bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet-derived growth factor is a principal inductive factor modulating mannose 6-phosphate/insulin-like growth factor-II receptor gene expression via a distal E-box in activated hepatic stellate cells
Authors: WEINER Joel A. , CHEN Anping , DAVIS Bernard H.
Published: August 2015

Hepatic stellate cells (HSCs) become activated during the early stages of hepatic injury associated with fibrogenesis. The mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGFIIR) plays an important role in early fibrogenesis by participating in the activation of latent transforming growth factor-β, a potent inducer of the matrix proteins in activated stellate cells that produce the hepatic nodule. Platelet-derived growth factor (PDGF), a potent HSC mitogen, is released early in hepatic injury and activates several signalling pathways in HSCs. In this study we examined the role of PDGF-BB in HSC regulation of M6P/IGFIIR gene expression. Several promoter elements were found and characterized that modulate M6P/IGFIIR expression in activated stellate cells. The presence of a distal CACGTG E-box at -2695 was required for M6P/IGFIIR expression in transfected stellate cells. When the distal E-box was removed there was no significant M6P/IGFIIR promoter activity. The distal E-box-binding protein responded specifically to PDGF-BB with increased binding. This coincided with PDGF-BB up-regulation of M6P/IGFIIR mRNA transcript levels. Downstream elements include two proximal (-2 to -48) CACGTG E-boxes that bind a different protein to the distal (-2695) E-box. The proximal E-boxes respond moderately to PDGF-BB. The promoter segment encompassing -144 to +109 is able to respond dramatically to serum but is refractory to PDGF-BB. However, a constitutively bound protein binding to the -611/-716 fragment appears to be a repressor that suppresses inductive changes in protein binding occurring downstream of -611. These results indicate that the M6P/IGFIIR promoter responds primarily and specifically to PDGF-BB through a distal E-box element and possibly through two proximal E-box elements.
Distinct signalling pathways mediate the cAMP response element (CRE)-dependent activation of the calcitonin gene-related peptide gene promoter by cAMP and nerve growth factor
Authors: FREELAND Karen , LIU Yu-Zhen , LATCHMAN David S.
Published: August 2015

The gene encoding the calcitonin gene-related peptide (CGRP) is activated in neuronal cells by treatment with cAMP and nerve growth factor (NGF). Both stimuli induce the phosphorylation of the cAMP response element (CRE)-binding protein (CREB) transcription factor on Ser-133 and require the CRE in the CGRP promoter to stimulate transcription. However, whereas the CRE is necessary and sufficient for promoter activation by cAMP, it is necessary but not sufficient for activation by NGF. We show that this difference is paralleled by a difference in the signalling pathways which are required for each stimulus to activate the CGRP promoter. Thus whilst cAMP-mediated activation requires the protein kinase A pathway, NGF-mediated stimulation requires the Ras/Raf mitogen-activated protein kinase kinase-1 (MEK-1)/p42/p44 mitogen-activated protein kinase (MAPK) pathway. Although NGF can activate the protein kinase C, p38 MAPK and c-Jun N-terminal kinase (JNK) pathways, these pathways are not involved in its effect on the CGRP promoter. The effect of the p42/p44 MAPK pathway on CREB and associated transcription factors, and the manner in which this results in activation of the CGRP promoter is discussed.
Inclusion of a furin-sensitive spacer enhances the cytotoxicity of ribotoxin restrictocin containing recombinant single-chain immunotoxins
Authors: GOYAL Anita , BATRA Janendra K.
Published: August 2015

Chimaeric toxins have considerable therapeutic potential to treat various malignancies. We have previously used the fungal ribonucleolytic toxin restrictocin to make chimaeric toxins in which the ligand was fused at either the N-terminus or the C-terminus of the toxin. Chimaeric toxins containing ligand at the C-terminus of restrictocin were shown to be more active than those having ligand at the N-terminus of the toxin. Here we describe the further engineering of restrictocin-based chimaeric toxins, anti-TFR(scFv)-restrictocin and restrictocin-anti-TFR(scFv), containing restrictocin and a single chain fragment variable (scFv) of a monoclonal antibody directed at the human transferrin receptor (TFR), to enhance their cell-killing activity. To promote the independent folding of the two proteins in the chimaeric toxin, a linear flexible peptide, Gly-Gly-Gly-Gly-Ser, was inserted between the toxin and the ligand to generate restrictocin-linker-anti-TFR(scFv) and anti-TFR(scFv)-linker-restrictocin. A 12-residue spacer, Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu, containing the recognition site for the protease furin, was incorporated between the toxin and the ligand to generate restrictocin-spacer-anti-TFR(scFv) and anti-TFR(scFv)-spacer-restrictocin. The incorporation of the proteolytically cleavable spacer enhanced the cell-killing activity of both constructs by 2-30-fold depending on the target cell line. However, the introduction of linker improved the cytotoxic activity only for anti-TFR(scFv)-linker-restrictocin. The proteolytically cleavable spacer-containing chimaeric toxins had similar cytotoxic activities irrespective of the location of the ligand on the toxin and they were found to release the restrictocin fragment efficiently on proteolysis in vitro.
Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria
Authors: ANTONSSON Bruno , MONTESSUIT Sylvie , LAUPER Sandra , ESKES Robert , MARTINOU Jean-Claude
Published: August 2015

Bax is a Bcl-2-family protein with pro-apoptotic activity that can form channels in lipid membranes. The protein has been shown to trigger cytochrome c release from mitochondria both in vitro and in vivo. Recombinant human Bax isolated in the presence of detergent was found to be present as an oligomer with an apparent molecular mass of approx. 160000 Da on gel filtration. When Bax was isolated in the absence of detergent the purified protein was monomeric with an apparent molecular mass of 22000 Da. Bax oligomers formed channels in liposomes and triggered cytochrome c release from isolated mitochondria, whereas monomeric Bax was inactive in both respects. Incubation of the monomeric Bax with 2% octyl glucoside induced formation of oligomers that displayed channel-forming activity in liposomes and triggered cytochrome c release from mitochondria. Triton X-100, Nonidet P-40 and n-dedecyl maltoside also activated monomeric Bax, whereas CHAPS had no activating effect. In cytosolic extracts from mouse liver, Bax migrated at a molecular mass of 24000 Da on gel filtration, whereas after incubation of the cytosol with 2% octyl glucoside Bax migrated at approximately 140000 Da. These results show that oligomeric Bax possesses channel-forming activity whereas monomeric Bax has no such activity.
Geldanamycin disrupts platelet-membrane structure, leading to membrane permeabilization and inhibition of platelet aggregation
Authors: SUTTITANAMONGKOL Sudawadee , GEAR Adrian R. L. , POLANOWSKA-GRABOWSKA Renata
Published: August 2015

Geldanamycin (GA), a benzoquinoid ansamycin antibiotic, has been used as a tyrosine kinase inhibitor and an anti-tumour agent and is known to bind to heat-shock protein 90. In the present study on human platelets we have found that GA inhibited platelet aggregation induced by ADP, thrombin and the thrombin-receptor-activating peptide and caused platelet plasma-membrane damage, detected by leakage of adenine nucleotides as well as serotonin. Scanning electron microscopy (SEM) revealed that platelet exposure to GA led to the formation of holes or fenestrations in the platelet plasma membrane, confirming GA's ability to initiate membrane damage. In addition, GA itself caused both the dephosphorylation and phosphorylation of proteins in resting platelets and prevented agonist-induced phosphorylation of pleckstrin, the 20-kDa myosin light chain and other proteins. Another ansamycin, herbimycin A, also inhibited platelet aggregation, but caused minimal membrane permeabilization, as detected by 3H release from platelets labelled previously with [3H]adenine, and much less membrane damage, revealed by SEM. Overall, GA is able to disrupt membrane structure and inhibit platelet aggregation, an ability which may be linked to alterations in the activity of protein kinases and phosphatases.
Four isoforms of serum response factor that increase or inhibit smooth-muscle-specific promoter activity
Authors: KEMP Paul R. , METCALFE James C.
Published: August 2015

Serum response factor (SRF) is a key transcriptional activator of the c-fos gene and of muscle-specific gene expression. We have identified four forms of the SRF coding sequence, SRF-L (the previously identified form), SRF-M, SRF-S and SRF-I, that are produced by alternative splicing. The new forms of SRF lack regions of the C-terminal transactivation domain by splicing out of exon 5 (SRF-M), exons 4 and 5 (SRF-S) and exons 3, 4 and 5 (SRF-I). SRF-M is expressed at similar levels to SRF-L in differentiated vascular smooth-muscle cells and skeletal-muscle cells, whereas SRF-L is the predominant form in many other tissues. SRF-S expression is restricted to vascular smooth muscle and SRF-I expression is restricted to the embryo. Transfection of SRF-L and SRF-M into C2C12 cells showed that both forms are transactivators of the promoter of the smooth-muscle-specific gene SM22α, whereas SRF-I acted as a dominant negative form of SRF.
Collagen fragments in urine derived from bone resorption are highly racemized and isomerized: a biological clock of protein aging with clinical potential
Authors: CLOOS Paul A. C. , FLEDELIUS Christian
Published: August 2015

Fragments of the α1 C-terminal telopeptide of type I collagen containing the sequence AHDGGR1209-1214 (CTx) can be measured in urine as an index of bone resorption. We report here that these molecules undergo racemization and isomerization of Asp1211in vitro and in vivo, generating a mixture of four isomers: the native peptide form (αL), an isomerized form containing a β-Asp bond (βL), a racemized form containing a D-Asp residue (αD) and an isomerized/racemized form (βD). To study these reactions at this specific site in collagen, we have employed four immunoassays, each specific for one of the isoforms, and developed HPLC methods for their separation. The kinetics of these reactions were studied in vitro under physiological conditions by incubation of synthetic AHDGGR hexapeptide or mineralized bone collagen. Reactions were found to be strongly shifted towards the β-Asp forms and slightly in favour of the D-enantiomeric forms. CTx isomers were measured in human urine and in enzymic digests of bovine bone collagen. The results indicated that the extent of racemization and isomerization were correlated with the age and turnover of collagen. The ratios between the native and age-related forms of CTx were elevated in urine from patients with Paget's disease or osteoporosis as compared with that from healthy adults. The αL/αD CTx ratio had the highest discriminatory power (T-score = 23.2; P < 0.0001 and T-score = 1.5; P < 0.0001 for Paget's disease and osteoporosis respectively). In conclusion, these findings indicate that an assessment of CTx ratios in urine may provide an estimate of bone turnover, aiding in the diagnosis of metabolic bone diseases.
Neuroserpin is expressed in the pituitary and adrenal glands and induces the extension of neurite-like processes in AtT-20 cells
Authors: HILL Rena M. , PARMAR Parmjeet K. , COATES Leigh C. , MEZEY Eva , PEARSON John F. , BIRCH Nigel P.
Published: August 2015

Two cDNAs encoding the serine protease inhibitor (serpin) neuroserpin were cloned from a rat pituitary cDNA library (rNS-1, 2922 bp; rNS-2, 1599 bp). In situ hybridization histochemistry showed neuroserpin transcripts in the intermediate, anterior and posterior lobes of the pituitary gland and medullary cells in the adrenal gland. Expression of rNS-1 mRNA was restricted to selected cells in the pituitary gland. Analysis of purified secretory-granule fractions from pituitary and adrenal tissues indicated that neuroserpin was found in dense-cored secretory granules. This result suggested that endocrine neuroserpin may regulate intragranular proteases or inhibit enzymes following regulated secretion. To investigate the function of neuroserpin in endocrine tissues we established stable anterior pituitary AtT-20 cell lines expressing neuroserpin. Cells with increased levels of neuroserpin responded by extending neurite-like processes. Extracellular proteolysis by serine protease plasminogen activators has been suggested to regulate neurite outgrowth. As neuroserpin inhibits tissue plasminogen activator (tPA) in vitro, we measured plasminogen-activator levels. Zymographic analysis indicated that AtT-20 cells synthesized and secreted a plasminogen activator identical in size to tPA. A higher-molecular-mass tPA-neuroserpin complex was also observed in AtT-20-cell conditioned culture medium. tPA levels were similar in parent AtT-20 cells and a stable cell line with increased levels of neuroserpin. There was no accumulation of a tPA-neuroserpin complex. Together these results identify endocrine cells as an important source of neuroserpin. Moreover they suggest that neuroserpin is released from dense-cored secretory granules to regulate cell-extracellular matrix interactions through a mechanism that may not directly involve tPA.
Quantitative assessment of complex formation of nuclear-receptor accessory proteins
Authors: GRAUMANN Klaus , JUNGBAUER Alois
Published: August 2015

Like other nuclear receptors, steroid hormone receptors form large protein hetero-complexes in their inactive, ligand-friendly state. Several heat-shock proteins, immunophilins and others have been identified as members of these highly dynamic complexes. The interaction kinetics and dynamics of hsp90, hsp70, p60 (Hop), FKBP52, FKBP51, p48 (Hip) and p23 have been assessed by a biosensor approach measuring the complex formation in real time. A core chaperone complex has been reconstituted from p60, hsp90 and hsp70. p60 forms a molecular bridge between hsp90 and hsp70 with an affinity in the range of 105 M-1. Dynamics of hsp90-p60 complex formation is modulated by ATP through changes in the co-operativity of interaction. At low protein concentrations ATP stabilizes the complex. Binding of p23 to hsp90 did not change the affinity of the hsp90-p60 complex and the stabilizing effect of ATP. Saturation of the p48-hsp70 interaction could not be achieved, suggesting multiple binding sites. A picture of the protein complex, including stoichiometric coefficients, co-operativity of interaction and equilibrium-binding constants, has been formed.
Characterization of a second member of the subfamily of calcium-binding mitochondrial carriers expressed in human non-excitable tissues
Authors: DEL ARCO Araceli , AGUDO Marta , SATRÚSTEGUI Jorgina
Published: August 2015

We have recently identified a subfamily of mitochondrial carriers that bind calcium, and cloned ARALAR1, a member of this subfamily expressed in human muscle and brain. We have now cloned a second human ARALAR gene (ARALAR2) coding for a protein 78.3% identical to Aralar1, but expressed in liver and non-excitable tissues. Aralar2 is identical to citrin, the product of the gene mutated in type-II citrullinaemia [Kobayashi, Sinasac, Iijima, Boright, Begum, Lee, Yasuda, Ikeda, Hirano, Terazono et al. (1999) Nat. Genet. 22, 159-163]. A related protein, DmAralar, 69% identical to Aralar1, was found in Drosophila melanogaster, the DMARALAR locus lying on the right arm of the third chromosome, band 99F. The N-terminal half of Aralar2/citrin is able to bind calcium and this requires the presence of the two most distal EF-hands. The localization of Aralar2/citrin expressed in human cell lines is mitochondrial, the C-terminal half containing sufficient information for import and assembly into mitochondria. The C-terminal half of Aralar proteins is related to the yeast YPR020c gene, with a very high sequence conservation (54.3% identity), suggesting that these proteins play an important role. Thus Aralar proteins are probably expressed in all tissues in an isoform-specific fashion, where they function as calcium-regulated metabolite (possibly anionic) carriers.
Direct and reversed amino acid sequence pattern analysis: structural reasons for activity of reversed sequence sites and results of kinase site mutagenesis
Authors: TORSHIN Ivan
Published: August 2015

During studies of kinase phosphorylation, not all functional kinase phosphorylation may be found using consensus sequence patterns. This type of phosphorylation is termed ‘non-consensus’ or ‘cryptic’ phosphorylation. Results presented here based on molecular dynamics of short peptides show that protein kinases may phosphorylate not only established consensus sequences (reading a sequence from N-terminus to C-terminus) but also reversed consensus sequences (reading from C- to N-terminus). Several protein sequences were analysed and corresponding biochemical data were presented. Similarity of molecular shapes of direct and reversed consensus peptides, and sequence conservation in the regions of reversed sites in the analysed proteins, indicate that at least part of the phosphorylation sites considered as ‘cryptic’ may be explained in terms of reversed consensus pattern occurrences.
Dye-ligand chromatographic purification of intact multisubunit membrane protein complexes: application to the chloroplast H+-F0F1-ATP synthase
Authors: SEELERT Holger , POETSCH Ansgar , ROHLFS Meino , DENCHER Norbert A.
Published: August 2015

n-Dodecyl-β-D-maltoside was used as a detergent to solubilize the ammonium sulphate precipitate of chloroplast FOF1-ATP synthase, which was purified further by dye-ligand chromatography. Upon reconstitution of the purified protein complex into phosphatidylcholine/phosphatidic acid liposomes, ATP synthesis, driven by an artificial ∆pH/∆ψ, was observed. The highest activity was achieved with ATP synthase solubilized in n-dodecyl-β-D-maltoside followed by chromatography with Red 120 dye. The optimal dye for purification with CHAPS was Green 5. All known subunits were present in the monodisperse proton-translocating ATP synthase preparation obtained from chloroplasts.
Genomic organization, chromosomal mapping and promoter analysis of the mouse selenocysteine tRNA gene transcription-activating factor (mStaf) gene
Authors: ADACHI Kazushige , KATSUYAMA Masato , SONG Shigeaki , OKA Takami
Published: August 2015

mStaf is a zinc-finger protein that activates the transcription of the mouse selenocysteine tRNA gene. The mStaf gene is approx. 35 kb long and split into 16 exons. All exon-intron junction sequences conform to the GT/AG rule. The transcription start site is located 83 bp upstream of the initiation codon. Chromosomal mapping localized the gene to mouse chromosome 7, region E3-F1. Sequence analysis of the proximal promoter region revealed several potential regulatory elements; these include the recognition elements of Sp1, Nkx, CP2, E2A, SIF (SIS-inducible factor), TFII-I and cAMP-responsive element (CRE), but no TATA sequences. Transfection experiments demonstrated that the 5ʹ-flanking region (-1894 to +37) of the mStaf gene drives transcription in mouse NMuMG cells and that a construct containing a fragment from -387 to +37 showed the highest transcriptional activity. Deletion and mutation experiments suggested that four Sp1 sites played an important role for the basal promoter activity. Furthermore, electrophoretic mobility-shift assays demonstrated that Sp3 but not other Sp (specificity protein) family members binds to three of the Sp1 sites. Our present study suggests that Sp3 is involved in the basal transcriptional activation of the mStaf gene.
Inactivation of cysteine proteases by peptidyl epoxides: characterization of the alkylation sites on the enzyme and the inactivator
Authors: ALBECK Amnon , KLIPER Sharon
Published: August 2015

Erythro peptidyl epoxides are selective inactivators of cysteine proteases. The alkylation site, both on the enzyme papain and on the epoxide itself, was characterized. The inactivation of papain with the peptidyl epoxide erythro benzyloxycarbonyl-Phe-Ala-epoxide was followed by total hydrolysis by acid. Mass spectral analysis of the hydrolysate revealed, in addition to the expected amino acids, a unique signal of m/z 209 (MH+). Its high-resolution mass spectrum and daughter peak analysis correspond to the product of alkylation on cysteine and the expected fragmentation. A similar MS pattern was obtained for a synthetic model compound corresponding to the expected hydrolysis product. A 13C NMR analysis of papain inactivated by a specifically 13C-labelled peptidyl epoxide indicated that the alkylation of the enzyme's cysteine residue occurs on the primary carbon of the epoxide moiety.
Histone synthesis in Leishmania infantum is tightly linked to DNA replication by a translational control
Authors: SOTO Manuel , QUIJADA Luis , ALONSO Carlos , REQUENA Jose M.
Published: August 2015

We have analysed the regulation of histone synthesis in Leishmania infantum following inhibition of DNA replication. Run-on experiments indicated that transcription rates of the genes coding for the four core histones (H2A, H2B, H3 and H4) were not affected by the inhibition with hydroxyurea of DNA synthesis. However, a dramatic decrease was observed in the newly synthesized histones after inhibition of DNA synthesis. Furthermore, the synthesis of both the histones and DNA resumed in promastigotes after removal of hydroxyurea, indicating that inhibition was reversible. Unlike most eukaryotes, in which the replication-dependent histone transcripts decrease upon a replication blockade, the levels of L. infantum histone mRNAs do not change under similar conditions. Thus the present data indicate that histone synthesis in Leishmania is tightly coupled to DNA replication by a mechanism operating at the translational level.
Endoproteolytic processing of integrin pro-α subunits involves the redundant function of furin and proprotein convertase (PC) 5A, but not paired basic amino acid converting enzyme (PACE) 4, PC5B or PC7
Authors: LISSITZKY Jean-Claude , LUIS José , MUNZER Jon Scott , BENJANNET Suzanne , PARAT Francis , CHRÉTIEN Michel , MARVALDI Jacques , SEIDAH Nabil Georges
Published: August 2015

Several integrin α subunits undergo post-translational endoproteolytic processing at pairs of basic amino acids that is mediated by the proprotein convertase furin. Here we ask whether other convertase family members can participate in these processing events. We therefore examined the endoproteolysis rate of the integrin subunits pro-α5, α6 and αv by recombinant furin, proprotein convertase (PC)5A, paired basic amino acid converting enzyme (PACE)4, PC1, PC2 and PC7 in vitro and/or ex vivo after overexpression in LoVo cells that were deficient in furin activity. We found that 60-fold more PC1 than furin was needed to produce 50% cleavage of pro-α subunit substrates in vitro; the defective pro-α chain endoproteolysis in LoVo cells was not rescued by overexpression of PC1 or PC2. No endoproteolysis occurred with PC7 either in vitro or ex vivo, although similar primary sequences of the cleavage site are found in integrins and in proteins efficiently processed by PC7, which suggests that a particular conformation of the cleavage site is required for optimal convertase-substrate interactions. In vitro, 50% cleavage of pro-α subunits was obtained with one-third of the amount of PC5A and PACE4 than of furin. In LoVo cells, PC5A remained more active than furin, PACE4 activity was quite low, and PC5B, which differs from PC5A by a C-terminal extension containing a transmembrane domain, was very inefficient in processing integrin α-subunit precursors. In conclusion, these results indicate that integrin α-subunit endoproteolytic processing involves the redundant function of furin and PC5A and to a smaller extent PACE4, but not of PC1, PC2, PC5B or PC7.
Subcellular localization of proteasomes and their regulatory complexes in mammalian cells
Authors: BROOKS Paul , FUERTES Graciela , MURRAY Rachael Z. , BOSE Suchira , KNECHT Erwin , RECHSTEINER Martin C. , HENDIL Klavs B. , TANAKA Keiji , DYSON Julian , RIVETT A. Jennifer
Published: August 2015

Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable γ-interferon-inducible catalytic β-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit specific antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core α-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some significant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immunofluorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following γ-interferon treatment of cultured cells but γ-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.
Tetratricopeptide repeat domain of Yarrowia lipolytica Pex5p is essential for recognition of the type 1 peroxisomal targeting signal but does not confer full biological activity on Pex5p
Authors: SZILARD Rachel K. , RACHUBINSKI Richard A.
Published: August 2015

Peroxins are proteins required for peroxisome assembly and are encoded by the PEX genes. The Yarrowia lipolytica pex5-1 mutant fails to import a subset of peroxisomal matrix proteins, including those with a type 1 peroxisomal targeting signal (PTS1). Pex5p family members interact with a PTS1 through their characteristic tetratricopeptide repeat (TPR) domain. We used binding assays in vitro to investigate the nature of the association of Y. lipolytica Pex5p (YlPex5p) with the PTS1 signal. A purified recombinant YlPex5p fusion protein interacted specifically, directly and autonomously with a protein terminating in a PTS1. Wild-type YlPex5p translated in vitro recognized functional PTS1s specifically. This activity is abrogated by the substitution of an aspartic residue for a conserved glycine residue in the TPR domain (G455D) of YlPex5p encoded by the pex5-1 allele. Deletion analysis demonstrated that an intact TPR domain of YlPex5p is necessary but not sufficient for both interaction with a PTS1 and functional complementation of a strain lacking YlPex5p.
Adult and fetal haemoglobin J-Sardegna [α50(CE8)His→Asp]: functional and molecular modelling studies
Authors: CORDA Marcella , DE ROSA Maria C. , PELLEGRINI Maria G. , SANNA Maria T. , OLIANAS Alessandra , FAIS Antonella , MANCA Laura , MASALA Bruno , ZAPPACOSTA Bruno , FICARRA Silvana , CASTAGNOLA Massimo , GIARDINA Bruno
Published: August 2015

Haemoglobin (Hb) J-Sardegna [α50(CE8)His → Asp] is a haemoglobin variant characteristic of subjects from the island of Sardinia. Here we report a study of the functional properties of both fetal and adult Hb J-Sardegna. The results indicate that adult Hb J-Sardegna displays an oxygen affinity that is higher than that of adult Hb only in the presence of 2,3-diphosphoglycerate (2,3-DPG). On the contrary, at 20 °C, the oxygen affinity of fetal Hb J-Sardegna is identical to that of normal fetal haemoglobin, both in the presence and in the absence of 2,3-DPG. A significant difference between these two systems (i.e. a higher oxygen affinity of fetal Hb J-Sardegna) shows up very clearly only when temperature is increased to 37 °C. Hence in fetal Hb, the main effect of the amino acid substitution is a decrease in the overall enthalpy change of oxygenation. The results outline the role of the α1-β1 interface in assessing the thermodynamics of oxygen binding. The functional properties of both adult and fetal Hb J-Sardegna have been interpreted at the structural level in light of the results obtained by a computational modelling approach performed in comparison with HbA and Hb Aichi, a variant characterized by a different mutation [α50(CE8)His → Arg] at the same position.
Structure–function studies of tryptophan mutants of equinatoxin II, a sea anemone pore-forming protein
Authors: MALOVRH Petra , BARLIĆ Ariana , PODLESEK Zdravko , MAĆEK Peter , MENESTRINA Gianfranco , ANDERLUH Gregor
Published: August 2015

Equinatoxin II (EqtII) is a eukaryotic cytolytic toxin that avidly creates pores in natural and model lipid membranes. It contains five tryptophan residues in three different regions of the molecule. In order to study its interaction with the lipid membranes, three tryptophan mutants, EqtII Trp45, EqtII Trp116/117 and EqtII Trp149, were prepared in an Escherichia coli expression system [here, the tryptophan mutants are classified according to the position of the remaining tryptophan residue(s) in each mutated protein]. They all possess a single intrinsic fluorescent centre. All mutants were less haemolytically active than the wild-type, although the mechanism of erythrocyte damage was the same. EqtII Trp116/117 resembles the wild-type in terms of its secondary structure content, as determined from Fourier-transform infrared (FTIR) spectra and its fluorescent properties. Tryptophans at these two positions are buried within the hydrophobic interior of the protein, and are transferred to the lipid phase during the interaction with the lipid membrane. The secondary structure of the other two mutants, EqtII Trp45 and EqtII Trp149, was altered to a certain extent. EqtII Trp149 was the most dissimilar from the wild-type, displaying a higher content of random-coil structure. It also retained the lowest number of nitrogen-bound protons after exchange with 2H2O, which might indicate a reduced compactness of the molecule. Tryptophans in EqtII Trp45 and EqtII Trp149 were more exposed to water, and also remained as such in the membrane-bound form.
On the preparation of β-haematin
Authors: BLAUER G. , AKKAWI M.
Published: August 2015

Synthetic β-haematin is considered to be identical, or at least very similar to, purified malaria pigment. Methods for its preparation use ferriprotoporphyrin IX at acid pH in the presence of acetic acid at different concentrations and degrees of ionization, elevated temperatures and long reaction times. Here we show that certain widely used reaction conditions, involving very high concentrations of acetic acid/acetate mixtures, do not produce substantial amounts of polymeric β-haematin on immediate isolation of the reaction products, but only during prolonged drying of the products at 37 °C after washing with water. Alternative, more convenient methods of preparation of pure β-haematin are suggested.
Organization and chromosomal localization of the human ECEL1 (XCE) gene encoding a zinc metallopeptidase involved in the nervous control of respiration
Authors: VALDENAIRE Olivier , ROHRBACHER Elisabeth , LANGEVELD An , SCHWEIZER Anja , MEIJERS Carel
Published: August 2015

ECEL1 (endothelin-converting enzyme-like 1; previously known as XCE) is a putative zinc metalloprotease that was identified recently on the basis of its strong identity with endothelin-converting enzyme. Although the physiological function of ECEL1 is unknown, inactivation of the corresponding gene in mice points to a critical role of this protein in the nervous control of respiration. In the present study we have characterized the human ECEL1 gene. It was located to region q36-q37 of chromosome 2 and shown to be composed of 18 exons spanning approx. 8 kb. The structure of the ECEL1 gene displays some striking similarities with those of genes of related metallopeptidases, supporting the hypothesis that they are all derived from a common ancestor. A short phylogenetic study describing the relationship between the various members of this gene family is also presented.
Characterization of AMP-activated protein kinase γ-subunit isoforms and their role in AMP binding
Authors: CHEUNG Peter C. F. , SALT Ian P. , DAVIES Stephen P. , HARDIE D. Grahame , CARLING David
Published: August 2015

The AMP-activated protein kinase (AMPK) cascade plays an important role in the regulation of energy homeostasis within the cell. AMPK is a heterotrimer composed of a catalytic subunit (α) and two regulatory subunits (β and γ). We have isolated and characterized two isoforms of the γ subunit, termed γ2 and γ3. Both γ2 (569 amino acids) and γ3 (492 amino acids) have a long N-terminal domain which is not present in the previously characterized isoform, γ1. As with γ1, mRNA encoding γ2 is widely expressed in human tissues, whereas significant expression of γ3 mRNA was only detected in skeletal muscle. Using isoform-specific antibodies, we determined the AMPK activity associated with the different γ isoforms in a number of rat tissues. In most tissues examined more than 80% of total AMPK activity was associated with the γ1 isoform, with the remaining activity being accounted for mainly by the γ2 isoform. Exceptions to this were testis and, more notably, brain where all three isoforms contributed approximately equally to activity. There was no evidence for any selective association between the α1 and α2isoforms and the various γ isoforms. However, the AMP-dependence of the kinase complex is markedly affected by the identity of the γ isoform present, with γ2-containing complexes having the greatest AMP-dependence, γ3 the lowest, and γ1 having an intermediate effect. Labelling studies, using the reactive AMP analogue 8-azido-[32P]AMP, indicate that the γ subunit may participate directly in the binding of AMP within the complex.
Neutral amino acid transporter ASCT2 displays substrate-induced Na+ exchange and a substrate-gated anion conductance
Authors: BRÖER Angelika , WAGNER Carsten , LANG Florian , BRÖER Stefan
Published: August 2015

The neutral amino acid transporter ASCT2 mediates electroneutral obligatory antiport but at the same time requires Na+ for its function. To elucidate the mechanism, ASCT2 was expressed in Xenopus laevis oocytes and transport was analysed by flux studies and two-electrode voltage clamp recordings. Flux studies with 22NaCl indicated that the uptake of one molecule of glutamine or alanine is accompanied by the uptake of four to seven Na+ ions. Similarly to the transport of amino acids, the Na+ uptake was mediated by an obligatory Na+ exchange mechanism that depended on the presence of amino acids but was not stoichiometrically coupled to the amino acid transport. Other cations could not replace Na+ in this transport mechanism. When NaCl was replaced by NaSCN in the transport buffer, the superfusion of oocytes with amino acid substrates resulted in large inward currents, indicating the presence of a substrate-gated anion channel in the ASCT2 transporter. The Km for glutamine derived from these experiments is in good agreement with the Km derived from flux studies; it varied between 40 and 90 μM at holding potentials of -60 and -20 mV respectively. The permeability of the substrate-gated anion conductance decreased in the order SCN- NO3- > I- > Cl- and also required the presence of Na+.
Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide
Authors: KOTSONIS Peter , FRÖHLICH Lothar G. , SHUTENKO Zhanna V. , HOREJSI Renate , PFLEIDERER Wolfgang , SCHMIDT Harald H. H. W.
Published: August 2015

The underlying mechanisms regulating the activity of the family of homodimeric nitric oxide synthases (NOSs) and, in particular, the requirement for (6R)-5,6,7,8-tetrahydro-L-biopterin (H4Bip) are not fully understood. Here we have investigated possible allosteric and stabilizing effects of H4Bip on neuronal NOS (NOS-I) during the conversion of substrate, L-arginine, into L-citrulline and nitric oxide. Indeed, in kinetic studies dual allosteric interactions between L-arginine and H4Bip activated recombinant human NOS-I to increase L-arginine turnover. Consistent with this was the observation that H4Bip, but not the pterin-based NOS inhibitor 2-amino-4,6-dioxo-3,4,5,6,8,8a,9,10-octahydro-oxazolo[1,2-f]-pteridine (PHS-32), caused an L-arginine-dependent increase in the haem Soret band, indicating an increase in substrate binding to recombinant human NOS-I. Conversely, L-arginine was observed to increase in a concentration-dependent manner H4Bip binding to pig brain NOS-I. Secondly, we investigated the stabilization of NOS quaternary structure by H4Bip in relation to uncoupled catalysis. Under catalytic assay conditions and in the absence of H4Bip, dimeric recombinant human NOS-I dissociated into inactive monomers. Monomerization was related to the uncoupling of reductive oxygen activation, because it was inhibited by both superoxide dismutase and the inhibitor NΩ-nitro-L-arginine. Importantly, H4Bip was found to react chemically with superoxide (O2-−) and enzyme-bound H4Bip was consumed under O2-−-generating conditions in the absence of substrate. These results suggest that H4Bip allosterically activates NOS-I and stabilizes quaternary structure by a novel mechanism involving the direct interception of auto-damaging O2-−.
GDP dissociation inhibitor D4-GDI (Rho-GDI 2), but not the homologous Rho-GDI 1, is cleaved by caspase-3 during drug-induced apoptosis
Authors: ESSMANN Frank , WIEDER Thomas , OTTO Albrecht , MÜLLER Eva-Christina , DÖRKEN Bernd , DANIEL Peter T.
Published: August 2015

Different cytotoxic drugs induce cell death by activating the apoptotic programme; a family of cysteinyl aspartate proteases named caspases has been shown to be involved in the initiation as well as the execution of this kind of cell death. In the present study, cleavage of D4-GDI (Rho-GDI 2), an abundant haemopoietic-cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, was demonstrated after treatment of BJAB Burkitt-like lymphoma cells with taxol or epirubicin. The cleavage of D4-GDI occurred simultaneously with the activation of caspase-3 but preceded DNA fragmentation and the morphological changes associated with apoptotic cell death. By using high-resolution two-dimensional gel electrophoresis it was shown that this cleavage is specific: whereas the level of the homologous protein Rho-GDI 1 was not significantly altered during drug-induced apoptosis and in cytochrome c/dATP-activated cellular extracts, D4-GDI disappeared owing to proteolytic cleavage. Inhibitor experiments with Z-DEVD-fmk (in which Z stands for benzyloxycarbonyl and fmk for fluoromethyl ketone) and microsequencing of the D4-GDI fragment revealed that this occurs at the caspase-3 cleavage site. Our results strongly suggest the differential regulation of the homologous GDP dissociation inhibitors Rho-GDI 1 and D4-GDI during drug-induced apoptosis by proteolysis mediated by caspase-3 but not by caspase-1. Owing to their crucial role as modulators of Rho GTPases, this might in turn have a significant impact on the mechanisms that induce the cytoskeletal and morphological changes in apoptotic cells.
Genomic organization of three novel toxins from the scorpion Buthus martensi Karsch that are active on potassium channels
Authors: DAI Li , WU Jing-Jiang , GU Yi-Hua , LAN Zheng-Dao , LING Min-Hua , CHI Cheng-Wu
Published: August 2015

The cDNA and genomic DNA of three novel toxins from the scorpion Buthus martensi Karsch that are active on K+ channels, designated BmKTX (where KTX is kaliotoxin), BmTX1 and BmTX2, were cloned and sequenced. On the basis of their known amino acid sequences, gene-specific primers for 3ʹ and 5ʹ rapid amplification of cDNA ends (RACE) were designed and synthesized. By overlapping the two partial cDNA sequences obtained by 3ʹ and 5ʹ RACE, their full-length cDNA sequences were completed. BmKTX encodes a signal peptide of 22 amino acid residues and a mature toxin of 38 residues, whereas BmTX1 and BmTX2 encode signal peptides of 20 and 21 residues respectively and a mature toxin of 38 residues for each. Their cDNA-deduced amino acid sequences were totally consistent with those determined except that the C-terminus of BmKTX had an additional Gly residue, which was removed during post-translational processing and was indispensable for the amidation of its C-terminal Lys residue. In addition, the first deduced amino acid for both BmTX1 and BmTX2 is Gln instead of pyro-Glu in the reported toxins, which obviously also undergoes post-translational processing. The genomic DNA species of these three toxins were also amplified by PCR, then cloned and sequenced. They all consisted of two exons disrupted by a small single intron. All of these introns were inserted within the signal peptides at position -6 for BmKTX and at position -5 for both BmTX1 and BmTX2 upstream of the mature toxins, and consisted of 87, 87 and 80 bp respectively.
The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain
Authors: LORENTSEN Rikke H. , GRAVERSEN Jonas H. , CATERER Nigel R. , THØGERSEN Hans C. , ETZERODT Michael
Published: August 2015

Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca2+-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca2+. Exon 2 encodes an α-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding.
Farnesylcysteine analogues inhibit store-regulated Ca2+ entry in human platelets: evidence for involvement of small GTP-binding proteins and actin cytoskeleton
Authors: ROSADO Juan A. , SAGE Stewart O.
Published: August 2015

We have investigated the mechanism of Ca2+ entry into fura-2-loaded human platelets by preventing the prenylation of proteins such as small GTP-binding proteins. The farnesylcysteine analogues farnesylthioacetic acid (FTA) and N-acetyl-S-geranylgeranyl-L-cysteine (AGGC), which are inhibitors of the methylation of prenylated and geranylgeranylated proteins respectively, significantly decreased thrombin-evoked increases in intracellular free Ca2+ concentration ([Ca2+]i) in the presence, but not in the absence, of external Ca2+, suggesting a relatively selective inhibition of Ca2+ entry over internal release. Both these compounds and N-acetyl-S-farnesyl-L-cysteine, which had similar effects to those of FTA, also decreased Ca2+ entry evoked by the depletion of intracellular Ca2+ stores with thapsigargin. The inactive control N-acetyl-S-geranyl-L-cysteine was without effect. Patulin, an inhibitor of prenylation that is inert with respect to methyltransferases, also decreased store-regulated Ca2+ entry. Cytochalasin D, an inhibitor of actin polymerization, significantly decreased store-regulated Ca2+ entry in a time-dependent manner. Both cytochalasin D and the farnesylcysteine analogues FTA and AGGC inhibited actin polymerization; however, when evoking the same extent of decrease in actin filament formation, FTA and AGGC showed greater inhibitory effects on Ca2+ entry, indicating a cytoskeleton-independent component in the regulation of Ca2+ entry by small GTP-binding-protein. These findings suggest that prenylated proteins such as small GTP-binding proteins are involved in store-regulated Ca2+ entry through actin cytoskeleton-dependent and cytoskeleton-independent mechanisms in human platelets.
C-terminal half of tetanus toxin fragment C is sufficient for neuronal binding and interaction with a putative protein receptor
Authors: HERREROS Judit , LALLI Giovanna , SCHIAVO Giampietro
Published: August 2015

Tetanus neurotoxin (TeNT) is a powerful bacterial protein toxin that cleaves VAMP/synaptobrevin, an essential protein of the synaptic vesicle fusion machinery, and consequently blocks neurotransmission. The extreme neurospecificity of TeNT is determined by the binding of its C-terminal domain (fragment C or HC) to neuronal receptors. Whereas polysialogangliosides are known acceptors for the toxin, the existence of additional protein receptors has also been suggested. We have reported previously on a 15 kDa cell-surface glycoprotein that interacts with TeNT in neuronal cell lines and motoneurons [Herreros, Lalli, Montecucco and Schiavo (2000) J. Neurochem., in the press]. Here, on the basis of the structural information provided by the crystallization of fragment C of TeNT, we have expressed its C-and N-terminal halves as recombinant proteins and analysed their binding abilities to rat phaeochromocytoma (PC12) cells differentiated with nerve growth factor. We found that the C-terminal subdomain of the fragment C of TeNT is necessary and sufficient for cell binding and for the interaction with the 15 kDa putative receptor. In contrast, the N-terminal half showed a very poor interaction with the cell surface. These results restrict the binding domain of TeNT to the C-terminal half of the fragment C and highlight the importance of this domain for the neurospecific interaction of the toxin with the synapse. Furthermore, these findings support the use of this portion of TeNT as a neurospecific targeting device, pointing to an involvement of the N-terminal subdomain in later steps of the intoxication pathway.