Highly purified Aplysia californica ADP-ribosyl cyclase was found to be a multifunctional enzyme. In addition to the known transformation of NAD+ into cADP-ribose this enzyme is able to catalyse the solvolysis (hydrolysis and methanolysis) of cADP-ribose. This cADP-ribose hydrolase activity, which becomes detectable only at high concentrations of the enzyme, is amplified with analogues such as pyridine adenine dinucleotide, in which the cleavage rate of the pyridinium-ribose bond is much reduced compared with NAD+. Although the specificity ratio Vmax/Km is in favour of NAD+ by 4 orders of magnitude, this multifunctionality allowed us to propose a ‘partitioning’ reaction scheme for the Aplysia enzyme, similar to that established previously for mammalian CD38/NAD+ glycohydrolases. This mechanism involves the formation of a single oxocarbenium-type intermediate that partitions to cADP-ribose and solvolytic products via competing pathways. In favour of this mechanism was the finding that the enzyme also catalysed the hydrolysis of NMN+, a substrate that cannot undergo cyclization. The major difference between the mammalian and the invertebrate enzymes resides in their relative cyclization/hydrolysis rate-constant ratios, which dictate their respective yields of cADP-ribose (ADP-ribosyl cyclase activity) and ADP-ribose (NAD+ glycohydrolase activity). For the Aplysia enzyme's catalysed transformation of NAD+ we favour a mechanism where the formation of cADP-ribose precedes that of ADP-ribose; i.e. macroscopically the invertebrate ADP-ribosyl cyclase conforms to a sequential reaction pathway as a limiting form of the partitioning mechanism.
Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I
Authors: MATSUMOTO Michi , IMAGAWA Masayoshi , AOKI Yasunobu
Published: August 2015

Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor α (TGFα), and 3,3ʹ,4,4ʹ,5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGFα stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGFα induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression.
FM1-43 reports plasma membrane phospholipid scrambling in T-lymphocytes
Authors: ZWEIFACH Adam
Published: August 2015

We have found using imaging techniques that stimulating Jurkat human leukaemic T-cells with ionomycin in the presence of FM1-43, a dye used to monitor exocytosis and endocytosis, causes large (6-10-fold) increases in FM1-43 fluorescence. These responses are too large to be caused by exocytosis. Instead, three lines of evidence suggest that FM1-43 is responding to phospholipid scrambling. First, ionomycin also stimulates increases in the fluorescence of annexin V, a phosphatidylserine-specific probe, while thapsigargin does not stimulate fluorescence increases of either probe. Secondly, cells that exhibit FM1-43 fluorescence increases after ionomycin stimulation stain with annexin V once FM1-43 is washed out. Thirdly, ionomycin stimulates uptake of 7-nitrobenz-2-oxa-1,3-diazole-labelled phosphatidylcholine, a specific assay for scramblase activity, whereas thapsigargin does not. We find that FM1-43 reports phospholipid scrambling with ‘better’ kinetics than annexin V, and does require extracellular Ca2+ to report phospholipid scrambling. We suggest that FM1-43 may be a useful probe to study the dynamics of phospholipid scrambling. The results are the first demonstration that FM1-43 can respond significantly to a biological process other than vesicular trafficking.
Ca2+ signals mediated by Ins(1,4,5)P3-gated channels in rat ureteric myocytes
Authors: BOITTIN François-Xavier , COUSSIN Frédéric , MOREL Jean-Luc , HALET Guillaume , MACREZ Nathalie , MIRONNEAU Jean
Published: August 2015

Localized Ca2+-release signals (puffs) and propagated Ca2+ waves were characterized in rat ureteric myocytes by confocal microscopy. Ca2+ puffs were evoked by photorelease of low concentrations of Ins(1,4,5)P3 from a caged precursor and by low concentrations of acetylcholine; they were also observed spontaneously in Ca2+-overloaded myocytes. Ca2+ puffs showed some variability in amplitude, time course and spatial spread, suggesting that Ins(1,4,5)P3-gated channels exist in clusters containing variable numbers of channels and that within these clusters a variable number of channels can be recruited. Immunodetection of Ins(1,4,5)P3 receptors revealed the existence of several spots of fluorescence in the confocal cell sections, supporting the existence of clusters of Ins(1,4,5)P3 receptors. Strong Ins(1,4,5)P3 photorelease and high concentrations of acetylcholine induced Ca2+ waves that originated from an initiation site and propagated in the whole cell by spatial recruitment of neighbouring Ca2+-release sites. Both Ca2+ puffs and Ca2+ waves were blocked selectively by intracellular applications of heparin and an anti-Ins(1,4,5)P3-receptor antibody, but were unaffected by ryanodine and intracellular application of an anti-ryanodine receptor antibody. mRNAs encoding for the three subtypes of Ins(1,4,5)P3 receptor and subtype 3 of ryanodine receptor were detected in these myocytes, and the maximal binding capacity of [3H]Ins(1,4,5)P3 was 10- to 12-fold higher than that of [3H]ryanodine. These results suggest that Ins(1,4,5)P3-gated channels mediate a continuum of Ca2+ signalling in smooth-muscle cells expressing a high level of Ins(1,4,5)P3 receptors and no subtypes 1 and 2 of ryanodine receptors.
Identification and expression of NEU3, a novel human sialidase associated to the plasma membrane
Authors: MONTI Eugenio , BASSI Maria T. , PAPINI Nadia , RIBONI Mirko , MANZONI Marta , VENERANDO Bruno , CROCI Gianluigi , PRETI Augusto , BALLABIO Andrea , TETTAMANTI Guido , BORSANI Giuseppe
Published: August 2015

Several mammalian sialidases have been described so far, suggesting the existence of numerous polypeptides with different tissue distributions, subcellular localizations and substrate specificities. Among these enzymes, plasma-membrane-associated sialidase(s) have a pivotal role in modulating the ganglioside content of the lipid bilayer, suggesting their involvement in the complex mechanisms governing cell-surface biological functions. Here we describe the identification and expression of a human plasma-membrane-associated sialidase, NEU3, isolated starting from an expressed sequence tag (EST) clone. The cDNA for this sialidase encodes a 428-residue protein containing a putative transmembrane helix, a YRIP (single-letter amino acid codes) motif and three Asp boxes characteristic of sialidases. The polypeptide shows high sequence identity (78%) with the membrane-associated sialidase recently purified and cloned from Bos taurus. Northern blot analysis showed a wide pattern of expression of the gene, in both adult and fetal human tissues. Transient expression in COS7 cells permitted the detection of a sialidase activity with high activity towards ganglioside substrates at a pH optimum of 3.8. Immunofluorescence staining of the transfected COS7 cells demonstrated the protein's localization in the plasma membrane.
Evidence that 12-lipoxygenase product 12-hydroxyeicosatetraenoic acid activates p21-activated kinase
Authors: WEN Yeshao , GU Jiali , KNAUS Ulla G. , THOMAS Lisa , GONZALES Noe , NADLER Jerry L.
Published: August 2015

The effect of 12-hydroxyeicosatetraenoic acid (12-HETE), an arachidonic acid metabolite of 12-lipoxygenase, to activate p21Rac/Cdc42-activated kinase (PAK1) was studied in a Chinese hamster ovary fibroblast cell line overexpressing the rat vascular type-1a angiotensin II receptor (CHO-AT1a). 12-HETE (0.1 μM) treatment induced a time-dependent activation of PAK1, with a peak effect at 10 min (335±16% of control; n = 3, P < 0.001). The stimulatory effect of 12-HETE on PAK1 activity was dose-dependent, with the maximal activation at 0.01 μM (350±15% of control; n = 3, P < 0.001). A PAK1 fragment encoding the Cdc42/Rac binding domain (amino acid residues 67-150 of hPAK1 termed PBD), was transfected into CHO-AT1a cells. PBD transfection markedly reduced 12-HETE-induced PAK1 activation. Furthermore, transfection of dominant negative Cdc42 and Rac1 inhibited 12-HETE-induced PAK1, strongly suggesting that Cdc42 and Rac1 are the upstream activators of 12-HETE-induced PAK1 activation. Low concentrations (1.5 μM) of LY294002, a highly specific inhibitor of phosphoinositide 3-kinase (PI-3K), abolished 12-HETE-induced PAK1 activation, suggesting that PI-3K activation is upstream of 12-HETE-induced PAK1 activation. Transfection of dominant negative PAK1 blocked 12-HETE-induced PAK1, cJun N-terminal kinase (JNK1) and extracellular-signal-regulated kinase (ERK) activity, while transfection of constitutively active PAK1 stimulated PAK1, JNK1 and ERK activity, suggesting that PAK1 is an upstream activator of 12-HETE-induced JNK1 and ERK activation in these cells. We conclude that 12-HETE can activate Cdc42, Rac1 and PI-3K, which then participate as upstream signalling molecules for PAK1 and JNK1 activation.
Novel application of S-nitrosoglutathione–Sepharose to identify proteins that are potential targets for S-nitrosoglutathione-induced mixed-disulphide formation
Authors: KLATT Peter , MOLINA Estela PINEDA , PÉREZ-SALA Dolores , LAMAS Santiago
Published: August 2015

Site-specific S-glutathionylation is emerging as a novel mechanism by which S-nitrosoglutathione (GSNO) may modify functionally important protein thiols. Here, we show that GSNO-Sepharose mimicks site-specific S-glutathionylation of the transcription factors c-Jun and p50 by free GSNO in vitro. Both c-Jun and p50 were found to bind to immobilized GSNO through the formation of a mixed disulphide, involving a conserved cysteine residue located in the DNA-binding domains of these transcription factors. Furthermore, we show that c-Jun, p50, glycogen phosphorylase b, glyceraldehyde-3-phosphate dehydrogenase, creatine kinase, glutaredoxin and caspase-3 can be precipitated from a mixture of purified thiol-containing proteins by the formation of a mixed-disulphide bond with GSNO-Sepharose. With few exceptions, protein binding to this matrix correlated well with the susceptibility of the investigated proteins to undergo GSNO- but not diamide-induced mixed-disulphide formation in vitro. Finally, it is shown that covalent GSNO-Sepharose chromatography of HeLa cell nuclear extracts results in the enrichment of proteins which incorporate glutathione in response to GSNO treatment. As suggested by DNA-binding assays, this group of nuclear proteins include the transcription factors activator protein-1, nuclear factor-ĸB and cAMP-response-element-binding protein. In conclusion, we introduce GSNO-Sepharose as a probe for site-specific S-glutathionylation and as a novel and potentially useful tool to isolate and identify proteins which are candidate targets for GSNO-induced mixed-disulphide formation.
Iron oxidation and hydrolysis reactions of a novel ferritin from Listeria innocua
Authors: YANG Xiaoke , CHIANCONE Emilia , STEFANINI Simonetta , ILARI Andrea , CHASTEEN Dennis N.
Published: August 2015

Iron deposition in the unusual 12-subunit ferritin from the bacterium Listeria innocua proceeds in three phases: a rapid first phase in which Fe2+ binds to the apoprotein, PZ of charge Z, according to the postulated reaction 2Fe2++PZ → [Fe2–P]Z+2+2H+, where [Fe2–P]Z+2 represents a dinuclear iron(II) complex formed at each of the 12 ferroxidase centres of the protein; a second phase corresponding to oxidation of this putative complex, i.e. [Fe2–P]Z+2+½O2 → [Fe2O–P]Z+2H+; and a third phase of iron(II) oxidation/mineralization, i.e. 4Fe2++O2+8H2O → 8FeOOH(s)+8H+ [where FeOOH(s) represents the hydrous ferric oxide mineral that precipitates from the solution], which occurs when iron is added in excess of 24Fe2+/protein. In contrast with other ferritins, the ferroxidation reaction in L. innocua ferritin proceeds more slowly than the oxidation/mineralization reaction. Water is the final product of dioxygen reduction in the 12-subunit L. innocua ferritin (the present work) and in the 24-subunit Escherichia coli bacterioferritin, whereas H2O2 is produced in 24-subunit mammalian ferritins. Possible reasons for this difference are discussed.
Structure–function studies on Hsp47: pH-dependent inhibition of collagen fibril formation in vitro
Authors: THOMSON Christy A. , ANANTHANARAYANAN Vettai S.
Published: August 2015

Hsp47, a 47kDa heat shock protein whose expression level parallels that of collagen, has been regarded as a collagen-specific molecular chaperone. Studies from other laboratories have established the association of Hsp47 with the nascent as well as the triple-helical procollagen molecule in the endoplasmic reticulum and its dissociation from procollagen in the Golgi. One of several roles suggested for Hsp47 in collagen biosynthesis is the prevention of aggregation of procollagen in the endoplasmic reticulum. However, no experimental evidence has been available to verify this suggestion. In the present study we have followed the aggregation of mature triple-helical collagen molecules into fibrils by using turbidimetric measurements in the absence and presence of Hsp47. In the pH range 6–7, fibril formation of type I collagen, as monitored by turbidimetry, proceeds with a lag of approx. 10min and levels off by approx. 60min. The addition of Hsp47 at pH 7 effectively inhibits fibril formation at and above a 1:1 molar ratio of Hsp47 to triple-helical collagen. This inhibition is markedly pH-dependent, being significantly diminished at pH 6. CD and fluorescence spectral data of Hsp47 in the pH range 4.2–7.4 reveal a significant alteration in its structure at pH values below 6.2, with a decrease in α-helix and an increase in β-structure. This conformational change is likely to be the basis of the decreased binding of Hsp47 to collagen in vitro at pH 6.3 as well as its inability to inhibit collagen fibril formation at this pH. Our results also provide a functional assay for Hsp47 that can be used in studies on collagen and Hsp47 interactions.
Identification of amino acid residues important in the cyclization reactions of chalcone and stilbene synthases
Authors: SUH Dae-Yeon , FUKUMA Kazuki , KAGAMI Junichi , YAMAZAKI Yasuyo , SHIBUYA Masaaki , EBIZUKA Yutaka , SANKAWA Ushio
Published: August 2015

Chalcone synthase (CHS) and stilbene synthase (STS) catalyse condensation reactions of p-coumaroyl-CoA and three C2 units from malonyl-CoA up to a common tetraketide intermediate but then catalyse different cyclization reactions to produce naringenin chalcone and resveratrol respectively. On the basis of sequence alignment with other condensing enzymes including 3-ketoacyl-(acyl carrier protein) synthases of polyketide and fatty-acid synthases, site-directed mutagenesis was performed on the active-site G372FGPG loops in CHS and STS. The CHS-P375G mutant showed a 6-fold decrease in overall condensing activity with selectively increased production of p-coumaroyltriacetic acid lactone (CTAL, the derailment product of the tetraketide intermediate). Meanwhile, resveratrol production by STS-P375G strongly decreased to give various products in the order CTAL > resveratrol≈ bisnoryangonin > naringenin. As a result, naringenin production (cross-reaction) by STS-P375G was close to 30% of resveratrol production. Both G374L mutants of CHS and STS showed no condensing activity with residual malonyl-CoA decarboxylase activity. These results suggested that the G372FGPG loop in CHS and STS contribute to a determination of the outcome during cyclization reactions by serving as a part of the active-site scaffold on which the stereochemistry of cyclization is performed. These observations provide the first biochemical indication that cyclization reactions are modulated by active-site geometry. The implications for the evolutionary relationship of these enzymes are also discussed.
Insulin effects on sterol regulatory-element-binding protein-1c (SREBP-1c) transcriptional activity in rat hepatocytes
Authors: AZZOUT-MARNICHE Dalila , BÉCARD Dominique , GUICHARD Colette , FORETZ Marc , FERRÉ Pascal , FOUFELLE Fabienne
Published: August 2015

The transcription factor sterol regulatory-element-binding protein-1c (SREBP-1c) plays a major role in the effect of insulin on the transcription of hepatic genes such as glucokinase and fatty acid synthase. We show here in cultured rat hepatocytes that insulin, through activation of the phosphatidylinositol 3-kinase pathway increases the abundance of the precursor form of SREBP-1c in endoplasmic reticulum. This precursor form is then rapidly cleaved, possibly irrespective of the continuous presence of insulin, leading to an increased content of the nuclear mature form of SREBP-1c. Nevertheless, the increased amount of the mature form of SREBP-1c in the nucleus is not a prerequisite for the rapid effect of insulin on the transcription of genes such as glucokinase, suggesting that additional actions of the hormone are involved, such as the activation of the nuclear form of SREBP-1c or of an unidentified SREBP-1c partner.
Activity and genomic organization of human glucose transporter 9 (GLUT9), a novel member of the family of sugar-transport facilitators predominantly expressed in brain and leucocytes
Authors: DOEGE Holger , BOCIANSKI Andreas , JOOST Hans-Georg , SCHÜRMANN Annette
Published: August 2015

The GLUT9 gene encodes a cDNA which exhibits significant sequence similarity with members of the glucose transporter (GLUT) family. The gene is located on chromosome 9q34 and consists of 10 exons separated by short introns. The amino acid sequence deduced from its cDNA predicts 12 putative membrane-spanning helices and all the motifs (sugar-transporter signatures) that have previously been shown to be essential for transport activity. A striking characteristic of GLUT9 is the presence of two arginines in the putative helices 7 and 8 at positions where the organic anion transporters harbour basic residues. The next relative of GLUT9 is the glucose transporter GLUT8/GLUTX1 (44.8% amino acid identity with GLUT9). A 2.6-kb transcript of GLUT9 was detected in spleen, peripheral leucocytes and brain. Transfection of COS-7 cells with GLUT9 produced expression of a 46-kDa membrane protein which exhibited reconstitutable glucose-transport activity and low-affinity cytochalasin-B binding. It is concluded that GLUT9 is a novel member of the family of sugar-transport facilitators with a tissue-specific function.
Role of Pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase
Authors: TALWAR Rashmi , LEELAVATHY Vijayapandian , KRISHNA RAO Jala V. , APPAJI RAO Naropantul , SAVITHRI Handanahal S.
Published: August 2015

Serine hydroxymethyltransferase belongs to the α class of pyridoxal-5´-phosphate enzymes along with aspartate aminotransferase. Recent reports on the three-dimensional structure of human liver cytosolic serine hydroxymethyltransferase had suggested a high degree of similarity between the active-site geometries of the two enzymes. A comparison of the sequences of serine hydroxymethyltransferases revealed the presence of several highly conserved residues, including Pro-297. This residue is equivalent to residue Arg-292 of aspartate aminotransferase, which binds the γ-carboxy group of aspartate. In an attempt to change the reaction specificity of the hydroxymethyltransferase to that of an aminotransferase and to assign a possible reason for the conserved nature of Pro-297, it was mutated to Arg. The mutation decreased the hydroxymethyltransferase activity significantly (by 85–90%) and abolished the ability to catalyse alternative reactions, without alteration in the oligomeric structure, pyridoxal 5´-phosphate content or substrate binding. However, the concentration of the quinonoid intermediate and the extent of proton exchange was decreased considerably (by approx. 85%) corresponding to the decrease in catalytic activity. Interestingly, mutant Pro-297 Arg was unable to perform the transamination reaction with l-aspartate. All these results suggest that although Pro-297 is indirectly involved in catalysis, it might not have any role in imparting substrate specificity, unlike the similarly positioned Arg-292 in aspartate aminotransferase.
Bradykinin activates the Janus-activated kinase/signal transducers and activators of transcription (JAK/STAT) pathway in vascular endothelial cells: localization of JAK/STAT signalling proteins in plasmalemmal caveolae
Authors: JU Hong , VENEMA Virginia J. , LIANG Haiying , HARRIS M. Brennan , ZOU Rong , VENEMA Richard C.
Published: August 2015

Bradykinin (BK) is an important physiological regulator of endothelial cell function. In the present study, we have examined the role of the Janus-activated kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in endothelial signal transduction through the BK B2 receptor (B2R). In cultured bovine aortic endothelial cells (BAECs), BK activates Tyk2 of the JAK family of tyrosine kinases. Activation results in the tyrosine phosphorylation and subsequent nuclear translocation of STAT3. BK also activates the mitogen-activated p44 and p42 protein kinases, resulting in STAT3 serine phosphorylation. Furthermore, Tyk2 and STAT3 form a complex with the B2R in response to BK stimulation. Under basal conditions, Tyk2, STAT3 and the B2R are localized either partially or entirely in endothelial plasmalemmal caveolae. Following BK stimulation of BAECs, however, the B2R and STAT3 are translocated out of caveolae. Taken together, these data suggest that BK activates the JAK/STAT pathway in endothelial cells and that JAK/STAT signalling proteins are localized in endothelial caveolae. Moreover, caveolar localization of the B2R and STAT3 appears to be regulated in an agonist-dependent manner.
Nitric oxide inhibits isoproterenol-stimulated adipocyte lipolysis through oxidative inactivation of the β-agonist
Authors: KLATT Peter , CACHO Judith , CRESPO M. Dolores , HERRERA Emilio , RAMOS Pilar
Published: August 2015

Nitric oxide has been implicated in the inhibition of catecholamine-stimulated lipolysis in adipose tissue by as yet unknown mechanisms. In the present study, it is shown that the nitric oxide donor, 2,2-diethyl-1-nitroso-oxyhydrazine, antagonized isoproterenol (isoprenaline)-induced lipolysis in rat adipocytes, freshly isolated from white adipose tissue, by decreasing the potency of the β-agonist without affecting its efficacy. These data suggest that nitric oxide did not act downstream of the β-adrenoceptor but reduced the effective concentration of isoproterenol. In support of the latter hypothesis, we found that pre-treatment of isoproterenol with nitric oxide abolished the lipolytic activity of the catecholamine. Spectroscopic data and HPLC analysis confirmed that the nitric oxide-mediated inactivation of isoproterenol was in fact because of the modification of the catecholamine through a sequence of oxidation reactions, which apparently involved the generation of an aminochrome. Similarly, aminochrome was found to be the primary product of isoproterenol oxidation by 3-morpholinosydnonimine and peroxynitrite. Finally, it was shown that nitric oxide released from cytokine-stimulated adipocytes attenuated the lipolytic effect of isoproterenol by inactivating the catecholamine. In contrast with very recent findings, which suggest that nitric oxide impairs the β-adrenergic action of isoproterenol through intracellular mechanisms and not through a chemical reaction between NO and the catecholamine, we showed that nitric oxide was able to attenuate the pharmacological activity of isoproterenol in vitro as well as in a nitric oxide-generating cellular system through oxidation of the β-agonist. These findings should be taken into account in both the design and interpretation of studies used to investigate the role of nitric oxide as a modulator of isoproterenol-stimulated signal transduction pathways.
Identification of multiple proteins expressed in murine embryos as binding partners for the WW domains of the ubiquitin-protein ligase Nedd4
Authors: JOLLIFFE Corina N. , HARVEY Kieran F. , HAINES Bryan P. , PARASIVAM Gayathri , KUMAR Sharad
Published: August 2015

Nedd4 is a member of a growing family of ubiquitin-protein ligases which consist of a lipid-binding domain, two to four WW domains and a C-terminal ubiquitin-protein ligase domain. The Nedd4 mRNA levels are developmentally regulated and Nedd4 protein is highly expressed in many mouse embryonic tissues. In this study we have used a far-Western screen to identify embryonic proteins that interact with the WW domains in mouse Nedd4. We report here identification of eight Nedd4 WW-domain-interacting proteins from mouse embryonic cDNA expression libraries. Two of the proteins are novel, while two have been identified previously as ligands for a WW domain. All of these proteins contain one or more PY motifs. In seven of the eight proteins, these PY motifs are necessary for their interaction with the WW domains of Nedd4. Using site-directed mutagenesis, and by using individual WW domains of Nedd4 as probes for far-Western analysis, we show that the three WW domains in Nedd4 interact with varying affinities with the PY motifs present in various Nedd4-binding proteins. These results provide evidence that Nedd4 can potentially interact with multiple proteins, possibly simultaneously, through its WW domains.
Aberrant phosphorylation of dentatorubral-pallidoluysian atrophy (DRPLA) protein complex in brain tissue
Authors: YAZAWA Ikuru
Published: August 2015

Dentatorubral-pallidoluysian atrophy (DRPLA) is caused by DRPLA protein which carries expansion of a glutamine repeat. Abnormal high-molecular-mass complex formation by DRPLA protein and its pathological ubiquitination comprise the disease processes in the brains of patients with DRPLA. In this study, DRPLA protein complex was isolated and shown to have pathologically stronger bond formation with DRPLA proteins in DRPLA brain tissue compared with control brain tissue. Immunochemical methods and an enzymic dephosphorylation technique were used to demonstrate that DRPLA protein complex is aberrantly phosphorylated in DRPLA brain tissue. Immunohistochemical studies show that both the ubiquitinated cytoplasmic inclusions and the nuclear membrane are aberrantly phosphorylated in DRPLA-affected neurons. This finding suggests that the nuclear membrane is another pathological focus of DRPLA neurodegeneration.
Activation of protein kinase B/cAkt in hepatocytes is sufficient for the induction of expression of the gene encoding glucokinase
Authors: IYNEDJIAN Patrick B. , ROTH Richard A. , FLEISCHMANN Mark , GJINOVCI Asllan
Published: August 2015

Inhibitors of signalling pathways were used to dissect the mechanism of insulin action on expression of the gene encoding glucokinase in cultured rat hepatocytes. Wortmannin and LY 294002 completely prevented the insulin-induced increase in glucokinase mRNA seen in unhibited cells, indicating that the phosphoinositide 3-kinase module has a key role. A ligand inducible protein kinase B (PKB, also termed cAkt) fusion protein was expressed by using adenoviral transduction of hepatocytes in primary culture. The PKB activity of this protein was shown to be activated in transduced hepatocytes within 30min of the addition of 4-hydroxytamoxifen and to stay high for 8h, as a result of serine phosphorylation at position 473 of PKB. The increase in PKB activity was reflected in the hyperphosphorylation of phosphorylated, heat and acid stable regulated by insulin protein (PHAS-I; also termed 4E-BP1, for eukaryotic initiation factor 4E-binding protein 1), a protein involved in the regulation of translation initiation. These effects were comparable to the insulin-induced activation of endogenous PKB and phosphorylation of PHAS-I in non-transduced hepatocytes. The addition of tamoxifen to transduced hepatocytes resulted in an induction of glucokinase mRNA with kinetics and magnitude similar to those of insulin-induced mRNA accumulation. The effect of tamoxifen depended on stimulated PKB activity because it did not occur in hepatocytes that were transduced with a mutant PKB fusion protein that was refractory to activation with tamoxifen. These results establish that acute activation of PKB is sufficient to produce an insulin-like induction of glucokinase in isolated hepatocytes. Together with the inhibition by phosphoinositide 3-kinase inhibitors, they suggest that the activation of PKB might be critical in mediating the induction of glucokinase by insulin. In addition, experiments showed that PD98059 decreased by half the increase in glucokinase mRNA brought about by insulin, suggesting a contributory role of the mitogen-activated protein kinase cascade.
Role of non-covalent enzyme–substrate interactions in the reaction catalysed by cellobiose phosphorylase from Cellulomonas uda
Authors: NIDETZKY Bernd , EIS Christian , ALBERT Martin
Published: August 2015

Steady-state kinetic studies of the enzymic glucosyl transfer to and from phosphate catalysed by cellobiose phosphorylase from Cellulomonas uda have shown that this enzyme operates by a ternary-complex kinetic mechanism in which β-cellobiose binds before phosphate, and β-d-glucose and α-d-glucopyranosyl phosphate are released in that order. α-d-Glucopyranosyl fluoride (but not β-d-glucopyranosyl fluoride) serves as alternative glucosyl donor for β-cellobiose synthesis with a specificity constant that is one-ninth that of the corresponding enzymic reaction with α-d-glucopyranosyl phosphate (≈ 20000 M-1·s-1 at 30°C). The kinetic parameters for a complete series of deoxy and deoxyfluoro analogues of d-glucose have been determined and the data yield estimates of the net strengths of hydrogen-bonding interactions with the non-reacting hydroxy groups of d-glucose at the transition state (0.8–4.0kcal/mol, where 1cal≡4.184J) and enable the prediction of the polarities of these hydrogen bonds. Each hydroxy group functions as donor of a hydrogen for bonding to probably a charged (at 3-OH) or neutral (at 2-OH and 6-OH) acceptor group on the enzyme. The equatorial 1-OH is essential for enzyme activity. Derivatives of d-glucose in which the 1-OH or the reacting 4-OH were replaced by hydrogen or fluorine have been tested as inhibitors to measure their affinities for the sugar-binding subsite +1 (numbered from the bond-cleaving/forming site). The data show that hydrogen-bonding interactions between the 1-OH and 4-OH and charged groups on the enzyme stabilize the ground-state ternary complex of the enzymic synthesis of β-cellobiose by 2.3 and 0.4kcal/mol, respectively, and assist the precise positioning of β-d-glucose for catalysis.
Cloning and expression of the human transient receptor potential 4 (TRP4) gene: localization and functional expression of human TRP4 and TRP3
Authors: McKAY Richard R. , SZYMECZEK-SEAY Caroline L. , LIEVREMONT Jean-Philippe , BIRD Gary St. J. , ZITT Christof , JÜNGLING Eberhard , LÜCKHOFF Andreas , PUTNEY James W.
Published: August 2015

Mammalian homologues of the Drosophila transient receptor potential (TRP) protein have been proposed to function as ion channels, and in some cases as store-operated or capacitative calcium entry channels. However, for each of the mammalian TRP proteins, different laboratories have reported distinct modes of cellular regulation. In the present study we describe the cloning and functional expression of the human form of TRP4 (hTRP4), and compare its activity with another well studied protein, hTRP3. When hTRP4 was transiently expressed in human embryonic kidney (HEK)-293 cells, basal bivalent cation permeability (barium) was increased. Whole-cell patch-clamp studies of hTRP4 expressed in Chinese hamster ovary cells revealed a constitutively active non-selective cation current which probably underlies the increased bivalent cation entry. Barium entry into hTRP4-transfected HEK-293 cells was not further increased by phospholipase C (PLC)-linked receptor activation, by intracellular calcium store depletion with thapsigargin, or by a synthetic diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In contrast, transient expression of hTRP3 resulted in a bivalent cation influx that was markedly increased by PLC-linked receptor activation and by OAG, but not by thapsigargin. Despite the apparent differences in regulation of these two putative channel proteins, green fluorescent protein fusions of both molecules localized similarly to the plasma-membrane, notably in discrete punctate regions suggestive of specialized signalling complexes. Our findings indicate that while both hTRP4 and hTRP3 can apparently function as cation channels, their putative roles as components of capacitative calcium entry channels are not readily demonstrable by examining their behaviour when exogenously expressed in cells.
Histone carbonylation in vivo and in vitro
Authors: WONDRAK Georg T. , CERVANTES-LAUREAN Daniel , JACOBSON Elaine L. , JACOBSON Myron K.
Published: August 2015

Non-enzymic damage to nuclear proteins has potentially severe consequences for the maintenance of genomic integrity. Introduction of carbonyl groups into histones in vivo and in vitro was assessed by Western blot immunoassay and reductive incorporation of tritium from radiolabelled NaBH4 (sodium borohydride). Histone H1 extracted from bovine thymus, liver and spleen was found to contain significantly elevated amounts of protein-bound carbonyl groups as compared with core histones. The carbonyl content of nuclear proteins of rat pheochromocytoma cells (PC12 cells) was not greatly increased following oxidative stress induced by H2O2, but was significantly increased following alkylating stress induced by N-methyl-N´-nitro-N-nitrosoguanidine or by combined oxidative and alkylating stress. Free ADP-ribose, a reducing sugar generated in the nucleus in proportion to DNA strand breaks, was shown to be a potent histone H1 carbonylating agent in isolated PC12 cell nuclei. Studies of the mechanism of histone H1 modification by ADP-ribose indicate that carbonylation involves formation of a stable acyclic ketoamine. Our results demonstrate preferential histone H1 carbonylation in vivo, with potentially important consequences for chromatin structure and function.
Calcineurin regulates ryanodine receptor/Ca2+-release channels in rat heart
Authors: BANDYOPADHYAY Arun , SHIN Dong Wook , AHN Jung On , KIM Do Han
Published: August 2015

The present study was undertaken to examine the physical and physiological interaction of protein phosphatase 2B, calcineurin, with the ryanodine receptor (RyR) in rat cardiac tissue and neonatal cardiomyocytes. The presence of calcineurin, the RyR and FK506-binding protein (FKBP)12.6 in rat cardiac sarcoplasmic reticulum (SR) was identified by Western blot analysis. The possible interactions between calcineurin, the RyR and FKBP12.6 were further studied by co-immunoprecipitation using CHAPS-solubilized cardiac-membrane fractions (CSMFs) or SR preparations. Physical interactions between the RyR and calcineurin were found in the CSMF in the presence of added 100µM Ca2+; however, the interactions were interrupted in the presence of 20mM EGTA, 1µM rapamycin or 1µM FK506, suggesting that the interaction is Ca2+-dependent, and is mediated by FKBP12.6. The Ca2+-dependent interaction between FKBP12.6 and the RyR was also found by co-immunoprecipitation. Effects of calcineurin inhibitors were tested on neonatal-rat-heart cardiomyocytes. Treatment of neonatal cardiomyocytes with 20µM deltamethrin, 10µM cyclosporin A (CsA), or 10µM FK506 led to Ca2+ oscillations in originally quiescent cardiomyocytes. Preincubation of cardiomyocytes with 20µM rapamycin which dissociates FKBP12.6 from the RyR, evoked Ca2+ oscillations, probably due to the leakiness of the RyR. However, Ca2+ oscillations by rapamycin were not further affected by 10µM CsA or 10µM deltamethrin, suggesting that only RyR-associated calcineurin could regulate the channel activities. In spontaneously Ca2+-oscillating cardiomyocytes, CsA or FK506 treatments increased the frequency of oscillations. In 10µM ryanodine-treated cardiomyocytes, CsA failed to induce Ca2+ oscillations. These data show evidence that calcineurin associated with the RyR could modulate Ca2+ release in rat heart.
3′-Untranslated regions of oxidative phosphorylation mRNAs function in vivo as enhancers of translation
Authors: LIEGRO Carlo M. Di , BELLAFIORE Marianna , IZQUIERDO José M. , RANTANEN Anja , CUEZVA José M.
Published: August 2015

Recent findings have indicated that the 3´-untranslated region (3´-UTR) of the mRNA encoding the β-catalytic subunit of the mitochondrial H+-ATP synthase has an in vitro translation-enhancing activity (TEA) [Izquierdo and Cuezva, Mol. Cell. Biol. (1997) 17, 5255–5268; Izquierdo and Cuezva, Biochem. J. (2000) 346, 849–855]. In the present work, we have expressed chimaeric plasmids that encode mRNA variants of green fluorescent protein in normal rat kidney and liver clone 9 cells to determine whether the 3´-UTRs of nuclear-encoded mRNAs involved in the biogenesis of mitochondria have an intrinsic TEA. TEA is found in the 3´-UTR of the mRNAs encoding the α- and β-subunits of the rat H+-ATP synthase complex, as well as in subunit IV of cytochrome c oxidase. No TEA is present in the 3´-UTR of the somatic mRNA encoding rat mitochondrial transcription factor A. Interestingly, the TEA of the 3´-UTR of mRNAs of oxidative phosphorylation is different, depending upon the cell type analysed. These data provide the first in vivo evidence of a novel cell-specific mechanism for the control of the translation of mRNAs required in mitochondrial function.
Charge reversal of ammodytoxin A, a phospholipase A2-toxin, does not abolish its neurotoxicity
Authors: PRIJATELJ Petra , ČOPIČ Alenka , KRIŽAJ Igor , GUBENŠEK Franc , PUNGERČAR Jože
Published: August 2015

The positive charge concentrated at the C-terminal region of ammodytoxin (Atx) A, which is involved in presynaptic toxicity, has been reversed. A six-site mutant of AtxA (K108N/K111N/K127T/K128E/E129T/K132E, where K108N = Lys108 → Asn etc.) was prepared, in which five out of seven C-terminal basic amino acid residues were substituted with neutral or acidic ones. The mutant was approximately 30-fold less lethal, but still neurotoxic. Consistent with this, its binding affinity for the neuronal receptors decreased by only a factor of five. Additionally, a single-site mutant of AtxA was prepared, with substitution at only one position (K127T) out of six mutated in the six-site mutant. Its toxicity indicated that most, if not all, of the six mutated residues partially contribute to the decreased lethality of the multiple-site mutant.
All natural DR3-type vitamin D response elements show a similar functionality in vitro
Authors: TOELL Andrea , POLLY Patsie , CARLBERG Carsten
Published: August 2015

The vitamin D3 receptor (VDR), which is the nuclear receptor for 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], acts primarily as a heterodimer with the retinoid X receptor (RXR) and binds preferentially to directly repeated arrangements of two hexameric binding sites with three spacing nucleotides [DR3-type vitamin D response elements (VDREs)]. In this study, all presently known natural DR3-type VDREs have been compared and classified on the basis of their complex-formation with VDR–RXR heterodimers and their ability to stabilize VDR–RXR heterodimer conformations. Based on the affinity of each VDRE for VDR–RXR heterodimers, the DR3-type VDREs were divided into three classes. The ligand sensitivity of this complex-formation and conformational stabilization was determined to be in the range of 0.1nM. No significant differences in the 1α,25(OH)2D3-modulated interactions of the DR3-type VDRE-complexed VDR–RXR heterodimer with the co-activator SRC-1 (steroid receptor co-activator-1) or the co-repressor NCoR (nuclear receptor co-repressor) were found. Taken together, the affinity for VDR–RXR heterodimers appears to be the major discriminating parameter between natural DR3-type VDREs. This will not only facilitate further investigation of the principles of DR3-type-VDRE-mediated gene regulation, but also strongly suggests that DR3-type VDREs alone cannot explain the pleiotropic genomic action of 1α,25(OH)2D3.