bluuebunny/crossref_metadata_2025 huggingface dataset:
Aggrecanase versus matrix metalloproteinases in the catabolism of the interglobular domain of aggrecan in vitro
Authors: LITTLE Chris B. , FLANNERY Carl R. , HUGHES Clare E. , MORT John S. , ROUGHLEY Peter J. , DENT Colin , CATERSON Bruce
Published: August 2015

The importance of aggrecanase versus matrix metalloproteinase (MMP) enzymic activities in the degradation of aggrecan in normal and osteoarthritic (OA) articular cartilage in vitro was studied in order to further our understanding of the potential role of these two enzyme activities in aggrecan catabolism during the pathogenesis of cartilage degeneration. Porcine and bovine articular cartilage was maintained in explant culture for up to 20 days in the presence or absence of the catabolic stimuli retinoic acid, interleukin-1 or tumour necrosis factor-α. Release of proteoglycan from cartilage was measured as glycosaminoglycan (GAG) release using a colorimetric assay. Analysis of proteoglycan degradation products, both released into culture media and retained within the cartilage matrix, was performed by Western blotting using antibodies specific for the N- and C-terminal neoepitopes generated by aggrecanase- and MMP-related catabolism of the interglobular domain of the aggrecan core protein (IGD). In addition, studies determining the mRNA expression for MMP-3 and MMP-13 in these same cultures were undertaken. These analyses indicated that all three catabolic agents stimulated the release of > 80% of the GAG from the articular cartilage over 4 days. The degree of GAG release corresponded to an increase in aggrecanase-generated aggrecan catabolites released into the media and retained within the cartilage. Importantly, there was no evidence for the release of MMP-generated aggrecan metabolites into the medium, nor the accumulation of MMP-generated catabolites within the tissue in these same cultures. Expression of the mRNAs for two MMPs known to be capable of degrading the aggrecan IGD, MMP-3 and MMP-13, was detected. However, increased expression of these MMPs was not correlated with aggrecan degradation. Analyses using porcine cartilage, cultured with or without catabolic stimulation for 12 h to 20 days, indicated that primary cleavage of the IGD by aggrecanase was responsible for release of aggrecan metabolites at both the early and late time points of culture. Cultures of late-stage OA human articular cartilage samples indicated that aggrecanase activity was upregulated in the absence of catabolic stimulation when compared with normal porcine or bovine cartilage. In addition, even in this late-stage degenerate cartilage, aggrecanase and not MMP activity was responsible for the release of the majority of aggrecan from the cartilage. This study demonstrates that the release of aggrecan from both normal and OA cartilage in response to catabolic stimulation in vitro involves a primary cleavage by aggrecanase and not MMPs.
Competition between Escherichia coli strains expressing either a periplasmic or a membrane-bound nitrate reductase: does Nap confer a selective advantage during nitrate-limited growth?
Authors: POTTER Laura C. , MILLINGTON Paul , GRIFFITHS Lesley , THOMAS Gavin H. , COLE Jeffrey A.
Published: August 2015

The physiological role of the periplasmic nitrate reductase, Nap, one of the three nitrate reductases synthesized by Escherichia coli K-12, has been investigated. A series of double mutants that express only one nitrate reductase were grown anaerobically in batch cultures with glycerol as the non-fermentable carbon source and nitrate as the terminal electron acceptor. Only the strain expressing nitrate reductase A grew rapidly under these conditions. Introduction of a narL mutation severely decreased the growth rate of the nitrate reductase A strain, but enhanced the growth of the Nap+ strain. The ability to use nitrate as a terminal electron acceptor for anaerobic growth is therefore regulated primarily by the NarL protein at the level of transcription. Furthermore, the strain expressing nitrate reductase A had a substantial selective advantage in competition with the strain expressing only Nap during nitrate-sufficient continuous culture. However, the strain expressing Nap was preferentially selected during nitrate-limited continuous growth. The saturation constants for nitrate for the two strains (which numerically are equal to the nitrate concentrations at half of the maximum specific growth rate and therefore reflect the relative affinities for nitrate) were estimated using the integrated Monod equation to be 15 and 50 μM for Nap and nitrate reductase A respectively. This difference is sufficient to explain the selective advantage of the Nap+ strain during nitrate-limited growth. It is concluded that one physiological role of the periplasmic nitrate reductase of enteric bacteria is to enable bacteria to scavenge nitrate in nitrate-limited environments.
Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose
Authors: THORNALLEY Paul J. , LANGBORG Annika , MINHAS Harjit S.
Published: August 2015

The glycation of proteins by glucose has been linked to the development of diabetic complications and other diseases. Early glycation is thought to involve the reaction of glucose with N-terminal and lysyl side chain amino groups to form Schiff's base and fructosamine adducts. The formation of the α-oxoaldehydes, glyoxal, methylglyoxal and 3-deoxyglucosone, in early glycation was investigated. Glucose (50 mM) degraded slowly at pH 7.4 and 37 °C to form glyoxal, methylglyoxal and 3-deoxyglucosone throughout a 3-week incubation period. Addition of t-BOC-lysine and human serum albumin increased the rate of formation of α-oxoaldehydes - except glyoxal and methylglyoxal concentrations were low with albumin, as expected from the high reactivity of glyoxal and methylglyoxal with arginine residues. The degradation of fructosyl-lysine also formed glyoxal, methylglyoxal and 3-deoxyglucosone. α-Oxoaldehyde formation was dependent on the concentration of phosphate buffer and availability of trace metal ions. This suggests that α-oxoaldehydes were formed in early glycation from the degradation of glucose and Schiff's base adduct. Since α-oxoaldehydes are important precursors of advanced glycation adducts, these adducts may be formed from early and advanced glycation processes. Short periods of hyperglycaemia, as occur in impaired glucose tolerance, may be sufficient to increase the concentrations of α-oxoaldehydes in vivo.
Histone H1- and other protein- and amino acid-hydroperoxides can give rise to free radicals which oxidize DNA
Authors: LUXFORD Catherine , MORIN Benedicte , DEAN Roger T. , DAVIES Michael J.
Published: August 2015

Exposure of amino acids, peptides and proteins to radicals, in the presence of oxygen, gives high yields of hydroperoxides. These materials are readily decomposed by transition metal ions to give further radicals. We hypothesized that hydroperoxide formation on nuclear proteins, and subsequent decomposition of these hydroperoxides to radicals, might result in oxidative damage to associated DNA. We demonstrate here that exposure of histone H1 and model compounds to γ-radiation in the presence of oxygen gives hydroperoxides in a dose-dependent manner. These hydroperoxides decompose to oxygen- and carbon-centred radicals (detected by electron paramagnetic resonance spectroscopy) on exposure to Cu+ and other transition metal ions. These hydroperoxide-derived radicals react readily with pyrimidine DNA bases and nucleosides to give adduct species (i.e. protein-DNA base cross-links). Product analysis has demonstrated that radicals from histone H1-hydroperoxides, and other protein and amino acid hydroperoxides, can also oxidize both free 2′-deoxyguanosine and intact calf thymus DNA to give the mutagenic oxidized base 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-hydroxy-2′-deoxyguanosine, 8-oxodG). The yield of 7,8-dihydro-8-oxo-2′-deoxyguanosine is proportional to the initial protein-hydroperoxide concentration, and corresponds (for histone H1-hydroperoxide, 280 μM) to approx. 1.4% conversion for free 2′-deoxyguanosine (200 μM), and 0.14% for 2′-deoxyguanosine in DNA (70 μg/ml). Evidence has also been obtained with DNA for reaction at cytosine and thymine, but not adenine; the lack of damage to the latter may result from damage transfer to 2′-deoxyguanosine residues. These studies demonstrate that initial radical-induced damage to nuclear proteins can give rise to subsequent DNA damage; the latter includes both DNA-protein cross-links and formation of oxidized DNA bases.
Cysteine residues in the Na+/dicarboxylate co-transporter, NaDC-1
Authors: PAJOR Ana M. , KRAJEWSKI Sally J. , SUN Nina , GANGULA Rama
Published: August 2015

The role of cysteine residues in the Na+/dicarboxylate co-transporter (NaDC-1) was tested using site-directed mutagenesis. The transport activity of NaDC-1 was not affected by mutagenesis of any of the 11 cysteine residues, indicating that no individual cysteine residue is necessary for function. NaDC-1 is sensitive to inhibition by the impermeant cysteine-specific reagent, p-chloromercuribenzenesulphonate (pCMBS). The pCMBS-sensitive residues in NaDC-1 are Cys-227, found in transmembrane domain 5, and Cys-476, located in transmembrane domain 9. Although cysteine residues are not required for function in NaDC-1, their presence appears to be important for protein stability or trafficking to the plasma membrane. There was a direct relationship between the number of cysteine residues, regardless of location, and the transport activity and expression of NaDC-1. The results indicate that mutagenesis of multiple cysteine residues in NaDC-1 may alter the shape or configuration of the protein, leading to alterations in protein trafficking or stability.
Reversible alkaline inactivation of lignin peroxidase involves the release of both the distal and proximal site calcium ions and bishistidine co-ordination of the haem
Authors: GEORGE Simon J. , KVARATSKHELIA Mamuka , DILWORTH Michael J. , THORNELEY Roger N. F.
Published: August 2015

Phanerochaete chrysosporium lignin peroxidase isoenzyme H2 (LiP H2) exhibits a transition to a stable, inactive form at pH 9.0 with concomitant spectroscopic changes. The Söret peak intensity decreases some 55% with a red shift from 408 to 412 nm; the bands at 502 nm and 638 nm disappear and the peak at 536 nm increases. The EPR spectrum changes from a signal typical of high spin ferric haem to an exclusively low spin spectrum with g = 2.92, 2.27, 1.50. These data indicate that the active pentaco-ordinated haem is converted into a hexaco-ordinated species at alkaline pH. Room temperature near-IR MCD data coupled with the EPR spectrum allow us to assign the haem co-ordination of alkali-inactivated enzyme as bishistidine. Re-acidification of the alkali-inactivated enzyme to pH 6 induces further spectroscopic changes and generates an irreversibly inactivated species. By contrast, a pH shift from 9.0 to 6.0 with simultaneous addition of 50 mM CaCl2 results in the recovery of the initial activity together with the spectroscopic characteristics of the native ferric enzyme. Incubating with 50 mM CaCl2 at a pH between 6.0 and 9.0 can also re-activate the enzyme. Divalent metals other than Ca2+ do not result in restoration of activity. Experiments with 45Ca indicate that two tightly bound calcium ions per enzyme monomer are lost during inactivation and reincorporated during subsequent re-activation, consistent with the presence of two structural Ca2+ ions in LiP H2. It is concluded that both the structural Ca2+ ions play key roles in the reversible alkaline inactivation of LiP H2.
Mutation of aspartic acid residues in the fructosyltransferase of Streptococcus salivarius ATCC 25975
Authors: SONG Donna D. , JACQUES Nicholas A.
Published: August 2015

The site-directed mutated fructosyltransferases (Ftfs) of Streptococcus salivarius ATCC 25975, D312E, D312S, D312N and D312K were all active at 37 °C, indicating that Asp-312 present in the ‘sucrose box’ was not the nucleophilic Asp residue responsible for the formation of a covalent fructosyl-enzyme intermediate required for enzyme activity. Analysis of the kinetic constants of the purified mutated forms of the enzyme showed that Asp-312 was most likely an essential amino acid involved in determining acceptor recognition and/or stabilizing a β-turn in the protein. In contrast, when the Asp-397 of the Ftf present in the conserved triplet RDP motif of all 60 bacterial and plant family-32 glycosylhydrolases was mutated to a Ser residue, both sucrose hydrolysis and polymerization ceased. Tryptophan emission spectra confirmed that this mutation did not alter protein structure. Comparison of published data from other site-directed mutated enzymes implicated the Asp residue in the RDP motif as the one that may form a transient covalent fructosyl intermediate during the catalysis of sucrose by the Ftf of S. salivarius.
Transcriptional analysis of human survivin gene expression
Authors: LI Fengzhi , ALTIERI Dario C.
Published: August 2015

The preservation of tissue and organ homoeostasis depends on the regulated expression of genes controlling apoptosis (programmed cell death). In this study, we have investigated the basal transcriptional requirements of the survivin gene, an IAP (inhibitor of apoptosis) prominently up-regulated in cancer. Analysis of the 5′ flanking region of the human survivin gene revealed the presence of a TATA-less promoter containing a canonical CpG island of ∼ 250 nt, three cell cycle dependent elements, one cell cycle homology region and numerous Sp1 sites. PCR-based analysis of human genomic DNA, digested with methylation-sensitive and -insensitive restriction enzymes, indicated that the CpG island was unmethylated in both normal and neoplastic tissues. Primer extension and S1 nuclease mapping of the human survivin gene identified two main transcription start sites at position -72 and within -57/-61 from the initiating ATG. Transfection of cervical carcinoma HeLa cells with truncated or nested survivin promoter-luciferase constructs revealed the presence of both enhancer and repressor sequences and identified a minimal promoter region within the proximal -230 nt of the human survivin gene. Unbiased mutagenesis analysis of the human survivin promoter revealed that targeting the Sp1 sequences at position -171 and -151 abolished basal transcriptional activity by ∼ 63-82%. Electrophoretic mobility-shift assay with DNA oligonucleotides confirmed formation of a DNA-protein complex between the survivin Sp1 sequences and HeLa cell extracts in a reaction abolished by mutagenesis of the survivin Sp1 sites. These findings identify the basal transcriptional requirements of survivin gene expression.
Cloning, expression and characterization of YSA1H, a human adenosine 5′-diphosphosugar pyrophosphatase possessing a MutT motif
Authors: GASMI Lakhdar , CARTWRIGHT Jared L. , MCLENNAN Alexander G.
Published: August 2015

The human homologue of the Saccharomyces cerevisiae YSA1 protein, YSA1H, has been expressed as a thioredoxin fusion protein in Escherichia coli. It is an ADP-sugar pyrophosphatase with similar activities towards ADP-ribose and ADP-mannose. Its activities with ADP-glucose and diadenosine diphosphate were 56% and 20% of that with ADP-ribose respectively, whereas its activity towards other nucleoside 5′-diphosphosugars was typically 2-10%. cADP-ribose was not a substrate. The products of ADP-ribose hydrolysis were AMP and ribose 5-phosphate. Km and kcat values with ADP-ribose were 60 μM and 5.5 s-1 respectively. The optimal activity was at alkaline pH (7.4-9.0) with 2.5-5 mM Mg2+ or 100-250 μM Mn2+ ions; fluoride was inhibitory, with an IC50 of 20 μM. The YSA1H gene, which maps to 10p13-p14, is widely expressed in all human tissues examined, giving a 1.4 kb transcript. The 41.6 kDa fusion protein behaved as an 85 kDa dimer on gel filtration. After cleavage with enterokinase, the 24.4 kDa native protein fragment ran on SDS/PAGE with an apparent molecular mass of 33 kDa. Immunoblot analysis with a polyclonal antibody raised against the recombinant YSA1H revealed the presence of a protein of apparent molecular mass 33 kDa in various human cells, including erythrocytes. The sequence of YSA1H contains a MutT sequence signature motif. A major proposed function of the MutT motif proteins is to eliminate toxic nucleotide metabolites from the cell. Hence the function of YSA1H might be to remove free ADP-ribose arising from NAD+ and protein-bound poly- and mono-(ADP-ribose) turnover to prevent the occurrence of non-enzymic protein glycation.
Signalling-competent truncated forms of ErbB2 in breast cancer cells: differential regulation by protein kinase C and phosphatidylinositol 3-kinase
Authors: ESPARÍS-OGANDO Azucena , DÍAZ-RODRÍGUEZ Elena , PANDIELLA Atanasio
Published: August 2015

Alterations that affect the ectodomain of receptor tyrosine kinases are often associated with constitutive activation of the enzymic activity of the mutant cell-associated receptor. Since the ectodomain of the ErbB2 receptor tyrosine kinase has been detected as a soluble fragment in the culture supernatant of cells and serum from patients with advanced breast cancer, the possible presence of cell-associated truncated forms of ErbB2 in cancer cells was investigated. Several cell-bound N-terminal truncated forms of ErbB2 were identified in breast cancer cells overexpressing this receptor. The presence of the truncated fragments was independent of lysosomal/proteasomal activity, indicating that classical receptor tyrosine kinase degradation systems were not involved in the N-terminal cleavages. The presence of these truncated forms of ErbB2 was up-regulated by protein kinase C and neuregulin; and down-regulated by phosphatidylinositol 3-kinase, and monoclonal antibodies that target the ectodomain of ErbB2, indicating that N-terminal cleavages of ErbB2 were regulated by multiple mechanisms. The truncated fragments were tyrosine-phosphorylated under resting conditions, and associated with the signalling intermediates Shc and Grb2. It is therefore likely that these truncated forms may be endowed with constitutive activity that allows them to permanently signal.
α5 subunit in Trypanosoma brucei proteasome can self-assemble to form a cylinder of four stacked heptamer rings
Authors: YAO Yi , TOTH Charles R. , HUANG Lan , WONG Mei-Lie , DIAS Prabha , BURLINGAME Alma L. , COFFINO Philip , WANG Ching C.
Published: August 2015

The proteasomes have a central role in catalysing protein degradation among both prokaryotes and eukaryotes. The 20 S proteasome constitutes their catalytic core. In studying the structure of Trypanosoma brucei 20 S proteasomes, we isolated by two-dimensional (2D) gel electrophoresis a 27 kDa subunit protein with an estimated pI of 4.7 and subjected it to mass spectrometric analysis. A tryptic peptide sequence from the protein was found identical with that of the rat α5 subunit. With the use of antiserum against T. brucei 20 S proteasomes to screen a T. b. rhodesiense λ expression cDNA library, we obtained a cDNA clone encoding a full-length protein of 246 amino acid residues with a calculated molecular mass of 27174 Da and a pI of 4.71. It bears 50.0% and 46.3% sequence identity with rat and yeast proteasome subunit α5 respectively, and matches all the peptide sequences derived from MS of the 2D gel-purified protein. The protein is thus designated the α5 subunit of T. brucei 20 S proteasome (TbPSA5). The recombinant protein, expressed in plasmid-transformed Escherichia coli, was found in a 27 kDa monomer form as well as polymerized forms with estimated molecular masses ranging from 190 to 800 kDa. Under the electron microscope, the most highly polymerized forms bear the appearance of cylinders of four-stacked heptamer rings with an estimated outer diameter of 14.5 nm and a length of 18 nm, which were immunoprecipitable by anti-(T. brucei 20 S proteasome) antiserum. In view of the documented self-assembly of the archaeon proteasome α subunit into double heptamer rings and the spontaneous assembly of the two α subunits from the 20 S proteasome of Rhodococcus erythropolis, the self-assembly of the T. brucei α subunit might reflect a common feature of proteasome biogenesis shared by prokaryotes and primitive eukaryotes such as the trypanosomes but apparently lost among the higher forms of eukaryote such as the yeast and the mammals.
Sp1 mediates cell proliferation-dependent regulation of rat DNA topoisomerase IIα gene promoter
Authors: YOON Jeong Ho , KIM Jeong Kee , RHA Geun Bae , OH Misook , PARK Se-Ho , SEONG Rho Hyun , HONG Seung Hwan , PARK Sang Dai
Published: August 2015

DNA topoisomerase IIα (topo IIα) is an essential nuclear enzyme required for chromosome segregation during mitosis. Consistent with its critical role in cell division is the fact that the expression of the gene for topo IIα is strongly regulated by the proliferation state of cells. Using a transient expression system, we determined the contribution of putative cis-acting elements in its promoter region to its basal level and cell proliferation-dependent transcription. Experiments with 5′ and/or 3′ serial deletion and site-directed mutation revealed that (1) maximal promoter activity resides in the fragment extending to position -663 bp from the ATG initiation codon, (2) minimal promoter activity is harboured at -195 bp, (3) the defined minimal promoter contains only two putative elements, inverted CCAAT box 4 (ICB4) (-166 to -162 bp) and the most proximal GC-rich box in the promoter (GC2) (-149 to -143 bp), and (4) ICB4 is most important in the basal-level transcription of the gene for rat topo IIα. The luciferase activities of the mutated reporter plasmids in G0-arrested and exponentially growing cells showed that proliferation-specific regulation is controlled mainly by GC2. Electrophoretic mobility-shift assays indicated that Sp1 binds specifically to the GC2 site. The extent of DNA-protein complex formation increases after the stimulation of cells to proliferate. These results indicate that the increased binding activity of Sp1 to GC2 is important in the up-regulation of the gene for topo IIα in growing cells.
A processing intermediate of a stromal chloroplast import protein in Chlamydomonas
Authors: SU Qingxiang , SCHUMANN Philipp , SCHILD Christof , BOSCHETTI Arminio
Published: August 2015

Proteins synthesized in the cytoplasm and destined for importation into the chloroplast across the double envelope membrane contain an N-terminal transit sequence which upon import is cleaved off by a stromal-processing peptidase. Since for stromal-residing proteins no intermediates have ever been found in vivo, it is assumed that precursor proteins are cleaved to the mature size by one proteolytic event which occurs immediately after translocation across both envelope membranes. During import of the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) into isolated chloroplasts of Chlamydomonas we identified an intermediate-sized product, called iSS. It might be identical to a previously described iSS obtained in vitro by a partially purified soluble chloroplast protease [Su and Boschetti (1993) Eur. J. Biochem. 217, 1039-1047]. The kinetics of the formation of iSS in chloroplasts suggest that pSS is processed to the mature small subunit (SS) not by one, but by two steps via this intermediate product. Since, after an induction period, the ratio of iSS/SS was constant under various experimental conditions of import, the formation of iSS was considered not to be a side-reaction. The location of iSS in the intermembrane space of the envelope, as suggested by protease treatment of chloroplasts, questions the one-step translocation mechanism of precursor import into chloroplasts.
Heat-shock protein 70 antisense oligomers enhance proteasome inhibitor-induced apoptosis
Authors: ROBERTSON John D. , DATTA Kaushik , BISWAL Shyam S. , KEHRER James P.
Published: August 2015

Recent evidence supports a role for heat-shock protein 70 (hsp70) and the 26 S proteasome in regulating apoptosis, although the precise nature of their involvement is not known. In the present study, control and Bcl-xL-overexpressing, interleukin-3-dependent FL5.12 cell lines were treated with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). Basal proteasome activity appeared to be ≈ 30% lower in bcl-xL cells compared with control cells using a substrate for the chymotrypsin-like activity. However, no difference in proteasome activity was detected using substrates for the trypsin-like or peptidylglutamyl peptide-hydrolysing activities. In addition, protein levels of the 20 S proteasome β-subunit, as determined by Western blot analyses, were similar in control and bcl-xL cells, leading to the conclusion that proteasome activities were the same in these two cell lines. At 24 h after treatment with 500 nM MG132, apoptosis in bcl-xL cells (22%) was less than that observed in control cells (34%). Concomitantly, caspase activity in control cells, as assessed by N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartyl-7-amino-4-methylcoumarin (Ac-DEVD-AMC), was twice that observed in bcl-xL cells. By 48 h after MG132 treatment, apoptosis and caspase activity in bcl-xLcells were similar to those observed in control cells at 24 h. Proteasome inhibition stimulated increases in hsp70 protein levels in control and bcl-xL cells by 12 h, although the maximal increases found in bcl-xL cells were less. Blocking this induction with hsp70 antisense oligonucleotides potentiated apoptosis after treatment with MG132. Inhibiting caspase activity with a broad-spectrum caspase inhibitor, t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone, prevented MG132-induced apoptosis. The more specific caspase-3 inhibitor, Ac-DEVD-aldehyde, afforded less protection, although both inhibitors completely inhibited Ac-DEVD-AMC cleavage. These data indicate that both hsp70 and Bcl-xL provide some protection against proteasome inhibitor-induced apoptosis.
Functional and conformational characterization of new mutants of heart fatty acid-binding protein
Authors: ZIMMERMAN Aukje W. , RADEMACHER Martin , RüTERJANS Heinz , LüCKE Christian , VEERKAMP Jacques H.
Published: August 2015

In this study we investigated the possible involvement of several amino acids (not located in the ligand-binding centre) in fatty acid binding and conformational stability of heart fatty acid-binding protein (H-FABP). We prepared recombinant human H-FABP proteins with mutations in the hydrophobic patch (Phe4, Trp8 and Phe64), portal region (Phe16), hinge region (Leu66, Gly67), second portal region (Glu72) and at the protein surface (Lys21) respectively. Oleic acid-binding affinity and conformational stability of human H-FABP are significantly decreased or completely lost by mutation of Trp8 or Phe16. NMR spectra confirmed that these residues are important for the stability of the protein fold. Substitution of Phe4 or Phe64 resulted in less stability, but oleic acid-binding affinity was not affected. Mutation of Lys21 had no effect on either structural integrity or fatty acid-binding affinity. Replacement of Leu66 or Gly67 did not affect fatty acid binding, but protein stability was reduced. Finally, mutation of Glu72 to Ser caused no change of affinity, but NMR spectra and urea-denaturation curves showed the extremely poor stability of this mutant. In conclusion, no relationship was observed between fatty acid-binding affinity and conformational stability.
Synapsins as major neuronal Ca2+/S100A1-interacting proteins
Authors: HEIERHORST Jörg , MITCHELHILL Ken I. , MANN Richard J. , TIGANIS Tony , CZERNIK Andrew J. , GREENGARD Paul , KEMP Bruce E.
Published: August 2015

The mammalian S100A1 protein can activate the invertebrate myosin-associated giant protein kinase twitchin in a Ca2+-dependent manner by more than 1000-fold in vitro; however, no mammalian S100-dependent protein kinases are known. In an attempt to identify novel mammalian Ca2+/S100A1-regulated protein kinases, brain extracts were subjected to combined Ca2+-dependent affinity chromatography with S100A1 and an ATP analogue. This resulted in the purification to near-homogeneity of the four major synapsin isoforms Ia, Ib, IIa and IIb. All four synapsins were specifically affinity-labelled with the ATP analogue 5′-p-fluorosulphonylbenzoyladenosine. S100A1 bound to immobilized synapsin IIa in BIAcore experiments in a Ca2+-dependent and Zn2+-enhanced manner with submicromolar affinity; this interaction could be competed for with synthetic peptides of the proposed S100A1-binding sites of synapsin. Double-labelling confocal immunofluorescence microscopy demonstrated that synapsins and S100A1 are both present in the soma and neurites of PC12 cells, indicating their potential to interact in neurons in vivo.
Interaction of the N-terminus of sterol carrier protein 2 with membranes: role of membrane curvature
Authors: HUANG Huan , BALL Judith M. , BILLHEIMER Jeffrey T. , SCHROEDER Friedhelm
Published: August 2015

Although neither the physiological function nor the mechanism of action of sterol carrier protein 2 (SCP2) is yet completely clear, it is thought that SCP2 interacts with membranes to elicit its biological effects. The results presented here show that the SCP2 N-terminus, composed of two amphipathic α-helices, interacted preferentially with highly curved but not lower-curvature membranes containing anionic phospholipid. CD spectra of SCP2 showed up to 1.2-fold increased α-helical content, on the interaction of SCP2 with small unilamellar vesicles (SUV) (median radius 10-14 nm) but less with large unilamellar vesicles (LUV) (median radius 52-60 nm). Although enhanced interaction with the SUV membranes was due in part to the radius of curvature and to the greater exposure of acidic phospholipid in the outer leaflet of the bilayer, simply increasing the molar percentage of acidic phospholipid in the LUV membranes had much less effect on SCP2 binding. A similar preferential interaction was observed with highly curved SUV as opposed to LUV for the SCP2 N-terminal peptide 1-32SCP2 as well as structurally modified peptides in the order 1-32SCP2 = 10-32SCP2 > 1-24SCP2 ≫ 1-E20-32SCP2. The CD results were confirmed with an independent filtration binding assay, which showed that SCP2 bound 5-fold more to SUV than LUV, whereas its N-terminal peptides bound up to 4-fold better in the order 1-32SCP2 = 10-32SCP2 > 1-24SCP2 > 1-E20-32SCP2. Finally, cholesterol potentiated the binding of SCP2 and N-terminal peptides to anionic-phospholipid-containing SUV but not LUV. These findings were consistent with the SCP2 N-terminus being a membrane-binding domain that was highly dependent on membrane surface curvature as well as on lipid composition.
Endocytosis and vesicular trafficking of immune complexes and activation of phospholipase D by the human high-affinity IgG receptor requires distinct phosphoinositide 3-kinase activities
Authors: GILLOOLY David J. , MELENDEZ Alirio J. , HOCKADAY Austin R. , HARNETT Margaret M. , ALLEN Janet M.
Published: August 2015

FcγRI, the human high-affinity IgG receptor, is responsible for the internalization of immune complexes and their subsequent targetting to the lysosomes for degradation. We show here that aggregation of FcγRI by surface immune complexes in interferon-γ-primed U937 cells causes the transient appearance of swollen vacuolar structures, probably swollen late endosomes, which disappear as the immune complexes are degraded. Wortmannin and LY294002, specific inhibitors of phosphoinositide 3-kinases (PI 3-kinases), delay the disappearance of these structures and also correspondingly inhibit degradation of FcγRI-mediated immune complexes. In addition these inhibitors delay the initial phase of FcγRI-mediated endocytosis of immune complexes and block the activity of FcγRI-stimulated phospholipase D, an enzyme that has previously been implicated in membrane-trafficking events. p85 is the regulatory subunit of PI 3-kinase. A p85-dependent PI 3-kinase was shown to be involved in the initial phase of FcγRI-mediated endocytosis, but not in the trafficking of immune complexes for degradation or the activation of phospholipase D. The results presented here show a role for a p85-independent PI 3-kinase in regulating the trafficking of FcγRI-mediated immune complexes, either directly or as a result of the activation of phospholipase D, and a distinct role for a p85-dependent PI 3-kinase isoform in the initial phases of FcγRI-mediated internalization of immune complexes.
Polyamine transport in bacteria and yeast
Authors: IGARASHI Kazuei , KASHIWAGI Keiko
Published: August 2015

The polyamine content of cells is regulated by biosynthesis, degradation and transport. In Escherichia coli, the genes for three different polyamine transport systems have been cloned and characterized. Two uptake systems (putrescine-specific and spermidine-preferential) were ABC transporters, each consisting of a periplasmic substrate-binding protein, two transmembrane proteins and a membrane-associated ATPase. The crystal structures of the substrate-binding proteins (PotD and PotF) have been solved. They consist of two domains with an alternating β-α-β topology, similar to other periplasmic binding proteins. The polyamine-binding site is in a cleft between the two domains, as determined by crystallography and site-directed mutagenesis. Polyamines are mainly recognized by aspartic acid and glutamic acid residues, which interact with the NH2- (or NH-) groups, and by tryptophan and tyrosine residues that have hydrophobic interactions with the methylene groups of polyamines. The precursor of one of the substrate binding proteins, PotD, negatively regulates transcription of the operon for the spermidine-preferential uptake system, thus providing another level of regulation of cellular polyamines. The third transport system, catalysed by PotE, mediates both uptake and excretion of putrescine. Uptake of putrescine is dependent on membrane potential, whereas excretion involves an exchange reaction between putrescine and ornithine. In Saccharomyces cerevisiae, the gene for a polyamine transport protein (TPO1) was identified. The properties of this protein are similar to those of PotE, and TPO1 is located on the vacuolar membrane.
The in vitro manipulation of carbohydrate metabolism: a new strategy for deciphering the cellular defence mechanisms against nitric oxide attack
Authors: LE GOFFE Claire , VALLETTE Geneviève , JARRY Anne , BOU-HANNA Chantal , LABOISSE Christian L.
Published: August 2015

This study was aimed at examining the effects of manipulating the carbohydrate source of the culture medium on the cellular sensitivity of epithelial cells to an oxidative attack. Our rationale was that substituting galactose for glucose in culture media would remove the protection afforded by glucose utilization in two major metabolic pathways, i.e. anaerobic glycolysis and/or the pentose phosphate pathway (PPP), which builds up cellular reducing power. Indeed, we show that the polarized human colonic epithelial cell line HT29-Cl.16E was sensitive to the deleterious effects of the NO donor PAPANONOate [3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine] only in galactose-containing medium. In such medium NO attack led to cytotoxic and apoptotic cell death, associated with formation of derivatives of NO auto-oxidation (collectively termed NOx) and peroxynitrite, leading to intracellular GSH depletion and nitrotyrosine formation. The addition of 2-deoxyglucose, a non-glycolytic substrate, to galactose-fed cells protected HT29-Cl.16E cells from NO attack and maintained control GSH levels through its metabolic utilization in the PPP, as shown by 14CO2 production from 2-deoxy[1-14C]glucose. Therefore, increasing the availability of reducing equivalents without interfering with energy metabolism is able to prevent NO-induced cell injury. Finally, this background provides the conceptual framework for establishing nutritional manipulation of cellular metabolic pathways that could provide new means for (i) deciphering the mechanisms of cell injury by reactive nitrogen species and reactive oxygen species at the whole-cell level and (ii) establishing the hierarchy of intracellular defence mechanisms against these attacks.
Stimulation of pancreatic β-cell proliferation by growth hormone is glucose-dependent: signal transduction via Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) with no crosstalk to insulin receptor substrate-mediated mitogenic signalling
Authors: COUSIN Sharon P. , HülGL Sigrun R. , MYERS JR. Martin G. , WHITE Morris F. , REIFEL-MILLER Anne , RHODES Christopher J.
Published: August 2015

Mitogenic signal-transduction pathways have not been well defined in pancreatic β-cells. In the glucose-sensitive rat β-cell line, INS-1, glucose (6-18 mM) increased INS-1 cell proliferation (> 20-fold at 15 mM glucose). Rat growth hormone (rGH) also induced INS-1 cell proliferation, but this was glucose-dependent in the physiologically relevant concentration range (6-18 mM glucose). The combination of rGH (10 nM) and glucose (15 mM) was synergistic, maximally increasing INS-1 cell proliferation by > 50-fold. Moreover, glucose-dependent rGH-induced INS-1 cell proliferation was increased further by addition of insulin-like growth factor 1 (IGF-1; 10 nM) to > 90-fold at 12 mM glucose. Glucose metabolism and phosphatidylinositol-3ʹ-kinase (PI3ʹK) activation were necessary for both glucose- and rGH-stimulated INS-1 cell proliferation. Glucose (> 3 mM) independently increased tyrosine-phosphorylation-mediated recruitment of growth-factor-bound protein 2 (Grb2)/murine sons of sevenless-1 protein (mSOS) and PI3ʹK to insulin receptor substrate (IRS)-1 and IRS-2, as well as SH2-containing protein (Shc) association with Grb2/mSOS and downstream activation of mitogen-activated protein kinase and 70 kDa S6 kinase. Glucose-induced IRS- and Shc-mediated signal transduction was enhanced further by the addition of IGF-1, but not rGH. In contrast, rGH was able to activate Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) signal transduction at glucose concentrations above 3 mM, but neither glucose independently, nor glucose with added IGF-1, were able to activate the JAK2/STAT5 signalling pathway. Thus rGH-mediated proliferation of β-cells is directly via the JAK2/STAT5 pathway without engaging the Shc or IRS signal-transduction pathways, although activation of PI3ʹK may play an important permissive role in the glucose-dependent aspect of rGH-induced β-cell mitogensis. The additive effect of rGH and IGF-1 on glucose-dependent β-cell proliferation is therefore reflective of rGH and IGF-1 activating distinctly different mitogenic signalling pathways in β-cells with minimal crosstalk between them.
Protein specific N-glycosylation of tyrosinase and tyrosinase-related protein-1 in B16 mouse melanoma cells
Authors: NEGROIU Gabriela , BRANZA-NICHITA Norica , PETRESCU Andrei J. , DWEK Raymond A. , PETRESCU Stefana M.
Published: August 2015

Tyrosinase and tyrosinase-related protein-1 (TRP-1) are two melanogenic enzymes that regulate melanin biosynthesis. Both are glycoproteins and belong to the TRP-1 gene family. They share a significant level of sequence similarity in several regions, including the catalytic domain and the potential N-glycosylation sites. We have recently shown that inhibition of the early steps of N-glycan processing in B16F1 cells dramatically affects tyrosinase activity and melanin synthesis. We present here results on N-glycan processing of TRP-1 and tyrosinase and compare the maturation process and activity of both glycoproteins in the presence of inhibitors of the endoplasmic reticulum stages of N-glycosylation.

N-glycan analysis reveals that each of these two glycoproteins contains a mixture of high-mannose and sialylated complex N-glycans. However, in contrast to TRP-1, tyrosinase presents a homogeneous high-mannose glycoform, also.

In the presence of α-glucosidases inhibitors, the maturation of tyrosinase N-glycans is completely inhibited, whereas TRP-1 is still able to acquire some complex glycans, indicating that endomannosidase acts preferentially on the later glycoprotein. In addition, the dopa-oxidase activity of tyrosinase is totally abolished, whereas for TRP-1 it is only partially affected. The results suggest that despite their structural similarity, tyrosinase is more sensitive than TRP-1 to perturbations of early N-glycan processing, in terms of maturation and catalytical activity.
Topology studies with biosynthetic fragments identify interacting transmembrane regions of the human red-cell anion exchanger (band 3; AE1)
Authors: GROVES Jonathan D. , TANNER Michael J. A.
Published: August 2015

The red-cell anion exchanger (band 3; AE1) is a multispanning membrane protein that traverses the bilayer up to 14 times and is N-glycosylated at Asn-642. We have shown that the integrity of six different loops are not essential for stilbene disulphonate-sensitive chloride uptake in Xenopus oocytes. We used an N-glycosylation mutagenesis approach to examine the orientation of the N-terminus and the endogenous glycosylation site of each C-terminal fragment by cell-free translation. The fragments initiating in the loops preceding spans 2, 9 and 11 did not insert into the membrane with the expected orientation. Furthermore, N-glycosylation of Asn-642 might facilitate the membrane integration of span 7. The correct integration of spans 2-3 required the presence of the region containing span 4 and that the luminal exposure of the C-terminus of span 7 is increased in the presence of the region including span 6 or span 8. The results suggest the span 8 region is required for the correct folding of spans 9-10, at least in the presence of the span 11-12 region. Our results suggest that there are intramolecular interactions between the regions of transmembrane spans 1 and 2, 2 and 4, 4 and 5, 7 and 8, 8 and 9-10, and 9-10 and 11-12. Spans 1, 4, 5, 6 and 8 might act as a scaffold for the assembly of spans 2-3, 7 and 9-10. This approach might provide a general method for dissecting the interactions between membrane-spanning regions of polytopic membrane proteins.
Identification of betacellulin as a major peptide growth factor in milk: purification, characterization and molecular cloning of bovine betacellulin
Authors: DUNBAR Andrew J. , PRIEBE Ilka K. , BELFORD David A. , GODDARD Chris
Published: August 2015

Betacellulin (BTC), a member of the epidermal growth factor (EGF) family of peptide growth factors, was purified from a growth-factor-enriched whey fraction of bovine milk by a combination of ion-exchange chromatography, gel-filtration chromatography, affinity chromatography and reverse-phase HPLC. Bovine BTC (bBTC) had an apparent molecular mass of 21-22 kDa on SDS/PAGE and exists in a glycosylated form. The cDNA encoding bBTC was obtained by a combination of 5ʹ and 3ʹ rapid amplification of cDNA ends (‘RACE’). The primary translation product consists of 178 amino acid residues containing a putative signal sequence, a transmembrane domain, the mature BTC domain and a cytoplasmic domain containing a highly hydrophilic Arg-Lys-rich region similar to that of mouse BTC and human BTC. The amino acid sequence of the bBTC precursor was 88% identical with human BTC and 79% identical with mouse BTC. The bBTC gene was found to be expressed in a wide range of tissues, including the mammary gland. The identification of BTC in milk raises the possibility that it has a major role in the growth and development of the neonatal gastrointestinal tract.
Synthesis of 8-epi-prostaglandin F2α by human endothelial cells: role of prostaglandin H2 synthase
Authors: WATKINS Michael T. , PATTON George M. , SOLER Hiram M. , ALBADAWI Hassan , HUMPHRIES Donald E. , EVANS James E. , KADOWAKI Hiroko
Published: August 2015

The experiments described in this paper were designed to determine the mechanism underlying the increase in 8-isoprostaglandin F2α (8-epi-PGF2α) production by cultured human endothelial cells during reoxygenation following hypoxia. Human umbilical artery endothelial cells were grown on microcarrier beads and exposed to sequential periods of normoxia, hypoxia, and reoxygenation. The amount of 8-epi-PGF2α in the medium was determined by ELISA. The production of 8-epi-PGF2α decreased by greater than 90% during hypoxia. Upon reoxygenation 8-epi-PGF2α production increased linearly for 90 min reaching nearly 3 times normoxic levels. When added to the medium during reoxygenation, neither superoxide dismutase nor Tiron, a cell-permeable superoxide scavenger, inhibited 8-epi-PGF2α production. However, 8-epi-PGF2α production was inhibited by catalase. The production of 8-epi-PGF2α was also inhibited by indomethacin and aspirin. Exogenous hydrogen peroxide stimulated 8-epi-PGF2α production by normoxic cells, and aspirin inhibited the hydrogen peroxide-mediated increase in 8-epi-PGF2α production. These results indicate that the reactive oxygen species responsible for 8-epi-PGF2α synthesis during reoxygenation is hydrogen peroxide and that in endothelial cells 8-epi-PGF2α synthesis is mediated by prostaglandin H2 synthase (PGHS). To verify the role of PGHS in 8-epi-PGF2α synthesis, human PGHS-1 was expressed in COS-7 cells, a PGHS negative cell line that does not synthesize 8-epi-PGF2α. In the presence of exogenous arachidonic acid the COS-7 cells expressing human PGHS-1 produced substantial amounts of PGE2 and 8-epi-PGF2α. These data indicate that human PGHS-1 can support the synthesis of 8-epi-PGF2α and that 8-epi-PGF2α synthesis by cultured human endothelial cells during reoxygenation is dependent on the activity of PGHS-1.
Proteophosphoglycans of Leishmania mexicana: Identification, purification, structural and ultrastructural characterization of the secreted promastigote proteophosphoglycan pPPG2, a stage-specific glycoisoform of amastigote aPPG
Authors: KLEIN Christian , GÖPFERT Ulrich , GOEHRING Nathan , STIERHOF York-Dieter , ILG Thomas
Published: August 2015

Protozoan parasites of the genus Leishmania secrete a range of proteophosphoglycans that appear to be important for successful colonization of the sandfly and for virulence in the mammalian host. A hallmark of these molecules is extensive phosphoglycosylation by phosphoglycan chains via the unusual linkage Manα1-PO4-Ser. In this study we have identified and purified to apparent homogeneity a novel proteophosphoglycan (pPPG2) which is secreted by Leishmania mexicana promastigotes (sandfly stage). Amino acid analysis and immunoblots using polypeptide-specific antisera suggest that pPPG2 shares a common protein backbone with a proteophosphoglycan (aPPG) secreted by Leishmania mexicana amastigotes (mammalian stage). Both pPPG2 and aPPG show a similar degree of Ser phosphoglycosylation (50.5 mol% vs. 44.6 mol%), but the structure of their phosphoglycan chains is developmentally regulated: in contrast to aPPG which displays unique, complex and highly branched glycan chains [Ilg, Craik, Currie, Multhaup, and Bacic (1998) J. Biol. Chem. 273, 13509-13523], pPPG2 contains short unbranched structures consisting of > 60 mol% neutral glycans, most likely (Manα1-2)0-5Man and Galβ1-4Man, as well as about 40 mol% monophosphorylated glycans of the proposed structures PO4-6Galβ1-4Man and PO4-6(Glcβ1-3)Galβ1-4Man. The major differences between pPPG2 and aPPG with respect to their apparent molecular mass, their ultrastructure and their proteinase sensitivity are most likely a consequence of this stage-specific glycosylation of their common protein backbone.