bluuebunny/crossref_metadata_2025 huggingface dataset:
17β-Oestradiol increases intracellular Ca2+ concentration in rat enterocytes
Authors: PICOTTO Gabriela , VAZQUEZ Guillermo , BOLAND Ricardo
Published: August 2015

The involvement of the phospholipase C (PLC) pathway in the non-genomic regulation of duodenal cell Ca2+ concentration by 17β-oestradiol was investigated. The PLC inhibitors neomycin (0.5 mM) and U-73122 (2 µM) suppressed the stimulatory effect of 0.1 nM 17β-oestradiol on the 45Ca2+ influx into enterocytes isolated from rat duodenum. The hormone (1 pM to 10 nM) increased the formation of 1,2-diacylglycerol in a biphasic pattern, characterized by an early peak at 45 s (+82%) and a later peak at 5 min (+46%). Both PLC inhibitors suppressed the first peak but were unable to block the 17β-oestradiol effect at 5 min. 17β-Oestradiol also increased the generation of inositol 1,4,5-trisphosphate within 15 s, with maximal stimulation at 30 s. 17β-Oestradiol induced a rapid (30 s) and sustained (up to 5 min) increase in the intracellular Ca2+ concentration ([Ca2+]i) of fura 2-loaded enterocytes. The fast rise in [Ca2+]i was specific because other sex steroid hormones were without effect and could be blocked to a great extent by U-73122 (by 86% at 1 min). The effects of 17β-oestradiol on enterocyte [Ca2+]i were decreased significantly (by 75%) in a Ca2+-free extracellular medium but a pronounced increase in [Ca2+]i was obtained after readmission of Ca2+ to the medium. The latter change was suppressed by 10 µM La3+, whereas nitrendipine (1 µM) and verapamil (10 µM) separately were without effect. The permeability of the 17β-oestradiol-induced Ca2+ influx pathway to Mn2+ was increased 2.8-fold by treatment with oestrogen. These results suggest the operation of a PLC-dependent store-operated Ca2+ channel mechanism in 17β-oestradiol regulation of enterocyte extracellular Ca2+ influx.
Complexes containing activating transcription factor (ATF)/cAMP-responsive-element-binding protein (CREB) interact with the CCAAT/enhancer-binding protein (C/EBP)–ATF composite site to regulate Gadd153 expression during the stress response
Authors: FAWCETT Timothy W. , MARTINDALE Jennifer L. , GUYTON Kathryn Z. , HAI Tsonwin , HOLBROOK Nikki J.
Published: August 2015

Gadd153, also known as chop, encodes a member of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family and is transcriptionally activated by cellular stress signals. We recently demonstrated that arsenite treatment of rat pheochromocytoma PC12 cells results in the biphasic induction of Gadd153 mRNA expression, controlled in part through binding of C/EBPβ and two uncharacterized protein complexes to the C/EBP–ATF (activating transcription factor) composite site in the Gadd153 promoter. In this report, we identified components of these additional complexes as two ATF/CREB (cAMP-responsive-element-binding protein) transcription factors having differential binding activities dependent upon the time of arsenite exposure. During arsenite treatment of PC12 cells, we observed enhanced binding of ATF4 to the C/EBP–ATF site at 2 h as Gadd153 mRNA levels increased, and enhanced binding of ATF3 complexes at 6 h as Gadd153 expression declined. We further demonstrated that ATF4 activates, while ATF3 represses, Gadd153 promoter activity through the C/EBP–ATF site. ATF3 also repressed ATF4-mediated transactivation and arsenite-induced activation of the Gadd153 promoter. Our results suggest that numerous members of the ATF/CREB family are involved in the cellular stress response, and that regulation of stress-induced biphasic Gadd153 expression in PC12 cells involves the ordered, sequential binding of multiple transcription factor complexes to the C/EBP–ATF composite site.
Transcriptional regulation of the human asparagine synthetase gene by carbohydrate availability
Authors: BARBOSA-TESSMANN Ione P. , PINEDA Van Leung , NICK Harry S. , SCHUSTER Sheldon M. , KILBERG Michael S.
Published: August 2015

Transcription of the asparagine synthetase (AS) gene is induced by amino acid deprivation. The present data illustrate that this gene is also under transcriptional control by carbohydrate availability. Incubation of human HepG2 hepatoma cells in glucose-free medium resulted in an increased AS mRNA content, reaching a maximum of about 14-fold over control cells after approx. 12 h. Extracellular glucose caused the repression of the content of AS mRNA in a concentration-dependent manner, with a k1/2 (concentration causing a half-maximal repression) of 1 mM. Fructose, galactose, mannose, 2-deoxyglucose and xylitol were found to maintain the mRNA content of both AS and the glucose-regulated protein GRP78 in a state of repression, whereas 3-O-methylglucose did not. Incubation in either histidine-free or glucose-free medium also resulted in adaptive regulation of the AS gene in BNL-CL.2 mouse hepatocytes, rat C6 glioma cells and human MOLT4 lymphocytes, in addition to HepG2 cells. In contrast, the steady-state mRNA content of GRP78 was unaffected by amino acid availability. Transient transfection assays using a reporter gene construct documented that glucose deprivation increases AS gene transcription via elements within the proximal 3 kbp of the AS promoter. These results illustrate that human AS gene transcription is induced following glucose limitation of the cells.
Structure and chromosomal assignment of the human lectin-like oxidized low-density-lipoprotein receptor-1 (LOX-1) gene
Authors: AOYAMA Takuma , SAWAMURA Tatsuya , FURUTANI Yoshiyuki , MATSUOKA Rumiko , YOSHIDA Michihiro C. , FUJIWARA Hisayoshi , MASAKI Tomoh
Published: August 2015

We have reported the cDNA cloning of a modified low-density-lipoprotein (LDL) receptor, designated lectin-like oxidized LDL receptor-1 (LOX-1), which is postulated to be involved in endothelial dysfunction and the pathogenesis of atherosclerosis. Here, we determined the organization of the human LOX-1 gene, including the 5´-regulatory region. The 5´-regulatory region contained several potential cis-regulatory elements, such as GATA-2 binding element, c-ets-1 binding element, 12-O-tetradecanoylphorbol 13-acetate-responsive element and shear-stress-responsive elements, which may mediate the endothelium-specific and inducible expression of LOX-1. The major transcription-initiation site was found to be located 29 nucleotides downstream of the TATA box and 61 nucleotides upstream from the translation-initiation codon. The minor initiation site was found to be 5 bp downstream from the major site. Most of the promoter activity of the LOX-1 gene was ascribed to the region (-150 to -90) containing the GC and CAAT boxes. The coding sequence was divided into 6 exons by 5 introns. The first 3 exons corresponded to the different functional domains of the protein (cytoplasmic, transmembrane and neck domains), and the residual 3 exons encoded the carbohydrate-recognition domain similar to the case of other C-type lectin genes. The LOX-1 gene was a single-copy gene and assigned to the p12.3–p13.2 region of chromosome 12. Since the locus for a familial hypertension has been mapped to the overlapping region, LOX-1 might be the gene responsible for the hypertension.
Regions 301–303 and 333–339 in the catalytic domain of blood coagulation Factor IX are Factor VIII-interactive sites involved in stimulation of enzyme activity
Authors: KOLKMAN Joost A. , LENTING Peter J. , MERTENS Koen
Published: August 2015

The contribution of the Factor IX catalytic domain to Factor VIIIa binding has been evaluated by functional analysis of Factor IX variants with substitutions in α-helix region 333–339 and region 301–303. These regions were found to play a prominent role in Factor VIIIa-dependent stimulation of Factor X activation, but do not contribute to the high-affinity interaction with Factor VIIIa light chain. We propose that complex assembly between Factor IXa and Factor VIIIa involves multiple interactive sites that are located on different domains of these proteins.
Aspartate-27 and glutamate-473 are involved in catalysis by Zymomonas mobilis pyruvate decarboxylase
Authors: CHANG Alan K. , NIXON Peter F. , DUGGLEBY Ronald G.
Published: August 2015

Zymomonas mobilis pyruvate decarboxylase (EC 4.1.1.1) was subjected to site-directed mutagenesis at two acidic residues near the thiamin diphosphate cofactor in the active site. Asp-27 was changed to Glu or Asn, and Glu-473 was mutated to Asp (E473D) or Gln (E473Q). Each mutant protein was purified to near-homogeneity, and the kinetic and cofactor-binding properties were compared with those of the wild-type protein. Despite the very conservative nature of these alterations, all mutants had a very low, but measurable, specific activity ranging from 0.025% (E473Q) to 0.173% (E473D) of the wild type. With the exception of E473Q, the mutants showed small decreases in the affinity for thiamin diphosphate, and binding of the second cofactor (Mg2+) was also weakened somewhat. With E473Q, both cofactors seemed to be very tightly bound so that they were not removed by the treatment that was effective for the wild-type enzyme and other mutant forms. All mutants showed minor changes in the Km for substrate, but these alterations did not account for the low activities. These low specific activities, accompanied by little change in the Km for pyruvate, are consistent with a quantitative model of the catalytic cycle in which the main effect of the mutations is to slow the decarboxylation step with a minor change in the rate constant for pyruvate binding.
A new scorpion toxin (BmK-PL) stimulates Ca2+-release channel activity of the skeletal-muscle ryanodine receptor by an indirect mechanism
Authors: KUNIYASU Akihiko , KAWANO Seiko , HIRAYAMA Yoshiyuki , JI Yong-Hua , XU Ke , OHKURA Masamichi , FURUKAWA Ken-Ichi , OHIZUMI Yasushi , HIRAOKA Masayasu , NAKAYAMA Hitoshi
Published: August 2015

A peptide toxin isolated from the Chinese scorpion Buthus martensiKarsch (BmK-PL) stimulated Ca2+-release channel activity in both triad membranes and reconstituted ryanodine receptors partially purified from rabbit skeletal muscle. In [3H]ryanodine binding experiments, the toxin increased the affinity of ryanodine for the receptor, from a Kd of 24.3 nM to 2.9 nM, which is an enhancement similar to that seen with known receptor activators, such as ATP and high concentrations of KCl. In contrast, toxin enhancement was not observed with purified receptors, although intrinsic binding activity and stimulation by the conventional receptor activators were retained. In single channel recordings of Ca2+-release activity, the toxin increased the open channel probability (Po) from 0.019 to 0.043 (226% of control) in triad preparations. Further toxin enhancement of Po from 0.07 to 0.37 (529% of control) was observed using partially-purified receptors in the presence of ATP. When purified receptors were assayed in the presence of ATP, however, they showed a high value of Po (0.33) and no further increase was observed following application of the toxin. Results derived from two different experimental methods consistently suggest that a molecule(s) required for toxin-induced enhancement is absent from the purified receptor preparation. Western blot analysis of receptors prepared using three different protocols showed that triadin was missing from the purified receptor preparation. The scorpion toxin minimally enhanced Ca2+-release channel activity of cardiac preparations. From these results, we conclude that the toxin preferentially increases the activity of skeletal-muscle ryanodine receptors by an indirect mechanism, possibly binding to associated protein molecule(s). Triadin is a strong candidate for such a molecule.
Biophysical activity of an artificial surfactant containing an analogue of surfactant protein (SP)-C and native SP-B
Authors: PALMBLAD Marie , JOHANSSON Jan , ROBERTSON Bengt , CURSTEDT Tore
Published: August 2015

Natural surfactant preparations containing phospholipids and the hydrophobic surfactant proteins B and C (SP-B and SP-C) are effective in the treatment of respiratory distress syndrome in premature infants. The limited supply, and the risk of infectious agents and immunological reactions have promoted the evaluation of synthetic peptides in surfactant preparations. However, the folding of synthetic SP-C into an α-helix is inefficient and α-helical SP-C analogues with Val → Leu substitutions form oligomers. In order to circumvent these problems we have synthesized an SP-C analogue, named SP-C(LKS), which differs from SP-C mainly by the exchange of most of the Val residues in positions 16–28 with Leu residues to promote an α-helical conformation, and by the introduction of Lys residues at positions 17, 22 and 27 in order to locate positive charges around the helical circumference and thereby avoid self polymerization. CD spectroscopy showed a spectrum typical for α-helical peptides and SDS/PAGE disclosed a single band. The biophysical activity of artificial surfactant preparations containing SP-C(LKS) and phospholipids, with and without native SP-B, was measured using a Wilhelmy balance and a pulsating bubble surfactometer. SP-C(LKS) (3%, w/w) in a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidylglycerol/palmitic acid (68:22:9, by wt.) suspended in 150 mM NaCl, showed rapid spreading at the air-liquid interface and produced a surface tension of < 1 mN/m at minimum bubble size (γmin) and 42 mN/m at maximum bubble size (γmax) in the pulsating bubble surfactometer. The addition of 2% (w/w) SP-B to the preparation reduced the maximum surface tension to 33–35 mN/m, i.e. both γmin and γmax values were similar to those of natural surfactant preparations. Optimal in vitro characteristics were also obtained from a preparation containing SP-C(LKS), SP-B, DPPC and phosphatidylglycerol, i.e. when palmitic acid was omitted from the lipid mixture. SP-B containing surfactant preparations made up in Hepes buffer at pH 6.9, instead of in 150 mM NaCl, had similar biophysical activity provided that palmitic acid was omitted, but decreased activity in the presence of palmitic acid.
Monomeric (glycine-proline-hydroxyproline)10 repeat sequence is a partial agonist of the platelet collagen receptor glycoprotein VI
Authors: ASSELIN Judith , KNIGHT C. Graham , FARNDALE Richard W. , BARNES Michael J. , WATSON Steve P.
Published: August 2015

We have previously reported that a triple-helical, collagen-related peptide (CRP; also known as CRP-XL) containing a glycine-proline-hydroxyproline (GPP*) repeat motif and cross-linked through cysteine residues at its N-terminus and C-terminus is a powerful stimulus of platelet aggregation and secretion through the surface receptor glycoprotein VI (GPVI). The activation of platelets is associated with tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase C γ2 (PLCγ2). We now report that the non-cross-linked backbone of CRP, monomeric CRP (mCRP), stimulates the tyrosine phosphorylation of Syk and PLCγ2 in platelets and induces the weak secretion of [3H]5-hydroxytryptamine ([3H]5-HT) and aggregation. The action of mCRP does not seem to be due to spontaneous cross-linking, because alkylation of the cysteine residues leads to an increase in activity. The tripeptide backbone of CRP, GPP*10 (in which P* represents hydroxyproline) also stimulates platelet shape change and the weak tyrosine phosphorylation of Syk and PLCγ2, but is unable to induce aggregation or secretion. The monomeric peptides partly inhibit the release of [3H]5-HT by CRP, suggesting that they are partial agonists of the collagen receptor GPVI. These results demonstrate that GPP* present as a repeat motif is sufficient to activate the platelet collagen receptor GPVI but that the cross-linking of monomers brings about an increase in activity.
Cellulose as an inert matrix for presenting cytokines to target cells: production and properties of a stem cell factor–cellulose-binding domain fusion protein
Authors: DOHENY J. Greg , JERVIS Eric J. , GUARNA M. Marta , HUMPHRIES R. Keith , WARREN R. Antony J. , KILBURN Douglas G.
Published: August 2015

A chimaera of stem cell factor (SCF) and a cellulose-binding domain from the xylanase Cex (CBDCex) effectively immobilizes SCF on a cellulose surface. The fusion protein retains both the cytokine properties of SCF and the cellulose-binding characteristics of CBDCex. When adsorbed on cellulose, SCF–CBDCex is up to 7-fold more potent than soluble SCF–CBDCex and than native SCF at stimulating the proliferation of factor-dependent cell lines. When cells are incubated with cellulose-bound SCF–CBDCex, activated receptors and SCF–CBDCex co-localize on the cellulose matrix. The strong binding of SCF–CBDCex to the cellulose surface permits the effective and localized stimulation of target cells; this is potentially significant for long-term perfusion culturing of factor-dependent cells. It also permits the direct analysis of the effects of surface-bound cytokines on target cells.
Insulin-like growth factor (IGF-I) induces myotube hypertrophy associated with an increase in anaerobic glycolysis in a clonal skeletal-muscle cell model
Authors: SEMSARIAN Christopher , SUTRAVE Pramod , RICHMOND David R. , GRAHAM Robert M.
Published: August 2015

Insulin-like growth factor-I (IGF-I) is an important autocrine/paracrine mediator of skeletal-muscle growth and development. To develop a definitive cultured cell model of skeletal-muscle hypertrophy, C2C12 cells were stably transfected with IGF-I and clonal lines developed and evaluated. Quantitative morphometric analysis showed that IGF-I-transfected myotubes had a larger area (2381±60 µm2 versus 1429±39 µm2; P< 0.0001) and a greater maximum width (21.4±0.6 µm versus 13.9±0.3 µm; P< 0.0001) than control C2C12 myotubes, independent of the number of cell nuclei per myotube. IGF-I-transfected myotubes had higher levels of protein synthesis but no difference in DNA synthesis when compared with control myotubes, indicating the development of hypertrophy rather than hyperplasia. Both lactate dehydrogenase and alanine aminotransferase activities were increased (3- and 5-fold respectively), and total lactate levels were higher (2.3-fold) in IGF-I-transfected compared with control myotubes, indicating an increase in anaerobic glycolysis in the hypertrophied myotubes. However, expression of genes involved in skeletal-muscle growth or hypertrophy in vivo, e.g. myocyte nuclear factor and myostatin, was not altered in the IGF-I myotubes. Finally, myotube hypertrophy could also be induced by treatment of C2C12 cells with recombinant IGF-I or by growing C2C12 cells in conditioned media from IGF-I-transfected cells. This quantitative model should be uniquely useful for elucidating the molecular mechanisms of skeletal-muscle hypertrophy.
Down-regulation of types I, II and III inositol 1,4,5-trisphosphate receptors is mediated by the ubiquitin/proteasome pathway
Authors: OBERDORF Jon , WEBSTER Jack M. , ZHU Chang Cheng , LUO Su Ge , WOJCIKIEWICZ Richard J. H.
Published: August 2015

Activation of certain phosphoinositidase-C-linked cell-surface receptors is known to cause an acceleration of the proteolysis of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors and, thus, lead to Ins(1,4,5)P3-receptor down-regulation. In the current study we have sought to determine whether the ubiquitin/proteasome pathway is involved in this adaptive response. The data presented show (i) that activation of phosphoinositidase-C-linked receptors causes Ins(1,4,5)P3-receptor ubiquitination in a range of cell types (AR4-2J cells, INS-1 cells and rat cerebellar granule cells), (ii) that the Ins(1,4,5)P3-receptor down-regulation induced by activation of these receptors is blocked by proteasome inhibitors, (iii) that all known Ins(1,4,5)P3 receptors (types I, II and III) are substrates for ubiquitination, (iv) that ubiquitination occurs while Ins(1,4,5)P3 receptors are membrane-bound, (v) that Ins(1,4,5)P3-receptor ubiquitination and down-regulation are stimulated only by those agonists that elevate Ins(1,4,5)P3 concentration persistently, and (vi) that a portion of cellular Ins(1,4,5)P3 receptors (those that are not type-I-receptor-associated) can be resistant to ubiquitination and degradation. In total these data indicate that the ubiquitin/proteasome pathway mediates Ins(1,4,5)P3-receptor down-regulation and suggest that ubiquitination is stimulated by the binding of Ins(1,4,5)P3 to its receptor.
Molecular and enzymic properties of recombinant 1,2-α-mannosidase from Aspergillus saitoi overexpressed in Aspergillus oryzae cells
Authors: ICHISHIMA Eiji , TAYA Noriyuki , IKEGUCHI Masamichi , CHIBA Yasunori , NAKAMURA Motoyoshi , KAWABATA Choko , INOUE Takashi , TAKAHASHI Koji , MINETOKI Toshiki , OZEKI Kenji , KUMAGAI Chieko , GOMI Katsuya , YOSHIDA Takashi , NAKAJIMA Tasuku
Published: August 2015

For the construction of an overexpression system of the intracellular 1,2-α-mannosidase (EC 3.2.1.113) gene (msdS) from Aspergillus saitoi (now designated Aspergillus phoenicis), the N-terminal signal sequence of the gene was replaced with that of the aspergillopepsin I (EC 3.4.23.18) gene (apnS) signal, one of the same strains as described previously. Then the fused 1,2-α-mannosidase gene (f-msdS) was inserted into the NotI site between P-No8142 and T-agdA in the plasmid pNAN 8142 (9.5 kbp) and thus the Aspergillus oryzae expression plasmid pNAN-AM1 (11.2 kbp) was constructed. The fused f-msdS gene has been overexpressed in a transformant A. oryzae niaD AM1 cell. The recombinant enzyme expressed in A. oryzae cells was purified to homogeneity in two steps. The system is capable of making as much as about 320 mg of the enzyme/litre of culture. The recombinant enzyme has activity with methyl-2-O-α-ᴅ-mannopyranosyl α-ᴅ-mannopyranoside at pH 5.0, while no activity was determined with methyl-3-O-α-ᴅ-mannopyranosyl α-ᴅ-mannopyranoside or methyl-6-O-α-ᴅ-mannopyranosyl α-ᴅ-mannopyranoside. The substrate specificity of the enzyme was analysed by using pyridylaminated (PA)-oligomannose-type sugar chains, Man9-6(GlcNAc)2-PA (Man is mannose; GlcNAc is N-acetylglucosamine). The enzyme hydrolysed Man8GlcNAc2-PA (type ‘M8A’) fastest, and ‘M6C’ {Manα1-3[Manα1-2Manα1-3(Manα1-6)Manα1-6]Manβ1-4GlcNAcβ1-4GlcNAc-PA} slowest, among the PA-sugar chains. Molecular-mass values of the enzyme were determined to be 63 kDa by SDS/PAGE and 65 kDa by gel filtration on Superose 12 respectively. The pI value of the enzyme was 4.6. The N-terminal amino acid sequence of the enzyme was GSTQSRADAIKAAFSHAWDGYLQY, and sequence analysis indicated that the signal peptide from apnS gene was removed. The molar absorption coefficient, ϵ, at 280 nm was determined as 91539 M-1·cm-1. Contents of the secondary structure (α-helix, β-structure and the remainder of the enzyme) by far-UV CD determination were about 55, 38 and 7% respectively. The melting temperature, Tm, of the enzyme was 71 °C by differential scanning calorimetry. The calorimetric enthalpy, ∆Hcal, of the enzyme was calculated as 13.3 kJ·kg of protein-1. Determination of 1 g-atom of Ca2+/mol of enzyme was performed by atomic-absorption spectrophotometry.
Identification of the amine–polyamine–choline transporter superfamily ‘consensus amphipathic region’ as the target for inactivation of the Escherichia coli GABA transporter GabP by thiol modification reagents. Role of Cys-300 in restoring thiol sensitivity to GabP lacking Cys
Authors: HU Liaoyuan A. , KING Steven C.
Published: August 2015

The Escherichia coli γ-aminobutyric acid transporter GabP (gab permease) contains a functionally significant cysteine residue (Cys-300) within its consensus amphipathic region (CAR), a putative channel-forming structure that extends out of transmembrane helix 8 and into the adjoining cytoplasmic loop 8-9 of transporters from the amine-polyamine-choline (APC) superfamily. Here we show that of the five cysteine residues (positions 158, 251, 291, 300 and 443) in the E. coli GabP, Cys-300 is the one that renders the transport activity sensitive to inhibition by thiol modification reagents: whereas substituting Ala for Cys-300 mimics the inhibitory effect of thiol modification, substituting Ala at position 158, 251, 291 or 443 preserves robust transport activity and confers no resistance to thiol inactivation; and whereas the robustly active Cys-300 single-Cys mutant is fully sensitive to thiol modification, other single-Cys mutants (Cys at 158, 251, 291 or 443) exhibit kinetically compromised transport activities that resist further chemical inactivation by thiol reagents. The present study reveals additionally that Cys-300 exhibits (1) sensitivity to hydrophobic thiol reagents, (2) general resistance to bulky (fluorescein 5-maleimide) and/or charged {2-sulphonatoethyl methanethiosulphonate or [2-(trimethylammonium)ethyl] methanethiosulphonate} thiol reagents and (3) a peculiar sensitivity to p-chloromercuribenzenesulphonate (PCMBS). The accessibility of PCMBS to Cys-300 (located midway through the lipid bilayer) might be related to the structural similarity that it shares with guvacine (1,2,3,6-tetrahydro-3-pyridinecarboxylic acid), a transported GabP substrate. These structural requirements for thiol sensitivity provide the first chemical evidence consistent with channel-like access to the polar surface of the CAR, a physical configuration that might provide a basis for understanding how this region impacts the function of APC transporters generally [Closs, Lyons, Kelly and Cunningham (1993) J. Biol. Chem. 268, 20796-20800] and the gab permease particularly [Hu and King (1998) Biochem. J. 300, 771-776].
Structure, function and regulation of pyruvate carboxylase
Authors: JITRAPAKDEE Sarawut , WALLACE John C.
Published: August 2015

Pyruvate carboxylase (PC; EC 6.4.1.1), a member of the biotin-dependent enzyme family, catalyses the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC has been found in a wide variety of prokaryotes and eukaryotes. In mammals, PC plays a crucial role in gluconeogenesis and lipogenesis, in the biosynthesis of neurotransmitter substances, and in glucose-induced insulin secretion by pancreatic islets. The reaction catalysed by PC and the physical properties of the enzyme have been studied extensively. Although no high-resolution three-dimensional structure has yet been determined by X-ray crystallography, structural studies of PC have been conducted by electron microscopy, by limited proteolysis, and by cloning and sequencing of genes and cDNA encoding the enzyme. Most well characterized forms of active PC consist of four identical subunits arranged in a tetrahedron-like structure. Each subunit contains three functional domains: the biotin carboxylation domain, the transcarboxylation domain and the biotin carboxyl carrier domain. Different physiological conditions, including diabetes, hyperthyroidism, genetic obesity and postnatal development, increase the level of PC expression through transcriptional and translational mechanisms, whereas insulin inhibits PC expression. Glucocorticoids, glucagon and catecholamines cause an increase in PC activity or in the rate of pyruvate carboxylation in the short term. Molecular defects of PC in humans have recently been associated with four point mutations within the structural region of the PC gene, namely Val145 → Ala, Arg451 → Cys, Ala610 → Thr and Met743 → Thr.
Angiostatin inhibits endothelial and melanoma cellular invasion by blocking matrix-enhanced plasminogen activation
Authors: STACK M. Sharon , GATELY Stephen , BAFETTI Lisa M. , ENGHILD Jan J. , SOFF Gerald A.
Published: August 2015

Angiostatin, a kringle-containing fragment of plasminogen, is a potent inhibitor of angiogenesis. The mechanism(s) responsible for the anti-angiogenic properties of angiostatin are unknown. We now report that human angiostatin blocks plasmin(ogen)-enhanced in vitro invasion of tissue plasminogen activator (t-PA)-producing endothelial and melanoma cells. Kinetic analyses demonstrated that angiostatin functions as a non-competitive inhibitor of extracellular-matrix (ECM)-enhanced, t-PA-catalysed plasminogen activation, with a Ki of 0.9±0.03 μM. This mechanism suggests that t-PA has a binding site for the inhibitor angiostatin, as well as for its substrate plasminogen that, when occupied, prevents ternary complex formation between t-PA, plasminogen and matrix protein. Direct binding experiments confirmed that angiostatin bound to t-PA with an apparent Kd [Kd(app)] of 6.7±0.7 nM, but did not bind with high affinity to ECM proteins. Together, these data suggest that angiostatin in the cellular micro-environment can inhibit matrix-enhanced plasminogen activation, resulting in reduced invasive activity, and suggest a biochemical mechanism whereby angiostatin-mediated regulation of plasmin formation could influence cellular migration and invasion.
Structure, alternative splicing and chromosomal localization of the cystatin-related epididymal spermatogenic gene
Authors: CORNWALL Gail A. , HSIA Nelson , SUTTON H. Gayle
Published: August 2015

The cystatin superfamily of cysteine protease inhibitors consists of three major families, including the stefins, cystatins and kininogens. However, the recent identification of several genes that possess sequence similarity with the cystatins but have different gene or protein structures indicates that several new cystatin families or subgroups of families might exist. We previously identified the cystatin-related epididymal spermatogenic (Cres) gene, which is related to the family 2 cystatins but exhibits highly tissue-specific expression in the reproductive tract. In the studies presented here, an analysis of gene structure as well as chromosomal mapping studies suggest that the Cres gene might represent a new subgroup within the family 2 cystatins. Although the Cres gene possesses an additional exon encoding 5ʹ untranslated sequences, its coding exons are similar in size to the three coding exons of the cystatin family 2 genes, and the Cres exon/intron splice junctions occur in identical locations as in the cystatin C gene. Furthermore, chromosomal mapping studies show that the Cres gene co-segregates with the cystatin C gene on mouse chromosome 2. Similar to the cystatin family 2 proteins, the Cres protein possesses the type A and B disulphide loops that are necessary for cystatin folding. Interestingly, Cres protein also possesses half of a type C disulphide loop. Although probably related to the cystatin genes, the Cres gene is distinct in that its promoter contains consensus motifs typical of regulated genes. Finally, reverse transcriptase-mediated PCR studies and the identification of new Cres cDNA clones indicate that the Cres mRNA is alternatively spliced, resulting in two Cres
Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease
Authors: CHAN Victor J. , SELZER Paul M. , McKERROW James H. , SAKANARI Judy A.
Published: August 2015

The mature form of the cathepsin B-like protease of Leishmania major (LmajcatB) is a 243 amino acid protein belonging to the papain family of cysteine proteases and is 54% identical to human-liver cathepsin B. Despite the high identity and structural similarity with cathepsin B, LmajcatB does not readily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg-Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrolyse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzymes. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly234 at the S2 subsite of LmajcatB, which forms a larger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastoris yeast expression system. The quality of the recombinant enzyme was confirmed by kinetic characterization, N-terminal sequencing, and Western blot analysis. Alteration of Gly234 to Glu, which is found at the corresponding site in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) activity toward Z-Arg-Arg-AMC 8-fold over the wild-type recombinant enzyme (kcat/Km = 3740±413 M-1·s-1 versus 472±72.4 M-1·s-1). The results of inhibition assays of rLmajcatB with an inhibitor of cathepsin L enzymes, K11002 (morpholine urea-Phe-homoPhe-vinylsulphonylphenyl, kinact/Ki = 208200±36000 M-1·s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-ʟ-isoleucyl-L-proline, kinact/Ki = 199200±32900 M-1·s-1], support the findings that this protozoan protease has the P2 specificity of cathepsin L-like enzymes while retaining structural homology to mammalian cathepsin B.
Inhibition of ubiquitin-proteasome pathway activates a caspase-3-like protease and induces Bcl-2 cleavage in human M-07e leukaemic cells
Authors: ZHANG Xue-min , LIN Hong , CHEN Catheryne , CHEN Ben D.-M.
Published: August 2015

The ubiquitin-proteasome pathway is the principal mechanism for the degradation of short-lived proteins in eukaryotic cells. Here we examine the possibility that ubiquitin-proteasome is involved in regulating the levels of Bcl-2, which is abundantly expressed in M-07e cells, a granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent human leukaemic cell line. Apoptosis in M-07e cells, induced by GM-CSF withdrawal, was associated with a gradual cleavage of Bcl-2 into a 22 kDa fragment. Treatment of M-07e cells with benzyloxycarbonyl-Leu-Leu-L-leucinal (Z-LLL-CHO; MG-132), a reversible ubiquitin-proteasome inhibitor, markedly accelerated the cleavage of Bcl-2 and promoted cell death through the apoptotic pathway. The cleavage of Bcl-2 was inhibited by a caspase-3 (CPP32)-specific inhibitor [acetyl-Asp-Glu-Val-Asp-CHO (DEVD-CHO)] but not caspase 1 inhibitor (acetyl-Tyr-Val-Ala-Asp-CHO), suggesting that Bcl-2 is a proteolytic substrate of a caspase-3-like protease activated during apoptosis. The simultaneous addition of recombinant human GM-CSF (rhGM-CSF) to M-07e cultures delayed the activation of caspase 3 and Bcl-2 cleavage triggered by Z-LLL-CHO, suggesting that the activation of the GM-CSF signalling pathway can partly overcome the apoptotic effect induced by Z-LLL-CHO. Apoptosis induced by inhibition of the proteasome pathway was verified in studies with lactacystin, a highly specific and irreversible proteasome inhibitor. Lactacystin-induced apoptosis in M-07e cells was remarkably similar to that induced by Z-LLL-CHO, which included caspase 3 activation, cleavage of Bcl-2 into a 22 kDa fragment and, ultimately, cell death. These results showed that inhibition of the ubiquitin-proteasome pathways can lead to the activation of a DEVD-CHO-sensitive caspase and induces Bcl-2 cleavage, which might have a role in mediating apoptosis in M-07e cells.
The activity of the activation function 2 of the human hepatocyte nuclear factor 4 (HNF-4α) is differently modulated by F domains from various origins
Authors: SUAUD Laurence , FORMSTECHER Pierre , LAINE Bernard
Published: August 2015

Hepatocyte nuclear factor 4 (HNF-4) is a member of the nuclear hormone-receptor superfamily, which plays an important role in the regulation of several genes involved in numerous metabolic pathways. HNF-4 contains a DNA-binding domain located in domain C and two activation-function domains, designated AF-1 and AF-2, located in domains A/B and E, respectively. The seven isoforms of human HNF-4, termed α1-α6 and γ, differ mainly by their A/B and F domains. The high sequence variability of the F domain led us to investigate whether this domain modulates the transcriptional activity of HNF-4. Using constructs having the same core receptor and different F domains, we observed that the F domains of HNF-4 modulate the transactivating activity of the full-length HNF-4. A more precise analysis using HNF-4α AF-2 fused to GAL4 protein and various F domains demonstrated that F domains of isoforms α3 and γ exhibited inhibitory effects on the activation function AF-2 but that their inhibition behaviours were weaker than that of HNF-4α2 F domain, which has been reported previously. The presence of domain F results in a decreased interaction with the co-activator glucocorticoid receptor-interacting protein 1. For a given F domain, the modulating effects on the full-length HNF-4 as well as on the AF-2 depended on the target promoters. Our results suggest that the presence of domain F results in conformation changes in HNF-4 AF-2 or in its spatial environment, which probably modify the interaction of the AF-2 activation domain with co-factors and transcription factors bound to cis-elements of the target promoters.
Fibrillin degradation by matrix metalloproteinases: implications for connective tissue remodelling
Authors: ASHWORTH Jane L. , MURPHY Gillian , ROCK Matthew J. , SHERRATT Michael J. , SHAPIRO Stephen D. , SHUTTLEWORTH C. Adrian , KIELTY Cay M.
Published: August 2015

Fibrillin is the principal structural component of the 10-12 nm diameter elastic microfibrils of the extracellular matrix. We have previously shown that both fibrillin molecules and assembled microfibrils are susceptible to degradation by serine proteases. In this study, we have investigated the potential catabolic effects of six matrix metalloproteinases (MMP-2, MMP-3, MMP-9, MMP-12, MMP-13 and MMP-14) on fibrillin molecules and on intact fibrillin-rich microfibrils isolated from ciliary zonules. Using newly synthesized recombinant fibrillin molecules, major cleavage sites within fibrillin-1 were identified. In particular, the six different MMPs generated a major degradation product of ~ 45 kDa from the N-terminal region of the molecule, whereas treatment of truncated, unprocessed and furin-processed C-termini also generated large degradation products. Introduction of a single ectopia lentis-causing amino acid substitution (E2447K; one-letter symbols for amino acids) in a calcium-binding epidermal growth factor-like domain, predicted to disrupt calcium binding, markedly altered the pattern of C-terminal fibrillin-1 degradation. However, the fragmentation pattern of a mutant fibrillin-1 with a comparable E → K substitution in an upstream calcium-binding epidermal growth factor-like domain was indistinguishable from wild-type molecules. Ultrastructural examination highlighted that fibrillin-rich microfibrils isolated from ciliary zonules were grossly disrupted by MMPs. This is the first demonstration that fibrillin molecules and fibrillin-rich microfibrils are degraded by MMPs and that certain amino acid substitutions change the fragmentation patterns. These studies have important implications for physiological and pathological fibrillin catabolism and for loss of connective tissue elasticity in ageing and disease.
Inhibition of interleukin-6 promoter activity by the 24 kDa isoform of fibroblast growth factor-2 in HeLa cells
Authors: DELRIEU Isabelle , FAYE Jean-Charles , BAYARD Francis , MARET Arlette
Published: August 2015

The expression of the interleukin (IL-6) gene can be regulated by various activating or inhibitory stimuli. This modulation involves several regulatory binding sites on the IL-6 promoter, and appears to be in general cell-specific. We have previously described that the nuclear 24 kDa isoform of fibroblast growth factor-2 (FGF-2) is able to increase IL-6 gene expression in NIH-3T3 cells. The transduction pathway involved was shown to be distinct from the extracellular mode of action of the smallest 18 kDa FGF-2 isoform. In the present study, we show that 24 kDa FGF-2-encoding vectors transfected into HeLa cells inhibit various co-transfected constructs incorporating the promoter element of the IL-6 gene and either the luciferase or the chloramphenicol acetyltransferase units. This down-regulation occurs dose-dependently with the 24 kDa FGF-2, is IL-6-promoter-specific, and does not involve an autocrine loop of the growth factor, since exogenously added FGF-2 fails to modulate the IL-6 promoter activity. Furthermore, 24 kDa FGF-2 inhibits the activity of both the co-transfected deletion mutants IL-6(-224) and IL-6(-158), and the point-mutated IL-6 promoter constructs in which the activating protein-1, nuclear factor (NF)-IL-6 and NF-ĸB elements are disrupted. We identify a responsive region to 24 kDa FGF-2 between positions -158 and -109 on the IL-6 promoter, which notably contains a retinoblastoma control element.
Mode of regulation of the extracellular signal-regulated kinases in the pancreatic β-cell line MIN6 and their implication in the regulation of insulin gene transcription
Authors: BENES Cyril , POITOUT Vincent , MARIE Jean-Claude , MARTIN-PEREZ Jorge , ROISIN Marie-Paule , FAGARD Remi
Published: August 2015

Physiological concentrations of glucose that lead to Ca2+ entry and insulin secretion activate extracellular signal-regulated protein kinases (ERK1 and ERK2) in the MIN6 pancreatic β-cell line. Here we show that this activation is inhibited by the down-regulation of protein kinase C (PKC) and by genistein, an inhibitor of protein tyrosine kinases. In contrast with results obtained in other cell types, neither the epidermal growth factor activity nor the Src family protein tyrosine kinases seem to be involved in the Ca2+-dependent activation of ERKs. inhibition of tyrosine phosphatases by vanadate leads to the activation of ERKs. As observed in the response to glucose, this activation is dependent on Ca2+ entry through L-type voltage-dependent Ca2+ channels. Thus the activation of ERKs in response to glucose depends on PKC and possibly on a tyrosine kinase/tyrosine phosphatase couple. To define the role of ERK activation by glucose we studied the regulation of transcription of the insulin gene. We found that this transcription is regulated in the MIN6 cells in the same range of glucose concentration as in primary islets, and that specific inhibition of mitogen-activated protein kinase kinase, the direct activator of ERK, impaired the response of the insulin gene to glucose. This was observed by analysis of the transfected rat insulin I gene promoter activity and a Northern blot of endogenous insulin mRNA.
Enhancing activity and phospholipase A2 activity: two independent activities present in the enhancing factor molecule
Authors: KADAM Shilpa , MULHERKAR Rita
Published: August 2015

Enhancing factor (EF), a molecule that increases the binding of epidermal growth factor (EGF) to A431 cells, was first isolated in our laboratory from mouse intestines, and subsequently shown to be a secretory form of phospholipase A2 (PLA2) [Mulherkar, Rao, Wagle, Patki and Deo (1993) Biochem. Biophys. Res. Commun. 195, 1254-1263]. We had proposed earlier that EF increases the binding of EGF by first binding to its own cell-surface receptor [identified as a 100 kDa molecule; Mulherkar and Deo (1986) J. Cell. Physiol. 127, 183-188], and then by creating a binding site for EGF. However, due to its PLA2 activity, there was a possibility that EF, by its phospholipase activity could be unmasking cryptic EGF receptors on the cell surface, thereby increasing the number of binding sites for EGF. To test whether enhancing activity and phospholipase activity are independent of each other, a series of mutations were created using the full-length EF cDNA as a template, expressed in 293 cells and the mutant recombinant proteins checked for EF as well as PLA2 activities. Our studies have shown that one of the mutant EF proteins, lacking PLA2 activity, retains EF activity. This demonstrates unambiguously that EF and PLA2 activities are two independent activities in the same molecule. Mutation in the Ca2+-binding loop resulted in loss of EF activity, thereby demonstrating that EF activity is Ca2+-dependent. The N-terminal region of the EF molecule appears to be crucial for the enhancing activity.
Inhibition of fibril formation in β-amyloid peptide by a novel series of benzofurans
Authors: HOWLETT David R. , PERRY Amanda E. , GODFREY Fiona , SWATTON Jane E. , JENNINGS Kevin H. , SPITZFADEN Claus , WADSWORTH Harry , WOOD Stephen J. , MARKWELL Roger E.
Published: August 2015

A series of benzofuran derivatives have been identified as inhibitors of fibril formation in the β-amyloid peptide. The activity of these compounds has been assessed by a novel fibril-formation-specific immunoassay and for their effects on the production of a biologically active fibril product. The inhibition afforded by the compounds seems to be associated with their binding to β-amyloid, as identified by scintillation proximity binding assay. Binding assays and NMR studies also indicate that the inhibition is associated with self-aggregation of the compounds. There is a close correlation between the activity of the benzofurans as inhibitors of fibril formation and their ability to bind to β-amyloid. Non-benzofuran inhibitors of the fibril formation process do not seem to bind to the same site on the β-amyloid molecule as the benzofurans. Thus a specific recognition site might exist for benzofurans on β-amyloid, binding to which seems to interfere with the ability of the peptide to form fibrils.
Structural elements within the methylation loop (residues 112–117) and EF hands III and IV of calmodulin are required for Lys115 trimethylation
Authors: COBB Jennifer A. , HAN Chang-Hoon , WILLS David M. , ROBERTS Daniel M.
Published: August 2015

Calmodulin is trimethylated by a specific methyltransferase on Lys115, a residue located in a six amino acid loop (LGEKLT) between EF hands III and IV. To investigate the structural requirements for methylation, domain exchange mutants as well as single point mutations of conserved methylation loop residues (E114A, Glu114 → Ala; L116T, Leu116 → Thr) were generated. E114A and L116T activated cyclic nucleotide phosphodiesterase (PDE) and NAD+ kinase (NADK) similar to wild-type calmodulin, but lost their ability to be methylated. Domain exchange mutants in which EF hand III or IV was replaced by EF hand I or II respectively (CaM1214 and CaM1232 respectively) showed a modest effect on PDE and NADK activation (50 to 100% of wild-type), but calmodulin methylation was abolished. A third domain exchange mutant, CaMEKL, has the methylation loop sequence placed at a symmetrical position between EF hands I and II in the N-terminal lobe [residues QNP(41-43) replaced by EKL]. CaMEKL activated PDE normally, but did not activate NADK. However, CaMEKL retained the ability to bind to NADK and inhibited activation by wild-type calmodulin. Site-directed mutagenesis of single residues showed that Gln41 and Pro43 substitutions had the strongest effect on NADK activation. Additionally, CaMEKL was not methylated, suggesting that the introduction of the methylation loop between EF hands I and II is not adequate for methyltransferase recognition. Overall the data indicate that residues in the methylation loop are essential but not sufficient for methyltransferase recognition, and that additional residues unique to EF hands III and IV are required. Secondly, the QNP sequence in the loop between EF hands I and II is necessary for NADK activation.
Expression of the rat liver carnitine palmitoyltransferase I (CPT-Iα) gene is regulated by Sp1 and nuclear factor Y: chromosomal localization and promoter characterization
Authors: STEFFEN Michelle L. , HARRISON Wilbur R. , ELDER Frederick F. B. , COOK George A. , PARK Edwards A.
Published: August 2015

Carnitine palmitoyltransferase (CPT)-I catalyses the transfer of long-chain fatty acids from CoA to carnitine for translocation across the mitochondrial inner membrane. Expression of the ‘liver’ isoform of the CPT-I gene (CPT-Iα) is subject to developmental, hormonal and tissue-specific regulation. To understand the basis for control of CPT-Iα gene expression, we have characterized the proximal promoter of the CPT-Iα gene. Here, we report the sequence of 6839 base pairs of the promoter and the localization of the rat CPT-Iα gene to region q43 on chromosome 1. Our studies show that the first 200 base pairs of the promoter are sufficient to drive transcription of the CPT-Iα gene. Within this region are two sites that bind both Sp1 and Sp3 transcription factors. In addition, nuclear factor Y (NF-Y) binds the proximal promoter. Mutation at the Sp1 or NF-Y sites severely decreases transcription from the CPT-Iα promoter. Other protein binding sites were identified within the first 200 base pairs of the promoter by DNase I footprinting, and these elements contribute to CPT-Iα gene expression. Our studies demonstrate that CPT-Iα is a TATA-less gene which utilizes NF-Y and Sp proteins to drive basal expression.