bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and molecular cloning of rat 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase
Authors: TANABE Atsushi , EGASHIRA Yukari , FUKUOKA Shin-Ichi , SHIBATA Katsumi , SANADA Hiroo
Published: August 2015

2-Amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD; EC 4.1.1.45) is one of the important enzymes regulating tryptophan—niacin metabolism. In the present study, we purified the enzyme from rat liver and kidney, and cloned the cDNA encoding rat ACMSD. The molecular masses of rat ACMSDs purified from the liver and kidney were both estimated to be 39kDa by SDS/PAGE. Analysis of N-terminal amino acid sequences showed that these two ACMSDs share the same sequence. An expressed sequence tag (EST) of the mouse cited from the DNA database was found to be identical with this N-terminal sequence. Reverse transcription-PCR (RT-PCR) was performed using synthetic oligonucleotide primers having the partial sequences of the EST, and then cDNAs encoding rat ACMSDs were isolated by using subsequent 3′-rapid amplification of cDNA ends and RT-PCR methods. ACMSD cDNAs isolated from liver and kidney were shown to be identical, consisting of a 1008bp open reading frame (ORF) encoding 336 amino acid residues with a molecular mass of 38091Da. The rat ACMSD ORF was inserted into a mammalian expression vector, before transfection into human hepatoma HepG2 cells. The transfected cells expressed ACMSD activity, whereas the enzyme activity was not detected in uninfected parental HepG2 cells. The distribution of ACMSD mRNA expression in various tissues was investigated in the rat by RT-PCR. ACMSD was expressed in the liver and kidney, but not in the other principal organs examined.
The human homologue of the yeast polyubiquitination factor Ufd2p is cleaved by caspase 6 and granzyme B during apoptosis
Authors: MAHONEY James A. , ODIN Joseph A. , WHITE Sarah M. , SHAFFER David , KOFF Andrew , CASCIOLA-ROSEN Livia , ROSEN Antony
Published: August 2015

In the present study, we demonstrate that a human homologue of Ufd2p (a yeast protein that catalyses the formation of long polyubiquitin chains, and is implicated in responses to environmental stress), UFD2 (ubiquitin fusion degradation protein-2), is cleaved during apoptosis induced by multiple stimuli, including UVB irradiation, Fas ligation, staurosporine treatment and cytotoxic lymphocyte granule-induced death. Caspase 6 and granzyme B efficiently cleave UFD2 [kcat/Km = (4–5)×104 M−1·s−1] at Asp123, whereas caspases 3 and 7 cleave UFD2 approx. 10-fold less efficiently immediately upstream at Asp109. Thus UFD2 is added to the growing list of proteins with closely spaced caspase and granzyme B cleavage sites, suggesting the presence of a previously unrecognized, conserved motif. Both cleavage sites are contained and conserved within a novel 300-amino-acid N-terminal domain present in apparent UFD2 orthologues in mice and zebrafish, but absent in all UFD2 family members in lower eukaryotes. Full-length recombinant UFD2 exhibited ubiquitin—protein ligase ('E3')-like ubiquitination activity in vitro, but this activity was abolished in recombinant UFD2 truncated at the granzyme B/caspase 6 cleavage site. Cleavage of UFD2 by caspases or granzyme B within this putative regulatory N-terminal domain might have important functional consequences within the apoptotic cascade.
Stabilin-1 and −2 constitute a novel family of fasciclin-like hyaluronan receptor homologues
Authors: POLITZ Oliver , GRATCHEV Alexei , McCOURT Peter A. G. , SCHLEDZEWSKI Kai , GUILLOT Pierre , JOHANSSON Sophie , SVINENG Gunbjorg , FRANKE Peter , KANNICHT Christoph , KZHYSHKOWSKA Julia , LONGATI Paola , VELTEN Florian W. , JOHANSSON Staffan , GOERDT Sergij
Published: August 2015

MS-1, a high-molecular-mass protein expressed by non-continuous and angiogenic endothelial cells and by alternatively activated macrophages (Mφ2), and the hepatic sinusoidal endothelial hyaluronan clearance receptor are similar with respect to tissue distribution and biochemical characteristics. In the present study we purified these proteins by immuno- and hyaluronan-affinity chromatography respectively, sequenced tryptic peptides and generated full-length cDNA sequences in both mouse and human. The novel genes, i.e. stabilin-1 and stabilin-2, code for homologous transmembrane proteins featuring seven fasciclin-like adhesion domains, 18–20 epidermal-growth-factor domains, one X-link domain and three to six B-(X7)-B hyaluronan-binding motifs. Northern-blotting experiments revealed the presence of both stabilins in organs with predominant endothelial sinuses such as liver, spleen and lymph node: stabilin-1 mRNA was also detected in organs with predominant Mφ2 cells, such as placenta, and in interleukin-4/glucocorticoid-stimulated Mφ2 cells in vitro. A polyclonal antibody made against human recombinant stabilin-1 confirmed the expression of stabilin-1 protein in splenic sinus endothelial cells in vivo and in Mφ2 in vitro. On the basis of high similarity at the protein level and the unique domain composition, which differs from that of all other known fasciclin-like proteins and hyaluronan receptors, stabilin-1 and stabilin-2 define a novel family of fasciclin-like hyaluronan receptor homologues that might play a role in cell—cell and cell—matrix interactions in vascular function and inflammatory processes.
Fluorescence resonance energy transfer between coumarin-derived mitochondrial F1-ATPase γ subunit and pyrenylmaleimide-labelled fragments of IF1 and c subunit
Authors: BARACCA Alessandra , BAROGI Silvia , PAOLINI Sara , LENAZ Giorgio , SOLAINI Giancarlo
Published: August 2015

We introduced a reporting group into a critical position of the mitochondrial F1-ATPase in order to gain structural information about enzyme—ligand complexes. Incubation of 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM) with bovine heart mitochondrial F1-ATPase pretreated with 1mM sodium arsenite modified the only cysteine residue in the γ subunit (γ-Cys78), resulting in an enzyme—CPM fluorescent complex (CPM—F1) with an ATPase activity similar to that of the native enzyme. Transferred fluorescence of F1-bound CPM occurred when different peptide fragments of naturally binding polypeptides carrying a pyrenylmaleimide (PM) moiety were bound to the enzyme. Fluorescence resonance energy transfer (RET) from PM bound to cysteine residues associated with Glu40, Lys47 and Lys58 of fragments of the inhibitor protein (IF1) with CPM—F1 occurred with an efficiency of approx. 20, 21 and 3% respectively. The distance at which the efficiency of energy transfer was 50%, R0, for the CPM and PM donor/acceptor pair was 4.1nm, indicating that the three IF1 fragments must be within 6.7nm of γ-Cys78. RET from the PM-bound hydrophilic fragment of c subunit (residues 37–42) of the F1F0-ATPase complex and CPM-bound γ-Cys78 occurred with an efficiency of approx. 30%, indicating a distance of 4.7nm between the two fluorophores. Based on previous observations and on the present RET measurements, the hydrophilic loop of c subunit was located at the base of the F1 foot, and the N-terminal region of IF1 was located on the surface of F1 in the lower part of the α3β3 hexamer ring.
Differentiation-dependent mechanisms of transcriptional regulation of the catalytic subunit of phosphorylase kinase
Authors: O'MAHONY Alison M. , WALSH Donal A.
Published: August 2015

The amount of phosphorylase kinase in skeletal muscle is exquisitely sensitive to developmental signals such as differentiation and innervation, and is clearly regulated in such a manner so as to always maintain the γ catalytic subunit under the control of its regulatory α, β and γ subunits. To identify how the transcription of the γ subunit is regulated, we have analysed 3.8kb of the upstream regulatory region using a luciferase reporter system. A complex sequence of interdependent regulations is evident. The γ catalytic subunit gene contains two inhibitory controls with very dominant features. Also evident are an array of multiple positive regulatory elements, prominent amongst which are four E-boxes, of which two are downstream, one is upstream and one is in the middle of the CAAT-TATA core promoter. Differentiation-dependent positive regulation arises as a consequence of both E-box regulation and the activation of at least one other regulatory element. The primary mode of transcriptional regulation of the γ catalytic subunit gene appears to occur by the relief of regulation of an otherwise default inhibitory status. It is noteworthy that such a mode of regulation mirrors the regulation of the enzymic activity of many protein kinases, including phosphorylase kinase. With phosphorylase kinase, both its transcriptional regulation as well as the regulation of the protein itself, are primed to maintain the γ catalytic subunit either unexpressed or inactivate respectively, until a positive signal occurs to override an otherwise dominant default inhibitory condition.
Metal imbalance and compromised antioxidant function are early changes in prion disease
Authors: THACKRAY Alana M. , KNIGHT Robert , HASWELL Stephen J. , BUJDOSO Raymond , BROWN David R.
Published: August 2015

The prion protein (PrP) has been shown to bind copper. In the present study we have investigated whether prion disease in a mouse scrapie model resulted in modification of metal concentrations. We found changes in the levels of copper and manganese in the brains of scrapie-infected mice prior to the onset of clinical symptoms. Interestingly, we noted a major increase in blood manganese in the early stages of disease. Analysis of purified PrP from the brains of scrapie-infected mice also showed a reduction in copper binding to the protein and a proportional decrease in antioxidant activity between 30 and 60 days post-inoculation. We postulate that alterations in trace-element metabolism as a result of changes in metal binding to PrP are central to the pathological modifications in prion disease.
Increasing the redox potential of isoform 1 of yeast cytochrome c through the modification of select haem interactions
Authors: LETT C. Marc , GUILLEMETTE J. Guy
Published: August 2015

The oxidation—reduction potential of eukaryotic cytochromes c varies very little from species to species. We have introduced point mutations into isoform 1 of yeast cytochrome c (iso-1-cytochrome c) to selectively engineer a protein with a higher redox potential. Of the ten different mutant proteins generated for the present investigation Y67R, Y67K and W59H were found to be non-functional. Three other mutant proteins, L32M, L32T and T49K, were functional, but too unstable for biophysical studies. Mutant cytochromes c K79S, K79T, Y48H and Y48K were purified and characterized. The Y48K mutant is the only one that exhibits a significant increase of +117mV in redox potential compared with the wild-type protein while still supporting oxidative phosphorylation invivo. Low temperature difference spectroscopy confirmed the formation of the holoprotein, while adsorption and CD spectroscopy reveal perturbations in the structure of Y48K iso-1-cytochrome c.
Phosphorylation of Xenopus transcription factor IIIA by an oocyte protein kinase CK2
Authors: WESTMARK Cara J. , GHOSE Romi , HUBER Paul W.
Published: August 2015

Transcription factor IIIA (TFIIIA), isolated from the cytoplasmic 7S ribonucleoprotein complex of Xenopus oocytes, is phosphorylated when incubated with [γ-32P]ATP. This modification is due to a trace kinase activity that remains associated with the factor through several steps of purification. The kinase can use either ATP or GTP, and will phosphorylate casein and phosvitin to the exclusion of TFIIIA. The kinase is reactive with a ten-amino-acid peptide that is a specific substrate for protein kinase CK2 (CK2; formerly casein kinase II). In addition, inhibition of phosphorylation by heparin and stimulation by spermidine indicate that the activity can be ascribed to CK2. Phospho amino acid analysis established that serine is the sole phosphoryl acceptor in TFIIIA. There are four consensus sites for CK2 in TFIIIA; all contain serine residues at the putative site of phosphorylation. TFIIIA immunoprecipitated from oocytes, which were incubated with [32P]orthophosphate, is also phosphorylated exclusively on serine residues. Only the cyanogen bromide fragment, which was derived from the N-terminal end of TFIIIA, is labelled in vivo. A recognition sequence for CK2, located at Ser16 in the β-turn of the first zinc-finger domain, is the only protein kinase consensus sequence present in this peptide. Assays in vitro with site-specific mutants of TFIIIA established that Ser16 is the preferred site of phosphorylation, with some secondary modification at Ser314.
Mutational analysis of the glucagon receptor: similarities with the vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (PACAP)/secretin receptors for recognition of the ligand's third residue
Authors: PERRET Jason , VAN CRAENENBROECK Mélanie , LANGER Ingrid , VERTONGEN Pascale , GREGOIRE Françoise , ROBBERECHT Patrick , WAELBROECK Magali
Published: August 2015

Receptor recognition by the Asp3 residues of vasoactive intestinal peptide and secretin requires the presence of a lysine residue close to the second transmembrane helix (TM2)/first extracellular loop junction and an ionic bond with an arginine residue in TM2. We tested whether the glucagon Gln3 residue recognizes the equivalent positions in its receptor. Our data revealed that the binding and functional properties of the wild-type glucagon receptor and the K188R mutant were not significantly different, whereas all agonists had markedly lower potencies and affinities at the I195K mutated receptor. In contrast, glucagon was less potent and the Asp3-, Asn3- and Glu3-glucagon mutants were more potent and efficient at the double-mutated K188R/I195K receptor. Furthermore, these alterations were selective for position 3 of glucagon, as shown by the functional properties of the mutant Glu9- and Lys15-glucagon. Our results suggest that although the Gln3 residue of glucagon did not interact with the equivalent binding pocket as the Asp3 residue of vasoactive intestinal peptide or secretin, the Asp3-glucagon analogue was able to interact with position 188 of the K188R/I195K glucagon receptor. Nevertheless, the Gln3 side chain of glucagon probably binds very close to this region in the wild-type receptor.
A natural protective mechanism against hyperglycaemia in vascular endothelial and smooth-muscle cells: role of glucose and 12-hydroxyeicosatetraenoic acid
Authors: ALPERT Evgenia , GRUZMAN Arie , TOTARY Hanan , KAISER Nurit , REICH Reuven , SASSON Shlomo
Published: August 2015

Bovine aortic endothelial and smooth-muscle cells down-regulate the rate of glucose transport in the face of hyperglycaemia, thus providing protection against deleterious effects of increased intracellular glucose levels. When exposed to high glucose concentrations these cells reduced the mRNA and protein content of their typical glucose transporter, GLUT-1, as well as its plasma-membrane abundance. Inhibition of the lipoxygenase (LO) pathway, and particularly 12-LO, reversed this glucose-induced down-regulatory process and restored the rate of hexose transport to the level seen in vascular cells exposed to normal glucose levels. This reversal was accompanied by increased levels of GLUT-1 mRNA and protein, as well as of its plasma-membrane content. Exposure of the vascular cells to elevated glucose concentrations increased by 2–3-fold the levels of cell-associated and secreted 12-hydroxyeicosatetraenoic acid (12-HETE), the product of 12-LO. Inhibition of 15- and 5-LO, cyclo-oxygenases 1 and 2, and eicosanoid-producing cytochrome P450 did not modify the hexose-transport system in vascular cells. These results suggest a role for HETEs in the autoregulation of hexose transport in vascular cells. 8-Iso prostaglandin F2α, a non-enzymic oxidation product of arachidonic acid, had no effect on the hexose-transport system in vascular cells exposed to hyperglycaemic conditions. Taken together, these findings show that hyperglycaemia increases the production rate of 12-HETE, which in turn mediates the down-regulation of GLUT-1 expression and the glucose-transport system in vascular endothelial and smooth-muscle cells.
Nitric oxide inactivation in brain by a novel O2-dependent mechanism resulting in the formation of nitrate ions
Authors: GRIFFITHS Charmaine , YAMINI Bakhtiar , HALL Catherine , GARTHWAITE John
Published: August 2015

In order for nitric oxide (NO) to function as a biological messenger it has to be inactivated, but little is known of how this is achieved. In cells from the brain, we have recently shown the existence of a powerful NO sink that ‘shapes’ NO signals for targeting its receptor, soluble guanylate cyclase, whilst simultaneously preventing NO rising to toxic concentrations [Griffiths and Garthwaite (2001) J. Physiol. (Cambridge, U.K.) 536, 855–862]. In the present study, the properties of this sink were investigated further. Inactivation of NO was preserved in rat brain homogenates. In both cerebellar cell suspensions and brain homogenates, NO inactivation required O2 and, from measurements in homogenates, the principal end-product was NO−3, which is also the main product of endogenously formed NO in vivo. Direct chemical reaction with O2, superoxide anions or haemoglobin was not responsible. Consumption of NO was, however, inhibited by heat or protease treatment. Pharmacological tests were negative for several candidate enzymes, namely cytochrome c oxidase, H2O2-dependent haem peroxidases, prostaglandin H synthase, 12/15-lipoxygenase and a flavohaemoglobin-like NO dioxygenase. The capacity of the NO sink in cells was limited because regeneration of the activity was slow (2h). It is concluded that NO is consumed in the brain through a novel protein, ultimately forming NO−3, and that the slow regeneration of the activity provides a scenario for NO to become toxic.
Dehydroascorbic acid uptake by coronary artery smooth muscle: effect of intracellular acidification
Authors: HOLMES Melanie E. , MWANJEWE James , SAMSON Sue E. , HAIST James V. , WILSON John X. , DIXON S. Jeffrey , KARMAZYN Morris , GROVER Ashok K.
Published: August 2015

Dehydroascorbic acid (DHAA) enters cells via Na+-independent glucose transporters (GLUT) and is converted to ascorbate. However, we found that Na+ removal inhibited [14C]DHAA uptake by smooth-muscle cells cultured from pig coronary artery. The uptake was examined for 2–12min at 10–200μM DHAA in either the presence of 134mM Na+ or in its absence (N-methyl d-glucamine, choline or sucrose replaced Na+). This inhibition of DHAA uptake by Na+ removal was paradoxical because it was inhibited by 2-deoxyglucose and cytochalasin B, as expected of transport via the GLUT pathway. We tested the hypothesis that this paradox resulted from an inefficient intracellular reduction of [14C]DHAA into [14C]ascorbate upon intracellular acidosis caused by the Na+ removal. Consistent with this hypothesis: (i) the Na+/H+-exchange inhibitors ethylisopropyl amiloride and cariporide also decreased the uptake, (ii) Na+ removal and Na+/H+-exchange inhibitors lowered cytosolic pH, with the decrease being larger in 12min than in 2min, and (iii) less of the cellular 14C was present as ascorbate (determined by HPLC) in cells in Na+-free buffer than in those in Na+-containing buffer. This inability to obtain ascorbate from extracellular DHAA may be detrimental to the coronary artery under hypoxia-induced acidosis during ischaemia/reperfusion.
Essential stations in the intracellular pathway of cytotoxic bovine seminal ribonuclease
Authors: BRACALE Aurora , SPALLETTI-CERNIA Daniela , MASTRONICOLA Mariarosaria , CASTALDI Francesco , MANNUCCI Roberta , NITSCH Lucio , D'ALESSIO Giuseppe
Published: August 2015

Bovine seminal RNase (BS-RNase) is a dimeric RNase selectively cytotoxic for malignant cells. No information is available on its pathway from the extracellular matrix through the cytosol, where it degrades rRNA. An investigation of this pathway is reported here, carried out by immunofluorescence studies, by assessing the effects on BS-RNase cytotoxicity of drugs that affect specific intracellular compartments and by assaying the behaviour of a protein variant, BS-RNase-KDEL (BS-RNase in which a Lys-Asp-Glu-Leu peptide segment is inserted at the C-terminal ends of the subunit chains), endowed with a consensus sequence that directs proteins to the endoplasmic reticulum. BS-RNase was found to bind both normal and malignant cells and to be internalized by both cell types in endosome vesicles. Non-cytotoxic RNases, such as RNase A and a monomeric derivative of BS-RNase, did not bind to the cell surface and were not internalized. However, an engineered, dimeric and cytotoxic variant of RNase A bound effectively and permeated cells. The results of immunofluorescence studies, the effects of nigericin, monensin and brefeldin A on the cytotoxic action of seminal RNase, and the behaviour of the BS-RNase-KDEL variant, led to the conclusion that the pathway of BS-RNase in malignant cells from the extracellular matrix to the cytosol has two essential intracellular stations: endosomes and the trans-Golgi network. In normal cells, however, the protein does not progress from the endosomal compartment to the Golgi complex.
GTP binds to Rab3A in a complex with Ca2+/calmodulin
Authors: PARK Jae-Bong , KIM Jun-Sub , LEE Jae-Yong , KIM Jaebong , SEO Ji-Yeon , KIM Ah-Ram
Published: August 2015

Ras-like small GTP-binding proteins of the Rab family regulate trafficking of the secretory or endocytic pathways. Rab3 proteins within the Rab family are expressed at high levels in neurons and endocrine cells, where they regulate release of dense-core granules and synaptic vesicles (SVs). Rab3A is present as either the soluble or the SV membrane-bound form in neurons that are dependent on the GDP- or GTP-bound states respectively. GDP dissociation inhibitor (GDI) is known to induce the dissociation of Rab3A from synaptic membranes when GTP is depleted. In an earlier study, Ca2+/calmodulin (CaM) was also shown to dissociate Rab3A from synaptic membranes by forming an equimolar complex with Rab3A in vitro. We have examined a possible role for Ca2+/CaM in modulating both the binding of guanine nucleotides to Rab3A and the GTPase activity of Rab3A. The basal level of Rab3A GTPase activity was not affected by an association with Ca2+/CaM. Ca2+/CaM—Rab3A complex that was formed in synaptic membranes was able to bind guanine nucleotides, whereas the Rab3A—GDI complex could not. In addition, Ca2+/CaM led to the replacement of the GDP molecule in the Rab3A—GDI complex with GTP in Rab3A. Taken together, these results suggest that CaM may have a role in stimulating GTP binding to Rab3A that is complexed with GDI, which leads to the formation of an active GTP-bound form of the Rab3A—Ca2+/CaM complex.
Sorbitol activates atypical protein kinase C and GLUT4 glucose transporter translocation/glucose transport through proline-rich tyrosine kinase-2, the extracellular signal-regulated kinase pathway and phospholipase D
Authors: SAJAN Mini P. , BANDYOPADHYAY Gautam , KANOH Yoshinori , STANDAERT Mary L. , QUON Michael J. , REED Brent C. , DIKIC Ivan , FARESE Robert V.
Published: August 2015

Sorbitol, ‘osmotic stress’, stimulates GLUT4 glucose transporter translocation to the plasma membrane and glucose transport by a phosphatidylinositol (PI) 3-kinase-independent mechanism that reportedly involves non-receptor proline-rich tyrosine kinase-2 (PYK2) but subsequent events are obscure. In the present study, we found that extracellular signal-regulated kinase (ERK) pathway components, growth-factor-receptor-bound-2 protein, son of sevenless (SOS), RAS, RAF and mitogen-activated protein (MAP) kinase/ERK kinase, MEK(−1), operating downstream of PYK2, were required for sorbitol-stimulated GLUT4 translocation/glucose transport in rat adipocytes, L6 myotubes and 3T3/L1 adipocytes. Furthermore, sorbitol activated atypical protein kinase C (aPKC) through a similar mechanism depending on the PYK2/ERK pathway, independent of PI 3-kinase and its downstream effector, 3-phosphoinositide-dependent protein kinase-1 (PDK-1). Like PYK2/ERK pathway components, aPKCs were required for sorbitol-stimulated GLUT4 translocation/glucose transport. Interestingly, sorbitol stimulated increases in phospholipase D (PLD) activity and generation of phosphatidic acid (PA), which directly activated aPKCs. As with aPKCs and glucose transport, sorbitol-stimulated PLD activity was dependent on the ERK pathway. Moreover, PLD-generated PA was required for sorbitol-induced activation of aPKCs and GLUT4 translocation/glucose transport. Our findings suggest that sorbitol sequentially activates PYK2, the ERK pathway and PLD, thereby increasing PA, which activates aPKCs and GLUT4 translocation. This mechanism contrasts with that of insulin, which primarily uses PI 3-kinase, D3-PO4 polyphosphoinositides and PDK-1 to activate aPKCs.
Mode of inhibition of HIV-1 reverse transcriptase by polyacetylenetriol, a novel inhibitor of RNA- and DNA-directed DNA polymerases
Authors: LOYA Shoshana , RUDI Amira , KASHMAN Yoel , HIZI Amnon
Published: August 2015

Polyacetylenetriol (PAT), a natural marine product from the Mediterranean sea sponge Petrosia sp., was found to be a novel general potent inhibitor of DNA polymerases. It inhibits equally well the RNA- and DNA-dependent DNA polymerase activities of retroviral reverse transcriptases (RTs) (i.e. of HIV, murine leukaemia virus and mouse mammary tumour virus) as well as cellular DNA polymerases (i.e. DNA polymerases α and β and Escherichia coli polymerase I). A study of the mode and mechanism of the polymerase inhibition by PAT has been conducted with HIV-1 RT. PAT was shown to be a reversible non-competitive inhibitor. PAT binds RT independently and at a site different from that of the primer-template and dNTP substrates with high affinity (Ki = 0.51μM and Ki = 0.53μM with dTTP and with dGTP as the variable substrates respectively). Blocking the polar hydroxy groups of PAT has only a marginal effect on the inhibitory capacity, thus hydrophobic interactions are likely to play a major role in inhibiting RT. Preincubation of RT with the primer-template substrate prior to the interaction with PAT reduces substantially the inhibition capacity, probably by preventing these contacts. PAT does not interfere with the first step of polymerization, the binding of RT to DNA, nor does the inhibitor interfere with the binding of dNTP to RT/DNA complex, as evident from the steady-state kinetic study, whereby Km remains unchanged. We assume, therefore, that PAT interferes with subsequent catalytic steps of DNA polymerization. The inhibitor may alter the optimal stereochemistry of the polymerase active site relative to the primer terminus, bound dNTP and the metal ions that are crucial for efficient catalysis or, alternatively, may interfere with the thumb sub-domain movement and, thus, with the translocation of the primer-template following nucleotide incorporation.
Evidence that cytosolic calcium increases are not sufficient to stimulate phospholipid scrambling in human T-lymphocytes
Authors: WURTH Georjeana A. , ZWEIFACH Adam
Published: August 2015

Phospholipid scrambling, the disruption of normal plasma-membrane asymmetry, occurs during apoptotic and necrotic cell death and during the activation of platelets and neutrophils. It is currently believed that phospholipid scrambling is triggered simply by increases in bulk cytosolic [Ca2+]. We have presented evidence previously that the styryl dye FM1-43 is sensitive to phospholipid scrambling in Jurkat human leukaemic T-lymphocytes. Here we have used FM1-43, in combination with fura 2 and the Ca2+-elevating agents ionomycin and thapsigargin, in imaging experiments to test the idea that increases in bulk cytosolic [Ca2+] stimulate scrambling. Intracellular Ca2+ increases of ≈ 2μM accompanied ionomycin-stimulated scrambling in ≈ 50% of cells, and scrambling occurred in > 99% of cells in which intracellular Ca2+ rose to 4μM. Chelating intracellular Ca2+ with bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid or EGTA suppressed both ionomycin-stimulated intra cellular Ca2+ increases and scrambling, demonstrating that intracellular Ca2+ increases are necessary for ionomycin-stimulated scrambling. However, elevating intracellular Ca2+ to 2–4μM with thapsigargin, a drug that depletes intracellular Ca2+ stores and triggers Ca2+ entry via Ca2+-release-activated Ca2+ channels, did not trigger scrambling, as assessed with either FM1-43 or FITC-labelled annexin V. These results suggest that increases in intracellular [Ca2+] are necessary but not sufficient to stimulate scrambling in lymphoyctes, and indicate that ionomycin has an additional effect that is required to stimulate scrambling.
Complete β-oxidation of valproate: cleavage of 3-oxovalproyl-CoA by a mitochondrial 3-oxoacyl-CoA thiolase
Authors: SILVA Margarida F. B. , RUITER Jos P. N. , OVERMARS Henk , BOOTSMA Albert H. , van GENNIP Albert H. , JAKOBS Cornelis , DURAN Marinus , TAVARES de ALMEIDA Isabel , WANDERS Ronald J. A.
Published: August 2015

The β-oxidation of valproic acid (VPA; 2-n-propylpentanoic acid) was investigated in vitro in intact rat liver mitochondria incubated with 3H-labelled VPA. The metabolism of [4,5-3H2]VPA and [2-3H]VPA was studied by analysing the different acyl-CoA intermediates formed by reverse-phase HPLC with radiochemical detection. Valproyl-CoA, Δ2(E)-valproyl-CoA,3-hydroxyvalproyl-CoA and 3-oxovalproyl-CoA (labelled and non-labelled) were determined using continuous on-line radiochemical and UV detection. The formation of these intermediates was investigated using the two tritiated precursors in respiratory states 3 and 4. Valproyl-CoA was present at highest concentrations under both conditions. Two distinct labelled peaks were found, which were identified as 3H2O and [4,5-3H2]3-oxo-VPA. The formation of 3H2O strongly suggested that VPA underwent complete β-oxidation and that [4,5-3H2]3-oxo-VPA was formed by hydrolysis of the corresponding thioester. The hypothesis that 3-oxovalproyl-CoA undergoes thiolytic cleavage was investigated further. For this purpose a mito chondrial lysate was incubated with synthetic 3-oxovalproyl-CoA, carnitine and carnitine acetyltransferase for subsequent monitoring of the formation of propionylcarnitine and pentanoylcarnitine using electrospray ionization tandem MS. The detection of these compounds demonstrated unequivocally that the intermediate 3-oxovalproyl-CoA is a substrate of a mitochondrial thiolase, producing propionyl-CoA and pentanoyl-CoA, thus demonstrating the complete β-oxidation of VPA in the mitochondrion. Our data should lead to a re-evaluation of the generally accepted concept that the biotransformation of VPA by mitochondrial β-oxidation is incomplete.
Actopaxin is phosphorylated during mitosis and is a substrate for cyclin B1/cdc2 kinase
Authors: CURTIS Michael , NIKOLOPOULOS Sotiris N. , TURNER Christopher E.
Published: August 2015

Prior to cell division, normal adherent cells adopt a round morphology that is associated with a loss of actin stress fibres and disassembly of focal adhesions. In this study, we investigate the mitotic phosphorylation of the recently described paxillin and actin-binding focal-adhesion protein actopaxin [Nikolopoulos and Turner (2000) J. Cell Biol. 151, 1435–1448]. Actopaxin is comprised of an N-terminus containing six putative cdc2 phosphorylation sites and a C-terminus consisting of tandem calponin homology domains. Here we show that the N-terminus of actopaxin is phosphorylated by cyclin B1/cdc2 kinase in vitro and that this region of actopaxin precipitates cdc2 kinase activity from mitotic lysates. Actopaxin exhibits reduced electrophoretic mobility during mitosis that is dependent on phosphorylation within the first two consensus cdc2 phosphorylation sites. Finally, as cells progress from mitosis to G1 there is an adhesion-independent dephosphorylation of actopaxin, suggesting that actopaxin dephosphorylation precedes cell spreading and the reformation of focal adhesions. Taken together, these results suggest a role for cyclin B1/cdc2-dependent phosphorylation of actopaxin in regulating actin cytoskeleton reorganization during cell division.
Identification of protein-derived tyrosyl radical in the reaction of cytochrome c and hydrogen peroxide: characterization by ESR spin-trapping, HPLC and MS
Authors: QIAN Steven Yue , CHEN Yeong-Renn , DETERDING Leesa J. , FANN Yang C. , CHIGNELL Colin F. , TOMER Kenneth B. , MASON Ronald P.
Published: August 2015

The reaction of cytochrome c and H2O2 is known to form a protein-centred radical that can be detected with the spin trap 2-methyl-2-nitrosopropane (MNP). To characterize the MNP/tyrosyl adduct structure that had previously been determined incorrectly [Barr, Gunther, Deterding, Tomer and Mason (1996) J. Biol. Chem. 271, 15498–15503], we eliminated unreasonable structure models by ESR studies with a series of 13C-labelled tyrosines, and photochemically synthesized an authentic MNP/tyrosyl adduct that has its trapping site on the C-3 position of the tyrosine phenyl ring. The observation of the identical ESR spectra for this radical adduct from the UV irradiation of 3-iodo-tyrosine and the adduct from the cytochrome c reaction demonstrated that the radical trapping site of MNP/tyrosyl is located on the equivalent C-3/C-5 positions instead of the C-1 position, as was proposed by Barr et al. In an on-line HPLC/ESR system, an identical retention time (17.7min) was observed for the ESR-active HPLC peak of the MNP/tyrosyl adduct from the following three reactions: (i) the tyrosine oxidation via horseradish peroxidase/H2O2; (ii) UV irradiation of 3-iodo-tyrosine and (iii) the reaction of cytochrome c with H2O2. This result demonstrated that the radical adducts of all three reactions are most probably the same. The mass spectrometric analysis of the HPLC fractions from reactions (i) and (ii) showed an ion at m/z 267 attributed to the MNP/tyrosyl adduct. We conclude that the cytochrome c-derived tyrosyl radical was trapped by MNP, leading to a persistent radical adduct at the C-3/C-5 positions of the tyrosine phenyl ring.
Modulation of Mycobacterium tuberculosis DnaA protein–adenine-nucleotide interactions by acidic phospholipids
Authors: YAMAMOTO Kohji , MUNIRUZZAMAN Syed , RAJAGOPALAN Malini , MADIRAJU Murty V.V.S.
Published: August 2015

The biochemical aspects of the initiation of DNA replication in Mycobacterium tuberculosis are unknown. To understand this process, we overproduced, purified and characterized the recombinant M. tuberculosis DnaA protein. The M. tuberculosis DnaA protein binds the origin of replication (oriC), ATP and ADP, and exhibited weak ATPase activity. ADP, after hydrolysis of ATP, remained strongly associated with DnaA and the exchange of ATP for bound ADP was weak. Vesicles prepared from acidic phospholipids, such as phosphatidylinositol, cardiolipin and phosphatidylglycerol, promoted dissociation of both ADP and ATP, whereas the neutral phospholipid phosphatidylethanolamine did not. The phospholipid-mediated dissociation of ATP was decreased in the presence of the M. tuberculosis oriC, whereas dissociation of ADP was stimulated in the presence of oriC. Acidic phospholipids in micelles, however, were not efficient in dissociating bound nucleotides from DnaA. Together, these results suggest that both polar head groups and membrane bilayer structure play an important role in M. tuberculosis DnaA—adenine-nucleotide interactions. We suggest that initiation of M. tuberculosis oriC involves intimate interactions between DnaA, adenine nucleotides and membrane phospholipids, and the latter helps to ensure that only the ATP form of the DnaA protein interacts continuously with oriC.
Substrate-binding recognition and specificity of trehalose phosphorylase from Schizophyllum commune examined in steady-state kinetic studies with deoxy and deoxyfluoro substrate analogues and inhibitors
Authors: EIS Christian , NIDETZKY Bernd
Published: August 2015

Trehalose phosphorylase is a component of the α-d-glucopyranosyl α-d-glucopyranoside (α,α-trehalose)-degrading enzyme system in fungi and it catalyses glucosyl transfer from α,α-trehalose to phosphate with net retention of the anomeric configuration. The enzyme active site has no detectable affinity for α,α-trehalose in the absence of bound phosphate and catalysis occurs from the ternary complex. To examine the role of non-covalent enzyme—substrate interactions for trehalose phosphorylase recognition, we used the purified enzyme from Schizophyllum commune and tested a series of incompetent structural analogues of the natural substrates and products as inhibitors of the enzyme. Equilibrium-binding constants (Ki) for deoxy- and deoxyfluoro derivatives of d-glucose show that loss of interactions with the 3-, 4- or 6-OH, but not the reactive 1- and the 2-OH, results in considerably (≥100-fold) weaker affinity for sugar-binding subsite +1, revealing the requirement for hydrogen bonding with hydroxyls, away from the site of chemical transformation to position precisely the d-glucose-leaving group/nucleophile for catalysis. The high specificity of trehalose phosphorylase for the sugar aglycon during binding and conversion of O-glycosides is in contrast with the observed α-retaining phosphorolysis of α-d-glucose-1-fluoride (α-d-Glc-1-F) since the productive bonding capability of the fluoride-leaving group with subsite +1 is minimal. The specificity constant (19M−1·s−1) and catalytic-centre activity (0.1s−1) for the reaction with α-d-Glc-1-F are 0.10- and 0.008-fold the corresponding kinetic parameters for the enzymic reaction with α,α-trehalose. The non-selective-inhibition profile for a series of inactive α-d-glycopyranosyl phosphates shows that the driving force for the binary-complex formation lies mainly in interactions of the enzyme with the phosphate group and suggests that hydrogen bonding with hydroxyl groups at the catalytic site (subsite −1) contributes to catalysis by providing stabilization, which is specific to the transition state. Vanadate, a tight-binding phosphate mimic, inhibits the phosphorolysis of α-d-Glc-1-F by forming a ternary complex whose apparent dissociation constant of 120μM is approx. 160-fold greater than the dissociation constant of the same inhibitor complex with α,α-trehalose.
Phospholipase C-γ1 is required for cell survival in oxidative stress by protein kinase C
Authors: BAI Xiao-Chun , DENG Fan , LIU An-Ling , ZOU Zhi-Peng , WANG Yu , KE Zhi-Yong , JI Qun-Sheng , LUO Shen-Qiu
Published: August 2015

Phospholipase C-γ1 (PLC-γ1) activation has been reported to enhance cell survival during the cellular response to oxidative stress. We studied the role of protein kinase C (PKC) pathways in mediating PLC-γ1 survival signalling in oxidative stress by using mouse embryonic fibroblasts genetically deficient in PLC-γ1 (Plcg1−/−) and its wild type (Plcg1+/+). PLC-γ1 was activated by H2O2 treatment in a dose- and time-dependent manner. Activation of PKC was also markedly increased in both cell lines treated with H2O2 (1–5mM), but with low doses (50–200μM), PKC activation was considerably decreased in Plcg1−/− cells. After treatment with H2O2, PKC-dependent phosphorylation of Bcl-2 and cell viability of Plcg1−/− cells decreased dramatically and caspase-3-like activity increased significantly compared with that of the wild-type cells. Furthermore, pretreatment of Plcg1+/+ cells with PKC-specific inhibitor decreased levels of PKC-dependent Bcl-2 phosphorylation, enhanced caspase-3 activity and their sensitivity to H2O2. On the contrary, treatment of Plcg1−/− cells with PKC-specific activator increased the Bcl-2 phosphorylation, decreased caspase-3 activity and improved their survival. These results suggest that PLC-γ1 mediates survival signalling in oxidative-stress response by PKC-dependent phosphorylation of Bcl-2 and inhibition of caspase-3.
Probing the serpin structural-transition mechanism in ovalbumin mutant R339T by proteolytic-cleavage kinetics of the reactive-centre loop
Authors: ARII Yasuhiro , HIROSE Masaaki
Published: August 2015

A mutant ovalbumin (R339T), but not the wild-type protein, is transformed into the canonical loop-inserted, thermostabilized form after the P1—P1′ cleavage [Yamasaki, Arii, Mikami and Hirose (2002) J. Mol. Biol. 315, 113–120]. The loop-insertion mechanism in the ovalbumin mutant was investigated by proteolytic-cleavage kinetics. The nature of the inserted loop prevented futher cleavage of the P1—P1′ pre-cleaved R339T mutant by subtilisin, which cleaved the second P8—P7 loop site in the P1—P1′ pre-cleaved wild-type protein. After subtilisin proteolysis of the intact R339T, however, two final products that corresponded to the single P1—P1′ and double P1—P1′/P8—P7 cleavages were generated with variable ratios depending on the proteolysis conditions. This was accounted for by the occurrence of two mutually competitive reactions: the loop-insertion reaction and the proteolytic cleavage of the second P8—P7 site in the immediate intermediate after the P1—P1′ cleavage. The competitive nature of the two reactions enabled us to establish a kinetic method to determine the rate constants of the reactions. The first-order rate constant for the loop insertion was determined to be 4.0×10−3/s in the R339T mutant. The second-order rate constant for the P8—P7 cleavage in the immediate P1—P1′ cleavage product for the R339T mutant was >10 times compared with that for its wild-type counterpart. This highly accessible loop nature may play a crucial role in the loop-insertion mechanism for R339T mutant ovalbumin.
Expression of the two mRNA isoforms of the iron transporter Nrmap2/DMTI in mice and function of the iron responsive element
Authors: TCHERNITCHKO Dimitri , BOURGEOIS Monique , MARTIN Marie-Elise , BEAUMONT Carole
Published: August 2015

Nramp2/DMT1 is a transmembrane proton-coupled Fe2+ transporter. Two different mRNAs are generated by alternative splicing; isoform I contains an iron responsive element (IRE), whereas isoform II does not. They encode two proteins differing at their C-terminal end and by their subcellular localization. IRE-mediated stabilization of isoform I mRNA is thought to stimulate DMT1 expression in response to iron deficiency. We have measured the two mRNAs by real-time quantitative PCR in several mouse tissues, in normal conditions or following injection of phenylhydrazine, a potent haemolytic agent. Isoform I mRNA is expressed in the duodenum and is induced by stimulation of erythropoiesis, whereas the non-IRE isoform is mostly induced in erythropoietic spleen. Surprisingly, both isoforms are highly expressed in the kidney and are not regulated by erythropoiesis. To evaluate the role of the IRE in regulating isoform I mRNA stability, in response to variations in cell iron status, several constructs were made in pCDNA3 with either a normal or a mutated IRE placed at the 3′ end of a stable mRNA. These constructs were transfected into HT29 cells and mRNAs were analysed after growing cells in the presence or absence of exogenous iron. There was no difference in the level of expression of the different messages, suggesting that the IRE does not regulate stability of isoform I mRNA. The half-life of the endogenous IRE-mRNA was also measured following actinomycin D addition in iron- or desferrioxamine-treated cells. Decay of the mRNA was very similar in both conditions. These results suggest that additional transcriptional regulations at the promoter level, or iron-dependent regulation of alternative splicing are likely to participate in the induction of isoform I mRNA by iron deficiency.
Modulation of the electrostatic charge at the active site of foot-and-mouth-disease-virus leader proteinase, an unusual papain-like enzyme
Authors: SCHLICK Petra , KRONOVETR Jakub , HAMPOELZ Bernhard , SKERN Tim
Published: August 2015

The leader proteinase (Lpro) of foot-and-mouth-disease virus is an unusual papain-like cysteine proteinase. Synthesized without an N-terminal pro precursor region, it frees itself from the growing polypeptide chain by cleavage at its own C-terminus. It also possesses a unique electrostatic environment around the active site, essentially due to Asp163, which orients the catalytic histidine residue, and Asp164; the equivalent residues in papain are Asn175 and Ser176. The importance of these residues for Lpro activity was examined by site-directed mutagenesis. Replacement of Asp163 with asparagine reduced activity by five-fold towards a hexapeptide substrate and slightly delayed self-processing when expressed in rabbit reticulocyte lysates. However, no effect on the cleavage of the only known cellular substrate of Lpro, eukaryotic initiation factor 4GI (eIF4GI), was observed. In contrast, replacement of Asp164 by either alanine, asparagine or lysine abrogated activity towards the hexapeptide. Furthermore, in all cases, the onset of both self-processing and eIF4GI cleavage were significantly delayed; the reaction rates were also diminished compared with those of the wild-type enzyme. The alanine-substituted enzyme was least affected, followed by those substituted with asparagine and lysine. The double mutant protein in which both aspartate residues were replaced by asparagine was most severely affected; it failed to complete either self-processing or eIF4GI cleavage within 3 h, compared with the 8min required by the wild-type enzyme. Hence, we propose that the electrostatic charge of Asp164, and to a lesser extent that of Asp163, is extremely important for Lpro to attain full activity upon synthesis.
Gal3p and Gal1p interact with the transcriptional repressor Gal80p to form a complex of 1:1 stoichiometry
Authors: TIMSON David J. , ROSS Helen C. , REECE Richard J.
Published: August 2015

The genes encoding the enzymes required for galactose metabolism in Saccharomyces cerevisiae are controlled at the level of transcription by a genetic switch consisting of three proteins: a transcriptional activator, Gal4p; a transcriptional repressor, Gal80p; and a ligand sensor, Gal3p. The switch is turned on in the presence of two small molecule ligands, galactose and ATP. Gal3p shows a high degree of sequence identity with Gal1p, the yeast galactokinase. We have mapped the interaction between Gal80p and Gal3p, which only occurs in the presence of both ligands, using protease protection experiments and have shown that this involves amino acid residue 331 of Gal80p. Gel-filtration experiments indicate that Gal3p, or the galactokinase Gal1p, interact directly with Gal80p to form a complex with 1:1 stoichiometry.
The Drosophila odz/ten-m gene encodes a type I, multiply cleaved heterodimeric transmembrane protein
Authors: DGANY Orly , WIDES Ron
Published: August 2015

The product of the Drosophila melanogaster odd Oz (odz)/Tenascin-major (ten-m) pair-rule gene consists of eight epidermal growth factor (EGF)-like repeats followed by a novel 1800 amino acid polypeptide stretch unique to proteins of the Odz/Ten-m family. The structure and membrane orientation of this large enigmatic protein was characterized by raising and employing antibodies directed against discrete Odz polypeptide regions. Protein-modifying reagents impermeable to the plasma membrane were used in concert with the battery of antibodies to demonstrate that Odz is a type I transmembrane protein with the vast C-terminal portion in the intracellular space, and with the EGF repeats deployed extracellularly. The polypeptide was shown to undergo multiple cleavages at discrete intracellular and extracellular sites, and its extreme C-terminus was shown to undergo either processing at a very large number of sites or programmed degradation. The polypeptide is presented at the cell surface with additional post-translational modifications, and as two subunits of previously cleaved Odz joined by cysteine disulphide bridges maintaining their association. The model derived for the Odz protein is discussed in light of other models proposed for proteins of the Odz/Ten-m family, and in terms of functional implications.