bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular, functional and structural properties of the prolyl oligopeptidase ofTrypanosoma cruzi(POP Tc80), which is required for parasite entry into mammalian cells
Authors: BASTOS Izabela M. D. , GRELLIER Philippe , MARTINS Natalia F. , CADAVID-RESTREPO Gloria , de SOUZA-AULT Marian R. , AUGUSTYNS Koen , TEIXEIRA Antonio R. L. , SCHRÉVEL Joseph , MAIGRET Bernard , da SILVEIRA José F. , SANTANA Jaime M.
Published: May 2005

We have demonstrated that the 80 kDa POP Tc80 (prolyl oligopeptidase of Trypanosoma cruzi) is involved in the process of cell invasion, since specific inhibitors block parasite entry into non-phagocytic mammalian host cells. In contrast with other POPs, POP Tc80 is capable of hydrolysing large substrates, such as fibronectin and native collagen. In this study, we present the cloning of the POPTc80 gene, whose deduced amino acid sequence shares considerable identity with other members of the POP family, mainly within its C-terminal portion that forms the catalytic domain. Southern-blot analysis indicated that POPTc80 is present as a single copy in the genome of the parasite. These results are consistent with mapping of POPTc80 to a single chromosome. The active recombinant protein (rPOP Tc80) displayed kinetic properties comparable with those of the native enzyme. Novel inhibitors were assayed with rPOP Tc80, and the most efficient ones presented values of inhibition coefficient Ki≤1.52 nM. Infective parasites treated with these specific POP Tc80 inhibitors attached to the surface of mammalian host cells, but were incapable of infecting them. Structural modelling of POP Tc80, based on the crystallized porcine POP, suggested that POP Tc80 is composed of an α/β-hydrolase domain containing the catalytic triad Ser548–Asp631–His667 and a seven-bladed β-propeller non-catalytic domain. Docking analysis suggests that triple-helical collagen access to the catalytic site of POP Tc80 occurs in the vicinity of the interface between the two domains.
Identification and characterization of a second 4,4′-dibenzamido-2,2′-stilbenedisulphonate (DBDS)-binding site on band 3 and its relationship with the anion/proton co-transport function
Authors: SALHANY James M. , CORDES Karen S. , SLOAN Renee L.
Published: May 2005

Band 3 mediates both electroneutral AE (anion exchange) and APCT (anion/proton co-transport). Protons activate APCT and inhibit AE with the same pK (∼5.0). SDs (stilbenedisulphonates) bind to a primary, high-affinity site on band 3 and inhibit both AE and APCT functions. In this study, we present fluorescence and kinetic evidence showing that lowering the pH activates a second site on band 3, which binds DBDS (4,4′-dibenzamido-2,2′-stilbenedisulphonate) independently of chloride concentration, and that DBDS binding to the second site inhibits the APCT function of band 3. Activation of the second site correlated with loss of chloride binding to the transport site, thus explaining the lack of competition. The kinetics of DBDS binding at the second site could be simulated by a slow-transition, two-state exclusive binding mechanism (R0↔T0+D↔TD↔RD, where D represents DBDS, R0 and T0 represent alternate conformational states at the second DBDS-binding site, and TD and RD are the same two states with ligand DBDS bound), with a calculated overall Kd of 3.9 μM and a T0+D↔TD dissociation constant of 55 nM. DBDS binding to the primary SD site inhibited approx. 94% of the proton transport at low pH (KI=68.5±11.8 nM). DBDS binding to the second site inhibited approx. 68% of the proton transport (KI=7.27±1.27 μM) in a band 3 construct with all primary SD sites blocked through selective cross-linking by bis(sulphosuccinimidyl)suberate. DBDS inhibition of proton transport at the second site could be simulated quantitatively within the context of the slow-transition, two-state exclusive binding mechanism. We conclude that band 3 contains two DBDS-binding sites that can be occupied simultaneously at low pH. The binding kinetic and transport inhibition characteristics of DBDS interaction with the second site suggest that it may be located within a gated access channel leading to the transport site.
Truncated human serum albumin retains general anaesthetic binding activity
Authors: LIU Renyu , YANG Jinsheng , HA Chung-Eun , BHAGAVAN Nadhipuram V. , ECKENHOFF Roderic G.
Published: May 2005

Multiple binding sites for anaesthetics in HSA (human serum albumin) make solution studies difficult to interpret. In the present study, we expressed the wild-type HSA domain 3 (wtHSAd3), a peptide with two known anaesthetic binding sites in a yeast expression system. We also expressed a site-directed mutant of domain 3 (Y411Wd3). The stability and secondary structure of the constructed fragments were determined by HX (hydrogen–tritium exchange) and CD spectroscopy. The binding of two general anaesthetics, 2-bromo-2-chloro-1,1,1-trifluoroethane and propofol, to wtHSAd3 and Y411Wd3 was determined using isothermal titration calorimetry, HX and intrinsic tryptophan fluorescence quenching. Although the expressed fragments are less stable than intact wtHSA as indicated by both CD and HX, they retain the secondary structure and anaesthetic-binding characteristics of an intact HSA molecule, but with fewer binding sites. Y411Wd3 had decreased affinity for propofol but not for 2-bromo-2-chloro-1,1,1-trifluoroethane, consistent with steric hindrance. Retention of structural features and anaesthetic binding properties with fewer binding sites in this truncated protein provide feasibility for using scaled-down models of otherwise intractable systems to gain an understanding of anaesthetic binding requirements and binding–stability relationships.
Role of the ERC motif in the proximal part of the second intracellular loop and the C-terminal domain of the human prostaglandin F2α receptor (hFP-R) in G-protein coupling control
Authors: PATHE-NEUSCHÄFER-RUBE Andrea , NEUSCHÄFER-RUBE Frank , PÜSCHEL Gerhard P.
Published: May 2005

The human FP-R (F2α prostaglandin receptor) is a Gq-coupled heptahelical ectoreceptor, which is of significant medical interest, since it is a potential target for the treatment of glaucoma and preterm labour. On agonist exposure, it mediates an increase in intracellular inositol phosphate formation. Little is known about the structures that govern the agonist-dependent receptor activation. In other prostanoid receptors, the C-terminal domain has been inferred in the control of agonist-dependent receptor activation. A DRY motif at the beginning of the second intracellular loop is highly conserved throughout the G-protein-coupled receptor family and appears to be crucial for controlling agonist-dependent receptor activation. It is replaced by an ERC motif in the FP-R and no evidence for the relevance of this motif in ligand-dependent activation of prostanoid receptors has been provided so far. The aim of the present study was to elucidate the potential role of the C-terminal domain and the ERC motif in agonist-controlled intracellular signalling in FP-R mutants generated by site-directed mutagenesis. It was found that substitution of the acidic Glu132 in the ERC motif by a threonine residue led to full constitutive activation, whereas truncation of the receptor's C-terminal domain led to partial constitutive activation of all three intracellular signal pathways that had previously been shown to be activated by the FP-R, i.e. inositol trisphosphate formation, focal adhesion kinase activation and T-cell factor signalling. Inositol trisphosphate formation and focal adhesion kinase phosphorylation were further enhanced by ligand binding in cells expressing the truncation mutant but not the E132T (Glu132→Thr) mutant. Thus C-terminal truncation appeared to result in a receptor with partial constitutive activation, whereas substitution of Glu132 by threonine apparently resulted in a receptor with full constitutive activity.
Growth-factor-dependent phosphorylation of Bim in mitosis
Authors: GRÃOS Mário , ALMEIDA Alexandra D. , CHATTERJEE Sukalyan
Published: May 2005

The regulation of survival and cell death is a key determinant of cell fate. Recent evidence shows that survival and death machineries are regulated along the cell cycle. In the present paper, we show that BimEL [a BH3 (Bcl-2 homology 3)-only member of the Bcl-2 family of proteins; Bim is Bcl-2-interacting mediator of cell death; EL is the extra-long form] is phosphorylated in mitosis. This post-translational modification is dependent on MEK (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase) and growth factor signalling. Interestingly, FGF (fibroblast growth factor) signalling seems to play an essential role in this process, since, in the presence of serum, inhibition of FGF receptors abrogated phosphorylation of Bim in mitosis. Moreover, we have shown bFGF (basic FGF) to be sufficient to induce phosphorylation of Bim in serum-free conditions in any phase of the cell cycle, and also to significantly rescue cells from serum-deprivation-induced apoptosis. Our results show that, in mitosis, Bim is phosphorylated downstream of growth factor signalling in a MEK-dependent manner, with FGF signalling playing an important role. We suggest that phosphorylation of Bim is a decisive step for the survival of proliferating cells.
Characterization of two novel pacifastin-like peptide precursor isoforms in the desert locust (Schistocerca gregaria): cDNA cloning, functional analysis and real-time RT-PCR gene expression studies
Authors: SIMONET Gert , BREUGELMANS Bert , PROOST Paul , CLAEYS Ilse , VAN DAMME Jozef , DE LOOF Arnold , VANDEN BROECK Jozef
Published: May 2005

In the last decade, a new serine protease inhibitor family has been described in arthropods. Eight members of the family were purified from locusts and share a conserved cysteine array (Cys-Xaa9–12-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa2–3-Gly-Xaa3–6-Cys-Thr-Xaa3-Cys) with nine inhibitory domains of the light chain of the crayfish protease inhibitor, pacifastin (PLDs; pacifastin light chain domains). Using cDNA cloning, several pacifastin-related precursors have been identified, encoding additional PLD-related peptides in different insect species. In the present study, two isoforms of a novel pacifastin-related precursor (SGPP-4) have been identified in the desert locust, predicting the previously identified SGPI-5 (Schistocerca gregaria PLD-related inhibitor-5) peptide and two novel PLD-related peptide sequences. One novel peptide (SGPI-5A) was synthesized chemically, and its inhibitory activity was assessed in vitro. Although proteases from a locust midgut extract were very sensitive to SGPI-5A, the same peptide proved to be a relatively poor inhibitor of bovine trypsin. By an in silico datamining approach, a novel pacifastin-related precursor with seven PLD-related domains was identified in the mosquito, Aedes aegypti. As in other insect pacifastin-related precursors, the Aedes precursor showed a particular domain architecture that is not encountered in other serine protease inhibitor families. Finally, a comparative real-time RT-PCR analysis of SGPP-4 transcripts in different tissues of isolated- (solitarious) and crowded-reared (gregarious) locusts was performed. This showed that SGPP-4 mRNA levels are higher in the brain, testes and fat body of gregarious males than of solitarious males. These results have been compared with data from a similar study on SGPP-1–3 transcripts and discussed with respect to a differential regulation of serine-protease-dependent pathways as a possible mechanism underlying locust phase polymorphism.
The transcriptional co-activator TAZ interacts differentially with transcriptional enhancer factor-1 (TEF-1) family members
Authors: MAHONEY William M. , HONG Jeong-Ho , YAFFE Michael B. , FARRANCE Iain K. G.
Published: May 2005

Members of the highly related TEF-1 (transcriptional enhancer factor-1) family (also known as TEAD, for TEF-1, TEC1, ABAA domain) bind to MCAT (muscle C, A and T sites) and A/T-rich sites in promoters active in cardiac, skeletal and smooth muscle, placenta, and neural crest. TEF-1 activity is regulated by interactions with transcriptional co-factors [p160, TONDU (Vgl-1, Vestigial-like protein-1), Vgl-2 and YAP65 (Yes-associated protein 65 kDa)]. The strong transcriptional co-activator YAP65 interacts with all TEF-1 family members, and, since YAP65 is related to TAZ (transcriptional co-activator with PDZ-binding motif), we wanted to determine if TAZ also interacts with members of the TEF-1 family. In the present study, we show by GST (glutathione S-transferase) pull-down assays, by co-immunoprecipitation and by modified mammalian two-hybrid assays that TEF-1 interacts with TAZ in vitro and in vivo. Electrophoretic mobility-shift assays with purified TEF-1 and GST–TAZ fusion protein showed that TAZ interacts with TEF-1 bound to MCAT DNA. TAZ can interact with endogenous TEF-1 proteins, since exogenous TAZ activated MCAT-dependent reporter promoters. Like YAP65, TAZ interacted with all four TEF-1 family members. GST pull-down assays with increasing amounts of [35S]TEF-1 and [35S]RTEF-1 (related TEF-1) showed that TAZ interacts more efficiently with TEF-1 than with RTEF-1. This differential interaction also extended to the interaction of TEF-1 and RTEF-1 with TAZ in vivo, as assayed by a modified mammalian two-hybrid experiment. These data show that differential association of TEF-1 proteins with transcriptional co-activators may regulate the activity of TEF-1 family members.
Amino acids in the pore region of Kv1 potassium channels dictate cell-surface protein levels: a possible trafficking code in the Kv1 subfamily
Authors: ZHU Jing , GOMEZ Barbara , WATANABE Itaru , THORNHILL William B.
Published: May 2005

Kv1.1 and Kv1.4 potassium channels have different pore region determinants that were found to affect their cell-surface levels positively and negatively [Zhu, Watanabe, Gomez and Thornhill (2001) J. Biol. Chem. 276, 39419–39427; Zhu, Watanabe, Gomez and Thornhill (2003) J. Biol. Chem. 278, 25558–25567; Zhu, Watanabe, Gomez and Thornhill (2003) Biochem. J. 375, 761–768]. In the present study, we focused on the deep pore region of Kv1 members to test whether a cell-surface trafficking code was dictated by two amino acids. Kv1 channels with a threonine/lysine amino acid pair in a non-contiguous pore region promoted high surface levels, whereas a serine/tyrosine amino acid pair inhibited high surface expression by inducing a high level of partial endoplasmic reticulum retention. Our work suggests that a possible positive trafficking amino acid pair coding here for the Kv1 subfamily is Thr/Lys>Thr/Val>Thr/Tyr>Thr/Arg∼Thr/His>Ser/Val>Ser/Tyr>Ser/Lys. The Kv1 trafficking code was not transferable to a Kv2 family member and thus it appears that it only governs surface levels in the context of its Kv1 native pore loop region and/or its S5 and S6 regions. All members of a given Kv2, Kv3 or Kv4 potassium channel subfamily have identical amino acids at similar positions in their deep pore regions (Thr/Tyr or Thr/Val), which suggests that any difference in surface levels among members is not dictated by these amino acids. Thus a major determinant for cell-surface trafficking of Kv1 potassium channels is an amino acid pair in their deep pore regions, whereas the cell-surface levels of a given Kv2, Kv3 or Kv4 subfamily member are probably not affected by these amino acids.
A mammalian fatty acid hydroxylase responsible for the formation of α-hydroxylated galactosylceramide in myelin
Authors: ECKHARDT Matthias , YAGHOOTFAM Afshin , FEWOU Simon N. , ZÖLLER Inge , GIESELMANN Volkmar
Published: May 2005

Hydroxylation is an abundant modification of the ceramides in brain, skin, intestinal tract and kidney. Hydroxylation occurs at the sphingosine base at C-4 or within the amide-linked fatty acid. In myelin, hydroxylation of ceramide is exclusively found at the α-C atom of the fatty acid moiety. α-Hydroxylated cerebrosides are the most abundant lipids in the myelin sheath. The functional role of this modification, however, is not known. On the basis of sequence similarity to a yeast C26 fatty acid hydroxylase, we have identified a murine cDNA encoding FA2H (fatty acid 2-hydroxylase). Transfection of FA2H cDNA in CHO cells (Chinese-hamster ovary cells) led to the formation of α-hydroxylated fatty acid containing hexosylceramide. An EGFP (enhanced green fluorescent protein)–FA2H fusion protein co-localized with calnexin, indicating that the enzyme resides in the endoplasmic reticulum. FA2H is expressed in brain, stomach, skin, kidney and testis, i.e. in tissues known to synthesize fatty acid α-hydroxylated sphingolipids. The time course of its expression in brain closely follows the expression of myelin-specific genes, reaching a maximum at 2–3 weeks of age. This is in agreement with the reported time course of fatty acid α-hydroxylase activity in the developing brain. In situ hybridization of brain sections showed expression of FA2H in the white matter. Our results thus strongly suggest that FA2H is the enzyme responsible for the formation of α-hydroxylated ceramide in oligodendrocytes of the mammalian brain. Its further characterization will provide insight into the functional role of α-hydroxylation modification in myelin, skin and other organs.
Plasmin alters the activity and quaternary structure of human plasma carboxypeptidase N
Authors: QUAGRAINE Mercy O. , TAN Fulong , TAMEI Hironori , ERDÖS Ervin G. , SKIDGEL Randal A.
Published: May 2005

Human CPN (carboxypeptidase N) is a tetrameric plasma enzyme containing two glycosylated 83 kDa non-catalytic/regulatory subunits that carry and protect two active catalytic subunits. Because CPN can regulate the level of plasminogen binding to cell surface proteins, we investigated how plasmin cleaves CPN and the consequences. The products of hydrolysis were analysed by activity assays, Western blotting, gel filtration and sequencing. When incubated with intact CPN tetramer, plasmin rapidly cleaved the 83 kDa subunit at the Arg457–Ser458 bond near the C-terminus to produce fragments of 72 and 13 kDa, thereby releasing an active 142 kDa heterodimer, and also cleaved the active subunit, decreasing its size from 55 kDa to 48 kDa. Further evidence for the heterodimeric form of CPN was obtained by re-complexing the non-catalytic 72 kDa fragment with recombinant catalytic subunit or by immunoprecipitation of the catalytic subunit after plasmin treatment of CPN using an antibody specific for the 83 kDa subunit. Upon longer incubation, plasmin cleaved the catalytic subunit at Arg218–Arg219 to generate fragments of 27 kDa and 21 kDa, held together by non-covalent bonds, that were more active than the native enzyme. These data show that plasmin can alter CPN structure and activity, and that the C-terminal 13 kDa fragment of the CPN 83 kDa subunit is a docking peptide that is necessary to maintain the stable active tetrameric form of human CPN in plasma.
Flexibility in substrate recognition by thimet oligopeptidase as revealed by denaturation studies
Authors: SIGMAN Jeffrey A. , PATWA Tasneem H. , TABLANTE Ana V. , JOSEPH Calleen D. , GLUCKSMAN Marc J. , WOLFSON Adele J.
Published: May 2005

Thimet oligopeptidase (TOP) is a soluble metalloendopeptidase belonging to a family of enzymes including neurolysin and neprilysin that utilize the HEXXH metal-binding motif. TOP is widely distributed among cell types and is able to cleave a number of structurally unrelated peptides. A recent focus of interest has been on structure–function relationships in substrate selectivity by TOP. The enzyme's structural fold comprises two domains that are linked at the bottom of a deep substrate-binding cleft via several flexible loop structures. In the present study, fluorescence spectroscopy has been used to probe structural changes in TOP induced by the chemical denaturant urea. Fluorescence emission, anisotropy and collisional quenching data support a two-step unfolding process for the enzyme in which complete loss of the tertiary structure occurs in the second step. Complete loss of activity and loss of catalytic Zn(II) from the active site, monitored by absorption changes of the metal chelator 4-(2-pyridylazo)-resorcinol, are also connected with the second step. In contrast, the first unfolding event, which is linked to changes in the non-catalytic domain, leads to a sharp increase in kcat towards a 9-residue substrate and a sharp decrease in kcat for a 5-residue substrate. Thus a conformational change in TOP has been directly correlated with a change in substrate selectivity. These results provide insight into how the enzyme can process the range of structurally unrelated peptides necessary for its many physiological roles.
Characterization of human SCD2, an oligomeric desaturase with improved stability and enzyme activity by cross-linking in intact cells
Authors: ZHANG Shaobo , YANG Yanzhu , SHI Yuguang
Published: May 2005

SCD (stearoyl-CoA desaturase) catalyses the conversion of saturated fatty acids into mono-unsaturated fatty acids, a critical step involved in lipid metabolism and various other biological functions. In the present study, we report the identification and characterization of a human gene that encodes a novel SCD enzyme (hSCD2). The hSCD2 gene codes for a 37.5-kDa protein that shares 61% and 57% sequence identity with the human SCD1 and mouse SCD2 enzymes respectively. The recombinant hSCD2 enzyme expressed in mammalian and Sf9 insect cells efficiently catalysed desaturation of both stearoyl- and palmitoyl-CoAs to the corresponding mono-unsaturated fatty acids. In comparison with the hSCD1 gene that is predominantly expressed in liver, hSCD2 is most abundantly expressed in pancreas and brain. Additionally, hSCD2 transcripts from adult and foetal tissues exhibit different sizes because of alternative splicing in the non-coding region, suggesting that hSCD2 expression is developmentally regulated. The recombinant human SCD2 and SCD1 transiently expressed in COS-7 cells exhibited as oligomeric proteins that consist of homodimers and oligomers when resolved by SDS/PAGE. The complex formation was independent of SCD protein expression levels, as supported by a relatively constant ratio of the level of dimers and oligomers to that of the monomers from COS-7 cells transiently transfected with different amounts of SCD expression vectors. Furthermore, treatment of intact COS-7 cells with a cross-linking reagent resulted in dose-dependent increases in the levels of SCD protein and activity, suggesting that oligomerization may play an important role in regulating the stability of SCD enzymes.
IRP1 Ser-711 is a phosphorylation site, critical for regulation of RNA-binding and aconitase activities
Authors: FILLEBEEN Carine , CALTAGIRONE Annie , MARTELLI Alain , MOULIS Jean-Marc , PANTOPOULOS Kostas
Published: May 2005

In iron-starved cells, IRP1 (iron regulatory protein 1) binds to mRNA iron-responsive elements and controls their translation or stability. In response to increased iron levels, RNA-binding is inhibited on assembly of a cubane [4Fe-4S] cluster, which renders IRP1 to a cytosolic aconitase. Phosphorylation at conserved serine residues may also regulate the activities of IRP1. We demonstrate that Ser-711 is a phosphorylation site in HEK-293 cells (human embryonic kidney 293 cells) treated with PMA, and we study the effects of the S711E (Ser-711→Glu) mutation on IRP1 functions. A highly purified preparation of recombinant IRP1S711E displays negligible IRE-binding and aconitase activities. It appears that the first step in the aconitase reaction (conversion of citrate into the intermediate cis-aconitate) is more severely affected, as recombinant IRP1S711E retains approx. 45% of its capacity to catalyse the conversion of cis-aconitate into the end-product isocitrate. When expressed in mammalian cells, IRP1S711E completely fails to bind to RNA and to generate isocitrate from citrate. We demonstrate that the apparent inactivation of IRP1S711E is not related to mutation-associated protein misfolding or to alterations in its stability. Sequence analysis of IRP1 from all species currently deposited in protein databases shows that Ser-711 and flanking sequences are highly conserved in the evolutionary scale. Our results suggest that Ser-711 is a critical residue for the control of IRP1 activities.
Kinetic isotope effects and ligand binding in PQQ-dependent methanol dehydrogenase
Authors: HOTHI Parvinder , SUTCLIFFE Michael J. , SCRUTTON Nigel S.
Published: May 2005

The reaction of PQQ (2,7,9-tricarboxypyrroloquinoline quinone)-dependent MDH (methanol dehydrogenase) from Methylophilus methylotrophus has been studied under steady-state conditions in the presence of an alternative activator [GEE (glycine ethyl ester)] and compared with similar reactions performed with ammonium (used more generally as an activator in steady-state analysis of MDH). Studies of initial velocity with methanol (protiated methanol, C1H3O1H) and [2H]methanol (deuteriated methanol, C2H3O2H) as substrate, performed with different concentrations of GEE and PES (phenazine ethosulphate), indicate competitive binding effects for substrate and PES on the stimulation and inhibition of enzyme activity by GEE. GEE is more effective at stimulating activity than ammonium at low concentrations, suggesting tighter binding of GEE to the active site. Inhibition of activity at high GEE concentration is less pronounced than at high ammonium concentration. This suggests a close spatial relationship between the stimulatory (KS) and inhibitory (KI) binding sites in that binding of GEE to the KS site sterically impairs the binding of GEE to the KI site. The binding of GEE is also competitive with the binding of PES, and GEE is more effective than ammonium in competing with PES. This competitive binding of GEE and PES lowers the effective concentration of PES at the site competent for electron transfer. Accordingly, the oxidative half-reaction, which is second-order with respect to PES concentration, is more rate-limiting in steady-state turnover with GEE than with ammonium. The smaller methanol C-1H/C-2H kinetic isotope effects observed with GEE are consistent with a larger contribution made by the oxidative half-reaction to rate limitation. Cyanide is much less effective at suppressing ‘endogenous’ activity in the presence of GEE than with ammonium, which is attributed to impaired binding of cyanide to the catalytic site through steric interaction with GEE bound at the KS site. The kinetic model developed previously for reactions of MDH with ammonium [Hothi, Basran, Sutcliffe and Scrutton (2003) Biochemistry 42, 3966–3978] is consistent with data obtained with GEE, although a more detailed structural interpretation is given here. Molecular-modelling studies rationalize the kinetic observations in terms of a complex binding scenario at the molecular level involving two spatially distinct inhibitory sites (KI and KI′). The KI′ site caps the entrance to the active site and is interpreted as the PES binding site. The KI site is adjacent to, and, for GEE, overlaps with, the KS site, and is located in the active-site cavity close to the PQQ cofactor and the catalytic site for methanol oxidation.
Molecular and genetic characterization of the interactions between the Drosophila stoned-B protein and DAP-160 (intersectin)
Authors: KELLY Leonard E. , PHILLIPS A. Marie
Published: May 2005

The stoned locus of Drosophila produces a dicistronic transcript and encodes two proteins, stoned-A (STNA) and stoned-B (STNB). Both proteins are located at synaptic terminals. The STNB protein contains a domain that has homology with the μ-subunit of the AP (adaptor protein) complex, as well as a number of NPF (Asp-Pro-Phe) motifs known to bind EH (Eps15 homology) domains. Mutations at the stoned locus interact synergistically with mutations at the shibire (dynamin) locus and alter synaptic vesicle endocytosis. The STNB protein has also been shown to interact with synaptic vesicles via synaptogamin-I. We initiated an investigation of the possible interaction of DAP-160 (dynamin-associated protein of 160 kDa), a Drosophila member of the intersectin family, with the STNB protein. We show here that both of the viable stoned alleles interacted with a genetic construct that reduces DAP-160 levels to 25% of normal. One of these stoned alleles contains a substitution resulting in a stop codon in the open reading frame encoding STNB. This allele also shows markedly reduced levels of both DAP-160 and dynamin. As anticipated, the NPF motifs in STNB are found to be high-affinity binding motifs for the EH domains of DAP-160. One of the SH3 (Src homology 3) domains of DAP-160 also interacts with STNB. Finally, we show that immunoprecipitation of STNB from fly head extracts co-precipitates with DAP-160, and we conclude that the interaction of the STNB protein with both synaptotagmin I and DAP-160 may regulate synaptic vesicle recycling by recruiting dynamin to a pre-fission complex.
Interactions between Cdc42 and the scaffold protein Scd2: requirement of SH3 domains for GTPase binding
Authors: WHEATLEY Edward , RITTINGER Katrin
Published: May 2005

The multi-domain protein Scd2 acts as a scaffold upon which the small GTPase Cdc42 (cell division cycle 42), its nucleotide-exchange factor Scd1 and the p21-activated kinase Shk1 assemble to regulate cell polarity and the mating response in fission yeast. In the present study, we show using isothermal titration calorimetry that Scd2 binds two molecules of active GTP-bound Cdc42 simultaneously, but independently of one another. The two binding sites have significantly different affinities, 21 nM and 3 μM, suggesting that they play distinct roles in the Shk1 signalling network. Each of the Cdc42-binding sites includes one of the SH3 (Src homology 3) domains of Scd2. Our data indicate that complex formation does not occur in a conventional manner via the conserved SH3 domain ligand-binding surface. Neither of the isolated SH3 domains is sufficient to interact with the GTPase, and they both require adjacent regions to either stabilize their conformations or contribute to the formation of the Cdc42-binding surface. Furthermore, we show that there is no evidence for an intramolecular PX–SH3 domain interaction, which could interfere with SH3 domain function. This work suggests that SH3 domains might contribute directly to signalling through small GTPases and thereby adds another aspect to the diverse nature of SH3 domains as protein–protein-interaction modules.
Quantification of endogenous retinoic acid in limited biological samples by LC/MS/MS
Authors: KANE Maureen A. , CHEN Na , SPARKS Susan , NAPOLI Joseph L.
Published: May 2005

We report a sensitive LC (liquid chromatography)/MS/MS assay using selected reaction monitoring to quantify RA (retinoic acid), which is applicable to biological samples of limited size (10–20 mg of tissue wet weight), requires no sample derivatization, provides mass identification and resolves atRA (all-trans-RA) from its geometric isomers. The assay quantifies over a linear range of 20 fmol to 10 pmol, and has a 10 fmol limit of detection at a signal/noise ratio of 3. Coefficients of variation are: instrumental, 0.5–2.9%; intra-assay, 5.4±0.4%; inter-assay 8.9±1.0%. An internal standard (all-trans-4,4-dimethyl-RA) improves accuracy by confirming extraction efficiency and revealing handling-induced isomerization. Tissues of 2–4-month-old C57BL/6 male mice had atRA concentrations of 7–9.6 pmol/g and serum atRA of 1.9±0.6 pmol/ml (±S.E.M.). Tissue 13-cis-RA ranged from 2.9 to 4.2 pmol/g, and serum 13-cis-RA was 1.2±0.3 pmol/ml. CRBP (cellular retinol-binding protein)-null mouse liver had atRA ∼30% lower than wild-type (P<0.05), but kidney, testis, brain and serum atRA were similar to wild-type. atRA in brain areas of 12-month-old female C57BL/6 mice were (±S.E.M.): whole brain, 5.4±0.4 pmol/g; cerebellum, 10.7±0.3 pmol/g; cortex, 2.6±0.4 pmol/g; hippocampus, 8.4±1.2 pmol/g; striatum, 15.3±4.7 pmol/g. These data provide the first analytically robust quantification of atRA in animal brain and in CRBP-null mice. Direct measurements of endogenous RA should have a substantial impact on investigating target tissues of RA, mechanisms of RA action, and the relationship between RA and chronic disease.
Decreased lipid efflux and increased susceptibility to cholesterol-induced apoptosis in macrophages lacking phosphatidylcholine transfer protein
Authors: BAEZ Juan M. , TABAS Ira , COHEN David E.
Published: May 2005

Macrophages are the predominant cellular component of atherosclerotic lesions, where they scavenge oxidatively modified lipoproteins while defending themselves against cholesterol-induced cytotoxicity by adaptive mechanisms that depend in part on the synthesis, distribution and efflux of phosphatidylcholines. PC-TP (phosphatidylcholine transfer protein) is a START (steroidogenic acute regulatory protein-related lipid transfer) domain protein that catalyses the intermembrane transfer of phosphatidylcholines and promotes apolipoprotein AI-mediated lipid efflux when overexpressed in the cytosol of Chinese-hamster ovary cells. To explore a role for PC-TP in the adaptive responses of macrophages to cholesterol loading, we utilized peritoneal macrophages from mice with homozygous disruption of the gene encoding PC-TP (Pctp−/−) and wild-type littermate controls. PC-TP was abundantly expressed in macrophages from wild-type but not Pctp−/− mice. In cholesteryl ester-loaded macrophages from Pctp−/− mice, the apolipoprotein AI-mediated efflux of phospholipids and cholesterol was decreased. This could be attributed to proportional decreases in the expression levels of ATP-binding cassette A1. Also, in response to free cholesterol loading, the absence of PC-TP from macrophages was associated with marked increases in apoptotic cell death. These findings suggest that PC-TP in macrophages may serve an atheroprotective role by defending against cholesterol-induced cytotoxicity.
Cellular factors required for protection from hyperoxia toxicity in Saccharomyces cerevisiae
Authors: OUTTEN Caryn E. , FALK Robert L. , CULOTTA Valeria C.
Published: May 2005

Prolonged exposure to hyperoxia represents a serious danger to cells, yet little is known about the specific cellular factors that affect hyperoxia stress. By screening the yeast deletion library, we have identified genes that protect against high-O2 damage. Out of approx. 4800 mutants, 84 were identified as hyperoxia-sensitive, representing genes with diverse cellular functions, including transcription and translation, vacuole function, NADPH production, and superoxide detoxification. Superoxide plays a significant role, since the majority of hyperoxia-sensitive mutants displayed cross-sensitivity to superoxide-generating agents, and mutants with compromised SOD (superoxide dismutase) activity were particularly vulnerable to hyperoxia. By comparison, factors known to guard against H2O2 toxicity were poorly represented amongst hyperoxia-sensitive mutants. Although many cellular components are potential targets, our studies indicate that mitochondrial glutathione is particularly vulnerable to hyperoxia damage. During hyperoxia stress, mitochondrial glutathione is more susceptible to oxidation than cytosolic glutathione. Furthermore, two factors that help maintain mitochondrial GSH in the reduced form, namely the NADH kinase Pos5p and the mitochondrial glutathione reductase (Glr1p), are critical for hyperoxia resistance, whereas their cytosolic counterparts are not. Our findings are consistent with a model in which hyperoxia toxicity is manifested by superoxide-related damage and changes in the mitochondrial redox state.
Muscle endopin 1, a muscle intracellular serpin which strongly inhibits elastase: purification, characterization, cellular localization and tissue distribution
Authors: TASSY Caroline , HERRERA-MENDEZ Carlos H. , SENTANDREU Miguel A. , AUBRY Laurent , BRÉMAUD Laure , PÉLISSIER Patrick , DELOURME Didier , BRILLARD Michèle , GAUTHIER Francis , LEVÉZIEL Hubert , OUALI Ahmed
Published: May 2005

In the present work, an endopin-like elastase inhibitor was purified for the first time from bovine muscle. A three-step chromatography procedure was developed including successively SP-Sepharose, Q-Sepharose and EMD-DEAE 650. This procedure provides about 300 μg of highly pure inhibitor from 500 g of bovine diaphragm muscle. The N-terminal sequence of the muscle elastase inhibitor, together with the sequence of a trypsin-generated peptide, showed 100% similarity with the cDNA deduced sequence of chromaffin cell endopin 1. Hence, the muscle inhibitor was designated muscle endopin 1 (mEndopin 1). mEndopin 1 had a molecular mass of 70 kDa, as assessed by both gel filtration and SDS/PAGE. According to the association rates determined, mEndopin 1 is a potent inhibitor of elastase (kass=2.41×107 M−1·s−1) and trypsin (kass=3.92×106 M−1·s−1), whereas plasmin (kass=1.78×103 M−1·s−1) and chymotrypsin (kass=1.0×102 M−1·s−1) were only moderately inhibited. By contrast, no inhibition was detected against several other selected serine proteinases, as well as against cysteine proteinases of the papain family. The cellular location of mEndopin in muscle tissue and its tissue distribution were investigated using a highly specific rabbit antiserum. The results obtained demonstrate an intracellular location and a wide distribution in bovine tissues.
A proteomics approach for the identification of nucleophosmin and heterogeneous nuclear ribonucleoprotein C1/C2 as chromatin-binding proteins in response to DNA double-strand breaks
Authors: LEE Seung Yun , PARK Ji-Hye , KIM Sungsu , PARK Eun-Jung , YUN Yungdae , KWON Jongbum
Published: May 2005

Double-strand breaks (DSBs) of chromosomal DNA trigger the cellular response that activates the pathways for DNA repair and cell-cycle checkpoints, and sometimes the pathways leading to cell death if the damage is too severe to be tolerated. Evidence indicates that, upon generation of DNA DSBs, many nuclear proteins that are involved in DNA repair and checkpoints are recruited to chromatin around the DNA lesions. In the present study we used a proteomics approach to identify DNA-damage-induced chromatin-binding proteins in a systematic way. Two-dimensional gel analysis for protein extracts of chromatin from DNA-damage-induced and control HeLa cells identified four proteins as the candidates for DNA-damage-induced chromatin-binding proteins. MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS analysis identified these proteins to be NPM (nucleophosmin), hnRNP (heterogeneous nuclear ribonucleoprotein) C1, hnRNP C2 and 37-kDa laminin-receptor precursor, and the identity of these proteins was further confirmed by immunoblot analysis with specific antibodies. We then demonstrated with chromatin-binding assays that NPM and hnRNP C1/C2, the abundant nuclear proteins with pleiotropic functions, indeed bind to chromatin in a DNA-damage-dependent manner, implicating these proteins in DNA repair and/or damage response. Immunofluorescence experiments showed that NPM, normally present in the nucleoli, is mobilized into the nucleoplasm after DNA damage, and that neither NPM nor hnRNP C1/C2 is actively recruited to the sites of DNA breaks. These results suggest that NPM and hnRNP C1/C2 may function at the levels of the global context of chromatin, rather than by specifically targeting the broken DNA.
Residue 234 in glutathione transferase T1-1 plays a pivotal role in the catalytic activity and the selectivity against alternative substrates
Authors: SHOKEER Abeer , LARSSON Anna-Karin , MANNERVIK Bengt
Published: May 2005

GST (glutathione transferase) T1-1 plays an important role in the biotransformation of halogenated alkanes, which are used in large quantities as solvents and occur as environmental pollutants. Many reactions that are catalysed by GST T1-1 qualify as detoxification processes, but some reactions with dihalogenated alkanes lead to reactive products more toxic than the substrates. Murine GST T1-1 is particularly active with dichloromethane, which may explain the high carcinogenicity of dichloromethane in the mouse. Human GST T1-1 activity is considerably lower with halogenated hydrocarbons and some related substrates. Human GST T1-1 is polymorphic with a frequent null phenotype, suggesting that it is advantageous, under some circumstances, to lack the functional enzyme, which catalyses GSH conjugations that may cause bioactivation. The present study shows that amino acid residue 234 is a determinant of the differences in catalytic efficiency between the human and the rodent enzymes. The replacement of Trp234 in human GST T1-1 by arginine, found in the rodent enzyme, enhanced the alkyltransferase activity by an order of magnitude with a series of homologous iodoalkanes and some typical GST substrates. The specific activity of the alternative mutant Trp234→Lys was lower than for the parental human GST T1-1 with many substrates, showing that a positive charge is not sufficient for increased activity. The enhanced activity of Trp234→Arg with alkylating agents was dependent on the substrate tested, whereas no increase of the peroxidase activity with cumene hydroperoxide was noted. Residue 234 therefore is also involved in the control of the substrate selectivity of GST T1-1.
Molecular evidence that melatonin is enzymatically oxidized in a different manner than tryptophan: investigations with both indoleamine 2,3-dioxygenase and myeloperoxidase
Authors: FERRY Gilles , UBEAUD Caroline , LAMBERT Pierre-Hervé , BERTIN Sophie , COGÉ Francis , CHOMARAT Pascale , DELAGRANGE Philippe , SERKIZ Bernard , BOUCHET Jean-Paul , TRUSCOTT Roger J. W. , BOUTIN Jean A.
Published: May 2005

The catabolism of melatonin, whether naturally occurring or ingested, takes place via two pathways: ∼70% can be accounted for by conjugation (sulpho- and glucurono-conjugation), and ∼30% by oxidation. It is commonly thought that the interferon-induced enzyme indoleamine 2,3-dioxygenase (EC 1.13.11.42), which oxidizes tryptophan, is also responsible for the oxidation of 5-hydroxytryptamine (serotonin) and its derivative, melatonin. Using the recombinant enzyme expressed in Escherichia coli, we show in the present work that indoleamine 2,3-dioxygenase indeed cleaves tryptophan; however, under the same conditions, it is incapable of cleaving the two other indoleamines. By contrast, myeloperoxidase (EC 1.11.1.7) is capable of cleaving the indole moiety of melatonin. However, when using the peroxidase conditions of assay – with H2O2 as co-substrate – indoleamine 2,3-dioxygenase is able to cleave melatonin into its main metabolite, a kynurenine derivative. The present work establishes that the oxidative metabolism of melatonin is due, in the presence of H2O2, to the activities of both myeloperoxidase and indoleamine 2,3-dioxygenase (with lower potency), since both enzymes have Km values for melatonin in the micromolar range. Under these conditions, several indolic compounds can be cleaved by both enzymes, such as tryptamine and 5-hydroxytryptamine. Furthermore, melatonin metabolism results in a kynurenine derivative, the pharmacological action of which remains to be studied, and could amplify the mechanisms of action of melatonin.
Cyclic nucleotide phosphodiesterases in Drosophila melanogaster
Authors: DAY Jonathan P. , DOW Julian A. T. , HOUSLAY Miles D. , DAVIES Shireen-A.
Published: May 2005

Cyclic nucleotide PDEs (phosphodiesterases) are important enzymes that regulate intracellular levels of cAMP and cGMP. In the present study, we identify and characterize novel PDEs in the genetic model, Drosophila melanogaster. The Drosophila genome encodes five novel PDE genes in addition to dunce. Predicted PDE sequences of Drosophila show highly conserved critical domains when compared with human PDEs. Thus PDE-encoding genes of D. melanogaster are CG14940-PDE1C, CG8279-PDE6β, CG5411-PDE8A, CG32648-PDE9 and CG10231-PDE11. Reverse transcriptase–PCRs of adult tissues reveal widespread expression of PDE genes. Drosophila Malpighian (renal) tubules express all the six PDEs: Drosophila PDE1, dunce (PDE4), PDE6, PDE8, PDE9 and PDE11. Antipeptide antibodies were raised against PDE1, PDE6, PDE9 and PDE11. Verification of antibody specificity by Western blotting of cloned and expressed PDE constructs allowed the immunoprecipitation studies of adult Drosophila lysates. Biochemical characterization of immunoprecipitated endogenous PDEs showed that PDE1 is a dual-specificity PDE (Michaelis constant Km for cGMP: 15.3±1 μM; Km cAMP: 20.5±1.5 μM), PDE6 is a cGMP-specific PDE (Km cGMP: 37±13 μM) and PDE11 is a dual-specificity PDE (Km cGMP: 6±2 μM; Km cAMP: 18.5±5.5 μM). Drosophila PDE1, PDE6 and PDE11 display sensitivity to vertebrate PDE inhibitors, zaprinast (IC50 was 71±39 μM for PDE1, 0.65±0.015 μM for PDE6 and 1.6±0.5 μM for PDE11) and sildenafil (IC50 was 1.3±0.9 μM for PDE1, 0.025±0.005 μM for PDE6 and 0.12±0.06 μM for PDE11). We provide the first characterization of a cGMP-specific PDE and two dual-specificity PDEs in Drosophila, and show a high degree of similarity in structure and function between human and Drosophila PDEs.
Spotting new DNA damage-responsive chromatin-binding proteins
Authors: WARDLEWORTH Benjamin N. , DOWNS Jessica A.
Published: May 2005

In response to DNA damage, cells initiate multiple repair mechanisms that all contribute to the survival of both the cell and the organism. These responses are numerous and variable, and can include cell cycle arrest, transcriptional activation of DNA repair genes and relocalization of repair proteins to sites of DNA damage. If all else fails, in multicellular organisms the initiation of apoptosis is also a potential cellular response to DNA damage. Despite a wealth of information about these events, it is clear that we do not yet have a comprehensive picture of the cellular responses to DNA damage. In this issue of the Biochemical Journal, a proteomics approach was used by Lee et al. to identify proteins that bind to chromatin in a DNA damage-inducible manner. The proteins identified, nucleophosmin, hnRNP C1 (heterogeneous nuclear ribonucleoprotein C1) and hnRNP C2, were proteins that would not necessarily have been predicted to behave this way. These studies have the potential to be extended and contribute to our knowledge of the cellular response to DNA damage.
Maintaining Alignment over the Long-Term: Lessons from the Evolution of an Electronic Point of Sale System
Authors: Boddy David , Paton Robert
Published: May 2005

Research in both the organizational and information systems areas suggests that aligning information systems and dimensions of the organization improves performance. However, the dynamic nature of both information systems and organizations makes a close alignment more an aspiration than a state. This paper examines alignment from the perspective of the management processes through which companies may achieve and sustain alignment in dynamic environments. In so doing they may also develop core capabilities in managing IS innovation, which enable sustained competitive advantage. The paper traces the evolution of alignment in a company that used information systems to develop the business in new strategic directions, and shows how semi-structures contributed to this.