bluuebunny/crossref_metadata_2025 huggingface dataset:
A mitochondrial uncoupling artifact can be caused by expression of uncoupling protein 1 in yeast
Authors: STUART Jeff A. , HARPER James A. , BRINDLE Kevin M. , JEKABSONS Mika B. , BRAND Martin D.
Published: August 2015

Uncoupling protein 1 (UCP1) from mouse was expressed in yeast and the specific (GDP-inhibitable) and artifactual (GDP-insensitive) effects on mitochondrial uncoupling were assessed. UCP1 provides a GDP-inhibitable model system to help interpret the uncoupling effects of high expression in yeast of other members of the mitochondrial carrier protein family, such as the UCP1homologues UCP2 and UCP3. Yeast expressing UCP1 at modest levels (approx. 1μg/mg of mitochondrial protein) showed no growth defect, normal rates of chemically uncoupled respiration and an increased non-phosphorylating proton conductance that was completely GDP-sensitive. The catalytic-centre activity of UCP1 in these yeast mitochondria was similar to that in mammalian brown-adipose-tissue mitochondria. However, yeast expressing UCP1 at higher levels (approx. 11μg/mg of mitochondrial protein) showed a growth defect. Their mitochondria had depressed chemically uncoupled respiration rates and an increased proton conductance that was partly GDP-insensitive. Thus, although UCP1 shows native behaviour at modest levels of expression in yeast, higher levels (or rates) of expression can lead to an uncoupling that is not a physiological property of the native protein and is therefore artifactual. This observation might be important in the interpretation of results from experiments in which the functions of UCP1homologues are verified by their ability to uncouple yeast mitochondria.
Intracellular distribution of glycogen synthase and glycogen in primary cultured rat hepatocytes
Authors: GARCÍA-ROCHA Mar , ROCA Angela , de la IGLESIA Núria , BABA Otto , FERNÁNDEZ-NOVELL Josep M. , FERRER Juan C. , GUINOVART Joan J.
Published: August 2015

Changes in the intracellular distribution of liver glycogen synthase (GS) might constitute a new regulatory mechanism for the activity of this enzyme at cellular level. Our previous studies indicated that incubation of isolated hepatocytes with glucose activated GS and resulted in its translocation from a homogeneous cytosolic distribution to the cell periphery. These studies also suggested a relationship with insoluble elements of the cytoskeleton, in particular actin. Here we show the translocation of GS in a different experimental model that allows the analysis of this phenomenon in long-term studies. We describe the reversibility of translocation of GS and its effect on glycogen distribution. Incubation of cultured rat hepatocytes with glucose activated GS and triggered its translocation to the hepatocyte periphery. The relative amount of the enzyme concentrated near the plasma membrane increased with time up to 8h of incubation with glucose, when the glycogen stores reached their maximal value. The lithium-induced covalent activation of GS was not sufficient to cause its translocation to the cell periphery. The intracellular distribution of GS closely resembled that of glycogen. Our results showed an interaction between GS and an insoluble element of the hepatocyte matrix. Although no co-localization between actin filaments and GS was observed in any condition, disruption of actin cytoskeleton resulted in a significantly lower percentage of cells in which the enzyme translocated to the cell periphery in response to glucose. This observation suggests that the microfilament network has a role in the translocation of GS.
Glutathione S-transferases as antioxidant defence agents confer pyrethroid resistance in Nilaparvata lugens
Authors: VONTAS John G. , SMALL Graham J. , HEMINGWAY Janet
Published: August 2015

Selection of a laboratory colony of the brown planthopper Nilaparvata lugens with the pyrethroids permethrin and λ-cyhalothrin increased its resistance to both insecticides. Biochemical analysis and synergistic studies with metabolic inhibitors indicated that elevated glutathione S-transferases (GSTs) with a predominant peroxidase activity conferred resistance to both pyrethroids, whereas esterases conferred part of the resistance to permethrin. Purified esterases hydrolysed permethrin at a slow rate, but incubation of either pyrethroid or their primary metabolites with partially purified GSTs had no effect on the metabolic profile. Although GSTs were sensitive to inhibition by both pyrethroids, they did not serve as binding proteins, as previously hypothesized [Grant and Matsumura (1988) Insect Biochem. 18, 615–622]. We demonstrate that pyrethroids, in addition to their neurotoxic effect, induce oxidative stress and lipid peroxidation in insects. Pyrethroid exposure induced lipid peroxides, protein oxidation and depleted reduced glutathione. Elevated GSTs in the resistant strains attenuated the pyrethroid-induced lipid peroxidation and reduced mortality, whereas their in vivo inhibition eliminated their protective role. We therefore hypothesize that the main role of elevated GSTs in conferring resistance in N. lugens is through protecting tissues from oxidative damage. Our study extends the GSTs' range of efficacy to pyrethroid insecticides and possibly explains the role of elevated GSTs in other pyrethroid-resistant insects.
Activation of pro-(matrix metalloproteinase-2) (pro-MMP-2) by thrombin is membrane-type-MMP-dependent in human umbilical vein endothelial cells and generates a distinct 63 kDa active species
Authors: LAFLEUR Marc A. , HOLLENBERG Morley D. , ATKINSON Susan J. , KNÄUPER Vera , MURPHY Gillian , EDWARDS Dylan R.
Published: August 2015

Thrombin, a critical enzyme in the coagulation cascade, has also been associated with angiogenesis and activation of the zymogen form of matrix metalloproteinase-2 (MMP-2 or gelatinase-A). We show that thrombin activated pro-MMP-2 in a dose- and time-dependent manner in cultured human umbilical-vein endothelial cells (HUVECs) to generate a catalytically active 63kDa protein that accumulated as the predominant form in the conditioned medium. This 63kDa thrombin-activated MMP-2 is distinct from the 62kDa species found following concanavalin A or PMA stimulated pro-MMP-2 activation. Hirudin and leupeptin blocked thrombin-induced pro-MMP-2 activation, demonstrating that the proteolytic activity of thrombin is essential. However, activation was also dependent upon membrane-type-MMP (MT-MMP) action, since it was blocked by EDTA, o-phenanthroline, hydroxamate metalloproteinase inhibitors, tissue inhibitor of metalloproteinase-2 (TIMP-2) and TIMP-4, but not TIMP-1. Thrombin inefficiently cleaved recombinant 72kDa pro-MMP-2, but efficiently cleaved the 64kDa MT-MMP-processed intermediate form in the presence of cells. Thrombin also rapidly (within 1h) increased cellular MT-MMP activity, and at longer time points (>6h) it increased expression of MT1-MMP mRNA and protein. Thus signalling via proteinase-activated receptors (PARs) may play a role in thrombin-induced MMP-2 activation, though this does not appear to involve PAR1, PAR2, or PAR4 in HUVECs. These results indicate that in HUVECs the activation of pro-MMP-2 by thrombin involves increased MT-MMP activity and preferential cleavage of the MT-MMP-processed 64kDa MMP-2 form in the presence of cells. The integration of these proteinase systems in the vascular endothelium may be important during thrombogenesis and tissue remodelling associated with neovascularization.
Elevated extracellular [K+] inhibits death-receptor- and chemical-mediated apoptosis prior to caspase activation and cytochrome c release
Authors: THOMPSON Gwilym J. , LANGLAIS Claudia , CAIN Kelvin , CONLEY Edward C. , COHEN Gerald M.
Published: August 2015

Efflux of intracellular K+ and cell shrinkage are features of apoptosis in many experimental systems, and a regulatory role has been proposed for cytoplasmic [K+] in initiating apoptosis. We have investigated this in both death-receptor-mediated and chemical-induced apoptosis. Using Jurkat T cells pre-loaded with the K+ ion surrogate 86Rb+, we have demonstrated an efflux of intracellular K+ during apoptosis that was concomitant with, but did not precede, other apoptotic changes, including phosphatidylserine externalization, mitochondrial depolarization and cell shrinkage. To further clarify the role of K+ ions in apoptosis, cytoprotection by elevated extracellular [K+] was studied. Induction of apoptosis by diverse death-receptor and chemical stimuli in two cell lines was inhibited prior to phosphatidylserine externalization, mitochondrial depolarization, cytochrome c release and caspase activation. Using a cell-free system, we have demonstrated a novel mechanism by which increasing [K+] inhibited caspase activation. In control dATP-activated lysates, Apaf-1 oligomerized to a biologically active caspase processing ≈ 700kDa complex and an inactive ≈ 1.4 MDa complex. Increasing [K+] inhibited caspase activation by preventing formation of the ≈ 700kDa complex, but not of the inactive complex. Thus intracellular and extracellular [K+] markedly affect caspase activation and the initiation of apoptosis induced by both death-receptor ligation and chemical stress.
Inhibition of HIV-1 gene expression by novel macrophage-tropic DNA enzymes targeted to cleave HIV-1 TAT/Rev RNA
Authors: UNWALLA Hoshang , BANERJEA Akhil C.
Published: August 2015

Many regions of the HIV-1 genome have been targeted in earlier studies by RNA-cleaving DNA enzymes possessing the 10–23 catalytic motif, and efficient inhibition of HIV-1 gene expression was reported. All these studies employed charged synthetic lipids to introduce the catalytic DNA into the mammalian cells, which severely limits its practical application and usefulness in vivo. Taking advantage of the ability of G residues to interact directly with the scavenger receptors on the macrophages, we synthesized a DNA enzyme 5970 that contained 10 G residues at the 3′ end. With the aim of improving the intracellular stability of the DNA enzyme 5970, we added two short stretches of stem-loop structures that were 12 bases long on either side of the DNA enzyme 5970. DNA enzyme 5970 without the poly-G tracts cleaved the synthetic RNA of HIV-1 TAT/Rev, two important regulatory proteins of HIV, very efficiently in a sequence-specific manner. Addition of 10 G residues at the 3′ end of the DNA enzyme affected the cleavage efficiency only marginally whereas the same DNA enzyme with stem-loop structures on either end was significantly less efficient. The DNA enzyme with the poly-G tract at its 3′ end was taken up specifically by a human macrophage-specific cell line directly in the absence of Lipofectin and was also able to inhibit HIV-1 gene expression in a transient-expression system as well as when challenged with the virus. The potential applications of these novel macrophage-tropic DNA enzymes are discussed.
Hsp70 regulates the interaction between the peroxisome targeting signal type 1 (PTS1)-receptor Pex5p and PTS1
Authors: HARANO Tomoyuki , NOSE Shizuko , UEZU Rumiko , SHIMIZU Nobuyoshi , FUJIKI Yukio
Published: August 2015

The peroxisome targeting signal type 1 (PTS1) receptor, Pex5p, of the tetratricopeptide repeat (TPR) motif family is located mostly in the cytosol and mediates the translocation of PTS1 proteins to peroxisomes. As a step towards understanding the mechanisms of protein import into peroxisomes, we investigated the molecular mechanisms involved in PTS1 recognition by Pex5p with regard to requirement of energy and cytosolic factors, using cell-free synthesized acyl-CoA oxidase (AOx) as a PTS1 cargo protein, together with Pex5p and heat-shock protein (Hsp)70 from rat liver. Pex5p was partly associated with peroxisomes of rat liver, was resistant to washing with a high concentration of salt and to alkaline extraction and was inaccessible to protease added externally. Pex5p bound to AOx in an ATP-dependent manner. AOx synthesized in a cell-free translating system from rabbit reticulocyte lysate was imported into peroxisomes without being supplemented with Pex5p and Hsp70, implying that peroxisome-associated Pex5p was released from the membranes and functional in this in vitro import assay. Antibodies against Pex5p and Hsp70 inhibited AOx import. In contrast, AOx synthesized in a wheat-germ lysate required the external addition of Pex5p for import, in which Hsp70 augmented the AOx import. The TPR domain of Pex5p was revealed to bind to the N-terminal part in an Hsp70-independent manner, whereas mutual interaction of the TPR region was noted in the presence of Hsp70. Hsp70 interacted with the TPR domain of Pex5p. Moreover, Hsp70 and ATP synergistically enhanced the binding of Pex5p to the C-terminal PTS1-containing part of AOx, implying that Pex5p recognizes its cargo PTS1 protein by chaperone-assisted as well as energy-dependent mechanisms in vivo.
Presteady-state kinetics of Bacillus 1,3–1,4-β-glucanase: binding and hydrolysis of a 4-methylumbelliferyl trisaccharide substrate
Authors: ABEL Mireia , PLANAS Antoni , CHRISTENSEN Ulla
Published: August 2015

In the present study the first stopped-flow experiments performed on Bacillus 1,3–1,4-β-glucanases are reported. The presteady-state kinetics of the binding of 4-methylumbelliferyl 3-O-β-cellobiosyl-β-d-glucoside to the inactive mutant E134A, and the wild-type-catalysed hydrolysis of the same substrate, were studied by measuring changes in the fluorescence of bound substrate or 4-methylumbelliferone produced. The presteady-state traces all showed an initial lag phase followed by a fast monoexponential phase leading to equilibration (for binding to E134A) or to steady state product formation (for the wild-type reaction). The lag phase, with a rate constant of the order of 100s−1, was independent of the substrate concentration; apparently an induced-fit mechanism governs the formation of enzyme–substrate complexes. The concentration dependencies of the observed rate constant of the second presteady-state phase were analysed according to a number of reaction models. For the reaction of the wild-type enzyme, it is shown that the fast product formation observed before steady state is not due to a rate-determining deglycosylation step. A model that can explain the observed results involves, in addition to the induced fit, a conformational change of the productive ES complex into a form that binds a second substrate molecule in a non-productive mode.
Role of the cystine-knot motif at the C-terminus of rat mucin protein Muc2 in dimer formation and secretion
Authors: BELL Sherilyn L. , XU Gongqiao , FORSTNER Janet F.
Published: August 2015

DNA constructs based on the 534-amino-acid C-terminus of rat mucin protein Muc2 (RMC), were transfected into COS cells and the resultant 35S-labelled dimers and monomers were detected by SDS/PAGE of immunoprecipitates. The cystine-knot construct, encoding the C-terminal 115 amino acids, appeared in cell lysates as a 45kDa dimer, but was not secreted. A construct, devoid of the cystine knot, failed to form dimers. Site-specific mutagenesis within the cystine knot was performed on a conserved unpaired cysteine (designated Cys-X), which has been implicated in some cystine-knot-containing growth factors as being important for intermolecular disulphide-bond formation. Dimerization of RMC was effectively abolished. Each cysteine (Cys-1–Cys-6) comprising the three intramolecular disulphide bonds of the cystine knot was then mutated. Dimer formation was impaired in each case, although much less so for the Cys-3 mutant than the others. Abnormal high-molecular-mass, disulphide-dependent aggregates formed with mutations Cys-1, Cys-2, Cys-4 and Cys-5, and were poorly secreted. It is concluded that the intact cystine-knot domain is essential for dimerization of the C-terminal domain of rat Muc2, and that residue Cys-X in the knot plays a key role. The structural integrity of the cystine knot, maintained by intramolecular bonds Cys-1–Cys-4, Cys-2–Cys-5 and Cys-3–Cys-6, also appears to be important for dimerization, probably by allowing correct positioning of the unpaired Cys-X residue for stable intermolecular cystine-bond formation.
Conformational stability and warfarin-binding properties of human serum albumin studied by recombinant mutants
Authors: WATANABE Hiroshi , KRAGH-HANSEN Ulrich , TANASE Sumio , NAKAJOU Keisuke , MITARAI Maki , IWAO Yasunori , MARUYAMA Toru , OTAGIRI Masaki
Published: August 2015

Correctly folded recombinant wild-type human serum albumin and the single-residue mutants K199A, W214A, R218H and H242Q were produced with the use of a yeast expression system. The changes in R218H resulted in a pronounced decrease in intrinsic fluorescence. Thermodynamic parameters for thermal denaturation of the present mutants and of five additional mutants have been determined, showing small increases in stability for two mutants (R218H and H242Q) and a larger decrease in stability for one (W214A). In the last of these, denaturation was a heterogeneous process starting at physiological temperature. The high-affinity binding constant for warfarin at pH7.4 was determined by fluorescence spectroscopy: there was a significant increase in affinity for binding of warfarin to H242Q and K199A and a smaller decrease in affinity for W214A and R218H. The findings show that Trp-214 is not as essential for the high-affinity binding of warfarin as has previously been thought.
Asparagine synthetase expression alone is sufficient to induce l-asparaginase resistance in MOLT-4 human leukaemia cells
Authors: ASLANIAN Ara M. , FLETCHER Bradley S. , KILBERG Michael S.
Published: August 2015

Childhood acute lymphoblastic leukaemia (ALL) is treated by combination chemotherapy with a number of drugs, always including the enzyme l-asparaginase (ASNase). Although the initial remission rate is quite high, relapse and associated drug resistance are a significant problem. In vitro studies have demonstrated increased asparagine synthetase (AS) expression in ASNase-resistant cells, which has led to the hypothesis that elevated AS activity permits drug-resistant survival. The data presented show that not only is elevated AS expression a property of ASNase-resistant MOLT-4 human leukaemia cells, but that short-term (12h) treatment of the cells with ASNase causes a relatively rapid induction of AS expression. The results also document that the elevated expression of AS in ASNase-resistant cells is not fully reversible, even 6 weeks after ASNase removal from the culture medium. Furthermore, ASNase resistance, assessed as both drug-insensitive cell growth rates and decreased drug-induced apoptosis, parallels this irreversible AS expression. Mimicking the elevated AS activity in ASNase-resistant cells by overexpression of the human AS protein by stable retroviral transformation of parental MOLT4 cells is sufficient to induce the ASNase-resistance phenotype. These data document that ASNase resistance in ALL cells is a consequence of elevated AS expression and that although other drug-induced metabolic changes occur, they are secondary to the increased asparagine biosynthetic rate.
Metalloprotease-disintegrin ADAM 12 interacts with α-actinin-1
Authors: CAO Yi , KANG Qing , ZOLKIEWSKA Anna
Published: August 2015

ADAM 12, a member of the ADAM family of proteins (containing ADisintegrin And Metalloprotease domain), has been implicated in differentiation and fusion of myoblasts. While the extracellular domain of ADAM 12 contains an active metalloprotease and a region involved in cell adhesion, the function of the cytoplasmic tail of ADAM 12 has been less clear. Here we show that the cytoplasmic domain of ADAM 12 interacts in vitro and in vivo with α-actinin-1, an actin-binding and cross-linking protein. Green fluorescent protein fused to ADAM 12 cytoplasmic domain co-localizes with α-actinin-1-containing actin stress fibres in C2C12 cells. The interaction between ADAM 12 and α-actinin-1 is direct and involves the 58-amino acid C-terminal fragment of ADAM 12 and the 27kDa N-terminal domain of α-actinin-1. Consistently, expression of the 27kDa fragment of α-actinin-1 in C2C12 cells using a mitochondrial targeting system results in recruitment of the co-expressed ADAM 12 cytoplasmic domain to the mitochondrial surface. Moreover, α-actinin-1 co-purifies with a transmembrane, His6-tagged form of ADAM 12 expressed in C2C12 myoblasts, indicating that the transmembrane ADAM 12 forms a complex with α-actinin-1 in vivo. These results indicate that the actin cytoskeleton may play a critical role in ADAM 12-mediated cell–cell adhesion or cell signalling during myoblast differentiation and fusion.
FK506-binding protein of the hyperthermophilic archaeum, Thermococcus sp. KS-1, a cold-shock-inducible peptidyl-prolyl cis–trans isomerase with activities to trap and refold denatured proteins
Authors: IDENO Akira , YOSHIDA Takao , IIDA Toshii , FURUTANI Masahiro , MARUYAMA Tadashi
Published: August 2015

The FK506 (tacrolimus)-binding protein (FKBP) type peptidyl-prolyl cis–trans isomerase (PPIase) in the hyperthermophilic archaeum Thermococcus sp. KS-1 was shown to be induced by temperature downshift to growth temperatures lower than the optimum. This PPIase (TcFKBP18) showed chaperone-like protein refolding activity in addition to PPIase activity in vitro. It refolded unfolded citrate synthase (CS) and increased the yield of the refolded protein. At a molar ratio of 15:1 ([TcFKBP18] to [CS]) in the refolding mixture, the recovered yield of folded CS was maximal at 62%, whereas that of spontaneous refolding was 11%. Increasing FKBP above a 15:1 ratio decreased the final yield, whereas the aggregation of unfolded CS was suppressed. A cross-linking analysis showed the formation of a complex between TcFKBP18 and unfolded CS (1:1 complex) at molar ratios of 3:1 to 15:1. However, molar ratios of 15:1 or 60:1 induced the binding of multiple FKBP molecules to an unfolded CS molecule (multimeric complex). Disrupting hydrophobic interaction by adding ethylene glycol at a molar ratio of 60:1 ([TcFKBP18] to [CS]) suppressed the formation of this multimeric complex, simultaneously enhancing CS refolding. FK506 also suppressed the formation of the multimeric complex while increasing the chaperone-like activity. These results suggest that the hydrophobic region of TcFKBP18, probably the FK506-binding pocket, was important for the interaction with unfolded proteins. No cross-linked product was detected between TcFKBP18 and native dimeric CS. TcFKBP18 probably traps the unfolded protein, then refolds and releases it in a native form. This FKBP might be important at growth temperatures lower than the optimum in Thermococcus sp. KS-1 cells.
G-protein-coupled-receptor activation of the smooth muscle calponin gene
Authors: DULIN Nickolai O. , ORLOV Sergei N. , KITCHEN Chad M. , VOYNO-YASENETSKAYA Tatyana A. , MIANO Joseph M.
Published: August 2015

A hallmark of cultured smooth muscle cells (SMCs) is the rapid down-regulation of several lineage-restricted genes that define their in vivo differentiated phenotype. Identifying factors that maintain an SMC differentiated phenotype has important implications in understanding the molecular underpinnings governing SMC differentiation and their subversion to an altered phenotype in various disease settings. Here, we show that several G-protein coupled receptors [α-thrombin, lysophosphatidic acid and angiotensin II (AII)] increase the expression of smooth muscle calponin (SM-Calp) in rat and human SMC. The increase in SM-Calp protein appears to be selective for G-protein-coupled receptors as epidermal growth factor was without effect. Studies using AII showed a 30-fold increase in SM-Calp protein, which was dose- and time-dependent and mediated by the angiotensin receptor-1 (AT1 receptor). The increase in SM-Calp protein with AII was attributable to transcriptional activation of SM-Calp based on increases in steady-state SM-Calp mRNA, increases in SM-Calp promoter activity and complete abrogation of protein induction with actinomycin D. To examine the potential role of extracellular signal-regulated kinase (Erk1/2), protein kinase B, p38 mitogen-activated protein kinase and protein kinase C in AII-induced SM-Calp, inhibitors to each of the signalling pathways were used. None of these signalling molecules appears to be crucial for AII-induced SM-Calp expression, although Erk1/2 may be partially involved. These results identify SM-Calp as a target of AII-mediated signalling, and suggest that the SMC response to AII may incorporate a novel activity of SM-Calp.
Increase in class 2 aldehyde dehydrogenase expression by arachidonic acid in rat hepatoma cells
Authors: CANUTO Rosa A. , FERRO Margherita , SALVO Raffaella A. , BASSI Anna M. , TROMBETTA Antonella , MAGGIORA Marina , MARTINASSO Germana , LINDAHL Ronald , MUZIO Giuliana
Published: August 2015

Aldehyde dehydrogenase (ALDH) is a family of several isoenzymes important in cell defence against both exogenous and endogenous aldehydes. Compared with normal hepatocytes, in rat hepatoma cells the following changes in the expression of ALDH occur: cytosolic class 3 ALDH expression appears and mitochondrial class 2 ALDH decreases. In parallel with these changes, a decrease in the polyunsaturated fatty acid content in membrane phospholipids occurs. In the present study we demonstrated that restoring the levels of arachidonic acid in 7777 and JM2 rat hepatoma cell lines to those seen in hepatocytes decreases hepatoma cell growth, and increases class 2 ALDH activity. This latter effect appears to be due to an increased gene transcription of class 2 ALDH. To account for this increase, we examined whether peroxisome-proliferator-activated receptors (PPARs) or lipid peroxidation were involved. We demonstrated a stimulation of PPAR expression, which is different in the two hepatoma cell lines: in the 7777 cell line, there was an increase in PPARα expression, whereas PPARγ expression increased in JM2 cells. We also found increased lipid peroxidation, but this increase became evident at a later stage when class 2 ALDH expression had already increased. In conclusion, arachidonic acid added to the culture medium of hepatoma cell lines is able to partially restore the normal phenotype of class 2 ALDH, in addition to a decrease in cell growth.
Possible mechanisms involved in the down-regulation of translation during transient global ischaemia in the rat brain
Authors: MARTÍN de la VEGA Cristina , BURDA Jozef , NEMETHOVA Miroslava , QUEVEDO Celia , ALCÁZAR Alberto , MARTÍN M. Elena , DANIELISOVA Viera , FANDO Juan L. , SALINAS Matilde
Published: August 2015

The striking correlation between neuronal vulnerability and down-regulation of translation suggests that this cellular process plays a critical part in the cascade of pathogenetic events leading to ischaemic cell death. There is compelling evidence supporting the idea that inhibition of translation is exerted at the polypeptide chain initiation step, and the present study explores the possible mechanism/s implicated. Incomplete forebrain ischaemia (30min) was induced in rats by using the four-vessel occlusion model. Eukaryotic initiation factor (eIF)2, eIF4E and eIF4E-binding protein (4E-BP1) phosphorylation levels, eIF4F complex formation, as well as eIF2B and ribosomal protein S6 kinase (p70S6K) activities, were determined in different subcellular fractions from the cortex and the hippocampus [the CA1-subfield and the remaining hippocampus (RH)], at several post-ischaemic times. Increased phosphorylation of the α subunit of eIF2 (eIF2α) and eIF2B inhibition paralleled the inhibition of translation in the hippocampus, but they normalized to control values, including the CA1-subfield, after 4–6h of reperfusion. eIF4E and 4E-BP1 were significantly dephosphorylated during ischaemia and total eIF4E levels decreased during reperfusion both in the cortex and hippocampus, with values normalizing after 4h of reperfusion only in the cortex. Conversely, p70S6K activity, which was inhibited in both regions during ischaemia, recovered to control values earlier in the hippocampus than in the cortex. eIF4F complex formation diminished both in the cortex and the hippocampus during ischaemia and reperfusion, and it was lower in the CA1-subfield than in the RH, roughly paralleling the observed decrease in eIF4E and eIF4G levels. Our findings are consistent with a potential role for eIF4E, 4E-BP1 and eIF4G in the down-regulation of translation during ischaemia. eIF2α, eIF2B, eIF4G and p70S6K are positively implicated in the translational inhibition induced at early reperfusion, whereas eIF4F complex formation is likely to contribute to the persistent inhibition of translation observed at longer reperfusion times.
Knockout of mouse β1,4-galactosyltransferase-1 gene results in a dramatic shift of outer chain moieties of N-glycans from type 2 to type 1 chains in hepatic membrane and plasma glycoproteins
Authors: KOTANI Norihiro , ASANO Masahide , IWAKURA Yoichiro , TAKASAKI Seiichi
Published: August 2015

To understand the contribution of β1,4-galactosyltransferase (β4Gal-T)-1 to galactosylation in vivo, N-glycans of hepatic membrane glycoproteins and plasma glycoproteins from β4Gal-T1 wild-type (β4Gal-T1+/+) and β4Gal-T1 knockout mice were compared. Unexpectedly, glycoproteins from the knockout mice were found to express considerable amounts of sialylated, galactosylated N-glycans. A striking contrast was that galactose residues were largely β1,4-linked to GlcNAc residues in the β4Gal-T1+/+ mouse glycans but β1,3-linked in the knockout mouse glycans, thus resulting in the shift of the backbone structure from type 2 chain (Galβ1 → 4GlcNAc) to type 1 chain (Galβ1 → 3GlcNAc). Detailed analysis of plasma glycoproteins revealed that the expression of sialyl linkage in N-glycans was shifted from the Siaα2 → 6Gal to the Siaα2 → 3Gal, and oversialylated type 1 chains were, remarkably, found in the knockout mouse glycans. Thus β4Gal-T1 deficiency was primarily compensated for by β1,3-galactosyltransferases, which resulted in different sialyl linkages being formed on the outer chains and altered backbone structures, depending on the acceptor specificities of sialyltransferases. These results suggest that β4Gal-T1 in mouse liver plays a central role in the synthesis of type 2 chain and is also involved in the regulation of sialylation of N-glycans. The knockout mice may prove useful in investigation of the mechanism which regulates the tissue-dependent terminal glycosylation.
Type-1 ribosome-inactivating protein from iris (Iris hollandica var. Professor Blaauw) binds specific genomic DNA fragments
Authors: HAO Qiang , PEUMANS Willy J. , van DAMME Els J. M.
Published: August 2015

The capacity of IRIP, a type-1 ribosome-inactivating protein (RIP) isolated from the bulbs of Iris hollandica, to bind specific DNA sequences from a mixture of approx. 200bp (average length) fragments of total genomic DNA from Iris genome was studied. Fragments that were preferentially bound by IRIP were enriched by several cycles of affinity binding and PCR, and were cloned and sequenced. The selected DNA fragments do not share conserved sequences, indicating that IRIP does not bind DNA fragments in a strictly sequence-specific manner. According to sequence analysis, most IRIP-bound fragments contain one or more possible free energy-stable hairpin structure(s) in their secondary structure, which may be the basis for recognition between IRIP and these DNA fragments. Some, but not all, DNA fragments moderately lower the RNA N-glycosidase activity of IRIP towards rabbit reticulocyte lysate ribosomes. IRIP does not remove adenines from the binding fragments, which implies that it does not act as a polynucleotide:adenosine glycosidase towards these DNA fragments. The selective binding of IRIP to conspecific DNA fragments is also discussed in view of the novel concept that RIPs may act as DNA-binding proteins with a regulatory activity on gene expression.
Angiotensin II-responsive element is the insulin-responsive element in the adipocyte fatty acid synthase gene: role of adipocyte determination and differentiation factor 1/sterol-regulatory-element-binding protein 1c
Authors: KIM Suyeon , DUGAIL Isabelle , STANDRIDGE Melissa , CLAYCOMBE Kate , CHUN Joseph , MOUSTAÏD-MOUSSA Naïma
Published: August 2015

We have previously shown that angiotensin II (Ang II) increases the expression of the gene encoding adipocyte fatty acid synthase (FAS). Here we investigate the mechanism responsible for increased FAS gene transcription by Ang II. We demonstrate that Ang II increased luciferase activity by 3-fold in 3T3-L1 adipocytes transfected with fusion constructs linking the FAS promoter to the luciferase reporter gene. Interestingly, we mapped the Ang II regulatory sequences to the insulin-responsive region (E box) in the proximal FAS promoter. The E box alone was able to mediate Ang II responsiveness when linked to a heterologous promoter. However, this response was lost when mutations that abolished the binding of the E box to its transcription factors were introduced. Using adenoviral overexpression of a dominant-negative form of adipocyte determination and differentiation factor 1 (ADD1), a transcription factor that binds to the insulin-responsive E box, we demonstrated that ADD1 was required for Ang II regulation of the FAS gene in 3T3-L1 adipocytes. Furthermore, ADD1 expression was also up-regulated by Ang II. With the use of transfections as well as glucose transport assays, we further demonstrated that Ang II stimulation of the FAS gene was dependent on glucose. In conclusion, this is the first report that Ang II regulates adipocyte FAS gene transcription via insulin response sequences in a glucose-dependent manner and that this regulation is mediated at least in part via the ADD1 transcription factor.
The characterization of ligand-specific maize (Zea mays) profilin mutants
Authors: KOVAR David R. , DRØBAK Bj⊘rn K. , COLLINGS David A. , STAIGER Christopher J.
Published: August 2015

Profilins are low-molecular-mass (12–15kDa) cytosolic proteins that are major regulators of actin assembly in all eukaryotic cells. In general, profilins from evolutionarily diverse organisms share the ability to bind to G-actin, poly-(l-proline) (PLP) and proline-rich proteins, and polyphosphoinositides. However, the functional importance of each of these interactions remains unclear and might differ between organisms. We investigated the importance of profilin's interaction with its various ligands in plant cells by characterizing four maize (Zea mays) profilin 5 (ZmPRO5) mutants that had single amino acid substitutions in the presumed sites of ligand interaction. Comparisons in vitro with wild-type ZmPRO5 showed that these mutations altered ligand association specifically. ZmPRO5-Y6F had a 3-fold increased affinity for PLP, ZmPRO5-Y6Q had a 5-fold decreased affinity for PLP, ZmPRO5-D8A had a 2-fold increased affinity for PtdIns(4,5)P2 and ZmPRO5-K86A had a 35-fold decreased affinity for G-actin. When the profilins were microinjected into Tradescantia stamen hair cells, ZmPRO5-Y6F increased the rate of nuclear displacement in stamen hairs, whereas ZmPRO5-K86A decreased the rate. Mutants with a decreased affinity for PLP (ZmPRO5-Y6Q) or an enhanced affinity for PtdIns(4,5)P2 (ZmPRO5-D8A) were not significantly different from wild-type ZmPRO5 in affecting nuclear position. These results indicate that plant profilin's association with G-actin is extremely important and further substantiate the simple model that profilin acts primarily as a G-actin-sequestering protein in plant cells. Furthermore, interaction with proline-rich binding partners might also contribute to regulating profilin's effect on actin assembly in plant cells.
Selective induction of phospholipase D1 in pathogen-activated human monocytes
Authors: LOCATI Massimo , RIBOLDI Elena , BONECCHI Raffaella , TRANSIDICO Pietro , BERNASCONI Sergio , HARIBABU Bodduluri , MORRIS Andrew J. , MANTOVANI Alberto , SOZZANI Silvano
Published: August 2015

Phospholipase D (PLD) activation is part of the complex signalling cascade induced during phagocyte activation. Two PLD isoforms have been cloned, but their role in phagocyte functions is still poorly defined. We report that resting fresh circulating human monocytes expressed PLD1. PLD1 protein expression was rapidly down-regulated during cell culture. Lipopolysaccharide and pathogen-derived agonists (Candida albicans, arabinoside-terminated lipoarabinomannan and Gram-positive bacteria, but not mannose-capped lipoarabinomannan or double-stranded RNA) strongly induced PLD1 expression at both the mRNA and protein levels. Pro-inflammatory cytokines [interleukin (IL)-1β and tumour necrosis factor α] had only a weak effect, whereas immune cytokines (IL-6 and interferon γ), anti-inflammatory cytokines (IL-13 and IL-10) and chemoattractants (fMet-Leu-Phe and macrophage chemoattractant protein 1) were inactive. None of the agonists tested induced significant changes in the basal expression of PLD2 mRNA. Consistent with PLD1 up-regulation was the observation that PLD enzymic activity was higher in monocytes treated with active-pathogen-derived agonists than in control cells, when stimulated with PMA or with chemotactic agonists (fMet-Leu-Phe and C5a). Thus PLD2 seems to be a constitutive enzyme in circulating monocytes. Conversely, PLD1 is an inducible protein, rapidly regulated during culture conditions and selectively induced during cell activation. Therefore PLD1 might have a relevant role in immune responses against pathogens and in chronic inflammation.
Genomic distribution of three promoters of the bovine gene encoding acetyl-CoA carboxylase α and evidence that the nutritionally regulated promoter I contains a repressive element different from that in rat
Authors: MAO Jianqiang , MARCOS Sagrario , DAVIS Scott K. , BURZLAFF Jay , SEYFERT Hans-Martin
Published: August 2015

The enzyme acetyl-CoA carboxylase α (ACC-α) is rate-limiting for the synthesis of long-chain fatty acids de novo. As a first characterization of the bovine gene encoding this enzyme, we established the entire bovine ACC-α cDNA sequence (7041bp) and used experiments with 5′ rapid amplification of cDNA ends to determine the heterogeneous composition of 5′ untranslated regions, as expressed from three different promoters (PI, PII and PIII). The individual locations of these promoters have been defined within an area comprising 35kbp on Bos taurus chromosome 19 (‘BTA19’), together with the segmentation of the first 14 exons. Primer extension analyses reveal that the nutritionally regulated PI initiates transcription from at least four sites. PI transcripts are much more abundant in adipose and mammary-gland tissues than in liver or lung. A 2.6kb promoter fragment drives the expression of reporter genes only weakly in different model cells, irrespective of stimulation with insulin or dexamethasone. Thus bovine PI is basically repressed, like its analogue from rat. Finely graded deletions of PI map two separate elements, which have to be present together in cis to repress bovine PI. The distal component resides within a well-preserved Art2 retroposon element. Thus sequence, structure and evolutionary origin of the main repressor of PI in bovines are entirely different from its functional counterpart in rat, which had been identified as a (CA)28 microsatellite. We show that, in different mammalian species, unrelated genome segments of different origins have been recruited to express as functionally homologous PI the ancient and otherwise highly conserved ACC-α-encoding gene.
Calcium binding and translocation by the voltage-dependent anion channel: a possible regulatory mechanism in mitochondrial function
Authors: GINCEL Dan , ZAID Hilal , SHOSHAN-BARMATZ Varda
Published: August 2015

Mitochondria play a central role in energy metabolism, Ca2+ signalling, aging and cell death. To control cytosolic or mitochondrial Ca2+ concentration, mitochondria possess several Ca2+-transport systems across the inner membrane. However, the pathway for Ca2+ crossing the outer membrane has not been directly addressed. We report that purified voltage-dependent anion channel (VDAC) reconstituted into lipid bilayers or liposomes is highly permeable to Ca2+. VDAC contains Ca2+-binding sites that bind Ruthenium Red (RuR), La3+ and that RuR completely closed VDACs in single or multichannel experiments. Energized, freshly prepared mitochondria accumulate Ca2+ (500–700nmol/mg of protein), and subsequently released it. The release of Ca2+ is accompanied by cyclosporin A-inhibited swelling, suggesting activation of permeability transition pore (PTP). RuR and ruthenium amine binuclear complex, when added to mitochondria after Ca2+ accumulation has reached a maximal level and before PTP is activated, prevented the release of Ca2+ and the accompanied mitochondrial swelling. RuR also prevented PTP opening promoted by atractyloside, an adenine nucleotide translocase inhibitor. These results suggest that VDAC, located in the mitochondrial outer membrane, controls Ca2+ transport into and from the mitochondria, and that the inhibition of Ca2+ uptake by RuR and La3+ may result from their interaction with VDAC Ca2+-binding sites. Inhibition of PTP opening or assembly by RuR and ruthenium amine binuclear complex suggest the involvement of VDAC in PTP activity and/or regulation. The permeability of VDAC to Ca2+ and its binding of Ca2+, suggest that VDAC has a role in regulation of the mitochondrial Ca2+ homoeostasis.
Protein kinase C δ and η isoenzymes control the shedding of the interleukin 6 receptor α in myeloma cells
Authors: THABARD Wilfrid , COLLETTE Madeleine , BATAILLE Régis , AMIOT Martine
Published: August 2015

The soluble interleukin 6 receptor α is an agonistic molecule of interleukin 6 (IL-6) and is important in the biology of multiple myeloma. More precisely, it potentiates the deleterious effects of IL-6 during tumour progression, facilitating angiogenesis and bone resorption. Because the mechanisms involved in the shedding of the interleukin 6 receptor α (IL-6Rα) in multiple myeloma are not known, we have investigated them in the XG-6 human myeloma cell line. Here we provide evidence that PMA-induced IL-6Rα shedding is controlled by a metalloproteinase and by protein kinase C (PKC) isoenzymes that do not require Ca2+ for their activation. We show that XG-6 cells express PKC-δ, −η and −∊ isoenzymes. However, after stimulation with PMA, only PKC-δ and PKC-η are activated, as shown by their translocation to the membrane. Treatment with PMA induces an increase in PKC-δ phosphorylation in its active loop. In addition, by using rottlerin, a specific inhibitor of PKC-δ, we demonstrate that PKC-δ is involved in the PMA-induced shedding of IL-6Rα. With the use of UO126, a specific inhibitor of the mitogen-activated protein kinase (MAPK) pathway, we show that the PMA-induced IL-6Rα shedding is mediated in part by the MAPK pathway. Finally, whereas GF109203X, a general PKC inhibitor, inhibits the activation of ERK1/2 (extracellular signal-regulated protein kinase 1/2), rottlerin has no inhibitory effect, indicating that the Ras/MAPK activation is PKC-dependent but PKC-δ-independent. Taken together, these results suggest that the PMA-induced shedding of IL-6Rα is mediated by a PKC isoenzyme network.
Matrix-assisted in vitro refolding of Pseudomonas aeruginosa class II polyhydroxyalkanoate synthase from inclusion bodies produced in recombinant Escherichia coli
Authors: REHM Bernd H. A. , QI Qingsheng , BEERMANN Br. Bernd , HINZ Hans-Jürgen , STEINBÜCHEL Alexander
Published: August 2015

In order to facilitate the large-scale preparation of active class II polyhydroxyalkanoate (PHA) synthase, we constructed a vector pT7-7 derivative that contains a modified phaC1 gene encoding a PHA synthase from Pseudomonas aeruginosa possessing six N-terminally fused histidine residues. Overexpression of this phaC1 gene under control of the strong Ø10 promoter was achieved in Escherichia coli BL21(DE3). The fusion protein was deposited as inactive inclusion bodies in recombinant E. coli, and contributed approx. 30% of total protein. The inclusion bodies were purified by selective solubilization, resulting in approx. 70–80% pure PHA synthase, then dissolved and denatured by 6M guanidine hydrochloride. The denatured PHA synthase was reversibly immobilized on a Ni2+-nitrilotriacetate–agarose matrix. The matrix-bound fusion protein was refolded by gradual removal of the chaotropic reagent. This procedure avoided the aggregation of folding intermediates which often decreases the efficiency of refolding experiments. Finally, the refolded fusion protein was eluted with imidazole. The purified and refolded PHA synthase protein showed a specific enzyme activity of 10.8m-units/mg employing (R/S)-3-hydroxydecanoyl-CoA as substrate, which corresponds to 27% of the maximum specific activity of the native enzyme. The refolding of the enzyme was confirmed by CD spectroscopy. Deconvolution of the spectrum resulted in the following secondary structure prediction: 10% α-helix, 50% β-sheet and 40% random coil. Gel filtration chromatography indicated an apparent molecular mass of 69kDa for the refolded PHA synthase. However, light-scattering analysis of a 10-fold concentrated sample indicated a molecular mass of 128kDa. These data suggest that the class II PHA synthase is present in an equilibrium of monomer and dimer.
Mutation of yeast Eug1p CXXS active sites to CXXC results in a dramatic increase in protein disulphide isomerase activity
Authors: NØRGAARD Per , WINTHER Jakob R.
Published: August 2015

Protein disulphide isomerase (PDI) is an essential protein which is localized to the endoplasmic reticulum of eukaryotic cells. It catalyses the formation and isomerization of disulphide bonds during the folding of secretory proteins. PDI is composed of domains with structural homology to thioredoxin and with CXXC catalytic motifs. EUG1 encodes a yeast protein, Eug1p, that is highly homologous to PDI. However, Eug1p contains CXXS motifs instead of CXXC. In the current model for PDI function both cysteines in this motif are required for PDI-catalysed oxidase activity. To gain more insight into the biochemical properties of this unusual variant of PDI we have purified and characterized the protein. We have furthermore generated a number of mutant forms of Eug1p in which either or both of the active sites have been mutated to a CXXC sequence. To determine the catalytic capacity of the wild-type and mutant forms we assayed activity in oxidative refolding of reduced and denatured procarboxypeptidase Y as well as refolding of bovine pancreatic trypsin inhibitor. The wild-type protein showed very little activity, not only in oxidative refolding but also in assays where only isomerase activity was required. This was surprising, in particular since mutant forms of Eug1p containing a CXXC motif displayed activity close to that of genuine PDI. These results lead us to propose that general disulphide isomerization is not the main function of Eug1p in vivo.