bluuebunny/crossref_metadata_2025 huggingface dataset:
Tyrosine kinases activate store-mediated Ca2+ entry in human platelets through the reorganization of the actin cytoskeleton
Authors: ROSADO Juan A. , GRAVES Darren , SAGE Stewart O.
Published: August 2015

We have recently reported that store-mediated Ca2+ entry in platelets is likely to be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, a model termed ‘secretion-like coupling’. In this model the actin cytoskeleton plays a key regulatory role. Since tyrosine kinases have been shown to be important for Ca2+ entry in platelets and other cells, we have now investigated the possible involvement of tyrosine kinases in the secretion-like-coupling model. Treatment of platelets with thrombin or thapsigargin induced actin polymerization by a calcium-independent pathway. Methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor, prevented thrombin- or thapsigargin-induced actin polymerization. The effects of tyrosine kinases in store-mediated Ca2+ entry were found to be entirely dependent on the actin cytoskeleton. PP1, an inhibitor of the Src family of proteins, partially inhibited store-mediated Ca2+ entry. In addition, depletion of intracellular Ca2+ stores stimulated cytoskeletal association of the cytoplasmic tyrosine kinase pp60src, a process that was sensitive to treatment with cytochalasin D and PP1, but not to inhibition of Ras proteins using prenylcysteine analogues. Finally, combined inhibition of both Ras proteins and tyrosine kinases resulted in complete inhibition of Ca2+ entry, suggesting that these two families of proteins have independent effects in the activation of store-mediated Ca2+ entry in human platelets.
Phosphatidylcholine-specific phospholipase C and phospholipase D are respectively implicated in mitogen-activated protein kinase and nuclear factor κB activation in tumour-necrosis-factor-α-treated immature acute-myeloid-leukaemia cells
Authors: PLO Isabelle , LAUTIER Dominique , LEVADE Thierry , SEKOURI Hadef , JAFFRÉZOU JeanPierre , LAURENT Guy , BETTAÏEB Ali
Published: August 2015

Tumour necrosis factor-α (TNFα) has been reported to induce potent growth inhibition of committed myeloid progenitor cells, whereas it is a potential growth stimulator of human CD34+CD38- multipotent haematopoietic cells. The present study was aimed at evaluating the respective role of two phospholipases, phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD) in the response of the CD34+ CD38- KG1a cells to TNFα. In these cells TNFα triggered phosphoinositide 3-kinase (PI3K)-dependent PC hydrolysis within 4–8min with concomitant production of both diacylglycerol (DAG) and phosphocholine (P-chol). DAG and P-chol production was accompanied by extracellular-signal-related protein kinase-1 (‘ERK-1’) activation and DNA-synthesis stimulation. PC-PLC stimulation was followed by PI3K-independent PLD activation with concomitant phosphatidic acid (PA) production followed by PA-derived DAG accumulation and sustained nuclear factor κB (NF-κB) activation. PLD/NF-κB signalling activation played no role in the TNFα proliferative effect and conferred no consistent protection of KG1a cells towards antileukaemic agents. Altogether these results suggest that, in KG1a cells, TNFα can stimulate in parallel PC-PLC and PLD, whose lipid products activate in turn mitogen-activated protein kinase (MAP kinase) and NF-κB signalling respectively. Finally, our study suggests that PC-PLC, but not PLD, plays a role in the TNFα proliferative effect in immature myeloid cells.
Inhibition by etomoxir of rat liver carnitine octanoyltransferase is produced through the co-ordinate interaction with two histidine residues
Authors: MORILLAS Montserrat , CLOTET Josep , RUBÍ Blanca , SERRA Dolors , ARIÑO Joaquín , HEGARDT Fausto G. , ASINS Guillermina
Published: August 2015

Rat peroxisomal carnitine octanoyltransferase (COT), which facilitates the transport of medium-chain fatty acids through the peroxisomal membrane, is irreversibly inhibited by the hypoglycaemia-inducing drug etomoxir. To identify the molecular basis of this inhibition, cDNAs encoding full-length wild-type COT, two different variant point mutants and one variant double mutant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae, an organism devoid of endogenous COT activity. The recombinant mutated enzymes showed activity towards both carnitine and decanoyl-CoA in the same range as the wild type. Whereas the wild-type version expressed in yeast was inhibited by etomoxir in an identical manner to COT from rat liver peroxisomes, the activity of the enzyme containing the double mutation H131A/H340A was completely insensitive to etomoxir. Individual point mutations H131A and H340A also drastically reduced sensitivity to etomoxir. Taken together, these results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by etomoxir and that the full inhibitory properties of the drug will be shown only if both histidines are intact at the same time. Our data demonstrate that both etomoxir and malonyl-CoA inhibit COT by interacting with the same sites.
α2-Macroglobulin modulates the immunoregulatory function of the lipocalin placental protein 14
Authors: RIELY Gregory J. , RACHMILEWITZ Jacob , KOO Peter H. , TYKOCINSKI Mark L.
Published: August 2015

Human placental protein 14 (PP14; also known as glycodelin and progesterone-associated endometrial protein) is an immunosuppressive protein of the lipocalin structural superfamily. Mechanisms regulating serum PP14's immunosuppressive activity remain to be elucidated. In the present study, an interaction between PP14 and a major serum protein carrier, α2-macroglobulin (α2M), was documented for the first time. Using native gel electrophoresis, we showed that PP14, as well as its alternative splice variant PP14.2, binds to both α2M and methylamine-activated (MA)-α2M. Cross-competition studies demonstrated that the variants compete for binding to α2M. PP14 bound to α2M and MA-α2M with Kd values of 167±70 and 221±56nM (means±S.D.) respectively, as determined by surface plasmon resonance. Significantly, the addition of α2M or MA-α2M to a T-cell proliferation assay strongly potentiated the inhibitory capacity of PP14. On the basis of these findings, α2M emerges as the first serum protein that can physically associate with, and thereby regulate, PP14. Moreover, this represents the first documented interaction between the protein carrier α2M and a lipocalin protein.
Purified recombinant insulin-degrading enzyme degrades amyloid β-protein but does not promote its oligomerization
Authors: CHESNEAU Valérie , VEKRELLIS Konstantinos , ROSNER Marsha Rich , SELKOE Dennis J.
Published: August 2015

Amyloid β-protein (Aβ) has been implicated as an early and essential factor in the pathogenesis of Alzheimer's disease. Although its cellular production has been studied extensively, little is known about Aβ clearance. Recently, insulin-degrading enzyme (IDE), a 110-kDa metalloendopeptidase, was found to degrade both endogenously secreted and synthetic Aβ peptides. Surprisingly, IDE-mediated proteolysis of [125I]Aβ(1-40) in microglial cell-culture media was accompanied by the formation of 125I-labelled peptides with higher apparent molecular masses, raising the possibility that the degradation products act as ‘seeds’ for Aβ oligomerization. To directly address the role of IDE in Aβ degradation and oligomerization, we investigated the action of purified recombinant wild-type and catalytically inactive IDEs. Our data demonstrate that (i) IDE alone is sufficient to cleave purified Aβ that is either unlabelled, iodinated or 35S-labelled; (ii) the initial cleavage sites are His14–Gln15, Phe19–Phe20 and Phe20–Ala21; and (iii) incubation of IDE with [125I]Aβ, but not with [35S]-Aβ, leads to the formation of slower migrating species on gels. Since iodination labels N-terminal fragments of Aβ, and 35S labels C-terminal products, we analysed unlabelled synthetic fragments of Aβ and determined that only the N-terminal fragments migrate with anomalously high molecular mass. These results indicate that IDE alone is sufficient to degrade Aβ at specific sites, and that its degradation products do not promote oligomerization of the intact Aβ peptide.
Proteins of the endoplasmic-reticulum-associated degradation pathway: domain detection and function prediction
Authors: PONTING Chris P.
Published: August 2015

Sequence database searches, using iterative-profile and Hidden-Markov-model approaches, were used to detect hitherto-undetected homologues of proteins that regulate the endoplasmic reticulum (ER)-associated degradation pathway. The translocon-associated subunit Sec63p (Sec = secretory) was shown to contain a domain of unknown function found twice in several Brr2p-like RNA helicases (Brr2 = bad response to refrigeration 2). Additionally, Cue1p (Cue=coupling of ubiquitin conjugation to ER degradation), a yeast protein that recruits the ubiquitin-conjugating (UBC) enzyme Ubc7p to an ER-associated complex, was found to be one of a large family of putative scaffolding-domain-containing proteins that include the autocrine motility factor receptor and fungal Vps9p (Vps=vacuolar protein sorting). Two other yeast translocon-associated molecules, Sec72p and Hrd3p (Hrd = 3-hydroxy-3-methylglutaryl-CoA reductase degradation), were shown to contain multiple tetratricopeptide-repeat-like sequences. From this observation it is suggested that Sec72p associates with a heat-shock protein, Hsp70, in a manner analogous to that known for Hop (Hsp70/Hsp90 organizing protein). Finally, the luminal portion of Ire1p (Ire=high inositol-requiring), thought to convey the sensing function of this transmembrane kinase and endoribonuclease, was shown to contain repeats similar to those in β-propeller proteins. This finding hints at the mechanism by which Ire1p may sense extended unfolded proteins at the expense of compact folded molecules.
An examination of how structural changes can affect the rate of electron transfer in a mutated bacterial photoreaction centre
Authors: RIDGE Justin P. , FYFE Paul K. , McAULEY Katherine E. , E. VAN BREDERODE Marion , ROBERT Bruno , VAN GRONDELLE Rienk , ISAACS Neil W. , COGDELL Richard J. , JONES Michael R.
Published: August 2015

A series of reaction centres bearing mutations at the (Phe) M197 position were constructed in the photosynthetic bacterium Rhodobacter sphaeroides. This residue is adjacent to the pair of bacteriochlorophyll molecules (PL and PM) that is the primary donor of electrons (P) in photosynthetic light-energy transduction. All of the mutations affected the optical and electrochemical properties of the P bacteriochlorophylls. A mutant reaction centre with the change Phe M197 to Arg (FM197R) was crystallized, and a structural model constructed at 2.3 Å (1Å = 0.1nm) resolution. The mutation resulted in a change in the structure of the protein at the interface region between the P bacteriochlorophylls and the monomeric bacteriochlorophyll that is the first electron acceptor (BL). The new Arg residue at the M197 position undergoes a significant reorientation, creating a cavity at the interface region between P and BL. The acetyl carbonyl substituent group of the PM bacteriochlorophyll undergoes an out-of-plane rotation, which decreases the edge-to-edge distance between the macrocycles of PM and BL. In addition, two new buried water molecules partially filled the cavity that is created by the reorientation of the Arg residue. These waters are in a suitable position to connect the macrocycles of P and BL via three hydrogen bonds. Transient absorption measurements show that, despite an inferred decrease in the driving force for primary electron transfer in the FM197R reaction centre, there is little effect on the overall rate of the primary reaction in the bulk of the reaction-centre population. Examination of the X-ray crystal structure reveals a number of small changes in the structure of the reaction centre in the interface region between the P and BL bacteriochlorophylls that could account for this faster-than-predicted rate of primary electron transfer.
Sequencing, functional expression and characterization of rat NTPDase6, a nucleoside diphosphatase and novel member of the ecto-nucleoside triphosphate diphosphohydrolase family
Authors: BRAUN Norbert , FENGLER Sabine , EBELING Christian , SERVOS Jörg , ZIMMERMANN Herbert
Published: August 2015

We have isolated and characterized the cDNA encoding nucleoside triphosphate diphosphohydrolase 6 (NTPDase6), a novel member of the ecto-nucleoside triphosphate diphosphohydrolase family. The rat-brain-derived cDNA has an open reading frame of 1365bp encoding a protein of 455 amino acid residues, a calculated molecular mass of 49971Da and a predicted N-terminal hydrophobic sequence. It shares 86% sequence identity with the human CD39L2 sequence and 48% and 51% identity respectively with sequences of the two related human and murine nucleoside diphosphatases (CD39L4, NTPDase5/ER-UDPase). The mRNA was expressed in all tissues investigated, revealing two major transcripts with differing abundances. PCR analysis suggests a single open reading frame. A Myc-His-tagged NTPDase6 was expressed in Chinese hamster ovary (CHO) and PC12 cells for immunological analysis and protein isolation. The protein was contained in membrane fractions of transfected CHO cells and occurred in a soluble form in the cell culture supernatants. NTPDase6 preferentially hydrolysed nucleoside 5′-diphosphates. With different substrates the order of activity was GDP>IDP≫UDP,CDP≫ADP. Nucleoside 5′-triphosphates were hydrolysed only to a minor extent and no hydrolysis of nucleoside 5′-monophosphates was observed. The enzyme was strongly and equally activated by Ca2+ and Mg2+ and had a Km for GDP of 211µM. The immunohistochemical analysis of transfected CHO and PC12 cells suggests that NTPDase6 is associated with the Golgi apparatus and to a small extent also with the plasma membrane. The enzyme might support glycosylation reactions in the Golgi apparatus and, when released from cells, might catalyse the hydrolysis of extracellular nucleotides.
Characterization of the mouse dynamin I gene promoter and identification of sequences that direct expression in neuronal cells
Authors: YOO Jiyun , LEE Sang Seop , JEONG Moon-Jin , LEE Kyung Im , KWON Byoung-Mog , KIM Sung-Hoon , PARK Young-Mee , HAN Mi Young
Published: August 2015

Dynamin I is expressed at high levels in brain and its expression is regulated during the developmental stages of brain. To elucidate the molecular mechanism by which the expression is tissue-specifically regulated, we cloned the 5′-flanking region of the mouse dynamin I gene and determined the nucleotide sequence of 1036 bases upstream from the translation start site. Transient transfection studies with a chloramphenicol acetyltransferase reporter gene in neuroblastoma NS20Y and Lewis lung cells demonstrated that the 5′-flanking region has a cell-type-specific promoter activity. Deletion analyses demonstrated that the minimal promoter activity was detected in the proximal region 195bp upstream of the translation initiation codon (-90 to +105). The minimal promoter was embedded in a GC-rich region (75% GC content), in which an Sp1-binding motif and a nuclear factor (NF)-κB-like element (NE-1) were found, but it lacked TATA and CAAT boxes. Mutational analysis and electrophoretic mobility-shift assay analysis revealed that Sp1 binds to the Sp1 site and that this element is critical for the promoter activity of the dynamin I gene. We found that the NE-1 sequence is required for the expression of the dynamin I gene but NEBP (NE-1-binding protein), which binds to the NE-1 sequence, is not NF-κB. We also found that one base in the NE-1 sequence (the underlined G residue in GGGATTCG̲CGGA) is critical for binding specificity to discriminate between NEBP and NF-κB. By UV cross-linking analysis, we found that NEBP is an approx. 104kDa nuclear protein.
Very low surfactant protein C contents in newborn Belgian White and Blue calves with respiratory distress syndrome
Authors: DANLOIS Fabien , ZALTASH Shahparak , JOHANSSON Jan , ROBERTSON Bengt , HAAGSMAN Henk P. , VAN EIJK Martin , BEERS Michael F. , ROLLIN Frédéric , RUYSSCHAERT Jean-Marie , VANDENBUSSCHE Guy
Published: August 2015

We have studied a respiratory distress syndrome (RDS) occurring in newborn calves of the Belgian White and Blue (BWB) breed that represents the large majority of beef cattle in Belgium. Pulmonary surfactant isolated from 14 BWB newborn calves that died from RDS and from 7 healthy controls was analysed for composition and surface activity. An extremely low content or, in some instances, an absence of surfactant protein C (SP-C) was detected in the RDS samples by Western blotting and differential amino acid analysis [0.03±0.01% (w/w) relative to total phospholipids, compared with 0.39±0.06% for healthy controls (means±S.E.M., P < 0.001)]. The contents of surfactant protein B (SP-B) were similar in RDS and control samples. The crude surfactant samples isolated from RDS calves had higher ratios of total protein to total phospholipid, altered phospholipid profiles and lower SP-A contents. Both crude and organic extracts of RDS surfactant samples showed increased dynamic surface tension compared with healthy controls when evaluated with a pulsating-bubble surfactometer. The addition of purified SP-C to organic extracts of RDS surfactant samples lowered surface tension. Strongly decreased levels of mature SP-C associated with fatal RDS and altered surface activity in vitro have, to the best of our knowledge, not been previously reported. The mechanisms underlying RDS and the decrease in SP-C in BWB calves remain to be established.
Endurance training increases stimulation of uncoupling of skeletal muscle mitochondria in humans by non-esterified fatty acids: an uncoupling-protein-mediated effect?
Authors: TONKONOGI Michail , KROOK Anna , WALSH Brandon , SAHLIN Kent
Published: August 2015

Uncoupled respiration (UCR) is an essential property of muscle mitochondria and has several functions in the cell. We hypothesized that endurance training may alter the magnitude and properties of UCR in human muscle. Isolated mitochondria from muscle biopsies taken before and after 6 weeks of endurance exercise training (n = 8) were analysed for UCR. To investigate the role of uncoupling protein 2 (UCP2) and UCP3 in UCR, the sensitivity of UCR to UCP-regulating ligands (non-esterified fatty acids and purine nucleotides) and UCP2 and UCP3 mRNA expression in muscle were examined. Oleate increased the mitochondrial oxygen consumption rate, an effect that was not attenuated by GDP and/or cyclosporin A. The effect of oleate was significantly greater after compared with before training. Training had no effect on UCP2 or UCP3 mRNA levels, but after training the relative increase in respiration rate induced by oleate was positively correlated with the UCP2 mRNA level. In conclusion, we show that the sensitivity of UCR to non-esterified fatty acids is up-regulated by endurance training. This suggests that endurance training causes intrinsic changes in mitochondrial function, which may enhance the potential for regulation of aerobic energy production, prevent excess free radical generation and contribute to a higher basal metabolic rate.
Cloning, expression and gene organization of a human Neu5Acα2–3Galβ1–3GalNAc α2,6-sialyltransferase: hST6GalNAc IV
Authors: HARDUIN-LEPERS Anne , STOKES David C. , STEELANT Wim F.A. , SAMYN-PETIT Bénédicte , KRZEWINSKI-RECCHI Marie-Ange , VALLEJO-RUIZ Veronica , ZANETTA Jean-Pierre , AUGÉ Claudine , DELANNOY Philippe
Published: August 2015

On the basis of the detection of expressed sequence tag (‘EST’) similar to the rat N-acetylgalactosamine α2,6-sialyltransferase (ST6GalNAc) III cDNA, we have identified a novel member of the human ST6GalNAc family. We have isolated a cDNA clone containing an open reading frame that codes for a type II membrane protein of 302 amino acids with a seven-amino-acid cytoplasmic domain, an 18-amino-acid transmembrane domain and the smallest described catalytic domain of 277 amino acids. This predicted sialyltransferase sequence is similar to the rat ST6GalNAc III (46.6%), but was found to be even more similar to the recently reported mouse ST6GalNAc IV (88.1%) on the basis of amino acid sequence identity. Northern-blot analysis showed that the newly identified gene is expressed constitutively in various adult human tissues as a 2.2kb transcript, but was also found to be expressed at lower levels in brain, heart and skeletal muscle as a 2.5kb transcript. Expression of the hST6GalNAc IV gene was investigated by reverse transcription PCR in various human cancer cells, and was found to be present in the majority of cell types with the exception of the carcinoma cell line T47D and pro-monocyte THP cells. The transient expression in COS-7 cells of the full-length cDNA led to the production of an active enzyme sharing the acceptor specificity of the ST6GalNAc family towards Neu5Acα2–3Galβ1–3GalNAcα-O-R (where ‘R’denotes H, benzyl, or a peptidic chain). Detailed analysisin vitro of substrate specificity revealed that the enzyme required the trisaccharide Neu5Acα2–3Galβ1–3GalNAc found on O-glycans and arylglycosides. In addition, we have clarified the genomic organization of ST6GalNAc IV gene.
Polydom: a secreted protein with pentraxin, complement control protein, epidermal growth factor and von Willebrand factor A domains
Authors: GILGÈS Delphine , VINIT Marie-Antoinette , CALLEBAUT Isabelle , COULOMBEL Laure , CACHEUX Valère , ROMEO Paul-Henri , VIGON Isabelle
Published: August 2015

To identify extracellular proteins with epidermal growth factor (EGF) domains that are potentially involved in the control of haemopoiesis, we performed degenerate reverse-transcriptase-mediated PCR on the murine bone-marrow stromal cell line MS-5 and isolated a new partial cDNA encoding EGF-like domains related to those in the Notch proteins. Cloning and sequencing of the full-length cDNA showed that it encoded a new extracellular multi-domain protein that we named polydom. This 387kDa mosaic protein contained a signal peptide followed by a new association of eight different protein domains, including a pentraxin domain and a von Willebrand factor type A domain, ten EGF domains, and 34 complement control protein modules. The human polydom mRNA is strongly expressed in placenta, its expression in the other tissues being weak or undetectable. The particular multidomain structure of the encoded protein suggests an important biological role in cellular adhesion and/or in the immune system.
Thermodynamic mixing of molecular states of the epidermal growth factor receptor modulates macroscopic ligand binding affinity
Authors: HOLBROOK Michael R. , SLAKEY Linda L. , GROSS David J.
Published: August 2015

The epidermal growth factor receptor (EGFr), when expressed on the cell surface, has long been known to display two distinct affinities for epidermal growth factor (EGF) binding. In addition, the treatment of cells expressing the EGFr with phorbol esters has been shown to cause a loss of the high-affinity binding capacity of the receptor. In the present study, point mutations that alter acidic or phosphorylation sites have been made in an intracellular domain near Tyr-992 (residues 988–992) of the EGFr. Equilibrium 125I-EGF binding studies demonstrate that the conversion of Tyr-992 into glutamate induces a 4-fold decrease in the EGFr apparent low-affinity dissociation constant, whereas the mutation of two acidic residues, Asp-988 and Glu-991, or the conversion of Tyr-992 into phenylalanine does not alter EGFr affinity. Phorbol ester treatment of EGFr-expressing Chinese hamster ovary cells results in a loss of high-affinity binding and an increase in the apparent low-affinity dissociation constant of the receptor, similar to the effect of a truncation mutant in which the C-terminal 190 residues are deleted. These results are examined in the context of a new model for regulation of the affinity of the EGFr for EGF in which a cytosolic particle stabilizes the high-affinity conformation of the EGFr and a rapid equilibrium exists between EGFr high-affinity and low-affinity conformations. This model demonstrates that the macroscopic affinities of the EGFr can differ from the affinities of individual EGFr molecules and provides a theoretical framework whereby the measured affinities of the EGFr are modulated by intracellular interactions.
In vitro membrane-inserted conformation of the cytochrome b5 tail
Authors: HANLON Michael R. , BEGUM Rukhsana R. , NEWBOLD Richard J. , WHITFORD David , WALLACE B. A.
Published: August 2015

The cytochrome b5 tail is a 43-residue membrane-embedded domain that is responsible for anchoring the catalytic domain of cytochrome b5 to the endoplasmic reticulum membrane. Different models for the structure of the membrane domain of cytochrome b5 have been proposed, including a helical hairpin and a single transmembrane helix. In the present study, CD spectroscopy was used to investigate the conformation of the tail in different environments, and as a function of temperature, with the goal of understanding the nature of the membrane-bound conformation. Whereas residue property profiling indicates that bending of a helix in the middle of the peptide might be possible, the experimental results in small unilamellar vesicles and lysophosphatidylcholine micelles are more consistent with a single transmembrane helix. Furthermore, although there is evidence for some refolding of the polypeptide with temperature, this is not consistent with a hairpin-to-transmembrane transition. Rather, it appears to represent an increase in helical content in fluid lipid environments, perhaps involving residues at the ends of the transmembrane segment.
Synergistic activation of stress-activated protein kinase 1/c-Jun N-terminal kinase (SAPK1/JNK) isoforms by mitogen-activated protein kinase kinase 4 (MKK4) and MKK7
Authors: FLEMING Yvonne , ARMSTRONG Christopher G. , MORRICE Nick , PATERSON Andrew , GOEDERT Michel , COHEN Philip
Published: August 2015

Stress-activated protein kinase 1 (SAPK1), also called c-Jun N-terminal kinase (JNK), becomes activated in vivo in response to pro-inflammatory cytokines or cellular stresses. Its full activation requires the phosphorylation of a threonine and a tyrosine residue in a Thr-Pro-Tyr motif, which can be catalysed by the protein kinases mitogen-activated protein kinase kinase (MKK)4 and MKK7. Here we report that MKK4 shows a striking preference for the tyrosine residue (Tyr-185), and MKK7 a striking preference for the threonine residue (Thr-183) in three SAPK1/JNK1 isoforms tested (JNK1α1, JNK2α2 and JNK3α1). For this reason, MKK4 and MKK7 together produce a synergistic increase in the activity of each SAPK1/JNK isoform in vitro. The MKK7β variant, which is several hundred-fold more efficient in activating all three SAPK1/JNK isoforms than is MKK7α´, is equally specific for Thr-183. MKK7 also phosphorylates JNK2α2 at Thr-404 and Ser-407 in vitro, Ser-407 being phosphorylated much more rapidly than Thr-183 in vitro. Thr-404/Ser-407 are phosphorylated in unstimulated human KB cells and HEK-293 cells, and phosphorylation is increased in response to an osmotic stress (0.5M sorbitol). However, in contrast with Thr-183 and Tyr-185, the phosphorylation of Thr-404 and Ser-407 is not increased in response to other agonists that activate MKK7 and SAPK1/JNK, suggesting that phosphorylation of these residues is catalysed by another protein kinase, such as CK2, which also phosphorylates Thr-404 and Ser-407 in vitro. MKK3, MKK4 and MKK6 all show a strong preference for phosphorylation of the tyrosine residue of the Thr-Gly-Tyr motifs in their known substrates SAPK2a/p38, SAPK3/p38γ and SAPK4/p38δ. MKK7 also phosphorylates SAPK2a/p38 at a low rate (but not SAPK3/p38γ or SAPK4/p38δ), and phosphorylation occurs exclusively at the tyrosine residue, demonstrating that MKK7 is intrinsically a ‘dual-specific’ protein kinase.
Endocytic uptake of advanced glycation end products by mouse liver sinusoidal endothelial cells is mediated by a scavenger receptor distinct from the macrophage scavenger receptor class A
Authors: MATSUMOTO Kenshi , SANO Hiroyuki , NAGAI Ryoji , SUZUKI Hiroshi , KODAMA Tatsuhiko , YOSHIDA Masaki , UEDA Shoichi , SMEDSRØD Bård , HORIUCHI Seikoh
Published: August 2015

Previous studies with peritoneal macrophages obtained from macrophage scavenger receptor class A (MSR-A) knock-out mice showed that the endocytic uptake of advanced glycation end products (AGE) by macrophages was mediated mainly by MSR-A. However, it is controversial whether the endocytic uptake of intravenously injected AGE proteins by liver sinusoidal endothelial cells (LECs) is similarly explained by receptor-mediated endocytosis via MSR-A. The present study was conducted to compare the capacity to endocytose AGE proteins in LECs and peritoneal macrophages obtained from MSR-A knock-out and littermate wild-type mice. The endocytic degradation capacity of MSR-A knock-out LECs for AGE–BSA was indistinguishable from that of wild-type LECs, whereas that of MSR-A knock-out peritoneal macrophages for AGE–BSA was decreased to 30% of that in wild-type cells. Similarly, the endocytic degradation of MSR-A knock-out LECs for acetylated low-density lipoprotein (acetyl-LDL) did not differ from that of wild-type LECs, whereas the endocytic degradation of acetyl-LDL by MSR-A knock-out peritoneal macrophages was less than 20% of that in wild-type cells. Furthermore, formaldehyde-treated serum albumin (f-Alb), a ligand known to undergo scavenger-receptor-mediated endocytosis by LECs, was effectively taken up by MSR-A knock-out LECs at a capacity that did not differ from that of wild-type LECs. Moreover, the endocytic uptake of AGE–BSA by LECs was effectively competed for by unlabelled f-Alb or acetyl-LDL. These results indicate that the scavenger-receptor ligands AGE proteins, acetyl-LDL and f-Alb are endocytosed by LECs through a non-MSR-A pathway.
Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase
Authors: LEADBEATER Claire , MCIVER Lisa , CAMPOPIANO Dominic J. , WEBSTER Scott P. , BAXTER Robert L. , KELLY Sharon M. , PRICE Nicholas C. , LYSEK Dominikus A. , NOBLE Michael A. , CHAPMAN Stephen K. , MUNRO Andrew W.
Published: August 2015

The structure of the Escherichia coli flavodoxin NADP+ oxidoreductase (FLDR) places three arginines (R144, R174 and R184) in the proposed NADPH-binding site. Mutant enzymes produced by site-directed mutagenesis, in which each arginine was replaced by neutral alanine, were characterized. All mutants exhibited decreased NADPH-dependent cytochrome c reductase activity (R144A, 241.6min-1; R174A, 132.1min-1; R184A, 305.5min-1 versus wild type, 338.9min-1) and increased Km for NADPH (R144A, 5.3µM; R174A, 20.2µM; R184A, 54.4µM versus wild type, 3.9µM). The kcat value for NADH-dependent cytochrome c reduction was increased for R174A (42.3min-1) and R184A (50.4min-1) compared with the wild type (33.0min-1), consistent with roles for R174 and R184 in discriminating between NADPH/NADH by interaction with the adenosine ribose 2′-phosphate. Stopped-flow studies indicated that affinity (Kd) for NADPH was markedly reduced in mutants R144A (635µM) and R184A (2.3mM) compared with the wild type (< 5µM). Mutant R184A displays the greatest change in pyridine nucleotide preference, with the NADH/NADPH Kd ratio > 175-fold lower than for wild-type FLDR. The rate constant for hydride transfer from NADPH to flavin was lowest for R174A (kred = 8.82s-1 versus 22.63s-1 for the wild type), which also exhibited tertiary structure perturbation, as evidenced by alterations in CD and fluorescence spectra. Molecular modelling indicated that movement of the C-terminal tryptophan (W248) of FLDR is necessary to permit close approach of the nicotinamide ring of NADPH to the flavin. The positions of NADPH phosphates in the modelled structure are consistent with the kinetic data, with R174 and R184 located close to the adenosine ribose 2′-phosphate group, and R144 likely to interact with the nicotinamide ribose 5′-phosphate group.
Role for the microtubule cytoskeleton in GLUT4 vesicle trafficking and in the regulation of insulin-stimulated glucose uptake
Authors: FLETCHER Laura M. , WELSH Gavin I. , OATEY Paru B. , TAVARÉ Jeremy M.
Published: August 2015

Insulin stimulates glucose uptake into adipocytes by promoting the translocation of the glucose transporter isoform 4 (GLUT4) from intracellular vesicles to the plasma membrane. In 3T3-L1 adipocytes GLUT4 resides both in an endosomal pool, together with transferrin receptors, and in a unique pool termed ‘GLUT4 storage vesicles’(GSVs), which excludes endosomal proteins. The trafficking of GLUT4 vesicles was studied in living 3T3-L1 adipocytes by time-lapse confocal microscopy of GLUT4 tagged with green fluorescent protein. GLUT4 vesicles exhibited two types of motion: rapid vibrations around a point and short (generally less than 10µm) linear movements. The linear movements were completely blocked by incubation of the cells in the presence of microtubule-depolymerizing agents. This suggests that a subpopulation of GLUT4 vesicles can exhibit motor-driven movements along microtubules. Upon further examination, microtubule depolymerization inhibited insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane by approx. 40%, but had no effect on insulin-induced translocation of the transferrin receptor to the plasma membrane from endosomes. We propose that an intact microtubule cytoskeleton may be required for optimal trafficking of GLUT4 present in the GSV pool, but not that resident in the endosomal pool.
Oxidized low-density lipoprotein impairs the anti-coagulant function of tissue-factor-pathway inhibitor through oxidative modification by its high association and accelerated degradation in cultured human endothelial cells
Authors: HORIE Shuichi , HIRAISHI Sayuri , HIRATA Yukiyo , KAZAMA Mutsuyoshi , MATSUDA Juzo
Published: August 2015

We have examined whether oxidized low-density lipoprotein (ox-LDL) affects the function of tissue-factor-pathway inhibitor (TFPI), an anti-coagulant regulator in the extrinsic pathway of coagulation, in cultured human umbilical vein endothelial cells (HUVEC). Treatment of culture medium of HUVEC with ox-LDL, but not with native or acetylated LDLs, drastically decreased the reactivity of TFPI to its antibody specific for Kunitz domain 1 or one specific for the conformation between Kunitz 1 and 2 of TFPI, and caused a rapid, concentration-dependent decrease in the functional activity of TFPI to inhibit Factor X activation. When 5ng of recombinant TFPI (rTFPI) was mixed with 10µg of ox-LDL for 30min, almost all of the rTFPI was detected in the ox-LDL fraction and no free rTFPI was observed on non-denaturing PAGE, in contrast with the virtual absence of rTFPI in the native LDL fraction. Ox-LDL decreased the antigen level of TFPI in the lysate of HUVEC in a time-dependent manner. It did not affect the mRNA level, but ox-LDL-dependent reduction of the TFPI antigen level in HUVEC was reversed by the simultaneous treatment of ox-LDL with bafilomycin A1, an inhibitor of the lysosomal proton pump. These results indicate that ox-LDL lessens the anti-coagulant function of TFPI through both oxidative modification and accelerated degradation of the molecule outside and inside HUVEC respectively.
Human T-lymphotrophic virus type I nucleocapsid protein NCp15: structural study and stability of the N-terminal zinc-finger
Authors: BERTOLA Françoise , MANIGAND Claude , PICARD Philippe , BELGHAZI Maya , PRECIGOUX Gilles
Published: August 2015

An 18-residue peptide, corresponding to the minimum sequence of the N-terminal zinc-finger domain in the nucleocapsid of human T-lymphotrophic virus type I, was synthesized by a solid-phase method and fully characterized. Its ability to complex metal ions (Co2+ and Zn2+) was clearly established by UV–visible spectroscopy and MS. The stability of these complexes was investigated by an original method with HPLC chromatography. Our results show that, even in the presence of air, the Zn2+ complex is highly stable. In contrast, the Co2+ complex undergoes a relatively fast degradation due to an intramolecular oxidation leading to the formation of a disulphide bridge between two cysteine residues. The 1H-NMR analysis indicates that Zn2+ binds to the Nδ atom of the histidine residue rather than to the Nε atom. Two-dimensional NMR techniques were used to determine the solution structure of the zinc-finger, illustrated by the existence of turns in the overall conformation.
Role of cytosolic phospholipase A2 in the production of lipid mediators and histamine release in mouse bone-marrow-derived mast cells
Authors: NAKATANI Noriaki , UOZUMI Naonori , KUME Kazuhiko , MURAKAMI Makoto , KUDO Ichiro , SHIMIZU Takao
Published: August 2015

Cytosolic phospholipase A2 (cPLA2) plays a critical role in mast-cell-related allergic responses [Uozumi, Kume, Nagase, Nakatani, Ishii, Tashiro, Komagata, Maki, Ikuta, Ouchi et al. (1997) Nature (London) 390, 618–622]. Bone-marrow-derived mast cells from mice lacking cPLA2 (cPLA-/- mice) were used in order to better define the role of cPLA2 in the maturation and degranulation of such cells. Cross-linking of high-affinity receptors for IgE (FcεRI) on cells from cPLA-/-mice led to the release of negligible amounts of arachidonic acid or its metabolites, the cysteinyl leukotrienes and prostaglandin D2, indicating an essential role for cPLA2 in the production of these allergic and pro-inflammatory lipid mediators. In addition, the histamine content of the mast cells and its release from the cells were reduced to 60%. While these results are in agreement with a reduced anaphylactic phenotype of cPLA-/- mice, the ratios of release of histamine and β-hexosaminidase were, paradoxically, significantly higher for cells from cPLA-/- mice than for those from wild-type mice. Consistently, IgE-induced calcium influx in mast cells was greater and more prolonged in cells from cPLA-/- mice than in those from wild-type mice. Thus the loss of cPLA2 not only diminishes the release of lipid mediators, but also alters degranulation. While the overall effect is still a decrease in the release of mast cell mediators, explaining the in vivo findings, the present study proposes a novel link between cPLA2 and the degranulation machinery.
A single amino acid substitution (N297A) in the conserved NPXXY sequence of the human N-formyl peptide receptor results in inhibition of desensitization and endocytosis, and a dose-dependent shift in p42/44 mitogen-activated protein kinase activation and chemotaxis
Authors: GRIPENTROG Jeannie M. , JESAITIS Algirdas J. , MIETTINEN Heini M.
Published: August 2015

The formyl peptide receptor (FPR) is a G-protein-coupled receptor (GPCR) that mediates chemotaxis and stimulates the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase pathway. We have examined the functional effects of substitutions of a conserved aspartic acid residue in the second transmembrane domain (D71A) and of residues in the conserved NPXXY motif in the seventh transmembrane domain (N297A and Y301A). These mutated receptors, expressed in Chinese hamster ovary (CHO) cells, bind ligand with affinities similar to wild-type FPR, but the D71A mutant is uncoupled from G-protein [Miettinen, Mills, Gripentrog, Dratz, Granger and Jesaitis (1997) J. Immunol 159, 4045–4054]. In the present study, we show that both the D71A and N297A mutations resulted in defective endocytosis. The N297A substitution also prevented desensitization, as determined by intracellular calcium mobilization by sequential stimulation with ligand. In chemotaxis assays, the N297A mutation resulted in cell migration towards gradients of up to 100nM N-formyl-methionyl-leucyl-phenylalanine (fMLF), whereas cells expressing the wild-type FPR and the Y301A mutant were no longer chemotactically responsive at 10–100nM fMLF. Maximal activation of p42/44 MAPK occurred in CHO cells expressing wild-type FPR at 10nM–100nM fMLF, whereas cells expressing the N297A mutant showed a dose-dependent increase in the amount of phosphorylated p42/44 MAPK up to 1–10µM fMLF. Since the MAPK kinase inhibitor PD98059 blocked fMLF-induced chemotaxis, our results suggest that the dose-dependent increase in p42/44 MAPK activation may correlate with the increased chemotactic migration of N297A transfectants at 10nM–100nM fMLF.
Reaction of dopa decarboxylase with L-aromatic amino acids under aerobic and anaerobic conditions
Authors: BERTOLDI Mariarita , VOLTATTORNI Carla BORRI
Published: August 2015

Analysis of the reaction of dopa decarboxylase (DDC) with L-dopa reveals that loss of decarboxylase activity with time is observed at enzyme concentrations approximately equal to the binding constant, Kd, of the enzyme for pyridoxal 5ƀ-phosphate (PLP). Instead, at enzyme concentrations higher than Kd the course of product formation proceeds linearly until complete consumption of the substrate. Evidence is provided that under both experimental conditions no pyridoxamine 5ƀ-phosphate (PMP) is formed during the reaction and that dissociation of coenzyme occurs at low enzyme concentration, leading to the formation of a PLP-L-dopa Pictet–Spengler cyclic adduct. Taken together, these results indicate that decarboxylation-dependent transamination does not accompany the decarboxylation of L-dopa proposed previously [O'Leary and Baughn (1977) J. Biol. Chem. 252, 7168–7173]. Nevertheless, when the reaction of DDC with L-dopa is studied under anaerobic conditions at an enzyme concentration higher than Kd, we observe that (1) the enzyme is gradually inactivated and inactivation is associated with PMP formation and (2) the initial velocity of decarboxylation is approximately half of that in the presence of O2. Similar behaviour is observed by comparing the reaction with L-5-hydroxytryptophan occurring in aerobiosis or in anaerobiosis. Therefore the reaction of DDC with L-aromatic amino acids seems to be under O2 control. In contrast, the reactivity of the enzyme with D-aromatic amino acids does not change in the presence or absence of O2. These and other results, together with previous results on the effect exerted by O2 on reaction specificity of DDC towards aromatic amines [Bertoldi, Frigeri, Paci and Borri Voltattorni (1999) J. Biol. Chem. 274, 5514–5521], suggest a productive effect of O2 on an intermediate complex of the reaction of the enzyme with L-aromatic amino acids or aromatic amines.
Defective localization of the Wilson disease protein (ATP7B) in the mammary gland of the toxic milk mouse and the effects of copper supplementation
Authors: MICHALCZYK Agnes A. , RIEGER Jennifer , ALLEN Katrina J. , MERCER Julian F. B. , ACKLAND M. Leigh
Published: August 2015

Toxic milk (tx) is a copper disorder of mice that causes a hepatic accumulation of copper similar to that seen in patients with Wilson disease. Both disorders are caused by a defect in the ATP7B copper-transporting ATPase. A feature of the tx phenotype is the production of copper-deficient milk by lactating dams homozygous for the tx mutation; the milk is lethal to the pups. It has not been determined whether the production of copper-deficient milk is a direct consequence of impaired expression of ATP7B protein in the mammary gland. With the use of immunohistochemistry, our study demonstrated that the ATP7B protein was mislocalized in the lactating tx mouse mammary gland, which would explain the inability of the tx mouse to secrete normal amounts of copper in milk. Confocal microscopy analysis showed that, in the lactating tx mammary gland, ATP7B was predominantly perinuclear in comparison with the diffuse, cytoplasmic localization of ATP7B in the lactating normal mammary gland. Lactating tx mice showed impaired delivery of copper from the mammary gland to the milk and this was not ameliorated by dietary copper supplementation. In contrast, the normal mouse mammary gland responded to increased dietary copper by increasing the amount of copper in milk. A change in the distribution of the ATP7B protein from perinuclear in the non-lactating gland to a diffuse, cytoplasmic localization in the lactating gland of the normal (DL) mouse suggests that the relocalization of APT7B is a physiological process that accompanies lactation. We conclude that the impaired copper transport from the mammary gland into milk in lactating tx mice is related to the mislocalization of ATP7B.
Steady-state kinetic mechanism of the NADP+- and NAD+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa
Authors: VELASCO-GARCÍA Roberto , GONZÁLEZ-SEGURA Lilian , MUÑOZ-CLARES Rosario A.
Published: August 2015

Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)+ to NADP(H). In Pseudomonas aeruginosa this reaction is a compulsory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. The kinetic mechanisms of the NAD+- and NADP+-dependent reactions were examined by steady-state kinetic methods and by dinucleotide binding experiments. The double-reciprocal patterns obtained for initial velocity with NAD(P)+ and for product and dead-end inhibition establish that both mechanisms are steady-state random. However, quantitative analysis of the inhibitions, and comparison with binding data, suggest a preferred route of addition of substrates and release of products in which NAD(P)+ binds first and NAD(P)H leaves last, particularly in the NADP+-dependent reaction. Abortive binding of the dinucleotides, or their analogue ADP, in the betaine aldehyde site was inferred from total substrate inhibition by the dinucleotides, and parabolic inhibition by NADH and ADP. A weak partial uncompetitive substrate inhibition by the aldehyde was observed only in the NADP+-dependent reaction. The kinetics of P. aeruginosa BADH is very similar to that of glucose-6-phosphate dehydrogenase, suggesting that both enzymes fulfil a similar amphibolic metabolic role when the bacteria grow in choline and when they grow in glucose.
Compartment-specific regulation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) by ERK-dependent and non-ERK-dependent inductions of MAPK phosphatase (MKP)-3 and MKP-1 in differentiating P19 cells
Authors: REFFAS Samia , SCHLEGEL Werner
Published: August 2015

Activation of mitogen-activated protein kinases (MAPKs), their upstream activators MAPK kinases (MAPKKs or MEKs) and induction of MKP-1 (CL100/3CH134) and MKP-3 (Pyst1/rVH6) dual-specificity MAPK phosphatases (MKPs) were studied in the mouse embryonic stem cell line P19 during the 7 day induction of neuronal differentiation triggered by aggregation and retinoic acid. ERK (extracellular signal-regulated kinase), but not JNK (c-Jun N-terminal kinase), was found activated with biphasic kinetics: a first transient phase on days 1 and 2, followed by a second activation that was sustained until the appearance of a neuronal phenotype. MEK activation appeared coincident with ERK activation. Cytosolic MKP-3 was induced in parallel to ERK activation, the induction being dependent on ERK activation, as was shown using the MEK-1 inhibitor PD98059. In contrast, nuclear MKP-1 was transiently elevated at 48h, coincident with ERK inactivation and independently of ERK activity. As shown by cell fractionation, activated ERK is translocated to the nucleus. The complementary induction of ERK-specific phosphatases MKP-1 and MKP-3 permits precise and independent control of cytoplasmic and nuclear ERK activity, most probably required to properly induce a complex cellular programme of differentiation.