bluuebunny/crossref_metadata_2025 huggingface dataset:
Glypican-3 is a binding protein on the HepG2 cell surface for tissue factor pathway inhibitor
Authors: MAST E. Alan , HIGUCHI A. Darryl , HUANG Zhong-Fu , WARSHAWSKY Ilka , SCHWARTZ L. Alan , BROZE George J.
Published: August 2015

Tissue factor pathway inhibitor (TFPI) is a primary regulator of the initiation of blood coagulation. TFPI is internalized and degraded by HepG2 cells through the low-density-lipoprotein receptor-related protein (LRP) but also binds another molecule present on the cell surface at approx. 10-fold the abundance of LRP [Warshawsky, Broze and Schwartz (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6664-6668]. When HepG2 cells are washed with heparin or dextran sulphate, a substance that binds TFPI is removed from the cell surface and can be detected in a slot-blot assay. Preincubation with trypsin destroys the reactivity of the TFPI-binding component in the slot-blot assay, suggesting that it is a protein. In addition, when the sulphation of glycosaminoglycans (GAGs) is prevented by growing the HepG2 cells in the presence of 30 mM sodium chlorate, TFPI binding is unaffected, whereas the binding of bovine lipoprotein lipase, a protein known to associate with cell-surface GAGs, falls to 50% of control levels. Dextran sulphate washes of HepG2 cells grown in sodium chlorate have an equal reactivity in slot-blot experiments to that of non-treated cells, suggesting that GAGs are not totally responsible for the binding activity observed. By using the slot blot to follow binding activity and conventional protein purification techniques, a protein species that migrates at 40 kDa after reduction was identified in the HepG2 cell wash. The binding of this protein to TFPI was confirmed with immobilized TFPI. Amino acid sequence analysis identified this protein species as a proteolytic fragment of glypican-3 (also called OCI-5), a member of the glypican family of glycosylphosphatidylinositol-anchored proteoglycans.
Characterization of chicken-liver glutathione S-transferase (GST) A1-1 and A2-2 isoenzymes and their site-directed mutants heterologously expressed in Escherichia coli: identification of Lys-15 and Ser-208 on cGSTA1-1 as residues interacting with ethacrynic acid
Authors: LIU Li-Fan , LIAW Yen-Chywan , TAM F. Ming
Published: August 2015

Escherichia coli-expressed chicken-liver glutathione S-transferase, cGSTA1-1, displays high ethacrynic acid (EA)-conjugating activity. Molecular modelling of cGSTA1-1 with EA in the substrate binding site reveals that the side chain of Phe-111 protrudes into the substrate binding site and possibly interacts with EA. Replacement of Phe-111 with alanine resulted in an enzyme (F111A mutant) with a 4.5-fold increase in EA-conjugating activity (9.2 mmol/min per mg), and an incremental Gibbs free energy (ΔΔG) of 4.0 kJ/mol lower than that of the wild-type cGSTA1-1. Two other amino acid residues that possibly interact with EA are Ser-208 and Lys-15. Substitution of Ser-208 with methionine generated a cGSTA1-1(F111AS208M) double mutant that has low EA-conjugating activity (2.0 mmol/min per mg) and an incremental Gibbs free energy of +3.9 kJ/mol greater than the cGSTA1-1(F111A) single mutant. The cGSTA1-1(F111A) mutant, with an additional Lys-15-to-leucine substitution, lost 90% of the EA-conjugating activity (0.55 mmol/min per mg). The Km values of the cGSTA1-1(F111A) and cGSTA1-1(F111AK15L) mutants for EA are nearly identical. The wild-type cGSTA2-2 isoenzyme has a low EA-conjugating activity (0.56 mmol/min per mg). The kcat of this reaction can be increased 2.5-fold by substituting Arg-15 and Glu-104 with lysine and glycine respectively. The KmEA of the cGSTA2-2(R15KE104G) double mutant is nearly identical with that of the wild-type enzyme. Another double mutant, cGSTA2-2(E104GL208S), has a KmEA that is 3.3-fold lower and a kcat that is 1.8-fold higher than that of the wild-type enzyme. These results, taken together, illustrate the interactions of Lys-15 and Ser-208 on cGSTA1-1 with EA.
α1-Adrenoceptor-mediated phosphorylation of myosin in rat-tail arterial smooth muscle
Authors: MITA Mitsuo , WALSH P. Michael
Published: August 2015

The mechanism of α1-adrenoceptor-mediated contraction was investigated in helical strips of the rat-tail artery. Muscle strips with the endothelium removed contracted in response to the α1-adrenoceptor agonist cirazoline, with half-maximal contraction at 0.23 μM. The contractile response to a submaximal concentration of cirazoline (0.3 μM) was biphasic, with a rapid phasic component peaking at approx. 30 s, followed by sustained tonic contraction. Phosphorylation of the 20 kDa light chain of myosin (LC20) in response to 0.3 μM cirazoline was also biphasic and closely matched the time-course of contraction. Resting LC20 phosphorylation levels were 0.22±0.06 mol of Pi/mol of LC20 (n = 3) and reached a maximum of 0.58±0.08 mol of Pi/mol of LC20 (n = 3). Phosphopeptide mapping and phosphoamino acid analysis revealed that LC20 phosphorylation occurred exclusively at serine-19. The sustained phase of contraction was eliminated by removal of extracellular Ca2+ and the phasic response was eliminated by depletion of endogenous Ca2+ stores. Both phases of the contractile response were restored by re-addition of Ca2+ to the bathing medium. LC20 phosphorylation and both phases of the contractile response to 0.3 μM cirazoline were inhibited by the myosin light-chain kinase inhibitor ML-9 (30 μM). Resting LC20 phosphorylation, however, was unaffected by ML-9. Finally, both phasic and tonic responses to 0.3 μM cirazoline were partially inhibited by chloroethylclonidine (50 μM), suggesting the involvement of both α1A and α1B adrenoceptors in these contractile responses.
ATP stimulates lysosomal sulphate transport at neutral pH: evidence for phosphorylation of the lysosomal sulphate carrier
Authors: CHOU Hsu-Fang , PASSAGE Merry , JONAS J. Adam
Published: August 2015

ATP markedly stimulated sulphate uptake by rat liver lysosomes that had been treated with N-ethylmaleimide to block the effects of the lysosomal proton-translocating ATPase (H+-ATPase). Maximal stimulation required millimolar concentrations of ATP and neutral buffer pH. ATP-stimulated transport exhibited saturation kinetics with a Km of 175 μM, identical with the Km for lysosomal sulphate uptake at pH 5.0, a process that does not require ATP. The requirement for ATP was specific: other nucleotides such as AMP, ADP, CTP, GTP, ITP and UTP failed to stimulate transport. Adenosine 5ʹ-[β,γ-imido]triphosphate, the non-hydrolysable analogue of ATP, also failed to stimulate sulphate uptake, suggesting a requirement for ATP hydrolysis. Lysosomal pH, membrane potential and glucose transport were unchanged by the presence of ATP under the experimental conditions, consistent with a direct effect of ATP on the sulphate transporter. Exposure of lysosomes to protein kinase A and protein kinase C inhibitors did not alter the stimulation of sulphate transport by ATP. The lysosomal sulphate transport protein might be subject to regulation by a phosphorylation pathway that is not dependent on protein kinase A or protein kinase C.
Kinetic mechanism of the glycogen-phosphorylase-catalysed reaction in the direction of glycogen synthesis: co-operative interactions of AMP and glucose 1-phosphate during catalysis
Authors: SERGIENKO A. Eduard , SRIVASTAVA D. K.
Published: August 2015

We employed our newly developed, continuous, spectrophotometric method [Sergienko and Srivastava (1994) Anal. Biochem. 221, 348-355] for measuring the glycogen-phosphorylase-catalysed reaction in the direction of glycogen synthesis, utilizing varied concentrations of AMP (2-400 μM) and glucose 1- phosphate (G1P; 4 μM to 41 mM). The experimental data revealed that the enzyme catalysis exhibits sigmoidal dependence on both AMP and G1P concentrations, with Hill coefficient and EC50 values (mutually) affected by the concentrations of the above substrates. A detailed kinetic analysis of the substrate-dependent activation, as well as glucose-inhibition data, lead us to propose the following mechanistic features of the glycogen-phosphorylase-catalysed reaction. (1) The enzyme exhibits catalytic activity when two molecules of AMP and two molecules of G1P are bound to the dimeric unit. (2) The binding of one molecule of glucose (the competitive inhibitor of G1P) per dimeric unit results into a complete loss of the enzyme activity. (3) There is no restriction of binding of AMP or G1P when one of the dimeric subunits is already bound with the other ligand. For example, one or two G1P molecules can bind to the enzyme dimer when zero, one or two molecules of AMP are already bound. The magnitudes of rate and equilibrium constants for the glycogen-phosphorylase-catalysed reaction, derived from analyses of the experimental data in the light of a few selected minimal models, are presented.
Mutagenesis of residue 157 in the active site of human glyoxalase I
Authors: RIDDERSTRÖM Marianne , CAMERON D. Alexander , JONES T. Alwyn , MANNERVIK Bengt
Published: August 2015

Met-157 in the active site of human glyoxalase I was changed by site-directed mutagenesis into alanine, glutamine or histidine in order to evaluate its possible role in catalysis. The glyoxalase I mutants were expressed in Escherichia coli and purified on an S-hexylglutathione affinity gel. The physicochemical properties of the mutant proteins were similar to those of the wild-type enzyme. The glutamine mutant exhibited the same high specific activity as wild-type glyoxalase I, whereas the alanine and histidine mutants had approx. 20% of wild-type activity. The kcat/Km values of the mutant glyoxalase I determined with the hemithioacetal adduct of glutathione and methylglyoxal were reduced to between 10 and 40% of the wild-type value. This reduction was due to lower kcat values for the alanine and histidine mutants and a twofold increase in the Km value for the glutamine mutant. With the hemithioacetal of glutathione and phenylglyoxal, the kinetic parameters of the mutants were also of the same magnitude as those of wild-type glyoxalase I. Studies with the competitive inhibitors S-hexyl- and S-benzyl-glutathione revealed that the affinity was reduced to 7-11% of the wild-type affinity for the glutamine and alanine mutants and to 30-40% for the histidine mutant, as measured by a comparison of Ki values. The results show that Met-157 has no direct role in catalysis, but is rather involved in forming the substrate-binding site of human glyoxalase I. The high activity of the glutamine mutant suggests that a structurally equivalent glutamine residue in the N-terminal half of Saccharomyces cerevisiae glyoxalase I may be part of a catalytically competent active site.
Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation
Authors: LUKACS L. Gergely , SEGAL Gersana , KARTNER Norbert , GRINSTEIN Sergio , ZHANG Fred
Published: August 2015

Although the cystic fibrosis transmembrane conductance regulator (CFTR) is primarily implicated in the regulation of plasma-membrane chloride permeability, immunolocalization and functional studies indicate the presence of CFTR in the endosomal compartment. The mechanism of CFTR delivery from the cell surface to endosomes is not understood. To delineate the internalization pathway, both the rate and extent of CFTR accumulation in endosomes were monitored in stably transfected Chinese hamster ovary (CHO) cells. The role of clathrin-dependent endocytosis was assessed in cells exposed to hypertonic medium, potassium depletion or intracellular acid-load. These treatments inhibited clathrin-dependent endocytosis by > 90%, as verified by measurements of 125I-transferrin uptake. Functional association of CFTR with newly formed endosomes was determined by an endosomal pH dissipation protocol [Lukacs, Chang, Kartner, Rotstein, Riordan and Grinstein (1992) J. Biol. Chem. 267, 14568-14572]. As a second approach, endocytosis of CFTR was determined after cell-surface biotinylation with the cleavable sulphosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate. Both the biochemical and the functional assays indicated that arresting the formation of clathrin-coated vesicles inhibited the retrieval of the CFTR from the plasma membrane to endosomes. An overall arrest of membrane traffic cannot account for the inhibition of CFTR internalization, since the fluid-phase endocytosis was not effected by the treatments used. Thus the efficient, constitutive internalization of surface CFTR (5% per min) occurs, predominantly by clathrin-dependent endocytosis. Stimulation of protein phosphorylation by cAMP-dependent protein kinase A and by protein kinase C decreased the rate of internalization of cell-surface biotinylated CFTR, and contributed to a substantial diminution of the internal CFTR pool compared with that of unstimulated cells. These results suggest that the rate of CFTR internalization may participate in the determination of the CFTR channel density, and consequently, of the cAMP-stimulated chloride conductance of the plasma membrane.
Kinase-related protein (telokin) is phosphorylated by smooth-muscle myosin light-chain kinase and modulates the kinase activity
Authors: SOBIESZEK Apolinary , ANDRUCHOV Y. Oleg , NIEZNANSKI Krzysztof
Published: August 2015

Telokin is an abundant smooth-muscle protein with an amino acid sequence identical with that of the C-terminal region of smooth-muscle myosin light-chain kinase (MLCK), although it is expressed as a separate protein [Gallagher and Herring (1991) J. Biol. Chem. 266, 23945-23952]. Here we demonstrate that telokin is also similar to smooth-muscle myosin regulatory light chain (ReLC) not only in its gross physical properties but also as an MLCK substrate. Telokin was slowly phosphorylated by MLCK in the presence of Ca2+ and calmodulin and could be readily dephosphorylated by myosin light-chain phosphatase. A threonine residue was phosphorylated with up to 0.25 mol/mol stoichiometry. This low stoichiometry, together with the observed dimerization of telokin [Sobieszek and Nieznanski (1997) Biochem. J. 322, 65-71], indicates that the telokin dimer was acting as the substrate with a single protomer being phosphorylated. Our enzyme kinetic analysis of the phosphorylation reaction confirms this interpretation. Because telokin phosphorylation also required micromolar concentrations of MLCK, which also facilitates the formation of kinase oligomers, we concluded that the oligomers are interacting with telokin. Thus it seems that telokin modulates the phosphorylation rate of myosin filaments by a mechanism that includes the direct or indirect inhibition of the kinase active site by the telokin dimer, and that removal of the inhibition is controlled by slow phosphorylation of the telokin dimer, which results in MLCK dimerization.
Characterization of the promoter of human adipocyte hormone-sensitive lipase
Authors: GROBER Jacques , LAURELL Henrik , BLAISE Régis , FABRY Béatrice , SCHAAK Stéphane , HOLM Cecilia , LANGIN Dominique
Published: August 2015

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The human HSL gene is composed of nine exons encoding the adipocyte form and a testis-specific coding exon. Northern blot analyses showed that human adipocytes express a 2.8 kb HSL mRNA, suggesting the presence of a short (20-150 bp) 5ʹ untranslated region (5ʹ-UTR). A single 5ʹ-UTR of approx. 70 nt was detected in RNase H mapping experiments. Two 5ʹ-UTRs of 70 and 170 nt respectively were obtained by rapid amplification of cDNA ends and cDNA library screenings. RNase protection experiments, with probes derived from the two products, showed that human adipocyte HSL mRNA contains only the 70 nt product. Primer extension analysis mapped the transcriptional start site 74 nt upstream of the start codon. In HT29, a human cell line expressing HSL, the presence of the short or the long 5ʹ-UTR is mutually exclusive. The short and long 5ʹ-UTR exons were located 1.5 and approx. 13 kb respectively upstream of the first coding exon. Various portions of the 5ʹ-flanking region upstream of the short product exon were linked to the luciferase gene and transfected into cells that express HSL (HT29 cells and rat adipocytes) and do not express HSL (HeLa cells). High luciferase activity was found for constructs containing the sequence between nt -2400 and -86, but not for shorter constructs. An analysis of 14 kb of genomic sequence revealed the presence of five DNase I hypersensitive sites associated with active gene transcription. Three of the sites are located in the vicinity of the transcriptional start site and could be linked to the minimal promoter activity. Two of the sites are located downstream of the exon containing the start codon, suggesting the presence of intronic regulatory elements.
Characterization of five different proteins produced by alternatively spliced mRNAs from the human cAMP-specific phosphodiesterase PDE4D gene
Authors: BOLGER B. Graeme , ERDOGAN Suat , JONES E. Randy , LOUGHNEY Kate , SCOTLAND Grant , HOFFMANN Ralf , WILKINSON Ian , FARRELL Cathy , HOUSLAY D. Miles
Published: August 2015

We have isolated and characterized complete cDNAs for two isoforms (HSPDE4D4 and HSPDE4A5) encoded by the human PDE4D gene, one of four genes that encode cAMP-specific rolipram-inhibited 3ʹ,5ʹ-cyclic nucleotide phosphodiesterases (type IV PDEs; PDE4 family). The HSPDE4D4 and HSPDE4D5 cDNAs encode proteins of 810 and 746 amino acids respectively. A comparison of the nucleotide sequences of these two cDNAs with those encoding the three other human PDE4D proteins (HSPDE4D1, HSPDE4D2 and HSPDE4D3) demonstrates that each corresponding mRNA transcript has a unique region of sequence at or near its 5ʹ-end, consistent with alternative mRNA splicing. Transient expression of the five cDNAs in monkey COS-7 cells produced proteins of apparent molecular mass under denaturing conditions of 68, 68, 95, 119 and 105 kDa for isoforms HSPDE4D1-5 respectively. Immunoblotting of human cell lines and rat brain demonstrated the presence of species that co-migrated with the proteins produced in COS-7 cells. COS-cell-expressed and native HSPDE4D1 and HSPDE4D2 were found to exist only in the cytosol, whereas HSPDE4D3, HSPDE4D4 and HSPDE4D5 were found in both cytosolic and particulate fractions. The IC50 values for the selective PDE4 inhibitor rolipram for the cytosolic forms of the five enzymes were similar (0.05-0.14 μM), whereas they were 2-7-fold higher for the particulate forms of HSPDE4D3 and HSPDE4D5 (0.32 and 0.59 μM respectively), than for the corresponding cytosolic forms. Our data indicate that the N-terminal regions of the HSPDE4D3, HSPDE4D4 and HSPDE4D5 proteins, which are derived from alternatively spliced regions of their mRNAs, are important in determining their subcellular localization, activity and differential sensitivity to inhibitors.
Regulation by glucocorticoids of angiotensinogen gene expression and secretion in adipose cells
Authors: AUBERT Jérôme , DARIMONT Christian , SAFONOVA Irina , AILHAUD Gérard , NEGREL Raymond
Published: August 2015

Adipose cells are an important source of angiotensinogen (AT). Its activation product, angiotensin II, stimulates in vitro and in vivo the production and release of prostacyclin which acts as a potent adipogenic signal in promoting the terminal differentiation of preadipocytes to adipocytes. Since glucocorticoids are known to promote adipose cell differentiation in vitro as well as in vivo, their role in the regulation of AT gene expression and secretion has been investigated in cultured Ob1771 mouse adipose cells. In contrast with liver cells, which are the major source of AT and the target of several hormones for the regulation of its expression, adipose cells are only responsive to glucocorticoids, which are able to up-regulate AT gene expression and AT secretion rapidly and dose-dependently. On exposure to glucocorticoids, accumulation of AT mRNA appears primarily to be due to transcriptional activation of the gene and is parallelled by secretion of the protein. Similar results on AT mRNA expression and AT secretion were obtained using explants of rat adipose tissue ex vivo demonstrating a major if not exclusive mechanism of regulation of AT production by glucocorticoids in mature adipose cells. Together these results provide a potential link between glucocorticoids, AT, the growth of adipose tissue and increased blood pressure.
Induction of the E-selectin promoter by interleukin 1 and tumour necrosis factor α, and inhibition by glucocorticoids
Authors: RAY P. Keith , FARROW Stuart , DALY Maria , TALABOT Francois , SEARLE Nicola
Published: August 2015

Cytokine-induced expression of the endothelial cell surface adhesion molecule E-selectin is inhibited by glucocorticoids (GCs). To investigate possible mechanisms for steroid inhibition, a reporter gene (ESAP) was constructed, comprising the cytokine responsive region of the E-selectin gene (nt -383 to +81) coupled to alkaline phosphatase (AP). In A549 cells stably transfected with the ESAP gene, AP production was highly responsive to the cytokines interleukin 1β (IL-1β) and tumour necrosis factor α, with ED50 values of 3 pM and 1000 pM respectively. Furthermore the cytokine-induced AP responses were inhibited by GCs, indicating that both transcriptional activation and GC suppression of the E-selectin gene were mediated via regulatory elements within the same region of the promoter. The relative potencies of GC drugs as inhibitors of IL-1β (10 pM)-stimulated ESAP-gene activation were fluticasone > beclomethasone > dexamethasone, with IC50 values of 0.13, 1.1 and 2.7 nM respectively. Inhibition by fluticasone was blocked by the GC receptor (GR) antagonist drug mifepristone (Ru486), which is consistent with the suppressive effects of GCs being mediated via the GR. However, because the E-selectin promoter lacks a consensus glucocorticoid responsive element, mechanisms for inhibition independent of GR-DNA binding were investigated. Evidence that GCs also inhibited cytokine activation of a synthetic nuclear factor κB (NFκB)-driven reporter gene transiently transfected into A549 cells suggested that interference with the activation and/or function of this transcription factor was important for GC inhibition of ESAP. However, in A549-ESAP cells, fluticasone (100 nM) did not affect IL-1β (10 pM)-induced IkBα degradation, NFκB-p65 nuclear translocation or the DNA-binding capacity of nuclear NFκB complexes, over a period during which cytokine-induced ESAP-gene activation was inhibited. Finally, there was no evidence to suggest that GC enhancement of IkBα gene expression contributed to the suppression of the cytokine response. We conclude that interference by GR with the transcriptional activation potential of DNA-bound NFκB complexes might contribute to mechanisms underlying the anti-inflammatory effects of GCs.
Structure and physiological function of calpains
Authors: SORIMACHI Hiroyuki , ISHIURA Shoichi , SUZUKI Koichi
Published: August 2015

For a long time now, two ubiquitously expressed mammalian calpain isoenzymes have been used to explore the structure and function of calpain. Although these two calpains, μ- and m-calpains, still attract intensive interest because of their unique characteristics, various distinct homologues to the protease domain of μ- and m-calpains have been identified in a variety of organisms. Some of these ‘novel’ calpain homologues are involved in important biological functions. For example, p94 (also called calpain 3), a mammalian calpain homologue predominantly expressed in skeletal muscle, is genetically proved to be responsible for limb-girdle muscular dystrophy type 2A. Tra-3, a calpain homologue in nematodes, is involved in the sex determination cascade during early development. PalB, a key gene product involved in the alkaline adaptation of Aspergillus nidulans, is the first example of a calpain homologue present in fungi. These findings indicate various important functional roles for intracellular proteases belonging to the calpain superfamily.
Endothelin attenuates apoptosis in human smooth muscle cells
Authors: WU-WONG Jinshyun R. , CHIOU J. William , DICKINSON Robert , OPGENORTH J. Terry
Published: August 2015

An imbalance between proliferation and apoptosis is an important causal factor for disorders involving abnormal cell accumulation. Endothelin (ET)-1, a 21-amino-acid peptide with mitogenic and vasoconstricting activities, not only acts as a mitogen, but also attenuates paclitaxel-induced apoptosis in smooth muscle cells. In both human pericardial and prostatic smooth muscle cells, addition of ET-1 reduced paclitaxel-induced DNA fragmentation and phosphatidylserine on the cell surface, two characteristics of apoptosis. By comparison, angiotensin II, another vasoactive peptide, did not have a significant effect on apoptosis. The effect of ET-1 was dose-dependent with an EC50 of 1 nM. These results suggest that ET is a potential survival factor for smooth muscle cells, and that altered activity of the ET system in disease states has potential to contribute to aberrant cell growth.
Separate bisphosphatase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: the role of the C-terminal tail in modulating enzyme activity
Authors: LI Lin , LING Song , WU Chun-lai , YAO Wei-zhe , XU Gen-jun
Published: August 2015

The separate bisphosphatase domain (amino acid residues 243-468) of the chicken liver bifunctional enzyme 6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase was expressed in Escherichia coli and purified to homogeneity. The fructose-2,6-bisphosphatase activity of the separate domain was 7-fold higher than that of the native bifunctional enzyme, and exhibited substrate inhibition characteristic of the native enzyme. The inhibition of the enzymes by fructose 2,6-bisphosphate could be overcome by Pi, glycerol 3-phosphate and GTP. Deletion of 30 amino acid residues from the C-terminus of the separate domain resulted in around a 5-fold increase in the Vmax of the bisphosphatase. Also, the truncated form was more accessible to chemical modification by diethyl pyrocarbonate and N-ethylmaleimide, suggesting a more open structure than the wild-type form. In addition, the mutation of cysteine-389 to alanine increased bisphosphatase activity by 20% and the Km value for fructose 2,6-bisphosphate by 3-fold, and both the point mutation at cysteine-389 and the deletional mutation led to the predominantly insoluble expression of the enzyme. The results indicated that the C-terminal tail plays a role in modulating the enzyme activity and suggested that the difference in the C-terminal tail sequence is responsible for the difference in activity of the chicken and rat liver fructose-2,6-bisphosphatases. It is postulated that an interaction between the C-terminal tail and the active site might be present.
Differential regulation of types-1 and -3 inositol trisphosphate receptors by cytosolic Ca2+
Authors: CARDY Thomas J. A. , TRAYNOR David , TAYLOR W. Colin
Published: August 2015

Biphasic regulation of inositol trisphosphate (IP3)-stimulated Ca2+ mobilization by cytosolic Ca2+ is believed to contribute to regenerative intracellular Ca2+ signals. Since cells typically express several IP3 receptor isoforms and the effects of cytosolic Ca2+ are not mediated by a single mechanism, it is important to resolve the properties of each receptor subtype. Full-length rat types-1 and -3 IP3 receptors were expressed in insect Sf9 cells at levels 10-40-fold higher than the endogenous receptors. The expressed receptors were glycosylated and assembled into tetramers, and binding of [3H]IP3 to each subtype was regulated by cytosolic Ca2+. The effects of increased [Ca2+] on native cerebellar and type-1 receptors expressed in Sf9 cells were indistinguishable. A maximally effective increase in [Ca2+] reversibly inhibited [3H]IP3 binding by approx. 50% by decreasing the number of IP3-binding sites (Bmax) without affecting their affinity for IP3. The effects of Ca2+ on type-3 receptors were more complex: increasing [Ca2+] first stimulated [3H]IP3 binding by increasing Bmax, and then inhibited it by causing a substantial decrease in the affinity of the receptor for IP3. The different effects of Ca2+ on the receptor subtypes were not a consequence of limitations in the availability of accessory proteins or of artifactual effects of Ca2+ on membrane structure. We conclude that Ca2+ can inhibit IP3 binding to types-1 and -3 IP3 receptors although by different mechanisms, and that IP3 binding to type-3 receptors is stimulated at intermediate [Ca2+]. A consequence of these differences is that, at resting cytosolic [Ca2+], type-3 receptors are more sensitive than type-1 receptors to IP3, but the situation reverses at higher cytosolic [Ca2+]. Such differences may be important in generating the spatially and temporally complex changes in cytosolic [Ca2+] evoked by receptors linked to IP3 formation.
Reconstitution, morphology and crystallization of a fatty acid β-oxidation multienzyme complex from Pseudomonas fragi
Authors: ISHIKAWA Momoyo , MIKAMI Yuriko , USUKURA Jiro , IWASAKI Hiroshi , SHINAGAWA Hideo , MORIKAWA Kosuke
Published: August 2015

The fatty acid β-oxidation multienzyme complex from Pseudomonas fragi, HDT, exhibits predominantly the three enzymic activities of 2-enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The HDT complex is encoded by the faoAB operon, consisting of the faoA and faoB genes that encode two individual constituents, the α-subunit and the β-subunit. We have constructed Escherichia coli overexpression systems for the faoAB gene product (coexpression of the α- and β-subunits), the α-subunit alone and the β-subunit alone, and have purified the three respective products. Gel-filtration analysis revealed that the faoAB gene product forms a heterotetrameric structure, α2β2, identical with the native HDT oligomeric state from P. fragi, whereas the α-subunit and β-subunit individually form dimers. Electron microscopy demonstrated that each protein morphologically adopts the above oligomeric structures. The HDT complex, reconstituted in vitro from the isolated α- and β-subunits, exhibits the three original enzymic activities and yields the same crystal as those from the native enzyme. CD measurements indicated that the α- and β-dimers hardly alter their global conformations upon the formation of the HDT complex. Interestingly, the β-dimer alone does not exhibit 3-oxoacyl-CoA thiolase activity, whereas the α-dimer alone exhibits both the 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities. These results suggest that the contact between the α- and β-subunits is essential for the thiolase activity. We have identified several structurally important proteolytic sites within each subunit, which are protected in the intact heterotetrameric molecule. These findings allow the possible location of the interface between the two subunits, which should be crucial for the exhibition of thiolase activity.
Serratia marcescens chitobiase is a retaining glycosidase utilizing substrate acetamido group participation
Authors: DROUILLARD Sophie , ARMAND Sylvie , DAVIES J. Gideon , VORGIAS E. Constantin , HENRISSAT Bernard
Published: August 2015

The stereochemistry of the reaction catalysed by Serratia marcescens chitobiase was determined by HPLC separation of the anomers of N-acetylglucosamine produced during the hydrolysis of p-nitrophenyl N-acetyl-β-D-glucosaminide (PNP-GlcNAc). In the early stages of the reaction, the β-anomer was found to prevail, whereas the α-anomer dominated at mutarotation equilibrium. This established that chitobiase hydrolyses glycosidic bonds with overall retention of the anomeric configuration. Chitobiase-catalysed hydrolysis of PNP-GlcNAc was competitively inhibited by a series of chito-oligosaccharides (degree of polymerization 2-5) that were selectively de-N-acetylated at their non-reducing end. The results are in accord with the participation of the acetamido group at C-2 of the substrate in the catalytic mechanism of chitobiase and related enzymes.
Structure of the trigonal crystal form of bovine annexin IV
Authors: ZANOTTI Giuseppe , MALPELI Giorgio , GLIUBICH Francesca , FOLLI Claudia , STOPPINI Monica , OLIVI Luca , SAVOIA Adolfo , BERNI Rodolfo
Published: August 2015

The structure of a trigonal crystal form of N-terminally truncated [des-(1-9)] bovine annexin IV, an annexin variant that exhibits the distinctive property of binding both phospholipids and carbohydrates in a Ca2+-dependent manner, has been determined at 3 Å (0.3 nm) resolution [space group: R3; cell parameters: a = b = 118.560 (8) Å and c = 82.233 (6) Å]. The overall structure of annexin IV, crystallized in the absence of Ca2+ ions, is highly homologous to that of the other known members of the annexin family. The trimeric assembly in the trigonal crystals of annexin IV is quite similar to that found previously in non-isomorphous crystals of human, chicken and rat annexin V and to the subunit arrangement in half of the hexamer of hydra annexin XII. Moreover, it resembles that found in two-dimensional crystals of human annexin V bound to phospholipid monolayers. The propensity of several annexins to generate similar trimeric arrays supports the hypothesis that trimeric complexes of such annexins, including annexin IV, may represent the functional units that interact with membranes.
Cross-talk between transcriptional regulation by thyroid hormone and myogenin: new aspects of the Ca2+-dependent expression of the fast-type sarcoplasmic reticulum Ca2+-ATPase
Authors: THELEN H. M. Marc , SIMONIDES S. Warner , MULLER Alice , HARDEVELD Cornelis VAN
Published: August 2015

We have previously demonstrated an interaction between the major determinants of skeletal muscle phenotype by showing that continuous contractile activity represses the thyroid hormone (3,3ʹ,5-tri-iodothyronine; T3)-dependent transcriptional activity of fast-type sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase (SERCA1), a characteristic of the fast phenotype. Both the free cytosolic Ca2+ concentration ([Ca2+]i) and the myogenic determination factors MyoD and myogenin have been implicated as mediators of the effect of contractile activity on skeletal muscle phenotype. Using L6 cells we have shown that an increase in the steady-state [Ca2+]i above the resting level of 120 nM indeed can mimic the effect of contractile activity on T3-dependent SERCA1 expression. We now show that the repressing effect of increased [Ca2+]i on T3-dependent SERCA1 expression in L6 cells is exerted at a pre-translational level and is accompanied by increased myogenin mRNA expression. Myogenin overexpression in these cells revealed that increased expression of myogenin alone strongly decreases the T3-dependent stimulation of SERCA1 promoter activity. These results suggest a pathway for the regulation of skeletal muscle phenotype in which [Ca2+]i mediates the effect of contractile activity by regulating the expression of myogenin, which in turn interferes with transcriptional regulation by T3.
Starvation and diabetes increase the amount of pyruvate dehydrogenase kinase isoenzyme 4 in rat heart
Authors: WU Pengfei , SATO Juichi , ZHAO Yu , JASKIEWICZ Jerzy , POPOV M. Kirill , HARRIS A. Robert
Published: August 2015

This study investigated whether conditions known to alter the activity and phosphorylation state of the pyruvate dehydrogenase complex have specific effects on the levels of isoenzymes of pyruvate dehydrogenase kinase (PDK) in rat heart. Immunoblot analysis revealed a remarkable increase in the amount of PDK4 in the hearts of rats that had been starved or rendered diabetic with streptozotocin. Re-feeding of starved rats and insulin treatment of diabetic rats very effectively reversed the increase in PDK4 protein and restored PDK enzyme activity to levels of chow-fed control rats. Starvation and diabetes also markedly increased the abundance of PDK4 mRNA, and re-feeding and insulin treatment reduced levels of the message to that of controls. In contrast with the findings for PDK4, little or no changes in the amounts of PDK1 and PDK2 protein and the abundance of their messages occurred in response to starvation and diabetes. The observed shift in the relative abundance of PDK isoenzymes probably explains previous studies of the effects of starvation and diabetes on heart PDK activity. The results indicate that control of the amount of PDK4 is important in long-term regulation of the activity of the pyruvate dehydrogenase complex in rat heart.
Subcellular co-localization and potential interaction of glucuronosyltransferases with nascent proteochondroitin sulphate at Golgi sites of chondroitin synthesis
Authors: SUGUMARAN Geetha , KATSMAN Maya , SILBERT E. Jeremiah
Published: August 2015

Microsomal membranes from chick embryo epiphyseal cartilage were fractionated by equilibrium sucrose-density-gradient centrifugation and assayed for GlcA (glucuronic acid) transferase I (the enzyme that transfers GlcA from UDP-GlcA to Gal-Gal-Xyl of proteochondroitin linkage region), for comparison with GlcA transferase II (the GlcA transferase of chondroitin polymerization). Gal(β1-3)Galβ1-methyl (disaccharide) and GalNAc(β1-4)GlcA(β1-3)GalNAc(β1-4)GlcA(β1-3)GalNAc (pentasaccharide) were used respectively as acceptors of [14C]GlcA from UDP-[14C]GlcA. Distributions of the two GlcA transferase activities in the sucrose-density-gradient fractions were compared with each other and with the previously reported distribution of the activities of Gal transferases (UDP-Gal to ovalbumin, and to xylose of the proteochondroitin linkage region) and GalNAc (N-acetylgalactosamine) transferase II of chondroitin polymerization. The linkage-region GlcA transferase I had a dual Golgi distribution similar to that of chondroitin-polymerizing GlcA transferase II and distinctly different from the distribution of linkage-region Gal transferases I and II, which were found exclusively in the heavier fractions. Solubilized GlcA transferase I was partly purified by sequential use of Q-Sepharose, heparin-Sepharose and wheatgerm agglutinin-agarose and was accompanied at each step by some of the GlcA transferase II activity. Both GlcA transferase I and II bound to the Q-Sepharose as though they were highly anionic. However, treatment with chondroitin ABC lyase eliminated the binding while markedly decreasing enzyme stability. The enzyme activities could not be reconstituted by adding chondroitin or chondroitin pentasaccharide to the chondroitin ABC lyase-treated enzymes. Incubation of the partly purified enzymes with both UDP-GlcA and UDP-GalNAc resulted in a 40-fold greater incorporation than with just one sugar nucleotide, indicating the presence of bound, nascent proteochondroitin serving as the acceptor for chondroitin polymerization. These results, together with the membrane co-localization, indicate that GlcA transferase I and GlcA transferase II occur closely together with nascent proteochondroitin at the site of synthesis and that this complex with the nascent proteochondroitin stabilizes both enzymes during purification.
Signal transduction by the Wnt family of ligands
Authors: DALE C. Trevor
Published: August 2015

The Wnt genes encode a large family of secreted polypeptides that mediate cell-cell communication in diverse developmental processes. The loss or inappropriate activation of Wnt expression has been shown to alter cell fate, morphogenesis and mitogenesis. Recent progress has identified Wnt receptors and components of an intracellular signalling pathway that mediate Wnt-dependent transcription. This review will highlight this ‘core’ Wnt signal-transduction pathway, but also aims to reveal the potential diversity of Wnt signalling targets. Particular attention will be paid to the overlap between developmental biology and oncogenesis, since recent progress shows Wnt signalling forms a paradigm for an interdisciplinary approach.
Cloning and sequencing of four new mammalian monocarboxylate transporter (MCT) homologues confirms the existence of a transporter family with an ancient past
Authors: PRICE T. Nigel , JACKSON N. Vicky , HALESTRAP P. Andrew
Published: August 2015

Measurement of monocarboxylate transport kinetics in a range of cell types has provided strong circumstantial evidence for a family of monocarboxylate transporters (MCTs). Two mammalian MCT isoforms (MCT1 and MCT2) and a chicken isoform (REMP or MCT3) have already been cloned, sequenced and expressed, and another MCT-like sequence (XPCT) has been identified. Here we report the identification of new human MCT homologues in the database of expression sequence tags and the cloning and sequencing of four new full-length MCT-like sequences from human cDNA libraries, which we have denoted MCT3, MCT4, MCT5 and MCT6. Northern blotting revealed a unique tissue distribution for the expression of mRNA for each of the seven putative MCT isoforms (MCT1-MCT6 and XPCT). All sequences were predicted to have 12 transmembrane (TM) helical domains with a large intracellular loop between TM6 and TM7. Multiple sequence alignments showed identities ranging from 20% to 55%, with the greatest conservation in the predicted TM regions and more variation in the C-terminal than the N-terminal region. Searching of additional sequence databases identified candidate MCT homologues from the yeast Saccharomyces cerevisiae, the nematode worm Caenorhabditis elegans and the archaebacterium Sulfolobus solfataricus. Together these sequences constitute a new family of transporters with some strongly conserved sequence motifs, the possible functions of which are discussed.
A 27 kDa protein binds to a positive and a negative regulatory sequence in the promoter of the ICL1 gene from Saccharomyces cerevisiae
Authors: ORDIZ Isabel , HERRERO Pilar , RODICIO Rosaura , GANCEDO M. Juana , MORENO Fernando
Published: August 2015

Isocitrate lyase, encoded by ICL1, is one of the key enzymes of the glyoxylate pathway, which operates as an anaplerotic route for replenishing the tricarboxylic acid cycle; it is required for growth of Saccharomyces cerevisiae on carbon sources such as ethanol, but is dispensable when fermentable carbon sources are available. The positive regulation of the ICL1 gene by an upstream activating sequence (UAS) element located between -397 and -388 has been previously reported. In this paper we show that the ICL1 promoter sequence 5ʹ-AGTCCGGACTAGCATCCCAG-3ʹ located between -261 and -242 contains an upstream repressing sequence (URS) element. We have identified and partially purified a 27 kDa protein that binds specifically to both the UAS and URS sequences of the ICL1 promoter. For both UAS and URS, binding requires the protein Snf1 (Cat1), a protein kinase essential for the derepression of genes repressed by glucose. Binding does not take place with extracts from glucose-grown strains, unless they lack Mig1, a negative regulatory protein involved in glucose repression.
NADPH oxidase of chondrocytes contains an isoform of the gp91phox subunit
Authors: MOULTON J. Paul , GOLDRING B. Mary , HANCOCK T. John
Published: August 2015

Previously it has been reported that chondrocytic cells produce oxygen free radicals and express the cytosolic components of NADPH oxidase. Here we report the expression of large subunit of the flavocytochrome of NADPH oxidase in chondrocytes and, further, show that the cDNA sequence contains three single base pair differences compared with the phagocyte gp91phox gene sequence. These base-pair differences may account for the different activity profiles reported between phagocytic and non-phagocytic cells.
Endogenous syntaxins 2, 3 and 4 exhibit distinct but overlapping patterns of expression at the hepatocyte plasma membrane
Authors: FUJITA Hideaki , TUMA L. Pamela , FINNEGAN M. Catherine , LOCCO Louis , HUBBARD L. Ann
Published: August 2015

To investigate the mechanisms regulating polarized vesicle delivery to the cell surface in hepatocytes, we have characterized the endogenous plasma membrane (PM)-associated syntaxins. These integral membrane proteins are components of the membrane docking/fusion apparatus and are thought to function as vesicle receptors at the PM. In hepatocytes, the PM is divided into two domains, the apical and basolateral. If syntaxins are mediating the specific recognition of vesicles delivered to either membrane surface, the simple prediction is that each domain expresses one syntaxin isoform. However, we report that rat hepatocytes express three endogenous PM-associated syntaxin isoforms, syntaxins 2, 3 and 4. By biochemical subfractionation, we determined that the syntaxins exhibit distinct, but overlapping patterns of expression among the PM domains. Syntaxin 4 is primarily expressed at the basolateral surface while syntaxins 2 and 3 are enriched at the apical PM. The immunolocalization of syntaxins 2 and 4 in rat hepatocytes and PM sheets revealed similarly complex patterns of PM expression with enhanced apical staining for both. A significant proportion of syntaxin 3 (25%) was detected in subcellular fractions containing transport vesicles. We have used quantitative immunoblotting to determine that the syntaxins are relatively abundant PM molecules (11-260 nM) in rat liver, spleen and kidney. Also, we determined that the syntaxin binding protein, Munc-18, is present at concentrations from 1.5-20 nM in the same tissues. Although this fundamental quantitative and morphological information is lacking in other systems, it is critical not only for defining syntaxin function, but also for predicting the specific mechanisms that regulate vesicle targeting in hepatocytes and other tissues.