bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutation of threonine-241 to proline eliminates autocatalytic modification of human carbonyl reductase
Authors: SCIOTTI Michel A. , NAKAJIN Shizuo , WERMUTH Bendicht , BAKER Michael E.
Published: August 2015

Carbonyl reductase catalyses the reduction of steroids, prostaglandins and a variety of xenobiotics. An unusual property of human and rat carbonyl reductases is that they undergo modification at lysine-239 by an autocatalytic process involving 2-oxocarboxylic acids, such as pyruvate and 2-oxoglutarate. Comparison of human carbonyl reductase with the pig enzyme, which does not undergo autocatalytic modification, identified three sites, alanine-236, threonine-241 and glutamic acid-246, on human carbonyl reductase that could be important in the reaction of lysine-239 with 2-oxocarboxylic acids. Mutagenesis experiments show that replacement of threonine-241 with proline (T241P) in human carbonyl reductase eliminates the formation of carboxyethyl-lysine-239. In contrast, the T241A mutant has autocatalytic activity similar to wild-type carbonyl reductase. The T241P mutant retains catalytic activity towards menadione, although with one-fifth the catalytic efficiency of wild-type carbonyl reductase. Replacement of threonine-241 with proline is likely to disrupt the local structure near lysine-239. We propose that integrity of this local environment is essential for chemical modification of lysine-239, but not absolutely required for carbonyl reductase activity.
The cytosolic C-terminus of the glucose transporter GLUT4 contains an acidic cluster endosomal targeting motif distal to the dileucine signal
Authors: SHEWAN Annette M. , MARSH Brad J. , MELVIN Derek R. , MARTIN Sally , GOULD Gwyn W. , JAMES David E.
Published: August 2015

The insulin-responsive glucose transporter GLUT4 is targeted to a post-endocytic compartment in adipocytes, from where it moves to the cell surface in response to insulin. Previous studies have identified two cytosolic targeting motifs that regulate the intracellular sequestration of this protein: FQQI5–8 in the N-terminus and LL489,490 (one-letter amino acid notation) in the C-terminus. In the present study we show that a GLUT4 chimaera in which the C-terminal 12 amino acids in GLUT4 have been replaced with the same region from human GLUT3 is constitutively targeted to the plasma membrane when expressed in 3T3-L1 adipocytes. To further dissect this domain it was divided into three regions, each of which was mutated en bloc to alanine residues. Analysis of these constructs revealed that the targeting information is contained within the residues TELEYLGP498–505. Using the transferrin–horseradish peroxidase endosomal ablation technique in 3T3-L1 adipocytes, we show that mutants in which this C-terminal domain has been disrupted are more sensitive to chemical ablation than wild-type GLUT4. These data indicate that GLUT4 contains a targeting signal in its C-terminus, distal to the dileucine motif, that regulates its sorting into a post-endosomal compartment. Similar membrane-distal, acidic-cluster-based motifs are found in the cytosolic tails of the insulin-responsive aminopeptidase IRAP (insulin-regulated aminopeptidase) and the proprotein convertase PC6B, indicating that this type of motif may play an important role in the endosomal sequestration of a number of different proteins.
A natural sequence consisting of overlapping glucocorticoid-responsive elements mediates glucocorticoid, but not androgen, regulation of gene expression
Authors: MASSAAD Charbel , GARLATTI Michèle , WILSON Elizabeth M. , CADEPOND Françoise , BAROUKI Robert
Published: August 2015

Cytosolic aspartate aminotransferase (cAspAT) is regulated by glucocorticoids in rat liver and kidney. Part of this regulation is mediated by an unusual glucocorticoid-responsive element (GRE)-like sequence called GRE A. GRE A is composed of two overlapping imperfect GREs, each comprising a conserved half-site (half-sites 1 and 4 respectively) and a poorly conserved half-site (half-sites 2 and 3 respectively). The sequence binds co-operatively two dimers of the glucocorticoid receptor (GR) and mediates efficient glucocorticoid regulation of gene expression. Analysis of deletions of the cAspAT gene promoter and subcloning of GRE A upstream of the thymidine kinase promoter indicate that this sequence is responsive to glucocorticoids, but not to androgens. Electrophoretic mobility shift assays indicate that the GRE A unit does not bind the androgen receptor (AR). The modification of three nucleotides in the poorly conserved half-sites 2 and 3, converting GRE A into two overlapping high-affinity GREs (ov-cGRE), resulted in co-operative binding of the AR. Furthermore, ov-cGRE efficiently mediated androgen regulation of the thymidine kinase promoter. A single base modification in half-site 2 or 3 in GRE A allowed the binding of the AR as one or two dimers respectively, and restored transcriptional activation by androgens only in the latter case. Thus the poor affinity of the AR for half-sites 2 and 3 prevented its binding to GRE A, indicating that the overlapping GRE A sequence of the cAspAT gene promoter discriminates a glucocorticoid-mediated from an androgen-mediated response.
Fine mapping of the α-actinin binding site within cysteine-rich protein
Authors: HARPER Brooke D. , BECKERLE Mary C. , POMIÈS Pascal
Published: August 2015

The cysteine-rich proteins (CRPs) are a family of highly conserved LIM (an acronym derived from the three gene products lin-11, isl-1 and mec-3) domain proteins that have been implicated in muscle differentiation. All CRP family members characterized so far have been shown to interact with the filamentous actin cross-linker α-actinin. The region of CRP required for this interaction has previously been broadly mapped to the molecule's N-terminal half. Here we report that the α-actinin-binding region of CRP, which we have mapped by using a combination of blot overlay and Western immunoblot techniques, is confined to an 18-residue sequence occurring within the protein's N-terminal glycine-rich repeat. A site-directed mutagenesis analysis of the binding region has revealed the critical importance of a single lysine residue (lysine 65 in human CRP1). Alterations at this site lead to a 10-fold decrease in α-actinin binding in comparison with wild-type CRP. The critical lysine residue localizes within a short α-helix, raising the possibility that mutagenesis-induced alterations in α-actinin-binding capacity might be attributed to the disruption of a key structural element.
The analysis of intermolecular interactions in concentrated hyaluronan solutions suggest no evidence for chain–chain association
Authors: GRIBBON Phillip , HENG Boon Chin , HARDINGHAM Timothy E.
Published: August 2015

Confocal fluorescence recovery after photobleaching (confocal-FRAP) was used to examine the influence of electrolytes (NaCl, KCl, MgCl2, MnCl2 and CaCl2) on the network and hydrodynamic properties of fluoresceinamine-labelled hyaluronan (FA-HA) at concentrations up to 10mg/ml. Self and tracer lateral diffusion coefficients showed that in Ca2+ and Mn2+, FA-HA (830kDa) was more compact than in Mg2+, Na+ or K+. These results were correlated with changes in the hydrodynamic radius of HA, determined by multi-angle laser-light-scattering analysis in dilute solution, which was smaller in CaCl2 (36nm) than in NaCl (43nm). The permeability of more concentrated solutions of HA (< 10mg/ml) to FITC-dextran tracers (2000kDa) was higher in CaCl2. The properties of HA in urea (up to 6M) were investigated to test for hydrophobic interactions and also in ethanol/water (up to 62%, v/v). In both, there was reduced hydrodynamic size and increased permeability to FITC-dextran, suggesting increased chain flexibility, but it did not show the changes predicted if chain–chain association was disrupted by urea, or enhanced by ethanol. Oligosaccharides of HA (HA20–26) also had no effect on the self diffusion of high-molecular-mass FA-HA (830kDa) solutions, or on dextran tracer diffusion, showing that there were no chain–chain interactions open to competition by short-chain segments. The results suggest that the effects of electrolytes and solvent are determined primarily by their effect on HA chain flexibility, with no evidence for association between chain segments contributing significantly to the major properties.
Comparison of the kinetic properties of the lipid- and protein-kinase activities of the p110α and p110β catalytic subunits of class-Ia phosphoinositide 3-kinases
Authors: BEETON Carolyn A. , CHANCE Edwin M. , FOUKAS Lazaros C. , SHEPHERD Peter R.
Published: August 2015

Growth factors regulate a wide range of cellular processes via activation of the class-Ia phosphoinositide 3-kinases (PI 3-kinases). We directly compared kinetic properties of lipid- and protein-kinase activities of the widely expressed p110α and p110β isoforms. The lipid-kinase activity did not display Michaelis–Menten kinetics but modelling the kinetic data demonstrated that p110α has a higher Vmax and a 25-fold higher Km for PtdIns than p110β. A similar situation occurs with PtdIns(4,5)P2, because at low concentration of PtdIns(4,5)P2 p110β is a better PtdIns(4,5)P2 kinase than p110α, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110β functions better in areas of membranes containing low levels of substrate whereas p110α would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110β phosphorylated p85 to a lower degree than did p110α. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110α and p110β. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110α has a higher Km (550µM) than p110β (Km 8µM). Similarly, the relative Vmax towards peptide substrate of p110α was three times higher than that of p110β. This implies differences in the rates of regulatory autophosphorylation in vivo, which are likely to mean differential regulation of the lipid-kinase activities of p110α and p110β in vivo.
Stanniocalcin 1 and 2 are secreted as phosphoproteins from human fibrosarcoma cells
Authors: JELLINEK Derek A. , CHANG Andy C. , LARSEN Martin R. , WANG Xin , ROBINSON Phillip J. , REDDEL Roger R.
Published: August 2015

Stanniocalcin 1 (STC1) and stanniocalcin 2 (STC2) are two recently identified mammalian peptide hormones. STC1 plays a role in calcium and phosphate homoeostasis, while the role of STC2 is unknown. We examined a human fibrosarcoma cell line, HT1080, that has high steady-state STC1 and STC2 mRNA levels, to determine whether these proteins are secreted. Following incubation of HT1080 cells with 32P, labelled STC1 and STC2 were found to be secreted into the medium. STC1 was phosphorylated in vitro by protein kinase C (PKC). In vitro and in vivo phosphorylation both occurred exclusively on serine and the phosphopeptide maps were similar, suggesting that PKC might be the in vivo kinase. STC2 was phosphorylated in vitro by casein kinase II (CK2), in vitro and in vivo phosphorylation were exclusively on serine and the phosphopeptide maps were indistinguishable. Phosphorylation of STC2 in intact cells resulted from the action of an ecto-protein kinase, since exogenous STC2 was phosphorylated by HT1080 cells and no phosphorylated STC2 was detectable inside the cells. The ectokinase activity was abolished by heparin and GTP could substitute for ATP as the phosphate donor, indicative of an ecto-CK2-like activity. The in vitro CK2 phosphorylation site was shown by matrix-assisted laser-desorption ionization–time-of-flight MS to be a single serine located between Ser-285 and Ser-298 in the C-terminal region of STC2. This is the first report of the secretion of STC1 or STC2 from mammalian cells. We conclude that these human fibrosarcoma cells express both STC1 and STC2 as secreted phosphoproteins in vivo, with STC2 being phosphorylated by an ecto-CK2-like enzyme.
Monoclonal antibodies identify residues 199–216 of the integrin α2 vWFA domain as a functionally important region within α2β1
Authors: TUCKWELL Danny S. , SMITH Lyndsay , KORDA Michelle , ASKARI Janet A. , SANTOSO Sentot , BARNES Michael J. , FARNDALE Richard W. , HUMPHRIES Martin J.
Published: August 2015

Integrin α2β1 is the major receptor for collagens in the human body, and the collagen-binding site on the α2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant α2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse α2β1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr221 to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-β1 antibody Mab13, and binding of Gi9 and JA218 to α2β1 was inhibited by substituting Mn2+ for Mg2+, demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212–216, while JA208 bound between residues 199–216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain.
Functional analysis of tumour necrosis factor-α-related apoptosis-inducing ligand (TRAIL): cysteine-230 plays a critical role in the homotrimerization and biological activity of this novel tumoricidal cytokine
Authors: TRABZUNI Danyah , FAMULSKI Konrad S. , AHMAD Manzoor
Published: August 2015

We have determined that the mutation of the cysteine-230 residue to either glycine or serine in TRAIL (tumour necrosis factor-α-related apoptosis-inducing ligand) results in the formation of a structurally incompetent dimer and a consequent loss of apoptotic activity. Similarly, chemical modification of the thiol residues present in both reduced and Zn2+-depleted trimer converts TRAIL into an inactive dimer. We postulate that cysteine-230 plays a critical role in homotrimerization of this tumoricidal cytokine.
Role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1
Authors: PAJOR Ana M. , KAHN Esther S. , GANGULA Rama
Published: August 2015

The role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1 was investigated by site-directed mutagenesis and subsequent expression of mutant transporters in Xenopus oocytes. Of the ten residues chosen for mutagenesis, eight (Lys-34, Lys-107, Arg-108, Lys-333, Lys-390, Arg-368, Lys-414 and Arg-541) were found to be non-essential for function or targeting. Only two conserved residues, Lys-84 (at the cytoplasmic end of helix 3) and Arg-349 (at the extracellular end of helix 7), were found to be important for transport. Both mutant transporters were expressed at the plasma membrane. The mutation of Lys-84 to Ala resulted in an increased Km for succinate of 1.8mM, compared with 0.3mM in the wild-type NaDC-1. The R349A mutant had Na+ and citrate kinetics that were similar to those of the wild type. However, succinate handling in the R349A mutant was altered, with evidence of inhibition at high succinate concentrations. In conclusion, charge neutralization of Lys-84 and Arg-349 in NaDC-1 affects succinate handling, suggesting that these residues might have roles in substrate binding.
Characterization of homocysteine metabolism in the rat liver
Authors: STEAD Lori M. , BROSNAN Margaret E. , BROSNAN John T.
Published: August 2015

Recent evidence suggests that an increased plasma concentration of the sulphur amino acid homocysteine is a risk factor for the development of vascular disease. The tissue(s) responsible for homocysteine production and export to the plasma are not well known. However, given the central role of the liver in amino acid metabolism, we developed a rat primary hepatocyte model in which homocysteine (and cysteine) production and export were examined. The dependence of homocysteine export from incubated hepatocytes on methionine concentration fitted well to a rectangular hyperbola, with half-maximal homocysteine export achieved at methionine concentrations of approx. 0.44mM. Hepatocytes incubated with 1mM methionine and 1mM serine (a substrate for the transulphuration pathway of homocysteine removal) produced and exported significantly less homocysteine (25–40%) compared with cells incubated with 1mM methionine alone. The effects of dietary protein on homocysteine metabolism were also examined. Rats fed a 60% protein diet had a significantly increased total plasma homocysteine level compared with rats fed a 20% protein diet. Invitro effects of dietary protein were examined using hepatocytes isolated from animals maintained on these diets. When incubated with 1mM methionine, hepatocytes from rats fed the high protein diet exported significantly more homocysteine compared with hepatocytes from rats fed the normal protein diet. Inclusion of serine significantly lowered homocysteine export in the normal protein group, but the effect was more marked in the high protein group. Invivo effects of serine were also examined. Rats fed a high protein diet enriched with serine had significantly lower total plasma homocysteine (25–30%) compared with controls. These data indicate a significant role for the liver in the regulation of plasma homocysteine levels.
Biogenesis of endoplasmic reticulum proteins involved in Ca2+ signalling during megakaryocytic differentiation: an in vitro study
Authors: LACABARATZ-PORRET Christine , LAUNAY Sophie , CORVAZIER Elisabeth , BREDOUX Raymonde , PAPP Béla , ENOUF Jocelyne
Published: August 2015

The endoplasmic reticulum (ER) plays a key role in Ca2+ signalling through Ca2+ release via inositol 1,4,5-trisphosphate receptors (InsP3-Rs) and Ca2+ uptake by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs). Here, we investigated the organization of platelet ER and its biogenesis during megakaryocytopoiesis. First, erythro/megakaryoblastic MEG 01, UT7, M-O7e and CHRF 288-11 cell lines, platelets and thrombopoietin-induced UT7-Mpl cells were selected for the study of SERCA2b and SERCA3 proteins by Western blotting using the antibodies IID8 and PL/IM430, respectively. As judged by platelet glycoprotein IIIa (GPIIIa) expression, an increase in SERCA3 proteins was observed while that of SERCA2b remained unchanged throughout maturation. Second, these studies were extended to the newly described alternatively spliced SERCA3a–c RNAs and InsP3-Rs using the in vitro model of PMA-induced differentiation of MEG 01 cells. Time-course and dose–response studies showed a maximal approx. 4-fold up-regulation of SERCA3 proteins using 10-8 M PMA for 3 days, which paralleled induction of GPIIIa expression. SERCA3 induction was found to occur at the level of mRNA. The modulation of the different SERCA3 species (i.e. 3a, 3b and 3c) was isoform-specific: while SERCA3a was slightly increased, an approx. 3-fold induction of SERCA3b, and a 4-fold induction of SERCA3c, was observed after 24h of PMA treatment. Isoform-specific Western blotting and/or reverse transcriptase PCR studies showed that InsP3-R types I, II and III are expressed in MEG 01 cells, as well as in platelets. Study of the expression of these InsP3-R types in PMA-induced MEG 01 cells revealed that: (i) InsP3-RI protein and mRNA showed no changes; (ii) InsP3-RII mRNA was up-regulated and peaked at hour 48 and (iii) InsP3-RIII mRNA and protein showed a transitory maximal 3- and 2.3-fold increase at hours 6 and 30, respectively. Upon PMA treatment of CHRF 288-11 cells, in which GPIIIa is not induced upon treatment, a similar pattern of regulation of InsP3-R types II and III was seen, but a distinct pattern of SERCA3 regulation was observed. These results suggest a profound reorganization of ER-protein patterns during megakaryocytopoiesis and underline the role of SERCA3 gene regulation in the control of Ca2+-dependent platelet functions.
Novel triterpenoids inhibit both DNA polymerase and DNA topoisomerase
Authors: MIZUSHINA Yoshiyuki , IIDA Akira , OHTA Keisuke , SUGAWARA Fumio , SAKAGUCHI Kengo
Published: August 2015

As described previously, we found that new triterpenoid compounds, designated fomitellic acids A and B, which selectively inhibit the activities of mammalian DNA polymerases α and β [Mizushina, Tanaka, Kitamura, Tamai, Ikeda, Takemura, Sugawara, Arai, Matsukage, Yoshida and Sakaguchi (1998) Biochem. J. 330, 1325–1332; Tanaka, Kitamura, Mizushina, Sugawara and Sakaguchi (1998) J. Nat. Prod. 61, 193–197] and that a known triterpenoid, ursolic acid, is an inhibitor of human DNA topoisomerases I and II (A. Iida, Y. Mizushina and K. Sakaguchi, unpublished work). Here we report that all of these triterpenoids are potent inhibitors of calf DNA polymerase α, rat DNA polymerase β and human DNA topoisomerases I and II, and show moderate inhibitory effects on plant DNA polymerase II and human immunodeficiency virus reverse transcriptase. However, these compounds did not influence the activities of prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I or other DNA metabolic enzymes such as human telomerase, T7 RNA polymerase and bovine deoxyribonuclease I. These triterpenoids were not only mammalian DNA polymerase inhibitors but also inhibitors of DNA topoisomerases I and II even though the enzymic characteristics of DNA polymerases and DNA topoisomerases, including their modes of action, amino acid sequences and three-dimensional structures, differed markedly. These triterpenoids did not bind to DNA, suggesting that they act directly on these enzymes. Because the three-dimensional structures of fomitellic acids were shown by computer simulation to be very similar to that of ursolic acid, the DNA-binding sites of both enzymes, which compete for the inhibitors, might be very similar. Fomitellic acid A and ursolic acid prevented the growth of NUGC cancer cells, with LD50 values of 38 and 30µM respectively.
Contribution of steroidogenic factor 1 to the regulation of cholesterol synthesis
Authors: MASCARÓ Cristina , NADAL Alicia , HEGARDT Fausto G. , MARRERO Pedro F. , HARO Diego
Published: August 2015

Steroidogenic factor 1 (SF-1) is an orphan member of the nuclear receptor family expressed in steroidogenic tissues, where it has an essential role in the regulation of the steroid hormone biosynthesis, adrenal and gonadal development and endocrine responses fundamental for reproduction. Here we show that SF-1 regulates the transcription of cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene, which is essential for the endogenous synthesis of cholesterol. We have identified an element located 365bp upstream of the gene for cytosolic HMG-CoA synthase; SF-1 binds as a monomer to this element and confers SF-1 responsiveness to homologous and heterologous promoters. It has been shown that in tissues with a high demand for cholesterol to be used in steroid synthesis, there is a lack of correlation between the cholesterol levels and the activity of the limiting enzymes of the mevalonate pathway. In accord with those results, we observed that cholesterol synthesis from acetate and either cytosolic HMG-CoA mRNA expression or transcriptional activity were not changed in response to 25-hydroxycholesterol in the SF-1-expressing steroidogenic Leydig tumour MA-10 cells. Moreover, the overexpression of SF-1 in non-steroidogenic CV-1 cells renders them less sensitive to the regulatory effects of cholesterol. This observation led to the hypothesis that in steroidogenic tissues the expression of SF-1 permits high levels of endogenous synthesis of cholesterol irrespective of the intracellular levels of this metabolite.
Thapsigargin suppresses phorbol ester-dependent human involucrin promoter activity by suppressing CCAAT-enhancer-binding protein α (C/EBPα) DNA binding
Authors: BALASUBRAMANIAN Sivaprakasam , AGARWAL Chapla , EFIMOVA Tatiana , DUBYAK George R. , BANKS Eric , WELTER Jean , ECKERT Richard L.
Published: August 2015

Human involucrin (hINV) is a keratinocyte differentiation marker expressed in the suprabasal epidermal layers. In cultured keratinocytes hINV mRNA levels are increased 10-fold by a 24-h treatment with 50ng/ml PMA, an agent that promotes keratinocyte differentiation. Previous studies show that thapsigargin (TGN), an agent that depletes intracellular calcium stores, inhibits keratinocyte differentiation. In the present study we show that TGN inhibits the PMA-dependent, differentiation-associated, increase in hINV mRNA levels and hINV promoter activity. Inhibition is half-maximal at 10nM and maximal at 100nM TGN. Neither basal hINV promoter activity nor glyceraldehyde-3-phosphate dehydrogenase mRNA levels are inhibited. Mutation of a functionally important CAATT-enhancer-binding protein (C/EBP) site within the hINV promoter proximal regulatory region eliminates the regulation, suggesting that TGN may effect C/EBP-dependent promoter activation. Consistent with this hypothesis, TGN inhibits C/EBPα-dependent promoter activation via a mechanism that involves inhibition of C/EBPα binding to DNA without changing C/EBPα protein levels. These results suggest that TGN interferes with hINV expression by interfering with C/EBP transcription-factor function.
Role of endocytosis in the transactivation of nuclear factor-κB by oxidized low-density lipoprotein
Authors: HAN Chang-Yeop , PARK Soo-Young , PAK Youngmi Kim
Published: August 2015

Oxidized low-density lipoprotein (oxLDL) has been shown to modulate transactivation by the peroxisome proliferator-activated receptor (PPAR)-γ and by nuclear factor-κB (NF-κB). In the present study, the oxLDL signalling pathways involved in NF-κB transactivation were investigated by utilizing a reporter construct driven by three upstream NF-κB binding sites, and various pharmacological inhibitors. OxLDL and its constituent lysophophatidylcholine (lysoPC) induced a rapid and transient increase in intracellular calcium and stimulated NF-κB transactivation in resting RAW264.7 macrophage cells in an oxidation-dependent manner. NF-κB activation by oxLDL or lysoPC was inhibited by inhibitors of protein kinase C or by a chelator of intracellular calcium. Tyrosine kinase or phosphatidylinositol 3-kinase inhibitors did not block NF-κB transactivation. Furthermore, oxLDL-induced NF-κB activity was abolished by PPAR-γ ligands. When the endocytosis of oxLDL was blocked by cytochalasin B, NF-κB transactivation by oxLDL was synergistically increased, while PPAR transactivation was blocked. These results suggest that oxLDL activates NF-κB in resting macrophages via protein kinase C- and/or calcium-dependent pathways, and that this does not involve the endocytic processing of oxLDL. The endocytosis-dependent activation of PPAR-γ by oxLDL may function as a route of inactivation of the oxLDL-induced NF-κB signal.
Requirement of N-glycosylation of the prostaglandin E2 receptor EP3β for correct sorting to the plasma membrane but not for correct folding
Authors: BÖER Ulrike , NEUSCHÄFER-RUBE Frank , MÖLLER Ulrike , PÜSCHEL Gerhard P.
Published: August 2015

Eight heptahelical receptors have been characterized for prostaglandin (PG) D2, PGE2, PGF2α, prostacyclin and thromboxane A2. They share a sequence identity of 40%. All of them have potential N-glycosylation sites. The current study analysed the role of the two N-glycosylation sites in the rat EP3β-subtype PGE2 receptor for protein folding and sorting. The N-glycosylation consensus sequences were eliminated by site-directed mutagenesis and receptors expressed in HEK-293 cells. Both potential N-glycosylation sites were used. Their joint elimination resulted in the synthesis of a receptor protein with full binding competence, biological activity and no reduction of affinity; however, the half-life of the non-glycosylated receptor was slightly reduced. Ligand binding to intact stably transfected cells and confocal laser microscopic immunocytochemistry showed that the glycosylated receptor was correctly inserted into the plasma membrane to a much larger extent than the non-glycosylated receptor, which tended to accumulate in the perinuclear zone of the endoplasmic reticulum. Inhibition of N-glycosylation with tunicamycin resulted in a similar perinuclear distribution of the wild-type receptor. Therefore, glycosylation of the EP3β receptor seems not to be necessary for correct folding of the receptor protein but for the efficient transport of the receptor protein to the plasma membrane. This contrasts with a previous finding which described a reduction of the affinity for PGE2 of the EP3α receptor by elimination of the distal glycosylation site when the receptor protein was expressed in insect cells.
Amino acid regulation of gene expression
Authors: FAFOURNOUX Pierre , BRUHAT Alain , JOUSSE Céline
Published: August 2015

The impact of nutrients on gene expression in mammals has become an important area of research. Nevertheless, the current understanding of the amino acid-dependent control of gene expression is limited. Because amino acids have multiple and important functions, their homoeostasis has to be finely maintained. However, amino-acidaemia can be affected by certain nutritional conditions or various forms of stress. It follows that mammals have to adjust several of their physiological functions involved in the adaptation to amino acid availability by regulating the expression of numerous genes. The aim of the present review is to examine the role of amino acids in regulating mammalian gene expression and protein turnover. It has been reported that some genes involved in the control of growth or amino acid metabolism are regulated by amino acid availability. For instance, limitation of several amino acids greatly increases the expression of the genes encoding insulin-like growth factor binding protein-1, CHOP (C/EBP homologous protein, where C/EBP is CCAAT/enhancer binding protein) and asparagine synthetase. Elevated mRNA levels result from both an increase in the rate of transcription and an increase in mRNA stability. Several observations suggest that the amino acid regulation of gene expression observed in mammalian cells and the general control process described in yeast share common features. Moreover, amino acid response elements have been characterized in the promoters of the CHOP and asparagine synthetase genes. Taken together, the results discussed in the present review demonstrate that amino acids, by themselves, can, in concert with hormones, play an important role in the control of gene expression.
Is double C2 protein (DOC2) expressed in bovine adrenal medulla? A commercial anti-DOC2 monoclonal antibody recognizes a major bovine mitochondrial antigen
Authors: DUNCAN Rory R. , APPS David K. , LEARMONTH Michele P. , SHIPSTON Michael J. , CHOW Robert H.
Published: August 2015

We have examined the expression in bovine adrenal medulla of double C2 protein (DOC2), a vesicular protein which associates with intracellular phospholipid and Ca2+ and is implicated in the modulation of regulated exocytosis. Extensive reverse transcription–PCR, Northern blot analyses and in vitro translation reactions have been combined with immunological studies to provide data to suggest that neither DOC2α nor DOC2β is expressed at detectable levels in bovine adrenal chromaffin cells, and that a widely used monoclonal antibody directed against DOC2 also recognizes mitochondrial complex III core protein 2.
p53 protein oxidation in cultured cells in response to pyrrolidine dithiocarbamate: a novel method for relating the amount of p53 oxidation in vivo to the regulation of p53-responsive genes
Authors: WU Hsiao-Huei , THOMAS James A. , MOMAND Jamil
Published: August 2015

A novel method was developed to determine the oxidation status of proteins in cultured cells. Methoxy-polyethylene glycol-maleimide MW 2000 (MAL-PEG) was used to covalently tag p53 protein that was oxidized at cysteine residues in cultured cells. Treatment of MCF7 breast cancer cells with pyrrolidine dithiocarbamate (PDTC), a metal chelator, resulted in a minimum of 25% oxidation of p53. The oxidized p53 had an average of one cysteine residue oxidized per p53 protein molecule. The effect of PDTC treatment on downstream components of the p53 signal-transduction pathway was tested. PDTC treatment prevented actinomycin D-mediated up-regulation of two p53 effector gene products, murine double minute clone 2 oncoprotein and p21WAF1/CIP1 (where WAF1 corresponds to wild-type p53-activated fragment 1 and CIP1 corresponds to cyclin-dependent kinase-interacting protein 1). Actinomycin D treatment led to accumulation of p53 protein in the nucleus. However, when cells were simultaneously treated with PDTC and actinomycin D, p53 accumulated in both the nucleus and the cytoplasm. The data indicate that an average of one cysteine residue per p53 protein molecule is highly sensitive to oxidation and that p53 can be efficiently oxidized by PDTC in cultured cells. PDTC-mediated oxidation of p53 correlates with altered p53 subcellular localization and reduced activation of p53 downstream effector genes. The novel method for detecting protein oxidation detailed in the present study may be used to determine the oxidation status of specific proteins in cells.
The protein kinase A catalytic subunit Cβ2: molecular characterization and distribution of the splice variant
Authors: THULLNER Sandra , GESELLCHEN Frank , WIEMANN Stefan , PYERIN Walter , KINZEL Volker , BOSSEMEYER Dirk
Published: August 2015

Cβ2, a 46 kDa splice variant of the Cβ isoform, is the largest isoform so far described for catalytic subunits from cAMP-dependent protein kinase in mammals. It differs from Cβ in the first 15 N-terminal residues which are replaced with a 62-residue domain with no similarity to other known proteins. The Cβ2 protein was identified in cardiac tissue by MS, microsequencing and C-subunit-isoform-selective antibodies. The Cβ2 protein has a very low abundance of about 2% of total affinity-purified C subunits from bovine cardiac tissue. This, and the similarity of its biochemical properties to Cα and Cβ, are probably some of the reasons why the Cβ2 protein has escaped detection so far. The abundance of the Cβ2 protein differs dramatically between tissues, with most protein detected in heart, liver and spleen, and the lowest level in testis. Cβ2 protein shows kinase activity against synthetic substrates, and is inhibited by the protein kinase inhibitor peptide PKI(5-24). The degree of Cβ2 removal from tissue extracts by binding to PKI(5-24) depends on the cAMP level, i.e. on the dissociation state of the holoenzyme. Two sites in the protein are phosphorylated: Thr-244 in the activation segment and Ser-385 close to the C-terminus. By affinity purification and immunodetection Cβ2 was found in cattle, pig, rat, mouse and turkey tissue and in HeLa cells. In the cAMP-insensitive CHO 10260 cell line, which has normal Cβ but is depleted of Cα, stable transfection with Cβ2 restored most of the cAMP-induced morphological changes. Cβ2 is a ubiquitously expressed protein with characteristic properties of a cAMP-dependent protein kinase catalytic subunit.
Role of mRNA stability and translation in the expression of cytochrome c oxidase during mouse myoblast differentiation: instability of the mRNA for the liver isoform of subunit VIa
Authors: THAMES Elizabeth L. , NEWTON Danforth A. , BLACK Samuel A. , BOWMAN Lewis H.
Published: August 2015

The role of mRNA stability and translation in mediating the expression of selected subunits of cytochrome c oxidase (COX) was examined during the differentiation of mouse myoblasts into myotubes in cell culture. The expression of the liver (L) and heart (H) isoforms of COX VIa, which undergo an isoform switch during muscle development, as well as of the Va subunit, which is expressed in all tissues, was analysed. The translational efficiencies of COX Va, VIa-L and VIa-H, as well as of mitochondrially encoded COX mRNAs, were inferred from their distribution in polysome gradients. These experiments suggest that the translational efficiencies of these mRNAs do not change during myoblast differentiation, although the nuclear mRNAs for COX Va, VIa-L and VIa-H are translated more efficiently than the mitochondrial mRNAs. Analysis of mRNA stability using the tetracycline-repressible promoter system and/or actinomycin D indicates that COX VIa-L mRNA decays with a half-life of ∼ 5–6h in both myoblasts and myotubes, whereas COX VIa-H and Va mRNAs decay with half-lives of > 15h in myotubes. This relative instability of COX VIa-L mRNA serves to limit the accumulation of COX VIa-L mRNA in these myogenic cells, as compared with mRNAs for other COX subunits. Deletion/replacement mapping experiments suggest that the COX VIa-L 3´ untranslated region contains a destabilization element. Analysis of the rate of poly(A) tail shortening on COX VIa-L and stable α-globin mRNAs suggests that the overall rate of poly(A) shortening per se is not rate limiting for the degradation of COX VIa-L mRNA.
Phosphorylation of Cdc28 and regulation of cell size by the protein kinase CKII in Saccharomyces cerevisiae
Authors: RUSSO Gian Luigi , VAN DEN BOS Christian , SUTTON Ann , COCCETTI Paola , BARONI Maurizio D. , ALBERGHINA Lilia , MARSHAK Daniel R.
Published: August 2015

The CDK (cyclin-dependent kinase) family of enzymes is required for the G1-to-S-phase and G2-to-M-phase transitions during the cell-division cycle of eukaryotes. We have shown previously that the protein kinase CKII catalyses the phosphorylation of Ser-39 in Cdc2 during the G1 phase of the HeLa cell-division cycle [Russo, Vandenberg, Yu, Bae, Franza and Marshak (1992) J. Biol. Chem. 267, 20317–20325]. To identify a functional role for this phosphorylation, we have studied the homologous enzymes in the budding yeast Saccharomyces cerevisiae. The S. cerevisiae homologue of Cdc2, Cdc28, contains a consensus CKII site (Ser-46), which is homologous with that of human Cdc2. Using in vitro kinase assays, metabolic labelling, peptide mapping and phosphoamino acid analysis, we demonstrate that this site is phosphorylated in Cdc28 in vivo as well in vitro. In addition, S. cerevisiae cells in which Ser-46 has been mutated to alanine show a decrease in both cell volume and protein content of 33%, and this effect is most pronounced in the stationary phase. Because cell size in S. cerevisiae is regulated primarily at the G1 stage, we suggest that CKII contributes to the regulation of the cell cycle in budding yeast by phosphorylation of Cdc28 as a checkpoint for G1 progression.
The molecular interaction of 4′-iodo-4′-deoxydoxorubicin with Leu-55Pro transthyretin ‘amyloid-like’ oligomer leading to disaggregation
Authors: SEBASTIÃO Maria Paula , MERLINI Giampaolo , SARAIVA Maria João , DAMAS Ana Margarida
Published: August 2015

The crystal structure of the amyloidogenic Leu-55Pro transthyretin (TTR) variant has revealed an oligomer structure that may represent a putative amyloid protofibril [Sebastião, Saraiva and Damas (1998) J. Biol. Chem. 273, 24715–24722]. Here we report biochemical evidence that corroborates the isolation of an intermediate structure, an ‘amyloid-like’ oligomer, which is most probably present in the biochemical pathway that leads to amyloid deposition and that was isolated by the crystallization of the Leu-55Pro TTR variant. 4´-Iodo-4´-deoxydoxorubicin (IDOX) is a compound that interacts with amyloid fibrils of various compositions and it has been reported to reduce the amyloid load in immunoglobulin light chain amyloidosis [Merlini, Ascari, Amboldi, Bellotti, Arbustini, Perfetti, Ferrari, Zorzoli, Marinone, Garini et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 2959–2963]. In this work, we observed that the monoclinic Leu-55Pro TTR crystals, soaked with IDOX, undergo rapid dissociation. Moreover, under the same conditions, the orthorhombic wild-type TTR crystals are quite stable. This is explained by the different TTR conformations isolated upon crystallization of the two proteins; while the Leu-55Pro TTR exhibits the necessary conformation for IDOX binding, the same structure is not present in the crystallized wild-type protein. A theoretical model concerning the interaction of Leu-55Pro TTR with IDOX, which is consistent with the dissociation of the amyloid-like oligomer, is presented. In this model the IDOX iodine atom is buried in a pocket located between the two β-sheets of the Leu-55Pro TTR monomer with the IDOX aromatic-moiety long axis nearly perpendicular to the direction of the β-sheets.
Impaired reductive regeneration of ascorbic acid in the Goto–Kakizaki diabetic rat
Authors: KASHIBA Misato , OKA Jun , ICHIKAWA Rumi , KAGEYAMA Asako , INAYAMA Takayo , KAGEYAMA Haruaki , ISHIKAWA Takahiro , NISHIKIMI Morimitsu , INOUE Masayasu , INOUE Shuji
Published: August 2015

Ascorbic acid (AA) is a naturally occurring major antioxidant that is essential for the scavenging of toxic free radicals in both plasma and tissues. AA levels in plasma and tissues have been reported to be significantly lower than normal in diabetic animals and humans, and might contribute to the complications found at the late stages of diabetes. In this study, plasma and hepatic AA levels and AA regeneration were studied in the Goto–Kakizaki diabetic rat (GK rat) to elucidate the mechanism of decreasing plasma and hepatic AA levels in diabetes. AA concentrations in the plasma and liver were significantly lower in GK than in control rats. AA levels in primary cultured hepatocytes derived from GK rats were lower than those derived from control Wistar rats with or without dehydroascorbic acid (DHA) in the medium. Among various enzyme activities that reduce DHA to AA, the NADPH-dependent regeneration of AA in the liver was significantly suppressed in GK rats. Northern blot analysis revealed that only the expression of 3-α-hydroxysteroid dehydrogenase (AKR) was significantly suppressed in these rats. These results suggest that decreased AA-regenerating activity, probably through decreased expression of AKR, contributes to the decreased AA levels and increased oxidative stress in GK rats.
Anti-(herpes simplex virus) activity of 4′-thio-2′-deoxyuridines: a biochemical investigation for viral and cellular target enzymes
Authors: VERRI Annalisa , FOCHER Federico , DUNCOMBE Richard J. , BASNAK Ivan , WALKER Richard T. , COE Paul L. , DE CLERCQ Erik , ANDREI Graciela , SNOECK Robert , BALZARINI Jan , SPADARI Silvio
Published: August 2015

The antiviral activity of several nucleoside analogues is often limited by their rapid degradation by pyrimidine nucleoside phosphorylases. In an attempt to avoid this degradation, several modified nucleosides have been synthesized. A series of 4´-thio-2´-deoxyuridines exhibits an anti-[herpes simplex virus (HSV)] activity significantly higher (20–600 times) than that shown by the corresponding 4´-oxy counterpart. We investigated the mode of action of these compounds and we found that: (i) several 4´-thio-2´-deoxyuridines are phosphorylated to the mono- and di-phosphates by HSV-1 thymidine kinase (TK) more efficiently than their corresponding 4´-oxy counterpart; (ii) both are inhibitors of cellular thymidylate synthase; (iii) 4´-thio-2´-deoxyuridines are resistant to phosphorolysis by human thymidine phosphorylase; (iv) both 4´-oxy- and 4´-thio-2´-deoxyuridines are phosphorylated to deoxyribonucleotide triphosphate in HSV-1-infected cells and are incorporated into viral DNA; (v) 4´-thio-2´-deoxyuridines are better inhibitors than their 4´-oxy counterparts of [3H]thymidine incorporation in HSV-1-infected cells; (vi) 4´-thio-2´-deoxyuridines are not recognized by HSV-1 and human uracil-DNA glycosylases. Our data suggest that 4´-thio-2´-deoxyuridines, resistant to pyrimidine phosphorylase, can be preferentially or selectively phosphorylated by viral TK in HSV-infected cells, where they are further converted into triphosphate by cellular nucleotide kinases. Once incorporated into viral DNA, they are better inhibitors of viral DNA synthesis than their corresponding 4´-oxy counterpart, either because they are not recognized, and thus not removed, by viral uracil-DNA glycosylase, or because they preferentially interfere with viral DNA polymerase.
Regulation of calpain and calpastatin in differentiating myoblasts: mRNA levels, protein synthesis and stability
Authors: BARNOY Sivia , SUPINO-ROSIN Lia , KOSOWER Nechama S.
Published: August 2015

Calpain (Ca2+-dependent intracellular protease)-induced proteolysis has been considered to play a role in myoblast fusion to myotubes. We found previously that calpastatin (the endogenous inhibitor of calpain) diminishes transiently during myoblast differentiation. To gain information about the regulation of calpain and calpastatin in differentiating myoblasts, we evaluated the stability and synthesis of calpain and calpastatin, and measured their mRNA levels in L8 myoblasts. We show here that µ-calpain and m-calpain are stable, long-lived proteins in both dividing and differentiating L8 myoblasts. Calpain is synthesized in differentiating myoblasts, and calpain mRNA levels do not change during differentiation. In contrast, calpastatin (though also a long-lived protein in myoblasts), is less stable in differentiating myoblasts than in the dividing cells, and its synthesis is inhibited upon initiation of differentiation. Inhibition of calpastatin synthesis is followed by a diminution in calpastatin mRNA levels. A similar calpastatin mRNA diminution is observed upon drug-induced inhibition of protein translation. On the other hand, transforming growth factor β (which inhibits differentiation) allows calpastatin synthesis and prevents the diminution in calpastatin mRNA. The overall results suggest that at the onset of myoblast differentiation, calpastatin is regulated mainly at the level of translation and that an inhibition of calpastatin synthesis leads to the decrease in its mRNA stability. The existing calpastatin then diminishes, resulting in decreased calpastatin activity in the fusing myoblasts, allowing calpain activation and protein degradation required for fusion.