bluuebunny/crossref_metadata_2025 huggingface dataset:
Inactivation of creatine kinase by S-glutathionylation of the active-site cysteine residue
Authors: REDDY Sharanya , JONES A. Daniel , CROSS Carroll E. , WONG Patrick S.-Y. , VAN DER VLIET Albert
Published: August 2015

Protein S-thiolation, the formation of mixed disulphides of cysteine residues in proteins with low-molecular-mass thiols, occurs under conditions associated with oxidative stress and can lead to modification of protein function. In the present study, we examined the site of S-thiolation of the enzyme creatine kinase (CK), an important source of ATP in myocytes. Inactivation of this enzyme is thought to play a critical role in cardiac injury during oxidative stress, such as during reperfusion injury. Reaction of rabbit CK M isoenzyme with GSSG, used to model protein S-thiolation, was found to result in enzyme inactivation that could be reversed by GSH or dithiothreitol. Measurement of GSH that is released during the thiolation reaction indicated that the maximum extent of CK thiolation was approx. 1 mol of GSH/mol of protein, suggesting thiolation on one reactive cysteine residue. Accordingly, matrix-assisted laser-desorption ionization MS confirmed that the molecular mass of CK was increased, consistent with addition of one GSH molecule/molecule of CK. Using trypsin digestion, HPLC and MS analysis, the active-site cysteine residue (Cys283) was identified as the site of thiolation. Reversal of thiolation was shown to be rapid when GSH is abundant, rendering dethiolation of CK thermodynamically favoured within the cell. We conclude that S-glutathionylation of CK could be one mechanism to explain temporary reversible loss in activity of CK during ischaemic injury. The maintainance of GSH levels represents an important mechanism for regeneration of active CK from S-glutathionylated CK.
Identification of an involucrin promoter transcriptional response element with activity restricted to keratinocytes
Authors: PHILLIPS Marjorie A. , QIN Qin , RICE Robert H.
Published: August 2015

The involucrin proximal promoter was examined for response elements that confer cell-type specificity. Using a segment spanning positions -157 to +41, three possible response elements were identified by their protein-binding activity using DNase I footprinting. From distal to proximal, they were: an activator protein-1 (AP-1) site (previously identified) overlapping an Ets-like site; a second Ets-like site located 13 bp more proximally; and an extended region designated footprinted site A (FPA). Mutation of the distal Ets-like site had essentially no effect on the transcriptional activity in transfections, while mutation of the proximal site reduced the activity by half. FPA was shown by electrophoretic mobility-shift assay (EMSA) to be comprised of two separable binding sites, FPA1 (distal) and FPA2 (proximal). While mutation of FPA2 had only a modest effect on transcriptional activity in transient transfections, mutation of FPA1 reduced transcriptional activity to approx. 20% of that obtained with the intact promoter. Additional mutations of FPA1 indicated that the active region comprises positions -85 to -73 (GTGGTGAAACCTGT). The molecular masses of the major proteins binding to this site were shown by UV cross-linking to be approx. 40 and 50 kDa, while minor bands were observed at 80 and 110 kDa. Since the involucrin promoter exhibits much higher transcriptional activity in keratinocytes than in other cell types in transfection assays (indicating that cell type specificity of expression is retained), the comparative influence of FPA1 was examined. While mutation of the AP-1 site affected transcriptional activity similarly in all cell lines tested, mutation of FPA1 decreased activity substantially in keratinocytes, but not in NIH-3T3 and HeLa cells, evidence for a contribution to cell-type specificity of expression. Furthermore, a correlation between the sensitivity to FPA1 mutation and amount of involucrin expression in different keratinocyte cell lines was evident. EMSA showed that NIH-3T3 and HeLa cells lacked the same FPA1 DNA-protein complex as keratinocytes. However, the amount of complex formed with nuclear extracts from several keratinocyte lines did not correlate well with the level of involucrin expression. Other factors, such as differences in post-translational modification or co-activators, must account for varied transcriptional response mediated by this site among keratinocyte lines.
Autophosphorylation of Tyr397 and its phosphorylation by Src-family kinases are altered in focal-adhesion-kinase neuronal isoforms
Authors: TOUTANT Madeleine , STUDLER Jeanne-Marie , BURGAYA Ferran , COSTA Alicia , EZAN Pascal , GELMAN Michèle , GIRAULT Jean-Antoine
Published: August 2015

In brain, focal adhesion kinase (FAK) is regulated by neurotransmitters and has a higher molecular mass than in other tissues, due to alternative splicing. Two exons code for additional peptides of six and seven residues (‘boxes’ 6 and 7), located on either side of Tyr397, which increase its autophosphorylation. Using in situ hybridization and a monoclonal antibody (Mab77) which does not recognize FAK containing box 7, we show that, although mRNAs coding for boxes 6 and 7 have different patterns of expression in brain, FAK+6,7 is the main isoform in forebrain neurons. The various FAK isoforms fused to green fluorescent protein were all targeted to focal adhesions in non-neuronal cells. Phosphorylation-state-specific antibodies were used to study in detail the phosphorylation of Tyr397, a critical residue for the activation and function of FAK. The presence of boxes 6 and 7 increased autophosphorylation of Tyr397 independently and additively, whereas they had a weak effect on FAK kinase activity towards poly(Glu,Tyr). Src-family kinases were also able to phosphorylate Tyr397 in cells, but this phosphorylation was decreased in the presence of box 6 or 7, and abolished in the presence of both. Thus the additional exons characteristic of neuronal isoforms of FAK do not alter its targeting, but change dramatically the phosphorylation of Tyr397. They increase its autophosphorylation in vitro and in transfected COS-7 cells, whereas they prevent its phosphorylation when co-transfected with Src-family kinases.
Fractionation and characterization of oligomeric, protofibrillar and fibrillar forms of β-amyloid peptide
Authors: WARD Robin V. , JENNINGS Kevin H. , JEPRAS Robert , NEVILLE William , OWEN Davina E. , HAWKINS Julie , CHRISTIE Gary , DAVIS John B. , GEORGE Ashley , KARRAN Eric H. , HOWLETT David R.
Published: August 2015

The β-amyloid (Aβ) peptide, a major component of senile plaques in Alzheimer's disease brain, has been shown previously to undergo a process of polymerization to produce neurotoxic forms of amyloid. Recent literature has attempted to define precisely the form of Aβ responsible for its neurodegenerative properties. In the present study we describe a novel density-gradient centrifugation method for the isolation and characterization of structurally distinct polymerized forms of Aβ peptide. Fractions containing protofibrils, fibrils, sheet structures and low molecular mass oligomers were prepared. The fractionated forms of Aβ were characterized structurally by transmission electron microscopy. The effects on cell viability of these fractions was determined in the B12 neuronal cell line and hippocampal neurons. Marked effects on cell viability in the cells were found to correspond to the presence of protofibrillar and fibrillar structures, but not to monomeric peptide or sheet-like structures of polymerized Aβ. Biological activity correlated with a positive reaction in an immunoassay that specifically detects protofibrillar and fibrillar Aβ; those fractions that were immunoassay negative had no effect on cell viability. These data suggest that the effect of Aβ on cell viability is not confined to a single conformational form but that both fibrillar and protofibrillar species have the potential to be active in this assay.
Heterogeneous nuclear ribonucleoprotein D0 contains transactivator and DNA-binding domains
Authors: TOLNAY Mate , BARANYI Lajos , TSOKOS George C.
Published: August 2015

Heterogeneous nuclear ribonucleoprotein D0 (hnRNP D0) is an abundant, ubiquitous protein that binds RNA and DNA sequences specifically, and has been implicated in the transcriptional regulation of the human complement receptor 2 gene. We found that in vivo expression of hnRNP D0-GAL4 fusion proteins increased the transcriptional activity of a GAL4-driven reporter gene, providing direct proof that hnRNP D0 possesses a transactivator domain. We found, using truncated hnRNP D0 proteins fused to GAL4, that 29 amino acids in the N-terminal region are critical for transactivation. We established, using a series of recombinant truncated hnRNP D0 proteins, that the tandem RNA-binding domains alone were not able to bind double-stranded DNA. Nevertheless, 24 additional amino acids of the C-terminus imparted sequence-specific DNA binding. Experiments using peptide-specific antisera supported the importance of the 24-amino-acid region in DNA binding, and suggested the involvement of the 19-amino-acid alternative insert which is present in isoforms B and D. The N-terminus had an inhibitory effect on binding of hnRNP D0 to single-stranded, but not to double-stranded, DNA. Although both recombinant hnRNP D0B and D0D bound DNA, only the B isoform recognized DNA in vivo. We propose that the B isoform of hnRNP D0 functions in the nucleus as a DNA-binding transactivator and has distinct transactivator and DNA-binding domains.
Effects of magnesium and nucleotides on the proton conductance of rat skeletal-muscle mitochondria
Authors: CADENAS Susana , BRAND Martin D.
Published: August 2015

During oxidative phosphorylation most of the protons pumped out to the cytosol across the mitochondrial inner membrane return to the matrix through the ATP synthase, driving ATP synthesis. However, some of them leak back to the matrix through a proton-conductance pathway in the membrane. When the ATP synthase is inhibited with oligomycin and ATP is not being synthesized, all of the respiration is used to drive the proton leak. We report here that Mg2+ inhibits the proton conductance in rat skeletal-muscle mitochondria. Addition of Mg2+ inhibited both oligomycin-inhibited respiration and the proton conductance, while removal of Mg2+ using EDTA activated these processes. The proton conductance was inhibited by more than 80% as free Mg2+ was raised from 25 nM to 220 μM. Half-maximal inhibition occurred at about 1 μM free Mg2+, which is close to the contaminating free Mg2+ concentration in our incubations in the absence of added magnesium chelators. ATP, GTP, CTP, TTP or UTP at a concentration of 1 mM increased the oligomycin-inhibited respiration rate by about 50%. However, these NTP effects were abolished by addition of 2 mM Mg2+ and any NTP-stimulated proton conductance was explained completely by chelation of endogenous free Mg2+. The corresponding nucleoside diphosphates (ADP, GDP, CDP, TDP or UDP) at 1 mM had no effect on oligomycin-inhibited respiration. We conclude that proton conductance in rat skeletal-muscle mitochondria is very sensitive to free Mg2+ concentration but is insensitive to NTPs or NDPs at 1 mM.
Human Sug1/p45 is involved in the proteasome-dependent degradation of Sp1
Authors: SU Kaihong , YANG Xiaoyong , ROOS Mark D. , PATERSON Andrew J. , KUDLOW Jeffrey E.
Published: August 2015

The transcription factor Sp1 was previously shown to undergo proteasome-dependent degradation when cells were glucose-starved and stimulated with the adenylate cyclase inducer, forskolin. However, the control of the Sp1 degradation process is largely unknown. Using in vitro and in vivo interaction studies, we show in the present study that Sp1 interacts with human Sug1 [hSug1, also known as p45 or thyroid-hormone-receptor interacting protein (‘TRIP1’)], an ATPase subunit of the 26 S proteasome and a putative transcriptional modulator. This interaction with Sp1 occurs through the C-terminus of hSug1, the region that contains the conserved ATPase domain in this protein. Both in vitro studies, in reconstituted degradation assays, and in vivo experiments, in which hSug1 is overexpressed in normal rat kidney cells, show that full-length hSug1 is able to stimulate the proteasome-dependent degradation of Sp1. However, hSug1 truncations that lack either the N- or C-terminal domain of hSug1 act as dominant negatives, inhibiting Sp1 degradation in vitro. Also, an ATPase mutant of hSug1, while still able to bind Sp1, acts as a dominant negative, blocking Sp1 degradation both in vitro and in vivo. These results demonstrate that hSug1 is involved in the degradation of Sp1 and that ATP hydrolysis by hSug1 is necessary for this process. Our findings indicate that hSug1 is an exchangeable proteasomal component that plays a critical regulatory role in the proteasome-dependent degradation of Sp1. However, hSug1 is not the factor limiting Sp1 degradation in the cells treated with glucosamine. This and other considerations suggest that hSug1 co-operation with other molecules is necessary to target Sp1 for proteasome degradation.
Oxalomalate, a competitive inhibitor of aconitase, modulates the RNA-binding activity of iron-regulatory proteins
Authors: FESTA Michela , COLONNA Alfredo , PIETROPAOLO Concetta , RUFFO Alfredo
Published: August 2015

We investigated the effect of oxalomalate (OMA, α-hydroxy-β-oxalosuccinic acid), a competitive inhibitor of aconitase, on the RNA-binding activity of the iron-regulatory proteins (IRP1 and IRP2) that control the post-transcriptional expression of various proteins involved in iron metabolism. The RNA-binding activity of IRP was evaluated by electrophoretic mobility-shift assay of cell lysates from 3T3-L1 mouse fibroblasts, SH-SY5Y human cells and mouse livers incubated in vitro with OMA, with and without 2-mercaptoethanol (2-ME). Analogous experiments were performed in vivo by prolonged incubation (72 h) of 3T3-L1 cells with OMA, and by injecting young mice with equimolar concentrations of oxaloacetate and glyoxylate, which are the precursors of OMA synthesis. OMA remarkably decreased the binding activity of IRP1 and, when present, of IRP2, in all samples analysed. In addition, the recovery of IRP1 by 2-ME in the presence of OMA was constantly lower versus control values. These findings suggest that the severe decrease in IRP1 RNA-binding activity depends on: (i) linking of OMA to the active site of aconitase, which prevents the switch to IRP1 and explains resistance to the reducing agents, and (ii) possible interaction of OMA with some functional amino acid residues in IRP that are responsible for binding to the specific mRNA sequences involved in the regulation of iron metabolism.
Cytochrome c release from isolated rat liver mitochondria can occur independently of outer-membrane rupture: possible role of contact sites
Authors: DORAN Elena , HALESTRAP Andrew P.
Published: August 2015

Percoll-purified rat liver mitochondria were shown to contain BAX dimer and rapidly (< 2 min) release 5-10% of their cytochrome c when incubated in a standard KCl incubation medium under energized conditions. This release was not accompanied by release of adenylate kinase (AK), another intermembrane protein, and was not inhibited by Mg2+, dATP, inhibitors of the permeability transition or ligands of the peripheral benzodiazepine receptor. However, release was greatly reduced by the presence of 5% (w/v) dextran (40 kDa), which caused a decrease in the light scattering (A520) of mitochondrial suspensions. Dextran also inhibited both mitochondrial oxidation of exogenous ferrocytochrome c in the presence of rotenone and antimycin, and respiratory-chain-driven reduction of exogenous ferricytochrome c. Hypo-osmotic medium or digitonin treatment of mitochondria caused a large additional release of both cytochrome c and AK that was not blocked by dextran. Polyaspartate, which stabilizes the low conductance state of the voltage-dependent anion channel (VDAC), increased cytochrome c release. VDAC and BAX are both found at the contact sites between the inner and outer membranes and dextran is known to stabilize these contact sites in isolated mitochondria. Thus our data suggest that regulation of a specific permeability pathway for cytochrome c may be mediated by changes in protein-protein interactions within contact sites. The adenine nucleotide translocase is known to bind to VDAC and thus provides an additional link between the specific cytochrome c release pathway and the permeability transition.
Purification from rat liver of a novel constitutively expressed member of the aldo-keto reductase 7 family that is widely distributed in extrahepatic tissues
Authors: KELLY Vincent P. , IRELAND Linda S. , ELLIS Elizabeth M. , HAYES John D.
Published: August 2015

Antiserum raised against human aflatoxin B1 aldehyde reductase 1 (hAFAR1) has been used to identify a previously unrecognized rat aldo-keto reductase (AKR). This novel enzyme is designated rat aflatoxin B1 aldehyde reductase 2 (rAFAR2) and it characteristically migrates faster during SDS/PAGE than does the archetypal ethoxyquin-inducible rAFAR protein (now called rAFAR1). Significantly, rAFAR2 is essentially unreactive with polyclonal antibodies raised against rAFAR1. Besides its distinct electrophoretic and immunochemical properties, rAFAR2 appears to be regulated differently from rAFAR1 as it is expressed in most rat tissues and does not appear to be induced by ethoxyquin. Multiple forms of rAFAR2 have been identified. Anion-exchange chromatography on Q-Sepharose, followed by adsorption chromatography on columns of Matrex Orange A and Cibacron Blue, have been employed to purify rAFAR2 from rat liver cytosol. The Q-Sepharose chromatography step resulted in the resolution of rAFAR2 into three peaks of AKR activity, two of which were purified and shown to be capable of catalysing the reduction of 2-carboxybenzaldehyde, succinic semialdehyde, 4-nitrobenzaldehyde and 9,10-phenathrenequinone. The two most highly purified rAFAR2-containing preparations eluted from the Cibacron Blue column were 91 and 98% homogeneous. Analysis of these by SDS/PAGE indicated that the least anionic (peak CBA5) comprised a polypeptide of 37.0 kDa, whereas the most anionic (peak CBA6) contained two closely migrating polypeptides of 36.8 and 37.0 kDa; by contrast, in the present study, rAFAR1 was estimated by SDS/PAGE to be composed of 38.0 kDa subunits. Final purification of the 37 kDa polypeptide in CBA5 and CBA6 was accomplished by reversed-phase HPLC. Partial proteolysis of the two preparations of the 37 kDa polypeptide with Staphylococcus aureus V8 protease yielded fragments of identical size, suggesting that they represent the product of a single gene. Furthermore, the peptide maps from CBA5 and CBA6 differed substantially from that yielded by rAFAR1, indicating that they are genetically distinct from the inducible reductase. A peptide generated by CNBr digestion of the 37 kDa polypeptide from CBA6 was shown by Edman degradation to share 88% sequence identity with residues Tyr168-Leu183 of rAFAR1. This provides evidence that the rat protein identified by its cross-reactivity with anti-hAFAR1 serum is an additional member of the AKR7 family.
Signalling pathways of insulin-like growth factor-I that are augmented by cAMP in FRTL-5 cells
Authors: ARIGA Miyako , NEDACHI Taku , AKAHORI Masakazu , SAKAMOTO Hideki , ITO Yoshiaki , HAKUNO Fumihiko , TAKAHASHI Shin-Ichiro
Published: August 2015

We have reported that pretreatment of rat FRTL-5 thyroid cells with thyrotropin (TSH) markedly potentiates the mitogenic response to insulin-like growth factor-I (IGF-I). The present study was undertaken to determine whether the augmentation by cAMP of IGF-I-dependent tyrosine phosphorylation of known IGF-I receptor substrates plays an important role in the cAMP-dependent potentiation of DNA synthesis induced by IGF-I. Pretreatment with TSH or dibutyryl cAMP did not affect the IGF-I-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). In contrast, cAMP pretreatment potentiated the tyrosine phosphorylation of IRS-2 induced by IGF-I, but did not affect the amount of IRS-2. We found that the IGF-I-dependent tyrosine phosphorylation of 66 kDa Shc (Src homology collagen) was markedly increased by cAMP pretreatment, and that this change was mainly due to an increase in the levels of 66 kDa Shc protein. Under these conditions, cAMP pretreatment significantly increased binding of Grb2 (growth-factor-receptor-bound protein 2) to Shc in response to IGF-I, and activation of MAP kinase (mitogen-activated protein kinase) induced by IGF-I was also enhanced by cAMP. The presence of PD98059, an inhibitor of MEK (MAP-kinase/Erk kinase), during treatment with IGF-I partially inhibited the cAMP-dependent augmentation of DNA synthesis in response to IGF-I. On the other hand, cAMP pretreatment increased binding of the phosphoinositide 3-kinase (PI 3-kinase) p85 subunit to IRS-2, which was reflected in PI 3-kinase activity. LY294002, a PI 3-kinase inhibitor, strongly depressed IGF-I-dependent DNA synthesis after pretreatment with and without TSH or dibutyryl cAMP. Our results suggest that the interaction between cAMP-dependent and IGF-I-dependent pathways leads to an augmentation of cell proliferation, which is mediated, at least in part, through the MAP kinase and PI 3-kinase signalling pathways. These effects are mediated by changes in tyrosine phosphorylation of IGF-I receptor substrates, including IRS-2 and Shc.
Multiple conserved segments of E1 small nucleolar RNA are involved in the formation of a ribonucleoprotein particle in frog oocytes
Authors: RUHL Donald D. , PUSATERI Mary Ellen , ELICEIRI George L.
Published: August 2015

E1/U17 small nucleolar RNA (snoRNA) is a box H/ACA snoRNA. To identify E1 RNA elements required for its assembly into a ribonucleoprotein (RNP) particle, we have made substitution mutations in evolutionarily conserved sequences and structures of frog E1 RNA. After E1 RNA was injected into the nucleus of frog oocytes, assembly of this exogenous RNA into an RNP was monitored by non-denaturing gel electrophoresis. Unexpectedly, nucleotide substitutions in many phylogenetically conserved segments of E1 RNA produced RNPs with abnormal gel-electrophoresis patterns. These RNA segments were at least nine conserved sequences and an apparently conserved structure. In another region needed for RNP formation, the requirement may be sequence(s) and/or structure. Base substitutions in each of these and in one additional conserved E1 RNA segment reduced the stability of this snoRNA in frog oocytes. Nucleolar localization was assayed by fluorescence microscopy after injection of fluorescein-labelled RNA. The H box (ANANNA) and the ACA box are both needed for efficient nucleolar localization of frog E1 RNA.
Enzymic methylation of arginyl residues in -Gly-Arg-Gly- peptides
Authors: HYUN Young-Lan , LEW D. Betty , PARK Seung Hee , KIM Chan-Wha , PAIK Woon Ki , KIM Sangduk
Published: August 2015

N G-Methylation of arginine residues in many nucleic-acid-binding proteins are formed post-translationally, catalysed by S-adenosylmethionine:protein-arginine N-methyltransferase in their glycine-rich and arginine-rich motifs. The amino acid sequences of the stimulator of HIV-1 TAR (Tat-responsive element) RNA-binding protein (SRB) and fibronectin also show the presence of the internal -Gly-Arg-Gly- (-GRG-) sequence, which is potentially methylatable by the methyltransferase. To investigate the sequence requirement for methylation of these proteins, several synthetic oligopeptides with different chain lengths and sequences similar to the -GRG- regions of SRB and fibronectin were synthesized. Whereas the heptapeptide AGGRGKG (residues 16-22 in SRB) served as the methyl acceptor for the methyltransferase with a Km of 50 μM, the 19-mer peptide (residues 10-28 in SRB) was methylated with a Km of 8.3 μM, indicating that a greater peptide chain length yields a better methyl acceptor. Product analysis of the methylated [methyl-14C]SRB-peptide by HPLC indicated the formation of NG-monomethylarginine and NG,NG-dimethyl(asymmetric)arginine. Synthetic peptides containing the cell attachment sequence [Arg-Gly-Asp (‘RGD’)] in fibronectin, GRGDSPK, GGRGDSPK and GGGRGDSPK, were also studied; whereas GRGDSPK was a poor methyl acceptor, the longer peptides were better methyl acceptors. To provide an understanding of the effect of methylation on fibronectin peptide, arginine-unmethylated and methylated GGRGDSPK were compared for their effect on the mitogenesis induced by β-hexosaminidase A and an agonistic antibody (mAb15) in bovine tracheal smooth-muscle cells; whereas the former inhibited 35-67% of mitogenesis at a concentration of 5-10 μM, the latter did not block mitogenesis. This lack of inhibition by the insertion of a methyl group on the arginyl residue of the cell attachment sequence might be due to the hindrance of the binding of fibronectin peptide to integrins.
Contrasting effects of N5-substituted tetrahydrobiopterin derivatives on phenylalanine hydroxylase, dihydropteridine reductase and nitric oxide synthase
Authors: WERNER Ernst R. , HABISCH Hans-Jörg , GORREN Antonius C. F. , SCHMIDT Kurt , CANEVARI Laura , WERNER-FELMAYER Gabriele , MAYER Bernd
Published: August 2015

Tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H4biopterin] is one of several cofactors of nitric oxide synthases (EC 1.14.13.39). Here we compared the action of N5-substituted derivatives on recombinant rat neuronal nitric oxide synthase with their effects on dihydropteridine reductase (EC 1.6.99.7) and phenylalanine hydroxylase (EC 1.14.16.1), the well-studied classical H4biopterin-dependent reactions. H4biopterin substituted at N5 with methyl, hydroxymethyl, formyl and acetyl groups were used. Substitution at N5 occurs at a position critical to the redox cycle of the cofactor in phenylalanine hydroxylase/dihydropteridine reductase. We also included N2ʹ-methyl H4biopterin, a derivative substituted at a position not directly involved in redox cycling, as a control. As compared with N5-methyl H4biopterin, N5-formyl H4biopterin bound with twice the capacity but stimulated nitric oxide synthase to a lesser extent. Depending on the substituent used, N5-substituted derivatives were redox-active: N5-methyl- and N5-hydroxylmethyl H4biopterin, but not N5-formyl- and N5-acetyl H4biopterin, reduced 2,6-dichlorophenol indophenol. N5-Substituted H4biopterin derivatives were not oxidized to products serving as substrates for dihydropteridine reductase and, depending on the substituent, were competitive inhibitors of phenylalanine hydroxylase: N5-methyl- and N5-hydroxymethyl H4biopterin inhibited phenylalanine hydroxylase, whereas N5-formyl- and N5-acetyl H4biopterin had no effect. Our data demonstrate differences in the mechanism of stimulation of phenylalanine hydroxylase and nitric oxide synthase by H4biopterin. They are compatible with a novel, non-classical, redox-active contribution of H4biopterin to the catalysis of the nitric oxide synthase reaction.
Determinant of the extracellular location of the N-terminus of human multidrug-resistance-associated protein
Authors: ZHANG Jian-Ting
Published: August 2015

Multidrug-resistance-associated protein (MRP) is a member of the ATP-binding cassette (ABC) membrane-transport superfamily and is responsible for multidrug resistance in cancer cells. Distinct from other members of the ABC superfamily, MRP has three membrane-spanning domains (MSDs) and the N-terminus is located extracellularly. It has been shown that the first MSD (MSD1) with an extracellular N-terminus is important for MRP function. To address what ensures the generation of this structural organization of MRP and to understand in general the molecular mechanism of membrane folding of polytopic proteins with extracellular N-termini, the biogenesis of MSD1 in human MRP1 was examined using an in vitro expression system. Surprisingly, the second transmembrane segment (TM2) in MSD1 was found to play a critical role in the correct membrane translocation and folding of MSD1 in human MRP1. TM2 not only plays an essential role to ensure the N-terminus-outside/C-terminus-inside orientation of TM1 with an extracellular N-terminus, it can also translocate into membranes post-translationally in a signal-recognition particle and ribosome-dependent manner to provide an additional insurance for correct folding of MSD1 in MRP. These findings suggest that TM2 in a polytopic membrane protein with an extracellular N-terminus may play a critical role in controlling correct membrane translocation and folding of the protein in general.
Characterization of the actin filament capping state in human erythrocyte ghost and cytoskeletal preparations
Authors: KUHLMAN Philip A.
Published: August 2015

The narrow Gaussian-length-distribution of actin filaments forming the cytoskeleton of the human erythrocyte indicates the existence of strict mechanisms for length determination and maintenance. A similar regulation is achieved in striated muscle by the capping of both the ends of the thin filaments, which consequently prevents monomer exchange. However, the ability of erythroid cytoskeletal preparations to nucleate actin polymerization has led to the proliferation of the idea that at least the barbed ends of the actin filaments are uncapped. The mechanism by which the length of the filaments is thus maintained has been left open to debate. In an effort to resolve any doubt regarding length-maintenance in human erythrocytes we have characterized the capping state of the actin filaments in a number of different ghost and cytoskeletal preparations. Under conditions of sufficiently high bivalent-cation concentration the actin filaments retain functional caps at both the barbed and pointed ends. Hence filament capping at both ends prevents redistribution of the actin monomer in a similar manner to that proposed for the thin filaments of striated muscle. Actin filament uncapping is apparently caused by the centrifugal shearing stress imposed during ghost preparation. The uncapping is more pronounced when the bivalent-cation concentration is reduced or when the membrane is removed by detergents. The effects of bivalent cations seem to be mediated through the erythroid protein spectrin, consistent with the hypothesis of Wallis et al. [Wallis, Babitch and Wenegieme (1993) Biochemistry 32, 5045-5050] that the ability of spectrin to resist shearing stress is dependent on the degree of bound bivalent cations.
Regulation of ecdysteroid signalling: molecular cloning, characterization and expression of 3-dehydroecdysone 3α-reductase, a novel eukaryotic member of the short-chain dehydrogenases/reductases superfamily from the cotton leafworm, Spodoptera littoralis
Authors: TAKEUCHI Hajime , CHEN Jian-Hua , O'REILLY David R. , REES Huw H. , TURNER Philip C.
Published: August 2015

One route of inactivation of ecdysteroids in insects involves ecdysone oxidase-catalysed conversion into 3-dehydroecdysone (3DE), followed by irreversible reduction by 3DE 3α-reductase to 3-epiecdysone. The 3DE 3α-reductase has been purified and subjected to limited amino acid sequencing. It occurs as two distinct forms, including a probable trimer of subunit molecular mass of approx. 26 kDa. A reverse-transcriptase PCR-based approach has been used to clone the cDNA (1.2 kb) encoding the 26 kDa protein. Northern blotting showed that the mRNA transcript was expressed in Malpighian tubules during the early stage of the last larval instar. Conceptual translation of the 3DE 3α-reductase cDNA and database searching revealed that the enzyme belongs to the short-chain dehydrogenases/reductases superfamily. Furthermore, the enzyme is a novel eukaryotic 3-dehydrosteroid 3α-reductase member of that family, whereas vertebrate 3-dehydrosteroid 3α-reductases belong to the aldo-keto reductase (AKR) superfamily. Enzymically active recombinant 3DE 3α-reductase has been produced using a baculovirus expression system. Surprisingly, we observed no similarity between this 3DE 3α-reductase and a previously reported 3DE 3β-reductase, which acts on the same substrate and belongs to the AKR family.
Molecular modelling and site-directed mutagenesis of the inositol 1,3,4,5-tetrakisphosphate-binding pleckstrin homology domain from the Ras GTPase-activating protein GAP1IP4BP
Authors: COZIER Gyles , SESSIONS Richard , BOTTOMLEY Joanna R. , REYNOLDS Jon S. , CULLEN Peter J.
Published: August 2015

GAP1IP4BP is a Ras GTPase-activating protein (GAP) that in vitro is regulated by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. We have studied Ins(1,3,4,5)P4 binding to GAP1IP4BP, and shown that the inositol phosphate specificity and binding affinity are similar to Ins(1,3,4,5)P4 binding to Bruton's tyrosine kinase (Btk), evidence which suggests a similar mechanism for Ins(1,3,4,5)P4 binding. The crystal structure of the Btk pleckstrin homology (PH) domain in complex with Ins(1,3,4,5)P4 has shown that the binding site is located in a partially buried pocket between the β1/β2- and β3/β4-loops. Many of the residues involved in the binding are conserved in GAP1IP4BP. Therefore we generated a model of the PH domain of GAP1IP4BP in complex with Ins(1,3,4,5)P4 based on the Btk-Ins(1,3,4,5)P4 complex crystal structure. This model had the typical PH domain fold, with the proposed binding site modelling well on the Btk structure. The model has been verified by site-directed mutagenesis of various residues in and around the proposed binding site. These mutations have markedly reduced affinity for Ins(1,3,4,5)P4, indicating a specific and tight fit for the substrate. The model can also be used to explain the specificity of inositol phosphate binding.
Evidence for phosphorylation and ubiquitinylation of the turnip yellow mosaic virus RNA-dependent RNA polymerase domain expressed in a baculovirus–insect cell system
Authors: HÉRICOURT François , BLANC Stéphane , REDEKER Virginie , JUPIN Isabelle
Published: August 2015

All RNA viruses known to date encode an RNA-dependent RNA polymerase (RdRp) that is required for replication of the viral genome. We have expressed and purified the turnip yellow mosaic virus (TYMV) RdRp in insect cells using a recombinant baculovirus, either in its native form, or fused to an hexa-histidine tag. Phosphorylation of the protein was demonstrated by labelling experiments in vivo, as well as phosphatase treatment of the purified protein in vitro. Phospho amino acid analysis and immunoblotting experiments identified serine and threonine residues as being the subject of phosphorylation. Peptide mass mapping using MS analysis of a protein digest revealed that phosphorylation sites are localized within a putative PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] in the N-terminal region of the protein. Using monoclonal antibodies specific for ubiquitin conjugates, we were able to demonstrate that the TYMV RdRp is conjugated to ubiquitin molecules when expressed in insect cells. These observations suggest that the TYMV RdRp may be processed selectively by the ubiquitin/proteasome degradation system upon phosphorylation of the PEST sequence.
Tissue-specificity, functional characterization and subcellular localization of a rat ubiquitin-specific processing protease, UBP109, whose mRNA expression is developmentally regulated
Authors: PARK Kyung Chan , CHOI Eun Jung , MIN Sang Won , CHUNG Sung Soo , KIM Hyun , SUZUKI Toshiaki , TANAKA Keiji , CHUNG Chin Ha
Published: August 2015

A cDNA encoding an ubiquitin-specific processing protease, UBP109, in rat skeletal muscle was cloned and its product was characterized. Northern analysis revealed that UBP109 mRNA is highly expressed in testis and spleen, compared with other tissues. Furthermore, in situ hybridization showed that the level of UBP109 mRNA in liver, spinal cord and brain dramatically changed during embryonic development, indicating that the expression of UBP109 mRNA is developmentally regulated. UBP109 was expressed in Escherichia coli and purified to apparent homogeneity using a 125I-labelled ubiquitin-peptide fusion as a substrate. The purified enzyme cleaved at the C-terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their sizes. UBP109 also released free ubiquitin from poly-His-tagged penta-ubiquitin. Moreover, it released free ubiquitin from poly-ubiquitinated protein conjugates of rabbit reticulocytes. In addition, UBP109 localized to both the cytoplasm and the nucleus and, among three putative nuclear localization sequences, only the one located near the C-terminus is responsible for nuclear localization. These results suggest that UBP109 may play an important role in generation of free ubiquitin from its precursors and its recycling from poly-ubiquitinated protein conjugates, and hence in regulation of ubiquitin-mediated cellular processes, particularly related to embryonic development.
Kinetics and control of oxidative phosphorylation in rat liver mitochondria after chronic ethanol feeding
Authors: MARCINKEVICIUTE Ausra , MILDAZIENE Vida , CRUMM Sara , DEMIN Oleg , HOEK Jan B. , KHOLODENKO Boris
Published: August 2015

Changes in the kinetics and regulation of oxidative phosphorylation were characterized in isolated rat liver mitochondria after 2 months of ethanol consumption. Mitochondrial energy metabolism was conceptually divided into three groups of reactions, either producing protonmotive force (∆p) (the respiratory subsystem) or consuming it (the phosphorylation subsystem and the proton leak). Manifestation of ethanol-induced mitochondrial malfunctioning of the respiratory subsystem was observed with various substrates; the respiration rate in State 3 was inhibited by 27±4% with succinate plus amytal, by 20±4% with glutamate plus malate, and by 17±2% with N,N,Nʹ,Nʹ-tetramethyl-p-phenylenediamine/ascorbate. The inhibition of the respiratory activity correlated with the lower activities of cytochrome c oxidase, the bc1 complex, and the ATP synthase in mitochondria of ethanol-fed rats. The block of reactions consuming the ∆p to produce ATP (the phosphorylating subsystem) was suppressed after 2 months of ethanol feeding, whereas the mitochondrial proton leak was not affected. The contributions of ∆p supply (the respiratory subsystem) and ∆p demand (the phosphorylation and the proton leak) to the control of the respiratory flux were quantified as the control coefficients of these subsystems. In State 3, the distribution of control exerted by different reaction blocks over respiratory flux was not significantly affected by ethanol diet, despite the marked changes in the kinetics of individual functional units of mitochondrial oxidative phosphorylation. This suggests the operation of compensatory mechanisms, when control redistributes among the different components within the same subsystem.
Recovery of active medium-chain-length-poly-3-hydroxyalkanoate polymerase from inactive inclusion bodies using ion-exchange resin
Authors: REN Qun , DE ROO Guy , KESSLER Birgit , WITHOLT Bernard
Published: August 2015

A novel process for the purification of active medium-chain-length-polyhydroxyalkanoate (mcl-PHA) polymerase was developed. This process is based on solubilization and activation of inactive polymerase inclusion bodies by incubation with ion-exchange resin. The mcl-PHA polymerase 1 from Pseudomonas oleovorans was overproduced from the Palk promoter. Most of the polymerase produced was sequestered in the cytoplasm as an inactive form in insoluble aggregates. By incubating the protein aggregates with S-Sepharose ion-exchange resin in the presence of dithiothreitol and glycerol, the mcl-PHA polymerase could be extracted in an active and soluble form with a final yield of about 5.2 mg/g of cell dry weight. The solubilized polymerase was able to catalyse the in vitro synthesis of mcl-PHA without any additional cell components, suggesting its potential application for production of biopolymer. The procedure used here may be of general value in solubilizing and activating purified inactive labile enzymes.
Phosphoinositide 3-kinase-dependent phosphorylation of the dual adaptor for phosphotyrosine and 3-phosphoinositides by the Src family of tyrosine kinase
Authors: DOWLER Simon , MONTALVO Leire , CANTRELL Doreen , MORRICE Nick , ALESSI Dario R.
Published: August 2015

We recently identified a novel adaptor protein, termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1), that possesses a Src homology (SH2) domain and a pleckstrin homology (PH) domain. DAPP1 exhibits a high-affinity interaction with PtdIns(3,4,5)P3 and PtdIns(3,4)P2, which bind to the PH domain. In the present study we show that when DAPP1 is expressed in HEK-293 cells, the agonists insulin, insulin-like growth factor-1 and epidermal growth factor induce the phosphorylation of DAPP1 at Tyr139. Treatment of cells with phosphoinositide 3-kinase (PI 3-kinase) inhibitors or expression of a dominant-negative PI 3-kinase prevent phosphorylation of DAPP1 at Tyr139, and a PH-domain mutant of DAPP1, which does not interact with PtdIns(3,4,5)P3 or PtdIns(3,4)P2, is not phosphorylated at Tyr139 following agonist stimulation of cells. Overexpression of a constitutively active form of PI 3-kinase induced the phosphorylation of DAPP1 in unstimulated cells. We demonstrated that Tyr139 of DAPP1 is likely to be phosphorylated in vivo by a Src-family tyrosine kinase, since the specific Src-family inhibitor, PP2, but not an inactive variant of this drug, PP3, prevented the agonist-induced tyrosine phosphorylation of DAPP1. Src, Lyn and Lck tyrosine kinases phosphorylate DAPP1 at Tyr139in vitro at similar rates in the presence or absence of PtdIns(3,4,5)P3, and overexpression of these kinases in HEK-293 cells induces the phosphorylation of Tyr139. These findings indicate that, following activation of PI 3-kinases, PtdIns(3,4,5)P3 or PtdIns(3,4)P2 bind to DAPP1, recruiting it to the plasma membrane where it becomes phosphorylated at Tyr139 by a Src-family tyrosine kinase.
Targeting and insertion of C-terminally anchored proteins to the mitochondrial outer membrane is specific and saturable but does not strictly require ATP or molecular chaperones
Authors: LAN Ling , ISENMANN Sandra , WATTENBERG Binks W.
Published: August 2015

A distinct class of proteins contain a C-terminal membrane anchor and a cytoplasmic functional domain. A subset of these proteins is targeted to the mitochondrial outer membrane. Here, to probe for the involvement of a saturable targeting mechanism for this class of proteins, and to elucidate the roles of chaperone proteins and ATP, we have utilized an in vitro targeting system consisting of in vitro-synthesized proteins and isolated mitochondria. To establish the specificity of targeting we have used a closely related protein pair. VAMP-1A and VAMP-1B are splice variants of the vesicle-associated membrane protein/synaptobrevin-1 (VAMP-1) gene. In intact cells VAMP-1B is targeted to mitochondria whereas VAMP-1A is targeted to membranes of the secretory pathway, yet these isoforms differ by only five amino acids at the extreme C-terminus. Here we demonstrate that, in vitro, VAMP-1B is imported into both intact mitochondria and mitochondrial outer-membrane vesicles with a 15-fold greater efficiency than VAMP-1A. We generated and purified bacterially expressed fusion proteins consisting of the C-terminal two-thirds of VAMP-1A or -1B proteins fused to glutathione S-transferase (GST). Using these fusion proteins we demonstrate that protein targeting and insertion is saturable and specific for the VAMP-1B membrane anchor. To elucidate the role of cytosolic chaperones on VAMP-1B targeting, we also used the purified, Escherichia coli-derived fusion proteins. 33P-Labelled GST-VAMP-1B61-116, but not GST-VAMP-1A61-118, was efficiently targeted to mitochondria in a chaperone-free system. Thus the information required for targeting is contained within the targeted protein itself and not the chaperone or a chaperone-protein complex, although chaperones may be required to maintain a transport-competent conformation. Moreover, ATP was required for transport only in the presence of cytosolic chaperone proteins. Therefore the ATP requirement of transport appears to reflect the participation of chaperones and not any other ATP-dependent step. These data demonstrate that targeting of C-terminally anchored proteins to mitochondria is sequence specific and mediated by a saturable mechanism. Neither ATP nor chaperone proteins are strictly required for either specific targeting or membrane insertion.
Substrate specificity of the expressed carotenoid 3,4-desaturase from Rubrivivax gelatinosus reveals the detailed reaction sequence to spheroidene and spirilloxanthin
Authors: STEIGER Sabine , ASTIER Chantal , SANDMANN Gerhard
Published: August 2015

Carotenoid biosynthesis in the photosynthetic bacterium Rubrivivax gelatinosus leads to the formation of hydroxyspheroidene and spirilloxanthin as the products of a branched pathway. In this study we investigated the role of the desaturase encoded by crtD which catalyses the introduction of C-3,4 double bonds into acyclic carotenoids. The desaturase was expressed in Escherichia coli, and the activity and the substrate specificity of the enzyme were evaluated in vitro by application of structurally different carotenoids. The results indicate that the enzyme is a 3,4-desaturase that converts 1-hydroxy carotenoids. The 3,4-desaturation reaction can only occur with mono-1-hydroxy carotenoids at a ψ-end group or with 1,1ʹ-dihydroxy derivatives carrying a 3ʹ,4ʹ-double bond. In addition, 1-HO-ζ-carotene could also be converted by the desaturase. Enzyme kinetic studies showed a substrate preference of 1-HO-neurosporene over 1-HO-lycopene. Consequences from the biochemical data for the reaction sequence of hydroxyspheroidene and spirilloxanthin formation and the interconnection of both branches are discussed.
Cloning and characterization of the 5′ flanking region of the sialomucin complex/rat Muc4 gene: promoter activity in cultured cells
Authors: PRICE-SCHIAVI Shari A. , PEREZ Aymee , BARCO Roy , CARRAWAY Kermit L.
Published: August 2015

Sialomucin complex (SMC/Muc4) is a heterodimeric glycoprotein complex consisting of a mucin subunit ascites sialoglycoprotein-1 (ASGP-1) and a transmembrane subunit (ASGP-2), which is aberrantly expressed on the surfaces of a variety of tumour cells. SMC is transcribed from a single gene, translated into a large polypeptide precursor, and further processed to yield the mature ASGP-1/ASGP-2 complex. SMC has complex spatial and temporal expression patterns in the normal rat, suggesting that it has complex regulatory mechanisms. A crude exon/intron map of the 5ʹ regions of the SMC/Muc4 gene generated from clones isolated from a normal rat liver genomic DNA library reveals that this gene has a small first exon comprising the 5ʹ untranslated region and signal peptide, followed by a large intron. The second exon appears to be large, comprising the 5ʹ unique region and a large part (probably all) of the tandem repeat domain. This structure is strikingly similar to that reported for the human MUC4 gene. Using PCR-based DNA walking, 2.4 kb of the 5ʹ-flanking region of the SMC/Muc4 gene was cloned and characterized. Promoter-pattern searches yielded multiple motifs commonly found in tissue-specific promoters. Reporter constructs generated from this 2.4 kb fragment demonstrate promoter activity in primary rat mammary epithelial cells (MEC), the human colon tumour cell line HCT-116, and the human lung carcinoma cell line NCI-H292, but not in COS-7 cells, suggesting epithelial cell specificity. Deletion constructs of this sequence transfected into rat MEC or HCT-116 cells demonstrate greatly varying levels of activity, suggesting that there are positive and negative, as well as tissue-specific, regulatory elements in this sequence. Taken together, these data suggest that the rat SMC/Muc4 promoter has been identified, that it is tissue- (epithelial cell-) specific, and that there are both positive and negative, as well as tissue-specific, regulatory elements in the sequence.