bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative study of protein tyrosine phosphatase-ɛ isoforms: membrane localization confers specificity in cellular signalling
Authors: ANDERSEN Jannik N⊘rgaard , ELSON Ari , LAMMERS Reiner , RØMER John , CLAUSEN Jes Thorn , MØLLER Karin Bach , MØLLER Niels Peter Hundahl
Published: August 2015

To study the influence of subcellular localization as a determinant of signal transduction specificity, we assessed the effects of wild-type transmembrane and cytoplasmic protein tyrosine phosphatase (PTP) ε on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHK-IR). The efficiency by which differently localized PTPε and PTPα variants attenuated insulin-induced cell rounding and detachment was determined in a functional clonal-selection assay and in stable cell lines. Compared with the corresponding receptor-type PTPs, the cytoplasmic PTPs (cytPTPs) were considerably less efficient in generating insulin-resistant clones, and exceptionally high compensatory expression levels were required to counteract phosphotyrosine-based signal transduction. Targeting of cytPTPε to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTPε levels. Consistent with these results, expression levels and subcellular localization of PTPε were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTPε stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTPε inhibitory response to insulin-induced cell rounding and detachment in BHK-IR cells. Taken together, the present localization–function study indicates that transcriptional control of the subcellular localization of PTPε may provide a molecular mechanism that determines PTPε substrate selectivity and isoform-specific function.
Molecular interaction of dihydropyridine receptors with type-1 ryanodine receptors in rat brain
Authors: MOUTON Jérôme , MARTY Isabelle , VILLAZ Michel , FELTZ Anne , MAULET Yves
Published: August 2015

In striated muscles, Ca2+ release from internal stores through ryanodine receptor (RyR) channels is triggered by functional coupling to voltage-activated Ca2+ channels known as dihydropyridine receptors (DHPRs) located in the plasma membrane. In skeletal muscle, this occurs by a direct conformational link between the tissue-specific DHPR (Cav1.1) and RyR1, whereas in the heart the signal is carried from the cardiac-type DHPR (Cav1.2) to RyR2 by calcium ions acting as an activator. Subtypes of both channels are expressed in the central nervous system, but their functions and mechanisms of coupling are still poorly understood. We show here that complexes immunoprecipitated from solubilized rat brain membranes with antibodies against DHPR of the Cav1.2 or Cav1.3 subtypes contain RyR. Only type-1 RyR is co-precipitated, although the major brain isoform is RyR2. This suggests that, in neurons, DHPRs could communicate with RyRs by way of a strong molecular interaction and, more generally, that the physical link between DHPR and RyR shown to exist in skeletal muscle can be extended to other tissues.
Affinity of mismatch-binding protein MutS for heteroduplexes containing different mismatches
Authors: BROWN James , BROWN Tom , FOX Keith R.
Published: August 2015

We have used bandshift analysis to measure the interaction between the Escherichia coli mismatch-binding protein MutS and synthetic DNA fragments containing all possible DNA mismatches as well as an unpaired T (∆T). The order of affinity is found to be ∆T > GT > GG > AA ≈ TT ≈ TC>CA>GA > CC>GC. We find that the affinity for GT mismatches is affected by the flanking sequence and decreases in the order GnCn>(GC)n>AnTn>(AT)n. Studies with base analogues show good binding to φT (where φ represents 1′,2′-dideoxyribose), but much weaker binding to Gφ.
Mitogen-stimulated TIS21 protein interacts with a protein-kinase-Cα-binding protein rPICK1
Authors: LIN Wey-Jinq , CHANG Yaun-Fu , WANG Wei-Li , HUANG Chi-Ying F.
Published: August 2015

TIS21 is induced transiently by PMA and a number of extracellular stimuli. Yeast two-hybrid screening has identified three TIS21 interacting clones from a rat cDNA library [Lin, Gary, Yang, Clarke and Herschman (1996) J. Biol. Chem 271, 15034–15044]. The amino acid sequence deduced from clone 5A shows 96.9% identity with the murine PICK1, a protein kinase Cα (PKCα)-binding protein postulated to act as an intracellular receptor for PKC. A fusion protein of glutathione S-transferase and rPICK1 associates with the TIS21 translated in vitro, suggesting a direct physical interaction between these two proteins. TIS21 and rPICK1 are co-immunoprecipitated from NIH 3T3 cells overexpressing these two proteins. This indicates that the interaction also occurs in mammalian cells. Deletion of the PDZ domain at the N-terminus of rPICK1 abolishes its interaction with TIS21. A putative carboxylate-binding loop required for PICK1 to bind PKCα [Staudinger, Lu and Olson (1997) J. Biol. Chem 272, 32019–32024] is within this deleted region. Our results suggest a potential competition between TIS21 and PKC for binding to PICK1. We show that recombinant TIS21 is phosphorylated by PKC in vitro. The catalytic activity of PKC towards TIS21 is significantly decreased in the presence of rPICK1, whereas phosphorylation of histone by PKC is not affected. rPICK1 seems to modulate the phosphorylation of TIS21 through specific interactions between these two proteins. TIS21 might have a role in PKC-mediated extracellular signal transduction through its interaction with rPICK1.
‘Shed’ furin: mapping of the cleavage determinants and identification of its C-terminus
Authors: PLAIMAUER Barbara , MOHR Gabriele , WERNHART Waltraud , HIMMELSPACH Michèle , DORNER Friedrich , SCHLOKAT Uwe
Published: August 2015

The human endoprotease furin is involved in the proteolytic maturation of the precursor molecules of a wide variety of bioactive proteins. Despite its localization in the membranes of the trans-Golgi system by means of a transmembrane domain, it has repeatedly been reported to form a C-terminally truncated, naturally secreted form referred to as ‘shed’ furin. In order to identify the cleavage site, internal deletion mutants of increasing size, N-terminal to Leu708, and subsequently individual amino acid substitutions were introduced, and Arg683 was identified as the prime determinant for shedding. MS analysis determined Ser682 as the C-terminus of shed furin, suggesting that monobasic cleavage may occur N-terminal to Arg683. Alteration of Arg683 directs the shedding mechanism to alternative cleaving sites previously unused.
Increased inwardly rectifying potassium currents in HEK-293 cells expressing murine transient receptor potential 4
Authors: ZHANG Zongming , TANG Yufang , ZHU Michael Xi
Published: August 2015

Drosophila transient receptor potential (Trp) and its mammalian homologues are postulated to form capacitative Ca2+ entry or store-operated channels. Here we show that expression of murine Trp4 in HEK 293 cells also leads to an increase in inwardly rectifying K+ currents. No similar increase was found in cell lines expressing Trp1, Trp3 or Trp6. Consistent with typical characteristics of inward rectifiers, the K+ currents in Trp4-expressing cells were blocked by low millimolar concentrations of Cs+ and Ba2+, but not by 1.2mM Ca2+, and were only slightly inhibited by 5mM tetraethylammonium. Single channel recordings of excised inside-out patches revealed the presence of two conducting states of 51pS and 94pS in Trp4-expressing cells. The outward current in the excised patches was blocked by 1mM spermine, but not by 1mM Mg2+. How Trp4 expression causes the increase in the K+ currents is not known. We propose that Trp4 either participates in the formation of a novel K+ channel or up-regulates the expression or activity of endogenous inwardly rectifying K+ channels.
Selective recognition of inositol phosphates by subtypes of the inositol trisphosphate receptor
Authors: NEROU Edmund P. , RILEY Andrew M. , POTTER Barry V. L. , TAYLOR Colin W.
Published: August 2015

Synthetic analogues of inositol trisphosphate (IP3), all of which included structures equivalent to the 4,5-bisphosphate of (1,4,5)IP3, were used to probe the recognition properties of rat full-length type 1, 2 and 3 IP3 receptors expressed in insect Spodoptera frugiperda 9 cells. Using equilibrium competition binding with [3H](1,4,5)IP3 in Ca2+-free cytosol-like medium, the relative affinities of the receptor subtypes for (1,4,5)IP3 were type 3 (Kd = 11±2nM)>type 2 (Kd = 17±2nM) > type 1 (Kd = 24±4nM). (1,4,5)IP3 binding was reversibly stimulated by increased pH, but the subtypes differed in their sensitivity to pH (type 1 > type 2>type 3). For all three subtypes, the equatorial 6-hydroxy group of (1,4,5)IP3 was essential for high-affinity binding, the equatorial 3-hydroxy group significantly improved affinity, and the axial 2-hydroxy group was insignificant; a 1-phosphate (or in its absence, a 2-phosphate) improved binding affinity. The subtypes differed in the extents to which they tolerated inversion of the 3-hydroxy group of (1,4,5)IP3 (type 1>type 2>type 3), and this probably accounts for the selectivity of (1,4,6)IP3 for type 1 receptors. They also differed in their tolerance of inversion, removal or substitution (by phosphate) of the 2-hydroxy group (types 2 and 3>type 1), hence the selectivity of (1,2,4,5)IP4 for type 2 and 3 receptors. Removal of the 3-hydroxy group or its replacement by fluorine or CH2OH was best tolerated by type 3 receptors, and accounts for the selectivity of 3-deoxy(1,4,5)IP3 for type 3 receptors. Our results provide the first systematic analysis of the recognition properties of IP3 receptor subtypes and have identified the 2- and 3-positions of (1,4,5)IP3 as key determinants of subtype selectivity.
Enhancement of macrophage survival and DNA synthesis by oxidized-low-density-lipoprotein (LDL)-derived lipids and by aggregates of lightly oxidized LDL
Authors: HAMILTON John A. , JESSUP Wendy , BROWN Andrew J. , WHITTY Genevieve
Published: August 2015

Human atherosclerotic plaque contains a partially characterized range of normal and oxidized lipids formed mainly from free and esterified cholesterol and phospholipids, some of which can be located in macrophage-derived ‘foam’ cells. Oxidation of low-density lipoprotein (LDL) is often considered as an important event leading to subsequent foam-cell development, which may also include enhanced cell survival and/or proliferation. The active component(s) in oxidized LDL (ox.LDL) causing macrophage proliferation is debated. We report here that the lipid component of ox.LDL can promote macrophage survival and DNA synthesis, the latter response showing a synergistic effect in the presence of low concentrations of macrophage colony-stimulating factor. 7-Ketocholesterol showed some stimulation of macrophage DNA synthesis whereas hypochlorite-oxidized (i.e. apolipoprotein B-oxidized) LDL did not. Plaque-derived lipids could enhance macrophage survival. It has not been proven that LDL in lesions is oxidized sufficiently to be the dominant source of sterols in vivo or to be able to induce macrophage growth in vitro or in vivo; it has been suggested that aggregation of modified LDL in vivo is an important step in the deposition of intracellular lipid. We found that aggregation of lightly oxidized LDL potentiated dramatically its ability to stimulate macrophage DNA synthesis, indicating that extensive oxidation of LDL is not required for this response in vitro and perhaps in vivo.
The efficiency of N-linked glycosylation of bovine DNase I depends on the Asn-Xaa-Ser/Thr sequence and the tissue of origin
Authors: NISHIKAWA Atsushi , MIZUNO Sumi
Published: August 2015

Bovine DNase I contains two potential N-linked glycosylation sites with the sequences Asn18-Ala-Thr and Asn106-Asp-Ser. A previous report established that pancreatic DNase I has only one sugar chain at Asn18 [Liao, Salnikow, Moore and Stein (1973) J. Biol. Chem. 248, 1489–1495]. We found, however, that bovine DNase I expressed in COS-1 cells was glycosylated about 70% at Asn106 in addition to being completely glycosylated at Asn18. Glycosylation of Asn106 increased to 97% when Asp107 was mutated to Glu or when Ser108 was mutated to Thr. Mutation of Asp107 to Trp had no effect, whereas a substitution with Pro at this position abolished glycosylation of Asn106. Analysis of the state of glycosylation of DNase I purified from a variety of bovine tissues revealed that DNase I from spleen, submaxillary gland, lung and adrenal had two sugar chains, whereas enzyme from pancreas and kidney had only one sugar chain. These findings demonstrate a major difference in the ability of various tissues to utilize N-linked glycosylation signals that contain suboptimal residues in the second and third positions.
Identification and characterization of a novel sucrose-non-fermenting protein kinase/AMP-activated protein kinase-related protein kinase, SNARK
Authors: LEFEBVRE Diana L. , BAI Yahong , SHAHMOLKY Nazanin , SHARMA Monika , POON Raymond , DRUCKER Daniel J. , ROSEN Cheryl F.
Published: August 2015

Subtraction hybridization after the exposure of keratinocytes to ultraviolet radiation identified a differentially expressed cDNA that encodes a protein of 630 amino acid residues possessing significant similarity to the catalytic domain of the sucrose-non-fermenting protein kinase (SNF1)/AMP-activated protein kinase (AMPK) family of serine/threonine protein kinases. Northern blotting and reverse-transcriptase-mediated PCR demonstrated that mRNA transcripts for the SNF1/AMPK-related kinase (SNARK) were widely expressed in rodent tissues. The SNARK gene was localized to human chromosome 1q32 by fluorescent in situ hybridization. SNARK was translated in vitro to yield a single protein band of approx. 76kDa; Western analysis of transfected baby hamster kidney (BHK) cells detected two SNARK-immunoreactive bands of approx. 76-80kDa. SNARK was capable of autophosphorylation in vitro; immunoprecipitated SNARK exhibited phosphotransferase activity with the synthetic peptide substrate HMRSAMSGLHLVKRR (SAMS) as a kinase substrate. SNARK activity was significantly increased by AMP and 5-amino-4-imidazolecarboxamide riboside (AICAriboside) in rat keratinocyte cells, implying that SNARK might be activated by an AMPK kinase-dependent pathway. Furthermore, glucose deprivation increased SNARK activity 3-fold in BHK fibroblasts. These findings identify SNARK as a glucose- and AICAriboside-regulated member of the AMPK-related gene family that represents a new candidate mediator of the cellular response to metabolic stress.
Cloning and characterization of a fourth human lysyl oxidase isoenzyme
Authors: MÄKI Joni M. , KIVIRIKKO Kari I.
Published: August 2015

We report here the complete cDNA sequence and exon-intron organization of the human lysyl oxidase-like (LOXL)3 gene, a new member of the lysyl oxidase (LO) gene family. The predicted polypeptide is 753 amino acids in length, including a signal peptide of 25 residues. The C-terminal region, residues 529-729, contains a LO domain similar to those in the LOX (the first characterized LO isoenzyme), LOXL and LOXL2 polypeptides. It possesses the putative copper binding sequence, and the lysine and tyrosine residues that form the lysyltyrosyl quinone cofactor. The N-terminal region, which is similar to that in LOXL2 but not those in LOX and LOXL, contains four subregions similar to scavenger receptor cysteine-rich domains and a putative nuclear localization signal. Recombinant LOXL3, expressed in HT-1080 cells, was secreted into the culture medium but was not detected by immunofluorescence staining in nuclei. The LOXL3 mRNA is 3.1kb in size and is expressed in many tissues, the highest levels among the tissues studied being seen in the placenta, heart, ovary, testis, small intestine and spleen.
Interaction of plasminogen with dipeptidyl peptidase IV initiates a signal transduction mechanism which regulates expression of matrix metalloproteinase-9 by prostate cancer cells
Authors: GONZALEZ-GRONOW Mario , GRENETT Hernan E. , WEBER Michael R. , GAWDI Govind , PIZZO Salvatore V.
Published: August 2015

Both plasminogen (Pg) activation and matrix metalloproteinases (MMPs) are involved in the proteolytic degradation of extracellular matrix components, a requisite event for malignant cell metastasis. The highly invasive 1-LN human prostate tumour cell line synthesizes and secretes large amounts of Pg activators and MMPs. We demonstrate here that the Pg type 2 (Pg 2) receptor in these cells is composed primarily of the membrane glycoprotein dipeptidyl peptidase IV (DPP IV). Pg 2has six glycoforms that differ in their sialic acid content. Only the highly sialylated Pg 2γ, Pg 2δ and Pg 2ε glycoforms bind to DPP IV via their carbohydrate chains and induce a Ca2+ signalling cascade; however, Pg 2ε alone is also able to significantly stimulate expression of MMP-9. We further demonstrate that the Pg-mediated invasive activity of 1-LN cells is dependent on the availability of Pg 2ε. This is the first demonstration of a direct association between the expression of MMP-9 and the Pg activation system.
Recruitment of coat-protein-complex proteins on to phagosomal membranes is regulated by a brefeldin A-sensitive ADP-ribosylation factor
Authors: BERÓN Walter , MAYORGA Luis S. , COLOMBO Maria I. , STAHL Philip D.
Published: August 2015

Particle internalization in macrophages is followed by a complex maturation process. We have previously observed that proteins bound to phagocytosed particles are sorted from phagosomes into a heterogeneous population of vesicles that fuse with endosomes. However, the mechanism and the protein machinery involved in the formation of these phagosome-derived vesicles are largely unknown. It has been shown that vesicles coated with coat protein complex type I (COPI) have a role in both secretion and endocytosis. To address the possibility that COPI proteins might participate in the formation of phagosome-derived vesicles we studied the recruitment of β-COP to highly purified phagosomes. The binding of β-COP to phagosomal membranes was regulated by nucleotides and inhibited by brefeldin A (BFA). An ADP-ribosylation factor 1 (ARF1) mutant defective in GTP hydrolysis supported the binding of β-COP to phagosomes independently of added nucleotide. AlF4 and Gβγ subunits, agents known to modulate heterotrimeric G-protein activity, were tested in the β-COP binding assay. AlF4 increased β-COP association, whereas binding was inhibited by the addition of Gβγ subunits. Our results suggest that COP proteins are recruited to phagosomal membranes by a mechanism that involves heterotrimeric GTP-binding proteins and a BFA-sensitive ARF. In addition, our findings indicate that COPI proteins are involved in the recycling of components from phagosomes to the cell surface.
Detection of phospholipid oxidation in oxidatively stressed cells by reversed-phase HPLC coupled with positive-ionization electroscopy MS
Authors: SPICKETT Corinne M. , RENNIE Nicola , WINTER Helen , ZAMBONIN Laura , LANDI Laura , JERLICH Andreas , SCHAUR R. Jörg , PITT Andrew R.
Published: August 2015

Measurement of lipid peroxidation is a commonly used method of detecting oxidative damage to biological tissues, but the most frequently used methods, including MS, measure breakdown products and are therefore indirect. We have coupled reversed-phase HPLC with positive-ionization electrospray MS (LC-MS) to provide a method for separating and detecting intact oxidized phospholipids in oxidatively stressed mammalian cells without extensive sample preparation. The elution profile of phospholipid hydroperoxides and chlorohydrins was first characterized using individual phospholipids or a defined phospholipid mixture as a model system. The facility of detection of the oxidized species in complex mixtures was greatly improved compared with direct-injection MS analysis, as they eluted earlier than the native lipids, owing to the decrease in hydrophobicity. In U937 and HL60 cells treated in vitro with t-butylhydroperoxide plus Fe2+, lipid oxidation could not be observed by direct injection, but LC-MS allowed the detection of monohydroperoxides of palmitoyl-linoleoyl and stearoyl-linoleoyl phosphatidylcholines. The levels of hydroperoxides observed in U937 cells were found to depend on the duration and severity of the oxidative stress. In cells treated with HOCl, chlorohydrins of palmitoyloleoyl phosphatidylcholine were observed by LC-MS. The method was able to detect very small amounts of oxidized lipids compared with the levels of native lipids present. The membrane-lipid profiles of these cells were found to be quite resistant to damage until high concentrations of oxidants were used. This is the first report of direct detection by LC-MS of intact oxidized phospholipids induced in cultured cells subjected to oxidative stress.
Identification, characterization and leucocyte expression of Siglec-10, a novel human sialic acid-binding receptor
Authors: MUNDAY James , KERR Sheena , NI Jian , CORNISH Ann L. , ZHANG Jiquan Q. , NICOLL Gavin , FLOYD Helen , MATTEI Marie-Genevieve , MOORE Paul , LIU Ding , CROCKER Paul R.
Published: August 2015

Here we characterize Siglec-10 as a new member of the Siglec family of sialic acid-binding Ig-like lectins. A full-length cDNA was isolated from a human spleen library and the corresponding gene identified. Siglec-10 is predicted to contain five extracellular Ig-like domains and a cytoplasmic tail containing three putative tyrosine-based signalling motifs. Siglec-10 exhibited a high degree of sequence similarity to CD33-related Siglecs and mapped to the same region, on chromosome 19q13.3. The expressed protein was able to mediate sialic acid-dependent binding to human erythrocytes and soluble sialoglycoconjugates. Using specific antibodies, Siglec-10 was detected on subsets of human leucocytes including eosinophils, monocytes and a minor population of natural killer-like cells. The molecular properties and expression pattern suggest that Siglec-10 may function as an inhibitory receptor within the innate immune system.
GTP cyclohydrolase I mRNA: novel splice variants in the slime mould Physarum polycephalum and in human monocytes (THP-1) indicate conservation of mRNA processing
Authors: GOLDERER Georg , WERNER Ernst R. , HEUFLER Christine , STROHMAIER Wolfgang , GRÖBNER Peter , WERNER-FELMAYER Gabriele
Published: August 2015

GTP cyclohydrolase I (EC 3.5.4.16) is the first enzyme in the biosynthesis of tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H4-biopterin] in mammals and of folic acid in bacteria. Here we have characterized the GTP cyclohydrolase I gene structure and two mRNA species from Physarum polycephalum, an acellular slime mould that synthesizes H4-biopterin and metabolites of the folic acid biosynthetic pathway. Its GTP cyclohydrolase I gene consists of seven exons, and the two GTP cyclohydrolase I cDNA species isolated from Physarum encode for proteins with 228 (25.7kDa) and 195 (22.1kDa) amino acids. Furthermore, we identified two previously undescribed mRNA species in interferon-γ-treated human myelomonocytoma cells (THP-1) in addition to the cDNA coding for the fully functional 250-residue (27.9kDa) protein, which is identical with that in human phaeochromocytoma cells. One of the new splice variants codes for a 233-residue (25.7kDa) protein, whereas the other codes for the full-length protein but is alternatively spliced within the 3′-untranslated region. In heterologous expression, the shorter proteins of Physarum as well as of THP-1 cells identified here are degraded by proteolysis. Accordingly, only the 27.9kDa protein was detectable in Western blots from THP-1 cell extracts. Quantification of GTP cyclohydrolase I mRNA species in different human cell types with and without cytokine treatment showed that in addition to the correct mRNA the two splice variants isolated here, as well as the two splice variants known from human liver, are strongly induced by cytokines in cell types with inducible GTP cyclohydrolase I (THP-1, dermal fibroblasts), but not in cell types with constitutive GTP cyclohydrolase I expression (SK-N-SH, Hep-G2). As in human liver, splicing of the new mRNA variant found in THP-1 cells occurs at the boundary of exons 5 and 6. Strikingly, the 195-residue protein from Physarum is alternatively spliced at a homologous position, i.e. at the boundary of exons 6 and 7. Thus alternative splicing of GTP cyclohydrolase I at this position occurs in two species highly distant from each other in terms of evolution. It remains to be seen whether variant proteins encoded by alternatively spliced GTP cyclohydrolase I mRNA transcripts do occur in vivo and whether they participate in regulation of enzyme activity.
The hexokinase 2 protein regulates the expression of the GLK1, HXK1 and HXK2 genes of Saccharomyces cerevisiae
Authors: RODRÍGUEZ Aránzazu , CERA Tamara de la , HERRERO Pilar , MORENO Fernando
Published: August 2015

The key glycolytic HXK2 gene, coding for the enzyme hexokinase 2 (Hxk2p), is expressed when cells of the yeast Saccharomyces cerevisiae are grown on a fermentable medium using glucose, fructose or mannose as a carbon source. After shifting the cells to a non-fermentable carbon source, the HXK2 gene is repressed and the HXK1 and GLK1 genes are rapidly de-repressed, producing the enzymes hexokinase 1 (Hxk1p) and glucokinase (Glk1p) respectively. Because the in vivo functions of the Hxk1p and Glk1p enzymes have remained a mystery so far, we have investigated this glucose-induced regulatory process. Here we demonstrate the involvement of Hxk2p in the glucose-induced repression of the HXK1 and GLK1 genes and the glucose-induced expression of the HXK2 gene. We have also demonstrated the involvement of Hxk1p as a negative factor in the expression of the GLK1 and HXK2 genes. Further experimental evidence, using mutant cells expressing a truncated version of Hxk2p unable to enter the nucleus, shows that nuclear localization of Hxk2p is necessary for glucose-induced repression signalling of the HXK1 and GLK1 genes and for glucose-induced expression of the HXK2 gene. Gel mobility-shift analysis shows that Hxk2p-mediated regulation is exerted through ERA (ethanol repression autoregulation)-like regulatory sequences present in the HXK1 and GLK1 promoters and in two downstream repressing sequences of the HXK2 gene. These findings reveal a novel mechanism of gene regulation whereby the product of a glycolytic gene, normally resident in the cytosol, interacts directly with nuclear proteins to regulate the transcription of the HXK1 and GLK1 genes and to autoregulate its own transcription.
Reactive oxygen species involved in trichosanthin-induced apoptosis of human choriocarcinoma cells
Authors: ZHANG Chun-yang , GONG Yi-xuan , MA Hui , AN Cheng-cai , CHEN Die-yan
Published: August 2015

The type-I ribosome-inactivating protein trichosanthin (TCS) has a broad spectrum of biological and pharmacological activities, including abortifacient, anti-tumour and anti-HIV activities. We have found for the first time that TCS stimulated the production of reactive oxygen species (ROS) in JAR cells (a human choriocarcinoma cell line) in a time- and concentration-dependent manner by using the fluorescent probe 2′,7′-dichlorofluorescein diacetate with confocal laser scanning microscopy. ESR spectral studies and the inhibition of ROS formation by the superoxide radical anion (O2-P) scavenger superoxide dismutase, the H2O2 scavenger catalase and the hydroxyl radical (OHP) scavenger mannitol suggested the involvement of O2-P, H2O2 and OHP. TCS-induced ROS formation was shown to be dependent on the presence of both extracellular and intracellular Ca2+; moreover, ROS production paralleled the intracellular Ca2+ elevation induced by TCS, suggesting that ROS production might be a consequence of Ca2+ signalling. TCS-induced activation of caspase-3 was initiated within 2h; however, TCS-induced production of ROS was initiated within 5min, suggesting that the production of ROS preceded the activation of caspase-3. Simultaneous observation of the nuclear morphological changes via two-photon laser scanning microscopy and ROS production via confocal laser scanning microscopy revealed that ROS is involved in the apoptosis of JAR cells. The involvement of ROS was also confirmed by the inhibition of TCS-induced cell death by the antioxidant Trolox and the ROS scavengers catalase and mannitol. Diethylenetriaminepenta-acetic acid, an inhibitor of metal-facilitated OHP formation, markedly inhibited TCS-induced cell death, suggesting that TCS induced OHP formation via the Fenton reaction. The finding that ROS is involved in the TCS-induced apoptosis of JAR cells might provide new insight into the anti-tumour and anti-HIV mechanism of TCS.
The marks, mechanisms and memory of epigenetic states in mammals
Authors: RAKYAN Vardhman K. , PREIS Jost , MORGAN Hugh D. , WHITELAW Emma
Published: August 2015

It is well recognized that there is a surprising degree of phenotypic variation among genetically identical individuals, even when the environmental influences, in the strict sense of the word, are identical. Genetic textbooks acknowledge this fact and use different terms, such as ‘intangible variation’ or ‘developmental noise’, to describe it. We believe that this intangible variation results from the stochastic establishment of epigenetic modifications to the DNA nucleotide sequence. These modifications, which may involve cytosine methylation and chromatin remodelling, result in alterations in gene expression which, in turn, affects the phenotype of the organism. Recent evidence, from our work and that of others in mice, suggests that these epigenetic modifications, which in the past were thought to be cleared and reset on passage through the germline, may sometimes be inherited to the next generation. This is termed epigenetic inheritance, and while this process has been well recognized in plants, the recent findings in mice force us to consider the implications of this type of inheritance in mammals. At this stage we do not know how extensive this phenomenon is in humans, but it may well turn out to be the explanation for some diseases which appear to be sporadic or show only weak genetic linkage.
Arg-Tyr-Asp (RYD) and Arg-Cys-Asp (RCD) motifs in dendroaspin promote selective inhibition of β1 and β3 integrins
Authors: WATTAM Beth , SHANG Dazhuang , RAHMAN Salman , EGGLEZOU Sofia , SCULLY Mike , KAKKAR Vijay , LU Xinjie
Published: August 2015

Arg-Gly-Asp (RGD) is a unique minimal integrin-binding sequence that is found within several glycoprotein ligands. This sequence has also been found in snake-venom anti-platelet proteins, including the disintegrins and dendroaspin, a natural variant of short-chain neurotoxins isolated from the venom of Dendroaspis jamesonii. In the present study, the motifs RYD and RCD were introduced into the dendroaspin scaffold to replace RGD. Both motifs in dendroaspin caused inhibition of ADP-induced platelet aggregation with IC50 values of 200 and 300nM respectively, similar to that of the wild-type RGD motif (170nM). In comparison with wild-type dendroaspin, both RYD- and RCD-containing dendroaspins were more selective in the inhibition of the adhesion of K562 cells to laminin rather than to fibrinogen and fibronectin, even though they were 10–30-fold less potent at inhibiting K562 cell (containing α5β1 integrin) adhesion to laminin compared with wild-type. Interestingly, the RYD motif produced a similar IC50 value to the RGD motif at inhibiting A375-SM cell (β3 integrin) adhesion to collagen, whereas the RCD motif was approx. 2–6-fold less potent compared with either RGD or RYD. These findings show that the selectivity of dendroaspin binding to β1 and β3 integrins can be modulated by the introduction of alternative cell recognition sequences.
Cloning of a lymphatic peptide/histidine transporter
Authors: SAKATA Kazuko , YAMASHITA Toshihide , MAEDA Mitsuyo , MORIYAMA Yoshinori , SHIMADA Shoichi , TOHYAMA Masaya
Published: August 2015

Although peptide transport across the plasma membrane has been characterized well in the kidney and the intestine, the functional relevance of this transport in other organs has not been addressed. Here we report the cloning of a cDNA for a novel peptide/histidine transporter found in the rat (rPHT2), whose mRNA is expressed mainly in the lymphatic system. rPHT2 encodes a protein of 582 amino acids and showed 49% identity with the brain PHT (PHT1) [Yamashita, Shimada, Guo, Sato, Kohmura, Hayakawa, Takagi and Tohyama (1997) J. Biol. Chem. 272, 10205–10211]. rPHT2 mRNA was abundant in lung, spleen and thymus, and detected faintly in brain, liver, adrenal gland and heart by Northern-blot analysis and reverse transcriptase PCR (RT-PCR). Intense signals for the gene were found in immunocytes using in situ hybridization. Ectopic expression of rPHT2 protein in HEK-293T cells and BHK cells was not found on the cell surface, but was found on the lysosomal membrane using light- and electron-microscopic analysis. Recombinant rPHT2 protein reconstituted into liposomes showed proton-dependent transport activity with histidine and histidyl-leucine. These findings suggest that rPHT2 is involved in the protein catabolic pathway in the lymphatic system.
Human α1-acid glycoprotein binds to CCR5 expressed on the plasma membrane of human primary macrophages
Authors: ATEMEZEM Aurélie , MBEMBA Elisabeth , VASSY Roger , SLIMANI Hocine , SAFFAR Line , GATTEGNO Liliane
Published: August 2015

We have reported previously that human α1-acid glycoprotein (AGP) inhibits the infection of human monocyte-derived macrophages (MDM) by R5 HIV-1, and that a disulphide-bridged peptide mimicking the clade B HIV-1 gp120 consensus V3 domain (V3Cs) binds specifically to CCR5 (the major co-receptor of R5 HIV strains) on these cells [Seddiki, Rabehi, Benjouad, Saffar, Ferriere, Gluckman and Gattegno (1997) Glycobiology 7, 1229–1236]. The present study demonstrates that AGP binds specifically to MDM at high- and low-affinity binding sites with Kd values of 16nM and 4.9μM respectively. The fact that heat denaturation of AGP only partly inhibited this binding (43%) suggests that protein–protein interactions are involved, as well as AGP glycans which are resistant to heat denaturation. Mannan, but not dextran, is a significant inhibitor (52%) of this binding, and sequential exoglycosidase treatment of AGP, which exposes penultimate mannose residues, has a strong stimulatory effect (∼ 2.8-fold). Therefore AGP glycans (probably mannose residues) are involved, at least partly, in the binding of AGP to MDM. In addition, AGP inhibits the binding of V3Cs and macrophage inflammatory protein-1β (MIP-1β) to MDM. The anti-CCR5 monoclonal antibody 2D7, specific for the second extracellular loop of CCR5, also inhibited AGP binding (67%), whereas anti-CCR5 antibodies specific for the C-terminus of CCR5 region had no effect. Native AGP, like V3Cs (but not heat-denatured AGP), binds to 46 and 33–36kDa electroblotted AGP-bound MDM membrane ligands, characterized as CCR5 by their interactions with anti-CCR5 antibodies and with MIP-1β. Therefore both AGP glycans and MDM CCR5 are involved in the binding of AGP to MDM. This suggests that the inhibitory effect of AGP on the infection of human primary macrophages by R5 HIV-1 may be related to specific binding of AGP to a macrophage membrane lectin or lectin-like component and to CCR5.
Cation-dependent structural features of β-casein-(1–25)
Authors: CROSS Keith J. , HUQ N. Laila , BICKNELL Wendy , REYNOLDS Eric C.
Published: August 2015

Complete sequence-specific, proton-resonance assignments have been determined for the calcium phosphate-stabilizing tryptic peptide β-casein-(1–25) containing the phosphorylated sequence motif Ser(P)17-Ser(P)-Ser(P)-Glu-Glu21. Spectra of the peptide have been recorded, in separate experiments, in the presence of excess ammonium ions, sodium ions and calcium ions, and of the dephosphorylated peptide in the presence of excess sodium ions. We observed significant changes to chemical shifts for backbone and side-chain resonances that were dependent upon the nature of the cation present. Medium-range nuclear Overhauser effect (nOe) enhancements, characteristic of small structured regions in the peptide, were observed and also found to be cation dependent. The secondary structure of the peptide was characterized by sequential and medium-range (i, i+2/3/4, which denotes an interaction between residue i and residue i+2, i+3 or i+4 in the peptide) nOe connectivities, and Hα chemical shifts. Four structured regions were identified in the calcium-bound peptide: residues Arg1 to Glu4 were involved in a loop-type structure, and residues Val8 to Glu11, Ser(P)17 to Glu20 and Glu21 to Thr24 were implicated in β-turn conformations. Comparison of the patterns of medium-range nOe connectivities in β-casein-(1–25) with those in αS1-casein-(59–79) suggest that the two peptides have distinctly different conformations in the presence of calcium ions, despite having a high degree of sequential and functional similarity.
Phosphatidylinositol transfer proteins and protein kinase C make separate but non-interacting contributions to the phosphorylation state necessary for secretory competence in rat mast cells
Authors: PINXTEREN Jef A. , GOMPERTS Bastien D. , ROGERS Danise , PHILLIPS Scott E. , TATHAM Peter E. R. , THOMAS Geraint M. H.
Published: August 2015

Mast cells permeabilized by streptolysin O undergo exocytosis when stimulated with Ca2+ and guanosine 5′-[γ-thio]triphosphate but become progressively refractory to this stimulus if it is delayed. This run-down of responsiveness occurs over a period of 20–30min, during which the cells leak soluble and tethered proteins. We show here that withdrawal of ATP during the process of run-down is strongly inhibitory but that as little as 25μM ATP can extend responsiveness significantly; this effect is maximal at 50μM. When phosphatidylinositol transfer proteins (PITPs) are provided to cells at the time of permeabilization, run-down is retarded. We conclude that in the presence of ATP they convey substrates for phosphorylation that are essential for exocytosis and thus interact with the regulatory machinery. Furthermore, we show that PITPα and PITPβ have additive effects in this mechanism, suggesting that they are not functionally redundant. Alternatively, secretion from run-down cells can be inhibited by the aminoglycoside antibiotic neomycin, which is understood to bind to phosphoinositide headgroups, and by a PH (pleckstrin homology) domain polypeptide that binds phosphoinositides. The apparent displacement of neomycin by exogenous PITPs suggests that these proteins screen essential lipids. Secretion from run-down cells is also inhibited by 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG-C16), an inhibitor of protein kinase C. The lack of synergy between neomycin and AMG-C16 suggests that protein kinase C independently provides a second essential component through protein phosphorylation and that there are two independent phosphorylation pathways necessary for secretion competence.
Characterization of a differentially expressed protein that shows an unusual localization to intracellular membranes in Leishmania major
Authors: KNUEPFER Ellen , STIERHOF York-Dieter , McKEAN Paul G. , SMITH Deborah F.
Published: August 2015

The SHERP genes are found as a tandem pair within the differentially regulated LmcDNA16 locus of Leishmania major. The SHERP gene product (small hydrophilic endoplasmic reticulum-associated protein) is unusual in its small size (6.2kDa), its acidic pI (4.6) and its exclusive, high-level expression (≈ 100000 copies per cell) in infective non-replicative parasite stages. No homologues have been found to date. Secondary-structure predictions suggest that SHERP contains an amphiphilic α-helix that is presumably involved in protein–protein interactions. SHERP has been localized to the endoplasmic reticulum as well as to the outer mitochondrial membrane in both wild-type and over-expressing parasites. Given the absence of an N-terminal signal sequence, transmembrane-spanning domains or detectable post-translational modifications, it is likely that this hydrophilic molecule is a peripheral membrane protein on the cytosolic face of intracellular membranes. This weak membrane association has been confirmed in cell-fractionation assays, in which SHERP redistributes from the cytoplasmic to the membrane fraction after in vivo cross-linking. SHERP does not appear to be involved in rearrangements of the cytoskeleton or conservation of organelle morphology during parasite differentiation. The role of this novel protein, presumed to be part of a protein complex, in infective parasites that are nutrient-deficient and pre-adapted for intracellular survival in the mammalian host is under investigation.
Metabolism of S-nitrosoglutathione in intact mitochondria
Authors: STEFFEN Mark , SARKELA Theresa M. , GYBINA Anna A. , STEELE Terry W. , TRASSETH Nathaniel J. , KUEHL Douglas , GIULIVI Cecilia
Published: August 2015

S-nitrosation of protein thiol groups by nitric oxide (NO•) is a widely recognized protein modification. Only few intracellular S-nitrosated proteins have been identified and it has been reported that S-nitrosation/denitrosation can serve as a regulatory process in signal-transduction pathways. Given the potential physiological importance of S-nitrosothiols, and considering that mitochondria are endowed with high levels of thiols and the biochemical requisites for synthesizing NO•, we examined the occurrence of S-nitrosoglutathione (GSNO) in intact, coupled rat liver mitochondria. These organelles contained 0.34nmol of GSNO/mg of protein, detected by HPLC with UV–visible and electrochemical detections. This concentration was dynamically modulated by the availability of NO•; its decay was affected mainly by GSH and superoxide dismutase in a reaction that entailed the generation of GSSG. On the basis of the relatively long half-life of GSNO and the negligible recovery of NO• during its decay, roles for GSNO as a storage and transport molecule for NO• are discussed. Moreover, the formation of GSNO and its reaction with GSH can be considered to be partly responsible for the catabolism of NO• via a complex mechanism that might result in the formation of hydroxylamine, nitrite or nitrous oxide depending upon the availability of oxygen, superoxide dismutase and glutathione. Finally, the high concentrations of GSH in the cytosol and mitochondria might favour the formation of GSNO by reacting with NO• ‘in excess’, thereby avoiding damaging side reactions (such as peroxynitrite formation), and facilitate the inactivation of NO• by generating other nitrogen-related species without the chemical properties characteristic of NO•.
Control of the threonine-synthesis pathway in Escherichia coli: a theoretical and experimental approach
Authors: CHASSAGNOLE Christophe , FELL David A. , RAÏS Badr , KUDLA Bernard , MAZAT Jean-Pierre
Published: August 2015

A computer simulation of the threonine-synthesis pathway in Escherichia coli Tir-8 has been developed based on our previous measurements of the kinetics of the pathway enzymes under near-physiological conditions. The model successfully simulates the main features of the time courses of threonine synthesis previously observed in a cell-free extract without alteration of the experimentally determined parameters, although improved quantitative fits can be obtained with small parameter adjustments. At the concentrations of enzymes, precursors and products present in cells, the model predicts a threonine-synthesis flux close to that required to support cell growth. Furthermore, the first two enzymes operate close to equilibrium, providing an example of a near-equilibrium feedback-inhibited enzyme. The predicted flux control coefficients of the pathway enzymes under physiological conditions show that the control of flux is shared between the first three enzymes: aspartate kinase, aspartate semialdehyde dehydrogenase and homoserine dehydrogenase, with no single activity dominating the control. The response of the model to the external metabolites shows that the sharing of control between the three enzymes holds across a wide range of conditions, but that the pathway flux is sensitive to the aspartate concentration. When the model was embedded in a larger model to simulate the variable demands for threonine at different growth rates, it showed the accumulation of free threonine that is typical of the Tir-8 strain at low growth rates. At low growth rates, the control of threonine flux remains largely with the pathway enzymes. As an example of the predictive power of the model, we studied the consequences of over-expressing different enzymes in the pathway.
Mapping the Zap-70 phosphorylation sites on LAT (linker for activation of T cells) required for recruitment and activation of signalling proteins in T cells
Authors: PAZ Pedro E. , WANG Soujuan , CLARKE Holly , LU Xaiobin , STOKOE David , ABO Arie
Published: August 2015

T-cell-receptor (TCR)-mediated LAT (linker for activation of T cells) phosphorylation is critical for the membrane recruitment of signalling complexes required for T-cell activation. Although tyrosine phosphorylation of LAT is required for recruitment and activation of signalling proteins, the molecular mechanism associated with this event is unclear. In the present study we reconstituted the LAT signalling pathway by demonstrating that a direct tyrosine phosphorylation of LAT with activated protein-tyrosine kinase Zap70 is necessary and sufficient for the association and activation of signalling proteins. Zap-70 efficiently phosphorylates LAT on tyrosine residues at positions 226, 191, 171, 132 and 127. By substituting these tyrosine residues in LAT with phenylalanine and by utilizing phosphorylated peptides derived from these sites, we mapped the tyrosine residues in LAT required for the direct interaction and activation of Vav, p85/p110α and phospholipase Cγ1 (PLCγ1). Our results indicate that Tyr226 and Tyr191 are required for Vav binding, whereas Tyr171 and Tyr132 are necessary for association and activation of phosphoinositide 3-kinase activity and PLCγ1 respectively. Furthermore, by expression of LAT mutants in LAT-deficient T cells, we demonstrate that Tyr191 and Tyr171 are required for T-cell activation and Tyr132 is required for the activation of PLCγ1 and Ras signalling pathways.
Role for phosphatidylinositol in nuclear envelope formation
Authors: LARIJANI Banafshé , BARONA Teresa M. , POCCIA Dominic L.
Published: August 2015

PtdIns is a minor membrane phospholipid that is important in signal transduction. Recently, derivatives of PtdIns phosphorylated at the 3-position of the inositol ring have been implicated in the regulation of constitutive membrane traffic and in membrane fusion events. Assembly of the nuclear envelope (NE), a crucial step in the progress of mitosis, is also likely to involve membrane fusion reactions. We therefore investigated the role of PtdIns and phosphoinositide 3-kinase (PI-3K) activity in NE formation in vitro. GTP-induced NE formation was blocked by wortmannin and LY294002, two specific inhibitors of PI-3K, suggesting a role for PtdIns phosphorylated at the 3-position. PtdIns-specific phospholipase C mimicked GTP hydrolysis as an inducer of NE formation. This induction was dependent on a membrane vesicle subfraction (MV1) that was highly enriched in PtdIns, as determined by heteronuclear two-dimensional NMR spectroscopy. On the basis of these results, we suggest that the MV1 population serves as a source of membranes rich in PtdIns that might facilitate fusion, possibly through the production of the membrane-destabilizing lipid diacylglycerol.