bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple interactions between pituitary hormones and the mannose receptor
Authors: SIMPSON Daniel Z. , HITCHEN Paul G. , ELMHIRST Elizabeth L. , TAYLOR Maureen E.
Published: August 2015

The macrophage mannose receptor, which has a well-documented role in the innate immune system, has an additional function in the clearance of pituitary hormones. Clearance is mediated by the recognition of sulphated terminal N-acetylgalactosamine residues (SO4-4GalNAc) on the hormones. Previous studies with an SO4-4GalNAc-containing neoglycoprotein suggest that the SO4-4GalNAc-binding site is localized to the N-terminal cysteine-rich domain of the receptor, distinct from the mannose/N-acetylglucosamine/fucose-specific C-type carbohydrate-recognition domains (CRDs). The present study characterizes the binding of natural pituitary hormone ligands to a soluble portion of the mannose receptor consisting of the whole extracellular domain and to a truncated form containing the eight CRDs but lacking the N-terminal cysteine-rich domain and the fibronectin type II repeat. Both forms of the receptor show high-affinity saturable binding of lutropin and thyrotropin. Binding to the full-length receptor is dependent on pH and ionic strength and is inhibited effectively by SO4-4GalNAc but only partly by mannose. In contrast, binding to the truncated form of the receptor, which is also dependent on pH and ionic strength, is inhibited by mannose but not by SO4-4GalNAc. The results are consistent with the presence of an SO4-4GalNAc-specific binding site in the cysteine-rich domain of the mannose receptor but indicate that interactions between other sugars on the hormones and the CRDs are also important in hormone binding.
Sulphation of lithocholic acid in the colon-carcinoma cell line CaCo-2
Authors: HALVORSEN Bente , KASE Bengt Frode , PRYDZ Kristian , GARAGOZLIAN Sedegheh , ANDRESEN Marianne S. , KOLSET Svein Olav
Published: August 2015

High levels of bile acids in the colon may correlate with an increased risk of colon cancer, but the underlying mechanisms are not known. Proteoglycan structures have been shown to change when human colon cells differentiate in vitro. The expression of [35S]sulphated molecules was used as a phenotypic marker to study the effects of bile acids on the human-colon-carcinoma cell line CaCo-2. [35S]sulphated compounds were isolated from the medium of cell fractions of cells metabolically labelled with [35S]sulphate in the absence and presence of cholic acid, deoxycholic acid, chenodeoxycholic acid and lithocholic acid (LA). Labelled molecules were analysed by gel chromatography, HPLC and SDS/PAGE in combination with chemical and enzymic methods. The expression of 35S-labelled proteoglycans was not affected by any of the bile acids tested. However, the level of sulphated metabolites increased 7-18-fold in different experiments during a 22 h labelling period in the presence of an LA concentration of 10 μg/ml (26.6 nmol/ml) compared with controls. Further analyses showed that this was due, at least in part, to the sulphation of LA itself. This sulphation of LA was a rapid process followed by secretion back to the medium. Brefeldin A did not reduce the sulphation of LA, indicating that this conversion takes place in the cytosol, rather than in the Golgi apparatus of the CaCo-2 cells. LA in colon may be sulphated efficiently by the colonocytes to reduce the toxic effects of this particular bile acid. Sulphation may possibly be an important protective mechanism in the colon.
GLUT4 trafficking in insulin-stimulated rat adipose cells: evidence that heterotrimeric GTP-binding proteins regulate the fusion of docked GLUT4-containing vesicles
Authors: FERRARA Cynthia M. , CUSHMAN Samuel W.
Published: August 2015

Agents that activate the G-protein Gi (e.g. adenosine) increase, and agents that activate Gs [e.g. isoprenaline (isoproterenol)] decrease, steady-state insulin-stimulated glucose transport activity and cell-surface GLUT4 in isolated rat adipose cells without changing plasma membrane GLUT4 content. Here we have further examined the effects of RsGs and RiGi ligands (in which Rs and Ri are Gs- and Gi-coupled receptors respectively) on insulin-stimulated cell-surface GLUT4 and the kinetics of GLUT4 trafficking in these same cells. Rat adipose cells were preincubated for 2 min with or without isoprenaline (200 nM) and adenosine deaminase (1 unit/ml), to stimulate Gs and decrease the stimulation of Gi respectively, followed by 0-20 min with insulin (670 nM). Treatment with isoprenaline and adenosine deaminase decreased insulin-stimulated glucose transport activity by 58%. Treatment with isoprenaline and adenosine deaminase also resulted in similar decreases in insulin-stimulated cell-surface GLUT4 as assessed by both bis-mannose photolabelling of the substrate-binding site and biotinylation of the extracellular carbohydrate moiety when evaluated under similar experimental conditions. After stimulation with insulin in the absence of Gs and the presence of Gi agents, a distinct sequence of plasma membrane events took place, starting with an increase in immunodetectable GLUT4, then an increase in the accessibility of GLUT4 to bis-mannose photolabel, and finally an increase in glucose transport activity. Pretreatment with isoprenaline and adenosine deaminase before stimulation with insulin did not affect the time course of the increase in immunodetectable GLUT4 in the plasma membrane, but did delay both the increase in accessibility of GLUT4 to photolabel and the increase in glucose transport activity. These results suggest that RsGs and RiGi modulate insulin-stimulated glucose transport by influencing the extent to which GLUT4 is associated with occluded vesicles attached to the plasma membrane during exocytosis, perhaps by regulating the fusion process through which the GLUT4 in docked vesicles becomes exposed on the cell surface.
Glycosylphosphatidylinositol-anchor intermediates associate with Triton-insoluble membranes in subcellular compartments that include the endoplasmic reticulum
Authors: SEVLEVER Daniel , PICKETT Samuel , MANN Karl J. , SAMBAMURTI Kumar , MEDOF M. Edward , ROSENBERRY Terrone L.
Published: August 2015

Glycosylphosphatidylinositol (GPI)-anchored proteins are resistant to solubilization with Triton X-100 at 4 °C, and they can be recovered in Triton-insoluble membranes (TIMs) that float to a characteristic buoyant density. Because the GPI structure itself has been shown to target GPI-anchored proteins to TIMs, we investigated the association of GPI-anchor intermediates with TIMs. GPI-anchor biosynthesis involves a pathway of some 10 steps that take place in the endoplasmic reticulum (ER). These intermediates include glucosaminyl-acylphosphatidylinositol [GlcN-(acyl)PI] and later mannosylated GPIs, denoted H6, H7 and H8, that are present not only in the ER but also in other cell compartments, including the plasma membrane. At least two-thirds of the GlcN-(acyl)PI in HeLa D cells and mannosylated GPIs in K562 cells were found in TIMs. Although previous reports have considered TIMs to be derived primarily from the plasma membrane, we recovered TIMs from subcellular fractions enriched in ER membranes. The ER marker calnexin and GPI-anchored proteins as well as N-acetylglucosaminyl-phosphatidylinositol and mannosylated GPIs were present in ER-TIMs. Interestingly, GlcN-PI and H7 were less enriched in ER-TIM than the other GPIs, suggesting that ER-TIMs might reflect a compartmentalization of the GPI-anchor biosynthetic pathway in the ER.
Structural characterization of human and bovine lung surfactant protein D
Authors: LETH-LARSEN Rikke , HOLMSKOV Uffe , HØJRUP Peter
Published: August 2015

Human and bovine surfactant proteins D (SP-D) were purified from late amniotic fluid and bronchioalveolar lavage on the basis of its Ca2+-dependent affinity for maltose. The molecular mass of a trimeric subunit was determined by matrix-assisted laser desorption ionization MS to lie in the range 115-125 kDa for human SP-D and 110-123 kDa for bovine SP-D. A single polypeptide chain was determined at 37-41 and 36-40 kDa for the human and bovine species respectively. The major parts of the primary structures of both SP-D molecules were determined by a combination of MS and Edman degradation. The heterogeneity in SP-D was caused mainly by a high number of post-translational modifications in the collagen-like region. Proline and lysine residues were partly hydroxylated and lysine residues were further O-glycosylated with the disaccharide galactose-glucose. A partly occupied N-linked glycosylation site was characterized in human SP-D. The carbohydrate was determined as a complex type bi-antennary structure, with a small content of mono-antennary and tri-antennary structures. No sialic acid residues were present on the glycan, but some had an attached fucose and/or an N-acetylglucosamine residue linked to the core. Bovine SP-D was determined as having a similar structure.
Evidence for an interaction of the metalloprotease–disintegrin tumour necrosis factor α convertase (TACE) with mitotic arrest deficient 2 (MAD2), and of the metalloprotease–disintegrin MDC9 with a novel MAD2-related protein, MAD2β
Authors: NELSON Karen K. , SCHLÖNDORFF Johannes , BLOBEL Carl P.
Published: August 2015

Metalloprotease-disintegrins are a family of transmembrane glycoproteins that have a role in fertilization, sperm migration, myoblast fusion, neural development and ectodomain shedding. In the present study we used the yeast two-hybrid system to search for proteins that interact with the cytoplasmic domain of two metalloprotease-disintegrins, tumour necrosis factor α convertase (TACE; ADAM17) and MDC9 (ADAM9; meltrin γ). We have identified mitotic arrest deficient 2 (MAD2) as a binding partner of the TACE cytoplasmic domain, and a novel MAD2-related protein, MAD2β, as a binding partner of the MDC9 cytoplasmic domain. MAD2β has 23% sequence identity with MAD2, which is a component of the spindle assembly (or mitotic) checkpoint mechanism. Northern blot analysis of human tissues indicates that MAD2β mRNA is expressed ubiquitously. The interaction of the TACE and MDC9 cytoplasmic domains with their binding partners has been confirmed biochemically. The independent identification of MAD2 and MAD2β as potential interacting partners of distinct metalloprotease-disintegrins raises the possibility of a link between metalloprotease-disintegrins and the cell cycle, or of functions for MAD2 and MAD2β that are not related to cell cycle control.
Delineation of the insulin-responsive sequence in the rat cytosolic aspartate aminotransferase gene: binding sites for hepatocyte nuclear factor-3 and nuclear factor I
Authors: BEURTON Fadela , BANDYOPADHYAY Uday , DIEUMEGARD Barbara , BAROUKI Robert , AGGERBECK Martine
Published: August 2015

Expression of the rat cytosolic aspartate aminotransferase gene is stimulated by glucocorticoids and repressed by insulin in the liver. The regulation by insulin and part of the glucocorticoid effect are mediated by a distal region in the promoter. A 142 bp fragment (-1844 to -1702) confers hormonal sensitivity to the heterologous thymidine kinase promoter in transient-transfection assays in H4IIEC3 hepatoma cells. Footprinting and gel-shift assays showed that several nuclear proteins bind to this region at conserved CCAAT-enhancer binding protein (C/EBP), activator protein (AP-1) and E-box sequences. Hepatocyte nuclear factor-3α (HNF-3)α and β bind to sequences upstream of a glucocorticoid-responsive element (GRE) half-site as demonstrated by supershift experiments. Nuclear factor I (NFI)-like proteins bind downstream of the GRE half-site. These sites around the GRE motif overlap with five insulin responsive element (IRE) -like sequences (TG/ATTT). The effect of insulin was not prevented by any single mutation in the IRE-like sites. However, mutation of two IRE sites (namely IREc and d) prevented the insulin effect although only marginally affecting the glucocorticoid effect. The results suggest that the effect of insulin is due to a complex interplay of factors requiring the synergistic contribution of at least two sites and underline the contribution of HNF-3 and NFI-like proteins.
A novel chaperone-activity-reducing mechanism of the 90-kDa molecular chaperone HSP90
Authors: ITOH Hideaki , OGURA Masahito , KOMATSUDA Atsushi , WAKUI Hideki , MIURA Akira B. , TASHIMA Yohtalou
Published: August 2015

The 90-kDa heat shock protein (HSP90) acts as a specific molecular chaperone in the folding and regulates a wide range of associated proteins such as steroid hormone receptors. It is known that HSP90 possesses two different chaperone sites, both in the N- and C-domains, and that the chaperone activity of HSP90 is blocked by binding of geldanamycin (GA) to the N-domain, the same as the ATP-binding site. Here we show that Cisplatin [cis-diamminedichloroplatinum (II), CDDP], an antineoplastic agent, associates with HSP90 and reduces its chaperone activity. In order to analyse the binding proteins, bovine brain cytosols were applied to a CDDP-affinity column and binding proteins were eluted by CDDP. In the elutants, only 90-kDa protein bands were detected on SDS/PAGE, and the protein was cross-reacted with the anti-HSP90 antibody on immunoblotting. No protein bands were detected in the elutants from the control column on SDS/PAGE. These results indicated that CDDP has a high affinity for HSP90. On CD spectrum analysis, the binding of CDDP to HSP90 resulted in a conformational change in the protein. Although HSP90 inhibited the aggregation of citrate synthase as a molecular chaperone in vitro,the activity was suppressed almost completely in the presence of CDDP. Mg/ATP has an influence on the chaperone activity to some extent. The CDDP binding region of HSP90 is near the C-terminal which is quite different from the GA-binding site. Our results suggest that the chaperone activity of HSP90 may be inhibited by the binding of CDDP or GA by different mechanisms.
Binding energy and specificity in the catalytic mechanism of yeast aldose reductases
Authors: NIDETZKY Bernd , MAYR Peter , HADWIGER Philipp , STüTZ Arnold E.
Published: August 2015

Derivatives of D-xylose and D-glucose, in which the hydroxy groups at C-5, and C-5 and C-6 were replaced by fluorine, hydrogen and azide, were synthesized and used as substrates of the NAD(P)H-dependent aldehyde reduction catalysed by aldose reductases isolated from the yeasts Candida tenuis, C. intermedia and Cryptococcus flavus. Steady-state kinetic analysis showed that, in comparison with the parent aldoses, the derivatives were reduced with up to 3000-fold increased catalytic efficiencies (kcat/Km), reflecting apparent substrate binding constants (Km) decreased to as little as 1/250 and, for D-glucose derivatives, up to 5.5-fold increased maximum initial rates (kcat). The effects on Km mirror the relative proportion of free aldehyde that is available in aqueous solution for binding to the binary complex enzyme-NAD(P)H. The effects on kcat reflect non-productive binding of the pyranose ring of sugars; this occurs preferentially with the NADPH-dependent enzymes. No transition-state stabilization energy seems to be derived from hydrogen-bonding interactions between enzyme-NAD(P)H and positions C-5 and C-6 of the aldose. In contrast, unfavourable interactions with the C-6 group are used together with non-productive binding to bring about specificity (6-10 kJ/mol) in a series of D-aldoses and to prevent the reaction with poor substrates such as D-glucose. Azide introduced at C-5 or C-6 destabilizes the transition state of reduction of the corresponding hydrogen-substituted aldoses by approx. 4-9 kJ/mol. The total transition state stabilization energy derived from hydrogen bonds between hydroxy groups of the substrate and enzyme-NAD(P)H is similar for all yeast aldose reductases (yALRs), at approx. 12-17 kJ/mol. Three out of four yALRs manage on only hydrophobic enzyme-substrate interactions to achieve optimal kcat, whereas the NAD(P)H-dependent enzyme from C. intermedia requires additional, probably hydrogen-bonding, interactions with the substrate for efficient turnover.
Functional and structural properties of lipid-associated apolipoprotein J (clusterin)
Authors: CALERO Miguel , TOKUDA Takahiko , ROSTAGNO Agueda , KUMAR Asok , ZLOKOVIC Berislav , FRANGIONE Blas , GHISO Jorge
Published: August 2015

Apolipoprotein J (apoJ, clusterin) is a multifunctional protein normally associated with lipids in plasma and cerebrospinal fluid, and secreted as lipoparticles by hepatocytes and astrocytes. To investigate whether the structural and functional properties of apoJ are modulated upon binding to lipids, we prepared apoJ high-density lipoprotein (HDL)-like particles employing either synthetic or plasma HDL-derived lipids. The majority of the resulting lipoparticles contained one molecule of apoJ per particle and exhibited the same α2 electrophoretic mobility characteristic of apoJ-containing plasma HDL. Particle size seemed to be dependent on the presence of cholesterol in the lipid mixture and ranged from diameters of 10 nm in the presence of cholesterol to 20 nm in the absence of cholesterol. CD analysis and Fourier-transform infrared spectroscopy revealed similar changes in the apoJ secondary structure induced by its incorporation into lipoparticles, namely a decrease in α-helix content and an increase in β-turn structures. Two functional assays, the binding interaction with Alzheimer's amyloid β peptides and the inhibitory activity of the complement membrane-attack complex, did not detect any changes in apoJ activity following its lipidation (P > 0.05). On the contrary, the binding affinity to the cellular receptor megalin was enhanced significantly (P < 0.01) after the association with lipids; the Kd value decreased from 78.8±10.7 nM for the delipidated form to 37.0±7.3 nM for apoJ-HDL. Although it is not known whether the structural changes observed are directly responsible for the higher receptor-binding affinity, the data suggest that the complement inhibition and amyloid β-binding motifs are located in areas of the molecule different from those involved in the apoJ-megalin interaction.
Nutrients differentially regulate multiple translation factors and their control by insulin
Authors: CAMPBELL Linda E. , WANG Xuemin , PROUD Christopher G.
Published: August 2015

Eukaryotic initiation factor eIF2B and eukaryotic elongation factor eEF2 each mediate regulatory steps important for the overall regulation of mRNA translation in mammalian cells and are activated by insulin. Here, we demonstrate that their activation by insulin requires the presence, in the medium in which the cells are maintained, of both amino acids and glucose: insulin only induced activation of eIF2B and the dephosphorylation of eEF2 when cells were exposed to both types of nutrient. Other translational regulators, e.g. the 70 kDa ribosomal protein S6 kinase (p70 S6 kinase) and the eIF4E binding protein 1, 4E-BP1, are also regulated by insulin but their control does not require glucose, only amino acids. The effects of nutrients on the activation of eIF2B do not reflect changes in the phosphorylation of eIF2 (and, by inference, operation of a kinase analogous to yeast Gcn2p), or a requirement for nutrients for inactivation of glycogen synthase kinase-3 or dephosphorylation of eIF2B. Nutrients did not affect the ability of insulin to activate protein kinase B. These data show that activation by insulin of p70 S6 kinase, which modulates the translation of specific mRNAs, depends on the availability of amino acids whereas regulation of factors involved in overall activation of translation (eIF2B, eEF2) requires both amino acids and glucose. These results add substantially to the emerging evidence that nutrients themselves modulate functions of mammalian cells and indicate that (i) nutrients modulate the activation of eIF2B and eEF2 through as-yet unidentified mechanisms and (ii) regulation of p70 S6 kinase and 4E-BP1 by insulin requires other inputs in addition to protein kinase B.
Hypochlorous acid causes caspase activation and apoptosis or growth arrest in human endothelial cells
Authors: VISSERS Margret C. M. , PULLAR Juliet M. , HAMPTON Mark B.
Published: August 2015

We have investigated the effect of hypochlorous acid (HOCl) on cultured human umbilical-vein endothelial cells and shown that, whereas higher concentrations cause rapid necrosis, smaller amounts of this oxidant induce apoptosis or growth arrest. Exposure to 20-40 nmol of HOCl per 1.2×105 cells initiated apoptosis that was determined morphologically, by the identification of apoptotic nuclei with Hoechst 33342, and by detection of phosphatidylserine on the outer membrane. Degraded DNA was detected by flow cytometry. HOCl induced caspase activity; specific inhibition of caspases was shown to prevent apoptosis. No caspase activation could be detected with 50 nmol of HOCl per 1.2×105 cells, a dose that caused more extensive necrosis. Lower doses of HOCl, which did not cause cell death, resulted in a transient growth arrest that was apparent with as little as 5 nmol of HOCl per 1.2×105 cells. These results show that HOCl can modify cellular responses that are dependent on signal transduction pathways in a manner similar to that of other oxidants.
Design of an adenosine phosphorylase by active-site modification of murine purine nucleoside phosphorylase: Enzyme kinetics and molecular dynamics simulation of Asn-243 and Lys-244 substitutions of purine nucleoside phosphorylase
Authors: MAYNES Jason T. , YAM W.-S. , JENUTH Jack P. , YUAN R. Gang , LITSTER Steven A. , PHIPPS Barry M. , SNYDER Floyd F.
Published: August 2015

Our objective was to alter the substrate specificity of purine nucleoside phosphorylase such that it would catalyse the phosphorolysis of 6-aminopurine nucleosides. We modified both Asn-243 and Lys-244 in order to promote the acceptance of the C6-amino group of adenosine. The Asn-243-Asp substitution resulted in an 8-fold increase in Km for inosine from 58 to 484 μM and a 1000-fold decrease in kcat/Km. The Asn-243-Asp construct catalysed the phosphorolysis of adenosine with a Km of 45 μM and a kcat/Km 8-fold that with inosine. The Lys-244-Gln construct showed only marginal reduction in kcat/Km, 83% of wild type, but had no activity with adenosine. The Asn-243-Asp;Lys-244-Gln construct had a 14-fold increase in Km with inosine and 7-fold decrease in kcat/Km as compared to wild type. This double substitution catalysed the phosphorolysis of adenosine with a Km of 42 μM and a kcat/Km twice that of the single Asn-243-Asp substitution. Molecular dynamics simulation of the engineered proteins with adenine as substrate revealed favourable hydrogen bond distances between N7 of the purine ring and the Asp-243 carboxylate at 2.93 and 2.88 Å, for Asn-243-Asp and the Asn-243-Asp;Lys-244-Gln constructs respectively. Simulation also supported a favourable hydrogen bond distance between the purine C6-amino group and Asp-243 at 2.83 and 2.88 Å for each construct respectively. The Asn-243-Thr substitution did not yield activity with adenosine and simulation gave unfavourable hydrogen bond distances between Thr-243 and both the C6-amino group and N7 of the purine ring. The substitutions were not in the region of phosphate binding and the apparent S0.5 for phosphate with wild type and the Asn-243-Asp enzymes were 1.35±0.01 and 1.84±0.06 mM, respectively. Both proteins exhibited positive co-operativity with phosphate giving Hill coefficients of 7.9 and 3.8 respectively.
Identification of cAMP-dependent protein kinase holoenzymes in preantral- and preovulatory-follicle-enriched ovaries, and their association with A-kinase-anchoring proteins
Authors: CARR Daniel W. , CUTLER Richard E. , COTTOM Joshua E. , SALVADOR Lisa M. , FRASER Iain D. C. , SCOTT John D. , HUNZICKER-DUNN Mary
Published: August 2015

Undifferentiated cells from preantral (PA) follicles respond to high levels of cAMP in a different manner than do differentiated cells from preovulatory (PO) follicles. We hypothesized that this differential response of PA and PO cells to cAMP could be due, in part, to either a difference in the profile of isoforms that comprise the cAMP-dependent protein kinase (PKA) holoenzymes and/or a difference in the interaction of PKA with A-kinase-anchoring proteins (AKAPs). To test these hypotheses, PKA activity, PKA holoenzymes, PKA subunits and AKAPs from PA and PO ovaries were compared. Soluble PKA holoenzymes and regulatory (R) subunits were separated by DEAE-cellulose chromatography and sucrose-density-gradient centrifugation. PKA R subunits were distinguished by photoaffinity labelling, autophosphorylation, size, isoelectric point and immunoreactivity. AKAPs were identified by RII subunit overlay assays and immunoreactivity. The results showed that extracts from PA and PO ovaries exhibited equivalent PKA holoenzyme profiles and activities, characterized by low levels of PKA type I (PKAI) holoenzyme and two distinct PKAII holoenzyme peaks, one containing only RIIβ subunits (PKAIIβ) and one containing both PKAIIβ and PKAIIα holoenzymes. Both PA and PO ovarian extracts also contained PKA catalytic (C)-subunit-free RIα, while only PO ovaries exhibited C-subunit-free RIIβ. Consistent with the elevated levels of C-subunit-free RIIβ in PO cells, PKA activation in PO cells required higher concentrations of forskolin than that in PA cells. While extracts of PA and PO ovaries exhibited a number of similar AKAPs, including four prominent ones reactive with anti-AKAP-KL antisera (where AKAP-KL is an AKAP especially abundant in kidney and liver), cAMP-agarose affinity chromatography revealed two major differences in AKAP binding to purified R subunits. PO ovaries contained increased levels of AKAP80 (AKAP of 80 kDa) bound selectively to R subunits in DEAE-cellulose peak 2 (comprising PKAIIβ and RIα), but not to R subunits in DEAE-cellulose peak 3 (comprising PKAIIα, PKAIIβ and RIIβ). PO ovaries also showed increased binding of R subunits to AKAPs reactive with anti-AKAP-KL antisera at 210, 175, 150 and 115 kDa. Thus in PO ovaries, unlike in PA ovaries, the majority of AKAPs are bound to R subunits. These results suggest that altered PKA-AKAP interactions may contribute to the distinct responses of PA and PO follicles to high levels of cAMP, and that higher cAMP levels are required to activate PKA in PO ovaries.
Decorin endocytosis: structural features of heparin and heparan sulphate oligosaccharides interfering with receptor binding and endocytosis
Authors: HAUSSER Heinz , KRESSE Hans
Published: August 2015

Receptor-mediated endocytosis of decorin depends on its core-protein-mediated interaction with a 51 kDa membrane protein, which, in addition to its core-protein-binding site, carries a binding site for glycosaminoglycan chains. Membrane-associated heparan sulphate as well as heparin are known to have an inhibitory effect on decorin endocytosis by cultured skin fibroblasts. In this study, structural features of both glycosaminoglycans required for binding to the 51 kDa protein and for inhibiting decorin endocytosis, were investigated. Upon digestion of [3H]glucosamine-labelled heparan sulphate with heparinase III, dodeca- and higher saccharides were able to interact with the receptor protein. In comparison with unbound fragments of the same size, bound fragments were enriched in N-sulphated disaccharides carrying one or two sulphate ester groups. Using heparinase III-generated fragments from [35S]sulphate-labelled heparan sulphate chains, binding of fragments as small as octasaccharides could be detected. Competition experiments between dermatan sulphate and chemically modified heparin revealed that N- and 6-O-sulphation of glucosamine residues are important structural elements for binding to the receptor, whereas iduronate-2-O-sulphate groups contribute to binding only to a limited extent. However, with respect to the inhibition of decorin endocytosis, 2-O-desulphation had a quantitatively similar effect to 6-O-desulphation. Furthermore, for maximal inhibition of decorin endocytosis, longer fragments were required than for binding to the receptor. Thus, it appears that heparin/heparan sulphate has to interact with additional component(s) for effective inhibition of decorin uptake.
Function of human mitochondrial 2,4-dienoyl-CoA reductase and rat monofunctional Δ3-Δ2-enoyl-CoA isomerase in β-oxidation of unsaturated fatty acids
Authors: GURVITZ Aner , WABNEGGER Leila , YAGI Ahmed I. , BINDER Maximilian , HARTIG Andreas , RUIS Helmut , HAMILTON Barbara , DAWES Ian W. , HILTUNEN J. Kalervo , ROTTENSTEINER Hanspeter
Published: August 2015

Human 2,4-dienoyl-CoA reductase (2,4-reductase; DECR) and rat monofunctional ∆3-∆2-enoyl-CoA isomerase (rat 3,2-isomerase; ECI) are thought to be mitochondrial auxiliary enzymes involved in the β-oxidation of unsaturated fatty acids. However, their function during this process has not been demonstrated. Although they lack obvious peroxisomal targeting signals (PTSs), both proteins have been suggested previously to also occur in the mammalian peroxisomal compartment. The putative function and peroxisomal location of the two mammalian proteins can be examined in yeast, since β-oxidation of unsaturated fatty acids is a compartmentalized process in Saccharomyces cerevisiae requiring peroxisomal 2,4-dienoyl-CoA reductase (Sps19p) and peroxisomal 3,2-isomerase (Eci1p). A yeast sps19∆ mutant expressing human 2,4-reductase ending with the native C-terminus could not grow on petroselinic acid [cis-C18:1(6)] medium but could grow when the protein was extended with a PTS tripeptide, SKL (Ser-Lys-Leu). We therefore reason that the human protein is a physiological 2,4-reductase but that it is probably not peroxisomal. Rat 3,2-isomerase expressed in a yeast eci1∆ strain was able to re-establish growth on oleic acid [cis-C18:1(9)] medium irrespective of an SKL extension. Since we had shown that ∆2,4 double bonds could not be metabolized extra-peroxisomally to restore growth of the sps19∆ strain, we postulate that rat 3,2-isomerase acted on the ∆3 unsaturated metabolite of oleic acid by replacing the mutant's missing activity from within the peroxisomes. Immunoblotting of fractionated yeast cells expressing rat 3,2-isomerase in combination with electron microscopy supported our proposal that the protein functioned in peroxisomes. The results presented here shed new light on the function and location of human mitochondrial 2,4-reductase and rat monofunctional 3,2-isomerase.
Ligand-specific utilization of the extracellular membrane-proximal region of the gp130-related signalling receptors
Authors: HAMMACHER Annet , WIJDENES John , HILTON Douglas J. , NICOLA Nicos A. , SIMPSON Richard J. , LAYTON Judith E.
Published: August 2015

The receptor gp130 is used by the interleukin-6 (IL-6)-type cytokines, which include IL-6 and leukaemia-inhibitory factor (LIF). To investigate the role of the three extracellular membrane-proximal fibronectin-type-III-like (FNIII) modules of gp130 and the related receptor for granulocyte colony-stimulating factor (G-CSFR) in cytokine signal transduction we have transfected into murine myeloid M1-UR21 cells the chimaera (GR-FNIII)gp130, which contains the membrane-proximal FNIII modules of the G-CSFR on a gp130 backbone, and its complement, the chimaera (gp130-FNIII)GR. Whereas the binding affinities of 125I-labelled IL-6 to (GR-FNIII)gp130, or of 125I-Tyr1,3-G-CSF to (gp130-FNIII)GR, were similar to wild-type gp130 and wild-type G-CSFR, respectively, 125I-LIF failed to bind with high affinity to (GR-FNIII)gp130. In assays measuring differentiation the (gp130-FNIII)GR cells were fully responsive to G-CSF, whereas the (GR-FNIII)gp130 cells responded fully to the agonistic anti-gp130 monoclonal antibody (mAb) B-S12, but not to IL-6 or LIF. Neutralizing mAbs that recognize the membrane-proximal FNIII modules of gp130 or the G-CSFR differentially interfered with signalling by B-S12, LIF and G-CSF. The data suggest that B-S12 and G-CSF induce the correct orientation or conformation for signalling by the wild-type and chimaeric homodimeric receptors, that the membrane-proximal region of gp130 is important for the correct formation of the signalling IL-6-IL-6 receptor-gp130 complex and that this region is also involved in LIF-dependent receptor heterodimerization and signalling.
Characterization of class II apurinic/apyrimidinic endonuclease activities in the human malaria parasite, Plasmodium falciparum
Authors: HALTIWANGER Brett M. , KARPINICH Natalie O. , TARASCHI Theodore F.
Published: August 2015

We have reported that the human malaria parasite, Plasmodium falciparum, repairs apurinic/apyrimidinic (AP) sites on DNA by a long-patch base excision repair (BER) pathway. This biology is different from that in mammalian cells, which predominantly repair AP sites by a DNA-polymerase-β-dependent, one-nucleotide patch BER pathway. As a starting point for the identification and biochemical characterization of the enzymes involved in the parasite DNA BER pathway, we chose characterization of the AP endonuclease activity in a P. falciparum cell-free lysate. Evidence is provided for the presence of class II, Mg2+-dependent and independent AP endonucleases in the parasite lysate. The investigation of the processing of AP sites in Plasmodium will provide new information about long-patch BER pathways; if they are different from those in the human host they might provide a new target for anti-malarial chemotherapy.
Intrinsic alcohol dehydrogenase and hydroxysteroid dehydrogenase activities of human mitochondrial short-chain L-3-hydroxyacyl-CoA dehydrogenase
Authors: HE Xue-Ying , YANG Ying-Zi , SCHULZ Horst , YANG Song-Yu
Published: August 2015

The alcohol dehydrogenase (ADH) activity of human short-chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) has been characterized kinetically. The kcat of the purified enzyme was estimated to be 2.2 min-1, with apparent Km values of 280 mM and 22μM for 2-propanol and NAD+, respectively. The kcat of the ADH activity was three orders of magnitude less than the L-3-hydroxyacyl-CoA dehydrogenase activity but was comparable with that of the enzyme's hydroxysteroid dehydrogenase (HSD) activity for oxidizing 17β-oestradiol [He, Merz, Mehta, Schulz and Yang (1999) J. Biol. Chem. 274, 15014-15019]. However, the kcat values of intrinsic ADH and HSD activities of human SCHAD were found to be two orders of magnitude less than those reported for endoplasmic-reticulum-associated amyloid β-peptide-binding protein (ERAB) [Yan, Shi, Zhu, Fu, Zhu, Zhu, Gibson, Stern, Collison, Al-Mohanna et al. (1999) J. Biol. Chem. 274, 2145-2156]. Since human SCHAD and ERAB apparently possess identical amino acid sequences, their catalytic properties should be identical. The recombinant SCHAD has been confirmed to be the right gene product and not a mutant variant. Steady-state kinetic measurements and quantitative analyses reveal that assay conditions such as pH and concentrations of coenzyme and substrate do not account for the kinetic differences reported for ERAB and SCHAD. Rather problematic experimental procedures appear to be responsible for the unrealistically high catalytic rate constants of ERAB. Eliminating the confusion surrounding the catalytic properties of this important multifunctional enzyme paves the way for exploring its role(s) in the pathogenesis of Alzheimer's disease.
Reaction mechanism of recombinant 3-oxoacyl-(acyl-carrier-protein) synthase III from Cuphea wrightii embryo, a fatty acid synthase type II condensing enzyme
Authors: ABBADI Amine , BRUMMEL Monika , SCHüTT Burkhardt S. , SLABAUGH Mary B. , SCHUCH Ricardo , SPENER Friedrich
Published: August 2015

A unique feature of fatty acid synthase (FAS) type II of higher plants and bacteria is 3-oxoacyl-[acyl-carrier-protein (ACP)] synthase III (KAS III), which catalyses the committing condensing reaction. Working with KAS IIIs from Cuphea seeds we obtained kinetic evidence that KAS III catalysis follows a Ping-Pong mechanism and that these enzymes have substrate-binding sites for acetyl-CoA and malonyl-ACP. It was the aim of the present study to identify these binding sites and to elucidate the catalytic mechanism of recombinant Cuphea wrightii KAS III, which we expressed in Escherichia coli. We engineered mutants, which allowed us to dissect the condensing reaction into three stages, i.e. formation of acyl-enzyme, decarboxylation of malonyl-ACP, and final Claisen condensation. Incubation of recombinant enzyme with [1-14C]acetyl-CoA-labelled Cys111, and the replacement of this residue by Ala and Ser resulted in loss of overall condensing activity. The Cys111Ser mutant, however, still was able to bind acetyl-CoA and to catalyse subsequent binding and decarboxylation of malonyl-ACP to acetyl-ACP. We replaced His261 with Ala and Arg and found that the former lost activity, whereas the latter retained overall condensing activity, which indicated a general-base action of His261. Double mutants Cys111Ser/His261Ala and Cys111Ser/His261Arg were not able to catalyse overall condensation, but the double mutant containing Arg induced decarboxylation of [2-14C]malonyl-ACP, a reaction indicating the role of His261 in general-acid catalysis. Finally, alanine scanning revealed the involvement of Arg150 and Arg306 in KAS III catalysis. The results offer for the first time a detailed mechanism for a condensing reaction catalysed by a FAS type II condensing enzyme.
14-3-3 Isotypes facilitate coupling of protein kinase C-ζ to Raf-1: negative regulation by 14-3-3 phosphorylation
Authors: VAN DER HOEVEN Paulus C. J. , VAN DER WAL José C. M. , RUURS Paula , VAN DIJK Marc C. M. , VAN BLITTERSWIJK Wim J.
Published: August 2015

14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-ζ (PKC-ζ) can phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-ζ-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-ζ and Raf-1 is mediated strongly by the 14-3-3β and -θ isotypes, but not by 14-3-3ζ. Far-Western blotting revealed that 14-3-3 binds PKC-ζ directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3β and Raf-1. Treatment of PKC-ζ with lambda protein phosphatase also reduced its binding to 14-3-3β in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3β facilitated PKC-ζ binding. Together, the results suggest that 14-3-3 binds both PKC-ζ (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-ζ mutant than with wild-type PKC-ζ, supporting the idea that kinase activity leads to complex dissociation. 14-3-3β and -θ were substrates for PKC-ζ, whereas 14-3-3ζ was not. Phosphorylation of 14-3-3β by PKC-ζ negatively regulated their physical association. 14-3-3β with its putative PKC-ζ phosphorylation sites mutated enhanced co-precipitation between PKC-ζ and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-ζ weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-ζ to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and activation by PKC-ζ.
Molecular cloning, sequencing and expression studies of the human breast cancer cell glutaminase
Authors: GÓMEZ-FABRE Pedro M. , ALEDO Juan C. , DEL CASTILLO-OLIVARES Antonio , ALONSO Francisco J. , NÚÑEZ DE CASTRO Ignacio , CAMPOS José A. , MÁRQUEZ Javier
Published: August 2015

Phosphate-activated glutaminase (GA) is overexpressed in certain types of tumour but its exact role in tumour cell growth and proliferation is unknown. Here we describe the isolation of a full-length cDNA clone of human breast cancer ZR75 cells, by a combination of λgt10 cDNA library screening and the rapid amplification of cDNA ends (‘RACE’) technique. The cDNA of human GA is 2408 nt with a 1806-base open reading frame encoding a 602-residue protein with a predicted molecular mass of 66309 Da. The deduced amino acid sequence contains a putative mitochondrial import presequence of 14 residues at the N-terminal end. Heterologous expression and purification in Escherichia coli yielded a product of the expected molecular size that was recognized by using antibodies against the recombinant human GA. Sequence analyses showed that human GA was highly similar to the rat liver enzyme. Northern gel analysis revealed that the gene is present in human liver, brain and pancreas, in which a major transcript of 2.4 kb was demonstrated, but not in kidney, heart, skeletal muscle, lung or placenta. These results strongly suggest that the first human GA cloned, the GA from ZR-75 breast cancer cells, and presumably those from human liver and brain, are liver-type isoenzymes, in sharp contrast with the present view that considers the kidney type as the isoform expressed in all tissues with GA activity, with the exception of postnatal liver.
Lack of a phenotype in transgenic mice aberrantly expressing COL2A1 mRNA because of highly selective post-transcriptional down-regulation
Authors: YUAN Constance M. , ALA-KOKKO Leena , GUELLEC Dominique LE , FRANC Suzanne , FERTALA Andrzej , KHILLAN Jaspal S. , SOKOLOV Boris P. , PROCKOP Darwin J.
Published: August 2015

We reported previously that a 1.9-kb 5ʹ-fragment from the human COL1A1 gene drove transcription of a promoterless human COL2A1 gene in tissues of transgenic mice that normally express the COL1A1 but not the COL2A1 gene. In the present study, we have established that the aberrant transcription of the COL2A1 gene did not produce any gross or microscopic phenotype, because the transcripts were not efficiently translated in cells that do not normally express the COL2A1 gene. In two lines of transgenic mice, the mRNA levels from the transgene were 30% to 45% of the mRNA for the proα1(I) chain of type I procollagen, the most abundant mRNA in the same tissues. Analysis of collagens extracted from skin of the transgenic mice indicated that triple-helical type II collagen, with the normal pattern of cyanogen bromide peptides, was synthesized from the transgene. However, the level of type II collagen in skin was less than 2% of the level of type I collagen. Hybridization in situ indicated the presence of mRNA for both COL2A1 and COL1A1 in the same cells. Immunofluorescence staining for type II collagen, however, was negative in the same tissues. The results, therefore, indicated that many mesenchymal cells in the transgenic mice had high steady-state levels of the homologous mRNAs for type I and type II procollagen, but only the mRNAs for type I procollagen were efficiently translated.
Role of phosphoinositide 3-kinase and the Cbl adaptor protein in coupling the α4β1 integrin to mitogen-activated protein kinase signalling
Authors: FINKELSTEIN Lisa D. , SHIMIZU Yoji
Published: August 2015

Cell adhesion mediated by β1 integrin receptors leads to the initiation of intracellular signals that affect cell differentiation and survival. Here we have analysed the mechanism by which the α4β1 integrin activates the mitogen-activated protein kinase pathway in HL60 cells, a myelomonocytic cell line that lacks the expression of focal adhesion kinase. A role for phosphoinositide 3-kinase (PI-3K) in α4 integrin-mediated activation of extracellular signal-regulated protein kinase 2 (ERK2) is suggested by the ability of PI-3K inhibitors and a dominant-negative form of the p85 subunit of PI-3K to block the activation of ERK2 by integrin. Stimulation of α4β1 integrins on HL60 cells also leads to increased tyrosine phosphorylation of the 120 kDa adaptor protein Cbl. PI-3K activity associated with Cbl also increases on the stimulation of α4β1 integrins, although immunodepletion experiments suggest that Cbl-associated PI-3K does not account for all of the PI-3K activity induced on the stimulation of integrins in these cells. The expression of wild-type Cbl or the 70Z/3 Cbl mutant enhances basal ERK2 activity in transfectants with a minimal effect on α4 integrin-mediated ERK2 activity. In contrast, overexpression of the Hut Cbl truncation mutant, which does not associate with p85, has no effect on the ERK2 pathway. These results suggest that PI-3K has a major role in coupling α4β1 integrins to ERK2 activation in myeloid cells and that the Cbl adaptor protein has a role in basal, but not α4β1 integrin-mediated, activation of ERK2.
The regulation of AMP-activated protein kinase by phosphorylation
Authors: STEIN Silvie C. , WOODS Angela , JONES Neil A. , DAVISON Matthew D. , CARLING David
Published: August 2015

The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (α) of AMPK (Thr172) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr172 on AMPK activity. Mutation of Thr172 to an aspartic acid residue (T172D) in either α1 or α2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr172 to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr172 accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the α subunit and one site on the β subunit. Furthermore, we provide evidence that phosphorylation of Thr172 may be involved in the sensitivity of the AMPK complex to AMP.
Racemization and isomerization of type I collagen C-telopeptides in human bone and soft tissues: assessment of tissue turnover
Authors: GINEYTS Evelyne , CLOOS Paul A. C. , BOREL Olivier , GRIMAUD Laurent , DELMAS Pierre D. , GARNERO Patrick
Published: August 2015

Urinary excretion of the type I collagen C-telopeptide (CTx) has been shown to be a sensitive index of the rate of bone resorption. The human type I collagen sequence A1209HDGGR1214 of CTx can undergo racemization of the aspartic acid residue Asp1211 and isomerization of the bond between this residue and Gly1212. These spontaneous non-enzymic chemical reactions takes place in vivo in bone, and the degree of racemization and isomerization of CTx molecules may be an index of the biological age and the remodelling of bone. The aim of the present study was to investigate the degree of racemization and isomerization of type I collagen in human connective soft tissues, in order to estimate the rate of collagen turnover in adult tissues and compare it with that of bone. We also performed a systematic evaluation of the pyridinium cross-link content in adult human tissues. Using antibodies raised against the different CTx forms, we found that bone and dermis are the tissues that show most racemization and isomerization. The type I collagen of arteries, lung, intestine, kidney, skeletal muscle and heart shows significantly less racemization and isomerization than that of bone, suggesting that these soft tissues have a faster turnover than bone. We also found that pyridinoline and, to a lesser degree, deoxypyridinoline are distributed throughout the different tissues investigated. Because bone type I collagen is characterized by a high degree of both racemization/isomerization and deoxypyridinoline cross-linking, the concomitant assessment of these two post-translational modifications is likely to result in a highly specific marker of bone resorption.