bluuebunny/crossref_metadata_2025 huggingface dataset:
Maintenance of the filamentous actin cytoskeleton is necessary for the activation of store-operated Ca2+ channels, but not other types of plasma-membrane Ca2+ channels, in rat hepatocytes
Authors: WANG Ying-Jie , GREGORY Roland B. , BARRITT Greg J.
Published: August 2015

The roles of the filamentous actin (F-actin) cytoskeleton and the endoplasmic reticulum (ER) in the mechanism by which store-operated Ca2+ channels (SOCs) and other plasma-membrane Ca2+ channels are activated in rat hepatocytes in primary culture were investigated using cytochalasin D as a probe. Inhibition of thapsigargin-induced Ca2+ inflow by cytochalasin D depended on the concentration and time of treatment, with maximum inhibition observed with 0.1μM cytochalasin D for 3h. Cytochalasin D (0.1μM for 3h) did not inhibit the total amount of Ca2+ released from the ER in response to thapsigargin but did alter the kinetics of Ca2+ release. The effects of cytochalasin D (0.1μM) on vasopressin-induced Ca2+ inflow were similar to those on thapsigargin-induced Ca2+ inflow, except that cytochalasin D did inhibit vasopressin-induced release of Ca2+ from the ER. Cytochalasin D (0.1μM) inhibited vasopressin-induced Mn2+ inflow (predominantly through intracellular messenger-activated non-selective cation channels), but the degree of inhibition was less than that of vasopressin-induced Ca2+ inflow (predominantly through Ca2+-selective SOCs). Maitotoxin- and hypotonic shock-induced Ca2+ inflow were enhanced rather than inhibited by 0.1μM cytochalasin D. Treatment with 0.1μM cytochalasin D substantially reduced the amount of F-actin at the cell cortex, whereas 5μM cytochalasin D increased the total amount of F-actin and caused an irregular distribution of F-actin at the cell cortex. Cytochalasin D (0.1μM) caused no significant change in the overall arrangement of the ER {monitored using 3′,3′-dihexyloxacarbocyanine iodide [DiOC6(3)] in fixed cells} but disrupted the fine structure of the smooth ER and reduced the diffusion of DiOC6(3) in the ER in live hepatocytes after photobleaching. It is concluded that (i) the concentration of cytochalasin D is a critical factor in the use of this agent as a probe to disrupt the cortical F-actin cytoskeleton in rat hepatocytes, (ii) a reduction in the amount of cortical F-actin inhibits SOCs but not intracellular messenger-activated non-selective cation channels, and (iii) inhibition of the activation of SOCs and reduction in the amount of cortical F-actin is associated with disruption of the organization of the ER.
Comparative characterization of two DEAD-box RNA helicases in superfamily II: human translation-initiation factor 4A and hepatitis C virus non-structural protein 3 (NS3) helicase
Authors: DU Mark X. , JOHNSON Robert B. , SUN Xin-Lai , STASCHKE Kirk A. , COLACINO Joseph , WANG Q. May
Published: August 2015

Eukaryotic initiation factor 4A (eIF4A) is an ATP-dependent RNA helicase and is homologous to the non-structural protein 3 (NS3) helicase domain encoded by hepatitis C virus (HCV). Reported here is the comparative characterization of human eIF4A and HCV NS3 helicase in an effort to better understand viral and cellular helicases of superfamily II and to assist in designing specific inhibitors against HCV infections. Both eIF4A and HCV NS3 helicase domain were expressed in bacterial cells as histidine-tagged proteins and purified to homogeneity. Purified eIF4A exhibited RNA-unwinding activity and acted on RNA or RNA/DNA but not DNA duplexes. In the absence of cellular cofactors, eIF4A operated unwinding in both the 3′ to 5′ and 5′ to 3′ directions, and was able to unwind blunt-ended RNA duplex, suggesting that bidirectionality is an intrinsic property of eIF4A. In contrast, HCV NS3 helicase showed unidirectional 3′ to 5′ unwinding of RNA and RNA/DNA, as well as of DNA duplexes. With respect to NTPase activity, eIF4A hydrolysed only ATP or dATP in the presence of RNAs, whereas HCV NS3 helicase could hydrolyse all ribo- and deoxyribo-NTPs in an RNA-independent manner. In parallel, only ATP or dATP could drive the unwinding activity of eIF4A whereas HCV NS3 could function with all eight standard NTPs and dNTPs. The observed differences in their substrate specificity may prove to be useful in designing specific inhibitors targeting HCV NS3 helicase but not human eIF4A.
Nuclear receptor corepressor-dependent repression of peroxisome-proliferator-activated receptor δ-mediated transactivation
Authors: KROGSDAM Anne-M. , NIELSEN Curt A.F. , NEVE Søren , HOLST Dorte , HELLEDIE Torben , THOMSEN Bo , BENDIXEN Christian , MANDRUP Susanne , KRISTIANSEN Karsten
Published: August 2015

The nuclear receptor corepressor (NCoR) was isolated as a peroxisome-proliferator-activated receptor (PPAR) δ interacting protein using the yeast two-hybrid system. NCoR interacted strongly with the ligand-binding domain of PPARδ, whereas interactions with the ligand-binding domains of PPARγ and PPARα were significantly weaker. PPAR—NCoR interactions were antagonized by ligands in the two-hybrid system, but were ligand-insensitive in in vitro pull-down assays. Interaction between PPARδ and NCoR was unaffected by coexpression of retinoid X receptor (RXR) α. The PPARδ—RXRα heterodimer bound to an acyl-CoA oxidase (ACO)-type peroxisome-proliferator response element recruited a glutathione S-transferase—NCoR fusion protein in a ligand-independent manner. Contrasting with most other nuclear receptors, PPARδ was found to interact equally well with interaction domains I and II of NCoR. In transient transfection experiments, NCoR and the related silencing mediator for retinoid and thyroid hormone receptor (SMRT) were shown to exert a marked dose-dependent repression of ligand-induced PPARδ-mediated transactivation; in addition, transactivation induced by the cAMP-elevating agent forskolin was efficiently reduced to basal levels by NCoR as well as SMRT coexpression. Our results suggest that the transactivation potential of liganded PPARδ can be fine-tuned by interaction with NCoR and SMRT in a manner determined by the expression levels of corepressors and coactivators.
Characterization of the role of the AMP-activated protein kinase in the stimulation of glucose transport in skeletal muscle cells
Authors: FRYER Lee G.D. , FOUFELLE Fabienne , BARNES Kay , BALDWIN Stephen A. , WOODS Angela , CARLING David
Published: August 2015

Stimulation of AMP-activated protein kinase (AMPK) in skeletal muscle has been correlated with an increase in glucose transport. Here, we demonstrate that adenoviral-mediated expression of a constitutively active mutant of AMPKα leads to activation of glucose transport in a skeletal-muscle cell line, similar to that seen following treatment with 5-amino-imidazolecarboxamide (AICA) riboside, hyperosmotic stress or insulin. In contrast, expression of a dominant-negative form of AMPK blocked the stimulation of glucose transport by both AICA riboside and hyperosmotic stress, but was without effect on either insulin or phorbol-ester-stimulated transport. These results demonstrate that activation of AMPK is sufficient for stimulation of glucose uptake into muscle cells, and is a necessary component of the AICA riboside- and hyperosmotic-stress-induced pathway leading to increased glucose uptake. On the other hand, AMPK is not required in the insulin- or phorbol-ester-mediated pathways. Long-term (5 days) expression of the constitutively active AMPK mutant increased protein expression of GLUT1, GLUT4 and hexokinase II, consistent with previous reports on the chronic treatment of rats with AICA riboside. Expression of constitutively active AMPK had no detectable effect on p38 mitogen-activated protein kinase levels, although interestingly the level of protein kinase B was decreased. These results demonstrate that long-term activation of AMPK is sufficient to cause increased expression of specific proteins in muscle. Our results add further support to the hypothesis that long-term activation of AMPK is involved in the adaptive response of muscle to exercise training.
Interaction of the transforming acidic coiled-coil 1 (TACC1) protein with ch-TOG and GAS41/NuBI1 suggests multiple TACC1-containing protein complexes in human cells
Authors: LAUFFART Brenda , HOWELL Scott J. , TASCH Jason E. , COWELL John K. , STILL Ivan H.
Published: August 2015

Dysregulation of the human transforming acidic coiled-coil (TACC) proteins is thought to be important in the evolution of breast cancer and multiple myeloma. However, the exact role of these proteins in the oncogenic process is currently unknown. Using the full-length TACC1 protein as bait to screen a human mammary epithelial cDNA library, we have identified two genes that are also amplified and overexpressed in tumours derived from different cellular origins. TACC1 interacts with the C-terminus of both the microtubule-associated colonic and hepatic tumour overexpressed (ch-TOG) protein, and the oncogenic transcription factor glioma amplified sequence 41/NuMA binding protein 1 (GAS41/NuBI1; where NuMA stands for nuclear mitotic apparatus protein 1). This suggests that the TACC proteins can form multiple complexes, dysregulation of which may be an important step during tumorigenesis.
The role of syndecan-2 in regulation of actin-cytoskeletal organization of Lewis lung carcinoma-derived metastatic clones
Authors: MUNESUE Seiichi , KUSANO Yuri , OGURI Kayoko , ITANO Naoki , YOSHITOMI Yasuo , NAKANISHI Hayao , YAMASHINA Ikuo , OKAYAMA Minoru
Published: August 2015

Syndecans, a family of transmembrane heparan sulphate proteoglycans, contribute to various biological processes, including adhesion, motility, proliferation, differentiation and morphogenesis. We document here the involvement of syndecan-2 acting alone or co-operatively with integrin α5β1, for regulation of actin-cytoskeletal organization on cell adhesion to fibronectin, using fibronectin-recombinant polypeptides containing the ligands for either or both of these receptors as substrata. Lewis lung carcinoma-derived low-metastatic P29 cells binding to the substrata by both receptors formed actin stress fibres, whereas those binding by syndecan-2 or integrin α5β1 alone formed filopodia or cortex actin. In contrast, higher metastatic LM66-H11 cells formed cortex actin even on substrata containing both ligands. Northern-blot and flow-cytometric analyses revealed that syndecan-2 expression in LM66-H11 cells was significantly lower (1/4.5 in mRNA and 1/8 in cell-surface expression) than in P29 cells, whereas expression levels of integrin α5β1 and other syndecans were similar in both cell types. These results suggest that the failure of LM66-H11 to form stress fibres is due to a lower expression of syndecan-2 than that due to a threshold for its function. This was confirmed by the finding that overexpression of syndecan-2 by transfection of its cDNA into LM66-H11 cells caused the formation of stress fibres on the fibronectin substratum. These in vitro cellular responses of the two clones might reflect their in vivo situation in primary tumours in which P29 cells with a stroma-inducing capacity were immediately surrounded by fibronectin-rich matrix formed by the induced stromal cells, whereas LM66-H11 cells without such capacity were not surrounded by a similar matrix.
β1 and β2 integrins activate different signalling pathways in monocytes
Authors: REYES-REYES Merit , MORA Nancy , GONZALEZ Gerardo , ROSALES Carlos
Published: August 2015

Integrin-mediated signals play an important but poorly understood role in regulating many leucocyte functions. In monocytes and macrophages, integrins of the β2 subfamily are involved in cell—cell interactions that are important for migration of the cells through the endothelium and also for phagocytosis. On the other hand, in the same cells, β1 integrin-mediated adhesion to extracellular matrix proteins results in a strong induction of immediate early genes that are important in inflammation. To investigate the signalling pathways from these two types of integrin in monocytic cells, THP-1 cells were selectively stimulated via β1 or β2 integrins by cross-linking each type of receptor with specific monoclonal antibodies or their natural ligands. The involvement of extracellular signal-regulated kinase (ERK), Syk and phosphoinositide 3-kinase (PI-3K) was then analysed. Nuclear factor κB (NF-κB) activation was also detected in THP-1 cells transiently transfected with an NF-κB-driven luciferase reporter gene. We found that binding of both types of integrin to their natural ligands activated ERK in a Syk- and PI-3K-dependent manner. Yet, cross-linking of integrins by anti-β1 antibodies caused activation of ERK while that by anti-β2 antibodies did not. Also both types of integrin activated NF-κB. However, PI-3K was required for β1 integrin-, but not β2 integrin-, mediated NF-κB activation. In addition, inhibition of PI-3K with wortmannin and LY294002 blocked β1 integrin-mediated NF-κB activation, but did not affect that mediated by β2 integrin. These data suggest that distinct integrins activate different signalling pathways in monocytic cells.
Folate synthesis in higher-plant mitochondria: coupling between the dihydropterin pyrophosphokinase and the dihydropteroate synthase activities
Authors: MOUILLON Jean-Marie , RAVANEL Stéphane , DOUCE Roland , RÉBEILLÉ Fabrice
Published: August 2015

The plant enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase (HPPK/DHPS) is a mitochondrial bifunctional protein involved in tetrahydrofolate synthesis. The first domain (HPPK) catalyses the pyrophosphorylation of 6-hydroxymethyl-7,8-dihydropterin (dihydropterin) by ATP, leading to 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (dihydropterinPPi) and AMP. The second domain (DHPS) catalyses the next step, i.e. the condensation of p-aminobenzoic acid (p-ABA) with dihydropterinPPi to give 7,8-dihydropteroate (dihydropteroate) and PPi. In the present article we studied the coupling between these two reactions. Kinetic data obtained for the HPPK domain are consistent with an ordered Bi Bi mechanism where ATP binds first and dihydropterinPPi is released last, as proposed previously for the monofunctional Escherichia coli enzyme. In the absence of p-ABA, AMP and dihydropterinPPi accumulate and negatively regulate the reaction. In the presence of p-ABA, the rates of AMP and dihydropteroate synthesis are similar, indicating a good coupling between the two reactions. DihydropterinPPi, an intermediate of the two reactions, never accumulates in this situation. The high specific activity of DHPS relative to HPPK, rather than a preferential channelling of dihydropterinPPi between the two catalytic sites, could explain these kinetic data. The maximal velocity of the DHPS domain is limited by the availability of dihydropterinPPi. It is strongly feedback-inhibited by dihydropteroate and also dihydrofolate and tetrahydrofolate monoglutamate, two intermediates synthesized downstream in the folate biosynthetic pathway. Thus the HPPK domain of this bifunctional protein is the limiting factor of the overall reaction, but the DHPS domain is a potential key regulatory point of the whole folate biosynthetic pathway.
Copper supplementation restores cytochrome c oxidase activity in cultured cells from patients with SCO2 mutations
Authors: SALVIATI Leonardo , HERNANDEZ-ROSA Evelyn , WALKER Winsome F. , SACCONI Sabrina , DiMAURO Salvatore , SCHON Eric A. , DAVIDSON Mercy M.
Published: August 2015

Human SCO2 is a nuclear-encoded Cu-binding protein, presumed to be responsible for the insertion of Cu into the mitochondrial cytochrome c oxidase (COX) holoenzyme. Mutations in SCO2 are associated with cardioencephalomyopathy and COX deficiency. Studies in yeast and bacteria have shown that Cu supplementation can restore COX activity in cells harbouring mutations in genes involving Cu transport. Therefore we investigated whether Cu supplementation could restore COX activity in cultured cells from patients with SCO2 mutations. Our data demonstrate that the COX deficiency observed in fibroblasts, myoblasts and myotubes from patients with SCO2 mutations can be restored to almost normal levels by the addition of CuCl2 to the growth medium.
Bid induces cytochrome c-impermeable Bax channels in liposomes
Authors: ROUCOU Xavier , ROSTOVTSEVA Tatiana , MONTESSUIT Sylvie , MARTINOU Jean-Claude , ANTONSSON Bruno
Published: August 2015

Bax is a proapoptotic member of the Bcl-2 family of proteins. The Bax protein is dormant in the cytosol of normal cells and is activated upon induction of apoptosis. In apoptotic cells, Bax gets translocated to mitochondria, inserts into the outer membrane, oligomerizes and triggers the release of cytochrome c, possibly by channel formation. The BH3 domain-only protein Bid induces a conformational change in Bax before its insertion into the outer membrane. The mechanism by which Bid promotes Bax activation is not understood, and whether Bid is the only protein required for Bax activation is unclear. Here we report that recombinant full-length Bax (BaxFL) does not form channels in lipid bilayers when purified as a monomer. In contrast, in the presence of Bid cut with caspase 8 (cut Bid), Bax forms ionic channels in liposomes and planar bilayers. This channel-forming activity requires an interaction between cut Bid and Bax, and is inhibited by Bcl-xL. Moreover, in the absence of the putative transmembrane C-terminal domain, Bax does not form ionic channels in the presence of cut Bid. Cut Bid does not induce Bax oligomerization in liposomes and the Bax channels formed in the presence of cut Bid are not large enough to permeabilize vesicles to cytochrome c. In conclusion, our results suggest that monomeric BaxFL can form channels only in the presence of cut Bid. Cut Bid by itself is unable to induce Bax oligomerization in lipid membranes. It is suggested that another factor that might be present in mitochondria is required for Bax oligomerization.
Spectroscopic characterization of mutations at the Phe41 position in the distal haem pocket of horseradish peroxidase C: structural and functional consequences
Authors: HEERING Hendrik A. , SMITH Andrew T. , SMULEVICH Giulietta
Published: August 2015

Three mutants of horseradish peroxidase isoenzyme C (HRPC) have been constructed in which the conserved distal aromatic residue Phe41 has been substituted by Trp, Val or Ala and the properties of the mutant proteins have been compared with that of the wild-type. The ferric and ferrous states have been studied by resonance Raman, electronic absorption and Fourier-transform infrared spectroscopies, together with their respective fluoride and CO complexes as probes for the integrity of the distal haem-pocket hydrogen-bonding network. The catalytic properties of the mutants, most notably the HRPC-mutant Phe41→Trp (F41W) variant, were also affected. Structural modelling suggests that the bulky indole group of the F41W mutant blocks the distal cavity, inhibiting the binding of fluoride and CO to the haem iron, severely impairing the reaction of the enzyme with H2O2 to form Compound I. Substitution with the smaller side-chain residues Val or Ala resulted in a 2-fold increase in the affinity of the mutants for the aromatic donor benzhydroxamic acid (BHA) compared with the wild-type, whereas the sterically hindered F41W mutant was not able to bind BHA at all. All the mutations studied increased the amount of a ferric six-coordinate aquo-high-spin species. On the other hand, the similarity in the Fe—Im stretching frequencies of the mutants and wild-type protein suggests that the distal haem-pocket mutations do not cause any substantive changes on the proximal side of the haem. Spectra of the HRPC mutant Phe41→Ala—CO and the HRPC mutant Phe41→Val—CO complexes strongly suggested a weakening of the interaction between CO and Arg38 due to a secondary rearrangement of the haem relative to helix B. The effects observed for these HRP mutants were somewhat different from those noted recently for the analogous Coprinus cinereus peroxidase (CIP) mutants, particularly the Trp mutant. These differences can be reconciled in part as being due to the smaller size of the distal cavity of HRP compared with that of CIP.
The mouse Muc5b mucin gene: cDNA and genomic structures, chromosomal localization and expression
Authors: ESCANDE Fabienne , PORCHET Nicole , AUBERT Jean-Pierre , BUISINE Marie-Pierre
Published: August 2015

We report here the isolation and characterization of the mouse Muc5b mucin gene (mMuc5b). We determined its complete cDNA sequence, its genomic organization, and chromosomal localization. Moreover, we analyzed the expression of this gene by reverse-transcription PCR and in situ hybridization. The structure of the gene was determined from a genomic cosmid clone that encompasses the entire mMuc5b gene, including the 5′-flanking region. The mMuc5b gene spans approximately 36kb and contains 49 exons. It is located on mouse distal chromosome 7. mMuc5b encodes at least two transcripts by alternative splicing of the second exon, the longest one being 14.9kb in length. The deduced peptide contains 4782 amino acids. Its central region can be subdivided into 10 imperfect repeats, each composed of a cysteine-rich domain followed by a threonine, serine, and proline-rich mucin-type domain. It is flanked by cysteine-rich domains similar to cysteine-rich domains of pre-pro-von Willebrand factor. Comparison with its human homologue MUC5B revealed common features including high sequence similarities in the 5′ and 3′ regions, and the conservation of the genomic organization. In contrast, mMuc5b differs from its human homologue, since no highly tandemly repeated sequences could be identified within its central region. mMuc5b is expressed mainly in laryngeal mucous glands, and at a lesser extend in stomach and duodenum.
Identification of Ca2+-dependent binding partners for the neuronal calcium sensor protein neurocalcin δ: interaction with actin, clathrin and tubulin
Authors: IVINGS Lenka , PENNINGTON Stephen R. , JENKINS Roz , WEISS Jamie L. , BURGOYNE Robert D.
Published: August 2015

The neuronal calcium sensors are a family of EF-hand-containing Ca2+-binding proteins expressed predominantly in retinal photoreceptors and neurons. One of the family members is neurocalcin δ, the function of which is unknown. As an approach to elucidating the protein interactions made by neurocalcin δ, we have identified brain cytosolic proteins that bind to neurocalcin δ in a Ca2+-dependent manner. We used immobilized recombinant myristoylated neurocalcin δ combined with protein identification using MS. We demonstrate a specific interaction with clathrin heavy chain, α- and β-tubulin, and actin. These interactions were dependent upon myristoylation of neurocalcin δ indicating that the N-terminal myristoyl group may be important for protein—protein interactions in addition to membrane association. Direct binding of neurocalcin δ to clathrin, tubulin and actin was confirmed using an overlay assay. These interactions were also demonstrated for endogenous neurocalcin δ by co-immunoprecipitation from rat brain cytosol. When expressed in HeLa cells, neurocalcin δ was cytosolic at resting Ca2+ levels but translocated to membranes, including a perinuclear compartment (trans-Golgi network) where it co-localized with clathrin, following Ca2+ elevation. These data suggest the possibility that neurocalcin δ functions in the control of clathrin-coated vesicle traffic.
Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C δ1
Authors: WATT Stephen A. , KULAR Gursant , FLEMING Ian N. , DOWNES C. Peter , LUCOCQ John M.
Published: August 2015

Ptd(4,5)P2 is thought to promote and organize a wide range of cellular functions, including vesicular membrane traffic and cytoskeletal dynamics, by recruiting functional protein complexes to restricted locations in cellular membranes. However, little is known about the distribution of PtdIns(4,5)P2 in the cell at high resolution. We have used the pleckstrin homology (PH) domain of phospholipase δ1 (PLCδ1), narrowly specific for PtdIns(4,5)P2, to map the distribution of the lipid in astrocytoma and A431 cells. We applied the glutathione S-transferase-tagged PLCδ1 PH domain (PLCδ1PH—GST) in an on-section labelling approach which avoids transfection procedures. Here we demonstrate PtdIns(4,5)P2 labelling in the plasma membrane, and also in intracellular membranes, including Golgi (mainly stack), endosomes and endoplasmic reticulum, as well as in electron-dense structures within the nucleus. At the plasma membrane, labelling was more concentrated over lamellipodia, but not in caveolae, which contained less than 10% of the total cell-surface labelling. A dramatic decrease in signal over labelled compartments was observed on preincubation with the cognate headgroup [Ins(1,4,5)P3], and plasma-membrane labelling was substantially decreased after stimulation with thrombin-receptor-activating peptide (SFLLRN in the one-letter amino acid code), a treatment which markedly diminishes PtdIns(4,5)P2 levels. Thus we have developed a highly selective method for mapping the PtdIns(4,5)P2 distribution within cells at high resolution, and our data provide direct evidence for this lipid at key functional locations.
Molecular and immunological characterization and IgE epitope mapping of Pen n 18, a major allergen of Penicillium notatum
Authors: YU Chia-Jung , CHEN Yen-Ming , SU Song-Nan , FOROUHAR Farhad , LEE Shu-Hua , CHOW Lu-Ping
Published: August 2015

The mould genus, Penicillium, is a significant source of environmental aero-allergens. A major allergen from Penicillium notatum, Pen n 18, was identified by two-dimensional immunoblotting using monoclonal antibody G11A10, raised against the vacuolar serine protease of Penicillium citrinum, followed by matrix-assisted laser-desorption ionization—time-of-flight MS analysis of the peptide digest. Pen n 18 was then cloned and the amino acid sequence deduced from the cDNA sequence. The cDNA encoded a 494 amino acid protein, considerably larger than mature Pen n 18, the differences being due to the N- and C-terminal prosequences. The deduced amino acid sequence showed extensive similarity with those of vacuolar serine proteases from various fungi. The Pen n 18 coding sequence was expressed in Escherichia coli as a His-tagged fusion protein and purified by Ni2+-chelate affinity chromatography. On immunoblots, the purified recombinant protein specifically bound IgE from mould-allergic patients, and cross-inhibition assays demonstrated the presence of common IgE-binding epitopes on Pen n 18 and a major allergen of P. citrinum, Pen c 18. When mapping of the allergenic epitopes was performed, at least nine different linear IgE-binding epitopes, located throughout the Pen n 18 protein, were identified. Of these, peptide C12, located in the N-terminal region of the molecule, was recognized by serum from 75% of the patients tested and therefore appears to be an immunodominant IgE-binding epitope.
Threonine-124 and phenylalanine-448 in Citrobacter freundii tyrosine phenol-lyase are necessary for activity with l-tyrosine
Authors: DEMIDKINA Tatyana V. , BARBOLINA Maria V. , FALEEV Nicolai G. , SUNDARARAJU Bakthavatsalam , GOLLNICK Paul D. , PHILLIPS Robert S.
Published: August 2015

Thr-124 and Phe-448 are located in the active site of Citrobacter freundii tyrosine phenol-lyase (TPL) near the phenol ring of a bound substrate analogue, 3-(4′-hydroxyphenyl)propionic acid [Sundararaju, Antson, Phillips, Demidkina, Barbolina, Gollnick, Dodson and Wilson (1997) Biochemistry 36, 6502–6510]. Thr-124 is replaced by Asp and Phe-448 is replaced by His in the crystal structure of a structurally similar enzyme, Proteus vulgaris tryptophan indole-lyase, which has 50% identical residues. Hence, Thr-124 and Phe-448 in TPL were mutated to Ala or Asp, and His, respectively, in order to probe the role of these residues in the reaction specificity for l-Tyr. These mutant enzymes have little or no β-elimination activity with l-Tyr or 3-fluoro-l-Tyr as a substrate, but retain significant elimination activity with S-(o-nitrophenyl)-l-cysteine, S-alkyl-l-cysteines and β-chloroalanine. Furthermore, the binding of l-Tyr and other non-substrate amino acids is not significantly affected by the mutations. The mutant TPLs form intermediates in rapid-scanning stopped-flow experiments with l-Phe, l-Tyr and l-Trp, similar to those seen with wild-type TPL. These results demonstrate that Thr-124 and Phe-448 are necessary for the reaction specificity of TPL for l-Tyr, and probably play a role in the elimination stage of the reaction mechanism. Thr-124 is within hydrogen-bonding distance of the phenolic group of the bound substrate, and may help to orientate the ring for β-elimination to occur. Phe-448 may be important to allow the formation of the closed conformation during the reaction.
Inhibition of cell growth through inactivation of eukaryotic translation initiation factor 5A (eIF5A) by deoxyspergualin
Authors: NISHIMURA Kazuhiro , OHKI Yuji , FUKUCHI-SHIMOGORI Tomomi , SAKATA Kaori , SAIGA Kan , BEPPU Takanobu , SHIRAHATA Akira , KASHIWAGI Keiko , IGARASHI Kazuei
Published: August 2015

The mechanism of inhibition of cell growth by deoxyspergualin was studied using mouse mammary carcinoma FM3A cells. Results of studies using deoxyspergualin analogues showed that both the guanidinoheptanate amide and glyoxyspermidine moieties of deoxyspergualin were necessary to cause inhibition of cell growth. When deoxyspergualin was added to the medium, there was a strong inhibition of cell growth and formation of active eukaryotic translation initiation factor 5A (eIF5A) at the third day of culture. There was also a marked decrease in cellular putrescine content and a small decrease in spermidine content. Accumulation of decapped mRNA, which is typically associated with eIF5A deficiency in yeast, was also observed. The inhibition of cell growth and the formation of active eIF5A was not reversed by addition of spermidine. The activity of deoxyhypusine synthase, the first enzyme in the formation of active eIF5A, was inhibited by deoxyspergualin in a cell-free system. These results, taken together, indicate that inhibition of active eIF5A formation is strongly involved in the inhibition of cell growth by deoxyspergualin.
The role of α-methylacyl-CoA racemase in bile acid synthesis
Authors: CUEBAS Dean A. , PHILLIPS Christopher , SCHMITZ Werner , CONZELMANN Ernst , NOVIKOV Dmitry K.
Published: August 2015

According to current views, the second peroxisomal β-oxidation pathway is responsible for the degradation of the side chain of bile acid intermediates. Peroxisomal multifunctional enzyme type 2 [peroxisomal multifunctional 2-enoyl-CoA hydratase/(R)-3-hydroxyacyl-CoA dehydrogenase; MFE-2] catalyses the second (hydration) and third (dehydrogenation) reactions of the pathway. Deficiency of MFE-2 leads to accumulation of very-long-chain fatty acids, 2-methyl-branched fatty acids and C27 bile acid intermediates in plasma, but bile acid synthesis is not blocked completely. In this study we describe an alternative pathway, which allows MFE-2 deficiency to be overcome. The alternative pathway consists of α-methylacyl-CoA racemase and peroxisomal multifunctional enzyme type 1 [peroxisomal multifunctional 2-enoyl-CoA hydratase/(S)-3-hydroxyacyl-CoA dehydrogenase; MFE-1]. (24E)-3α,7α,12α-Trihydroxy-5β-cholest-24-enoyl-CoA, the presumed physiological isomer, is hydrated by MFE-1 with the formation of (24S,25S)-3α,7α,12α,24-tetrahydroxy-5β-cholestanoyl-CoA [(24S,25S)-24-OH-THCA-CoA], which after conversion by a α-methylacyl-CoA racemase into the (24S,25R) isomer can again be dehydrogenated by MFE-1 to 24-keto-3α,7α,12α-trihydroxycholestanoyl-CoA, a physiological intermediate in cholic acid synthesis. The discovery of the alternative pathway of cholesterol side-chain oxidation will improve diagnosis of peroxisomal deficiencies by identification of serum 24-OH-THCA-CoA diastereomer profiles.
Mo(V) co-ordination in the periplasmic nitrate reductase from Paracoccus pantotrophus probed by electron nuclear double resonance (ENDOR) spectroscopy
Authors: BUTLER Clive S. , FAIRHURST Shirley A. , FERGUSON Stuart J. , THOMSON Andrew J. , BERKS Ben C. , RICHARDSON David J. , LOWE David J.
Published: August 2015

The first electron nuclear double resonance (ENDOR) study of a member of the Mo-bis-molybdopterin guanine dinucleotide family of molybdoenzymes is presented, using the periplasmic nitrate reductase from Paracoccus pantotrophus. Rapid freeze-quenched time-resolved EPR revealed that during turnover the intensity of a Mo(V) EPR signal known as High-g [resting] increases. This signal is split by two interacting protons that are not solvent-exchangeable. X-band proton-ENDOR analysis resolved broad symmetrical resonance features that arose from four classes of protons weakly coupled to the Mo(V). Signals from two of these were lost upon exchange into deuterated buffer, suggesting that they may originate from OH− or H2O groups. One of these signals was also lost when the enzyme was redox-cycled in the presence of azide. Since these protons are very weakly coupled OH/H2O groups, they are not likely to be ligated directly to the Mo(V). This suggests that protonation of a Mo(VI)=O group does not occur on reduction to Mo(V), but most probably accompanies reduction of Mo(V) to Mo(IV). A resonance feature from a more strongly coupled proton, that was not lost following exchange into deuterated buffer, could also be resolved at 22–24MHz. The anisotropy of this feature, determined from ENDOR spectra collected at a range of field positions, indicated a Mo—proton distance of approx. 3.2Å, consistent with this being one of the β-methylene protons of a Mo—Cys ligand.
Characterization of Streptococcus pneumoniae thymidylate kinase: steady-state kinetics of the forward reaction and isothermal titration calorimetry
Authors: PETIT Chantal M. , KORETKE Kristin K.
Published: August 2015

Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis. The tmk gene from the bacterial pathogen Streptococcus pneumoniae was identified. The gene, encoding a 212-amino-acid polypeptide (23352Da), was cloned and overexpressed in Escherichia coli with an N-terminal hexahistidine tag. The enzyme was purified to homogeneity, and characterized in the forward reaction. The pH profile of TMK indicates that its activity is optimal at pH 8.5. The substrate specificity of the enzyme was examined; it was found that not only ATP, but also dATP and to a lesser extent CTP, could act as phosphate donors, and dTMP and dUMP could serve as phosphate acceptors. Furthermore, AZT-MP (3′-azido-3′-deoxythymidine 5′-monophosphate) was shown not to be a substrate for S. pneumoniae TMK. Steady-state kinetics and inhibition studies with adenosine 5′-[β-thio]diphosphate and dTDP in addition to isothermal titration calorimetry were performed. The data showed that binding follows an ordered pathway, in which ATP binds first with a Km of 235±46μM and a Kd of 116±3μM, and dTMP binds secondly with a Km of 66±12μM and a Kd of 53±2μM.
Characteristics of physiological inducers of the ethanol utilization (alc) pathway in Aspergillus nidulans
Authors: FLIPPHI Michel , KOCIALKOWSKA Janina , FELENBOK Béatrice
Published: August 2015

The ethanol utilization (alc) pathway in Aspergillus nidulans is one of the strongest expressed gene systems in filamentous fungi. The pathway-specific activator AlcR requires the presence of an inducing compound to activate transcription of genes under its control. We have demonstrated recently that acetaldehyde is the sole physiological inducer of ethanol catabolism. In the present study we show that compounds with catabolism related to that of ethanol, i.e. primary alcohols, primary monoamines and l-threonine, act as inducers because their breakdown results in the production of inducing aliphatic aldehydes. Such aldehydes were shown to induce the alc genes efficiently at low external concentrations. When ethanol is mixed with representatives of another class of strong direct inducers, ketones, the physiological inducer, acetaldehyde, prevails as effector. Although direct inducers essentially carry a carbonyl function, not all aldehydes and ketones act as inducers. Structural features discriminating non-inducing from inducing compounds concern: (i) the length of the aliphatic side group(s); (ii) the presence and nature of any non-aliphatic substituent. These characteristics enable us to predict whether or not a given carbonyl compound will induce the alc genes.
μ-1,2-Peroxobridged di-iron(III) dimer formation in human H-chain ferritin
Authors: BOU-ABDALLAH Fadi , PAPAEFTHYMIOU Georgia C. , SCHESWOHL Danielle M. , STANGA Sean D. , AROSIO Paolo , CHASTEEN N. Dennis
Published: August 2015

Biomineralization of the ferritin iron core involves a complex series of events in which H2O2 is produced during iron oxidation by O2 at a dinuclear centre, the ‘ferroxidase site’, located on the H-subunit of mammalian proteins. Rapid-freeze quench Mössbauer spectroscopy was used to probe the early events of iron oxidation and mineralization in recombinant human ferritin containing 24 H-subunits. The spectra reveal that a μ-1,2-peroxodiFe(III) intermediate (species P) with Mössbauer parameters δ (isomer shift) = 0.58mm/s and ΔEQ (quadrupole splitting) = 1.07mm/s at 4.2K is formed within 50ms of mixing Fe(II) with the apoprotein. This intermediate accounts for almost all of the iron in the sample at 160ms. It subsequently decays within 10s to form a μ-oxodiFe(III)—protein complex (species D), which partially vacates the ferroxidase sites of the protein to generate Fe(III) clusters (species C) at a reaction time of 10min. The intermediate peroxodiFe(III) complex does not decay under O2-limiting conditions, an observation suggesting inhibition of decay by unreacted Fe(II), or a possible role for O2 in ferritin biomineralization in addition to that of direct oxidation of iron(II).
Gastric MUC5AC and MUC6 are large oligomeric mucins that differ in size, glycosylation and tissue distribution
Authors: NORDMAN Henrik , DAVIES Julia R. , LINDELL Gert , de BOLÓS Carme , REAL Francisco , CARLSTEDT Ingemar
Published: August 2015

Gastric MUC5AC and MUC6 mucins were studied using polyclonal antibodies. Immunohistochemistry showed MUC5AC to originate from the surface epithelium, whereas MUC6 was produced by the glands. Mucins from the surface epithelium or glands of corpus and antrum were purified using CsCl/4M guanidinium chloride density-gradient centrifugation. MUC5AC appeared as two distinct populations at 1.4 and 1.3g/ml, whereas MUC6, which was enriched in the gland tissue, appeared at 1.45g/ml. Reactivity with antibodies against the Leb structure (where Le represents the Lewis antigen) followed the MUC5AC distribution, whereas antibodies against the Ley structure and reactivity with the GlcNAc-selective Solanum tuberosum lectin coincided with MUC6, suggesting that the two mucins are glycosylated differently. Rate-zonal centrifugation of whole mucins and reduced subunits showed that both gastric MUC5AC and MUC6 are oligomeric glycoproteins composed of disulphide-bond linked subunits and that oligomeric MUC5AC was apparently smaller than MUC6. A heterogeneous population of ‘low-density’ MUC5AC mucins, which were smaller than the ‘high-density’ ones both before and after reduction, reacted with an antibody against a variable number tandem repeat sequence within MUC5AC, suggesting that they represent precursor forms of this mucin. Following ion-exchange HPLC, both MUC5AC and MUC6 appeared as several distinct populations, probably corresponding to ‘glycoforms’ of the mucins, the most highly charged of which were found in the gland tissue.
Caspases: the enzymes of death
Authors: Zhivotovsky Boris
Published: August 2015

The caspases are a unique family of cysteine proteases, which cleave proteins next to an aspartate residue. Among all known mammalian proteases, only the serine protease granzyme B has similar substrate specificity. In addition to a central role of caspases in the initiation and execution phases of apoptosis, these enzymes have some other non-apoptotic functions in living cells. During apoptosis, upon activation, caspases cleave specific substrates and thereby mediate many of the typical biochemical and morphological changes in apoptotic cells, such as cell shrinkage, chromatin condensation, DNA fragmentation and plasma-membrane blebbing. Thus, detection of activated caspases can be used as a biochemical marker for apoptosis induced by diverse stimuli in many types of cells.
Death receptors
Authors: Wajant Harald
Published: August 2015

Death receptors [Fas/Apo-1/CD95, TNF-R1 [tumour necrosis factor (TNF) receptor 1], DR3 [death receptor 3], TRAIL-R1 [TNF-related apoptosis-inducing ligand receptor 1], TRAIL-R2, DR6, p75-NGFR [p75-nerve growth factor receptor], EDAR [ectodermal dysplasia receptor]] form a subgroup of the TNF-R superfamily that can induce apoptosis (programmed cell death) via a conserved cytoplasmic signalling module termed the death domain. Although death receptors have been recognized mainly as apoptosis inducers, there is growing evidence that these receptors also fulfil a variety of nonapoptotic functions. This review is focused on the molecular mechanisms of apoptotic and non-apoptotic death receptor signalling in light of the phenotype of mice deficient in the various death receptors.
Gene repression by nuclear hormone receptors
Authors: Moehren Udo , Eckey Maren , Baniahmad Aria
Published: August 2015

Repression by nuclear hormone receptors (NHRs) plays an important role in development, immune response and cellular function. We review mechanisms of how NHRs act as repressors of gene transcription either by direct contact with basal transcription factors or through recruitment of cofactors and enzymic activities that modulate chromatin accessibility. We describe also the role and biochemical mechanism of the cognate hormone that switches a NHR from a transcriptional silencer into an activator. This includes data from crystal structure, functional receptor domain analyses and the role of co-repressors in chromatin modification and remodelling. Furthermore, the comparison of negative response elements with classical response elements unravels the role of co-repressors in this context. We also describe the inhibition of the nuclear factor kappaB and Jun/Fos pathway by NHRs, as well as the molecular mechanism of anti-hormone therapies. Anti-hormones are commonly used in breast and prostate cancer therapy to inhibit cancer proliferation through repression of the oestrogen or androgen receptor, respectively. Here we provide a comprehensive overview of the various mechanism of NHR repression.
Receptor mechanisms of rapid extranuclear signalling initiated by steroid hormones
Authors: Boonyaratanakornkit Viroj , Edwards Dean P
Published: August 2015

In addition to their role as direct regulators of gene transcription mediated by classical nuclear hormone receptors, steroid hormones have also been described to exert rapid effects on intracellular signalling pathways independent of gene transcription. This chapter focuses on recent advances in our understanding of the receptors and mechanisms that mediate these rapid signalling actions of oestrogens and progesterone. Increasing evidence suggests that at least some of these rapid actions are mediated by a subpopulation of the classical nuclear oestrogen receptor (ER) and progesterone receptor (PR) that localize to the cytoplasm or associate with the plasma membrane. Human PR has been shown to mediate rapid progestin activation of the Src/Ras/Raf/mitogen-activated protein kinase signalling pathway in mammalian cells by a direct interaction with the Src homology 3 domain of Src tyrosine kinases through a Pro-Xaa-Xaa-Pro-Xaa-Arg motif located in the N-terminal domain of the receptor. Moreover, this is an extranuclear action of PR that is separable from its direct transcriptional activity. Additionally, a novel membrane protein unrelated to nuclear PR was recently identified that has properties of a G-protein-coupled receptor for progesterone and has been shown to be involved in mediating the extranuclear signalling actions of progesterone that promotes oocyte maturation in fish. The role of this membrane PR (mPR) in mammalian cells is less clear and the relationship of the membrane and classical nuclear PR in mediating rapid non-transcriptional signalling of progestins has not been explored. To date, a novel membrane ER unrelated to classical nuclear receptors has not been cloned and characterized, and many of the known rapid extranuclear signalling actions of oestrogen appear also to be mediated by a subpopulation of nuclear ER, or a closely related receptor. A novel protein termed modulator of non-genomic activity of ER (MNAR) has been identified that acts as an adaptor between ER and Src, and thus provides a mechanisms for coupling of oestrogen and ER with rapid oestrogen-induced activation of Src and the downstream mitogen-activated protein kinase signalling cascade. The physiological relevance of rapid extranuclear signalling by the classical ER has been provided by experiments showing that these actions contribute to the anti-apoptotic effect of oestrogen in bone in vivo and to the rapid effects of oestrogen on vasodilation and protection of endothelial cells against injury.
Orchestra for assembly and fate of polyubiquitin chains
Authors: Kirsten Kuhlbrodt , Julien Mouysset , Thorsten Hoppe
Published: August 2015

Selective protein degradation by the 26 S proteasome usually requires a polyubiquitin chain attached to the protein substrate by three classes of enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). This reaction can produce different polyubiquitin chains that, depending on size and linkage type, can provide distinct intracellular signals. Interestingly, polyubiquitination is sometimes regulated by additional conjugation factors, called E4s (polyubiquitin chain conjugation factors). Yeast UFD2 (ubiquitin fusion degradation protein-2), the first E4 to be described, binds to the ubiquitin moieties of preformed conjugates and catalyses ubiquitin-chain elongation together with E1, E2, and E3. Recent studies have illustrated that the E4 enzyme UFD2 co-operates with an orchestra of ubiquitin-binding factors in an escort pathway to transfer and deliver polyubiquitinated substrates to the 26 S proteasome. Here we propose a model in which E4-dependent polyubiquitination pathways are modulated by different ubiquitin-binding proteins, using ataxin-3 as an example.
Targeting of host-cell ubiquitin pathways by viruses
Authors: Shackelford Julia , Pagano Joseph S.
Published: August 2015

The ability of viruses to co-opt cell signalling pathways has, over millions of years of co-evolution, come to pervade nearly every facet of cellular functions. Recognition of the extent to which the ubiquitin–proteasome system can be directed or subverted by viruses is relatively recent. Viral products interact with, and adjust, the ubiquitin–proteasome machinery precisely and at many levels, and they do so at distinct stages of viral life-cycles. The implications for both cells and viruses are fundamental, and understanding viral strategies in this context opens up fascinating new areas for research that span from basic cell biology to therapeutic interventions against both viruses and malignancies.