bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of retinaldehyde dehydrogenase 3
Authors: Graham Caroline E. , Brocklehurst Keith , Pickersgill Richard W. , Warren Martin J.
Published: March 2006

RALDH3 (retinal dehydrogenase 3) was characterized by kinetic and binding studies, protein engineering, homology modelling, ligand docking and electrostatic-potential calculations. The major recognition determinant of an RALDH3 substrate was shown to be an eight-carbon chain bonded to the aldehyde group whose kinetic influence (kcat/Km at pH 8.5) decreases when shortened or lengthened. Surprisingly, the β-ionone ring of all-trans-retinal is not a major recognition site. The dissociation constants (Kd) of the complexes of RALDH3 with octanal, NAD+ and NADH were determined by intrinsic tryptophan fluorescence. The similarity of the Kd values for the complexes with NAD+ and with octanal suggests a random kinetic mechanism for RALDH3, in contrast with the ordered sequential mechanism often associated with aldehyde dehydrogenase enzymes. Inhibition of RALDH3 by tri-iodothyronine binding in competition with NAD+, predicted by the modelling, was established kinetically and by immunoprecipitation. Mechanistic implications of the kinetically influential ionizations with macroscopic pKa values of 5.0 and 7.5 revealed by the pH-dependence of kcat are discussed. Analogies with data for non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans, together with the present modelled structure of the thioacyl RALDH3, suggest (a) that kcat characterizes deacylation of this intermediate for specific substrates and (b) the assignment of the pKa of the major ionization (approximating to 7.5) to the perturbed carboxy group of Glu280 whose conjugate base is envisaged as supplying general base catalysis to attack of a water molecule. The macroscopic pKa of the minor ionization (5.0) is considered to approximate to that of the carboxy group of Glu488.
Transport of polyamines in Drosophila S2 cells: kinetics, pharmacology and dependence on the plasma membrane proton gradient
Authors: Romero-Calderón Rafael , Krantz David E.
Published: March 2006

Polyamine transport activities have been described in diverse multicellular systems, but their bioenergetic mechanisms and molecular identity remain unclear. In the present paper, we describe a high-affinity spermine/spermidine transport activity expressed in Drosophila S2 cells. Ion-replacement experiments indicate that polyamine uptake across the cell membrane is Na+-, K+-, Cl−- and Ca2+-independent, but pH-sensitive. Additional experiments using ionophores suggest that polyamine uptake may be H+-coupled. Pharmacological experiments show that polyamine uptake in S2 cells is selectively blocked by MGBG {methylglyoxal bis(guanylhydrazone) or 1,1′-[(methylethanediylidine)-dinitrilo]diguanidine} and paraquat (N,N-dimethyl-4,4′-bipyridylium), two known inhibitors of polyamine uptake in mammalian cells. In addition, inhibitors known to block the Slc22 (solute carrier 22) family of organic anion/cation transporters inhibit spermine uptake in S2 cells. These data and the genetic tools available in Drosophila will facilitate the molecular identification and further characterization of this activity.
Localization of a portion of the liver isoform of fatty-acid-binding protein (L-FABP) to peroxisomes
Authors: Antonenkov Vasily D. , Sormunen Raija T. , Ohlmeier Steffen , Amery Leen , Fransen Marc , Mannaerts Guy P. , Hiltunen J. Kalervo
Published: March 2006

The liver isoform of fatty-acid-binding protein (L-FABP) facilitates the cellular uptake, transport and metabolism of fatty acids and is also involved in the regulation of gene expressions and cell differentiation. Consistent with these functions, L-FABP is predominantly present in the cytoplasm and to a lesser extent in the nucleus; however, a significant portion of this protein has also been detected in fractions containing different organelles. More recent observations, notably on L-FABP-deficient mice, indicated a possible direct involvement of L-FABP in the peroxisomal oxidation of long-chain fatty acids. In order to clarify the links between L-FABP and peroxisomal lipid metabolism, we reinvestigated the subcellular distribution of the protein. Analytical subcellular fractionation by a method preserving the intactness of isolated peroxisomes, two-dimensional gel electrophoresis of peroxisomal matrix proteins combined with MS analysis, and immunoelectron microscopy of liver sections demonstrate the presence of L-FABP in the matrix of peroxisomes as a soluble protein. Peroxisomal L-FABP was highly inducible by clofibrate. The induction of L-FABP was accompanied by a marked increase in the binding capacity of peroxisomal matrix proteins for oleic acid and cis-parinaric acid. The peroxisomal β-oxidation of palmitoyl-CoA and acyl-CoA thioesterase activity were stimulated by L-FABP, indicating that the protein modulates the function of peroxisomal lipid-metabolizing enzymes. The possible role of intraperoxisomal L-FABP in lipid metabolism is discussed.
Biochemical characterization of RGS14: RGS14 activity towards G-protein α subunits is independent of its binding to Rap2A
Authors: Mittal Vivek , Linder Maurine E.
Published: March 2006

RGS (regulators of G-protein signalling) modulate signalling by acting as GAPs (GTPase-activating proteins) for α subunits of heterotrimeric G-proteins. RGS14 accelerates GTP hydrolysis by Giα family members through its RGS domain and suppresses guanine nucleotide dissociation from Giα1 and Giα3 subunits through its C-terminal GoLoco domain. Additionally, RGS14 binds the activated forms of the small GTPases Rap1 and Rap2 by virtue of tandem RBDs (Raf-like Ras/Rap binding domains). RGS14 was identified in a screen for Rap2 effectors [Traver, Splingard, Gaudriault and De Gunzburg (2004) Biochem. J. 379, 627–632]. In the present study, we tested whether Rap binding regulates RGS14's biochemical activities. We found that RGS14 activity towards heterotrimeric G-proteins, as either a GAP or a GDI (guanine nucleotide dissociation inhibitor), was unaffected by Rap binding. Extending our biochemical characterization of RGS14, we also examined whether RGS14 can suppress guanine nucleotide exchange on Giα1 in the context of the heterotrimer. We found that a heterotrimer composed of N-myristoylated Giα1 and prenylated Gβγ is resistant to the GDI activity of the GoLoco domain of RGS14. This is consistent with models of GoLoco domain action on free Gα and suggests that RGS14 alone cannot induce subunit dissociation to promote receptor-independent activation of Gβγ-mediated signalling pathways.
YZGD from Paenibacillus thiaminolyticus, a pyridoxal phosphatase of the HAD (haloacid dehalogenase) superfamily and a versatile member of the Nudix (nucleoside diphosphate x) hydrolase superfamily
Authors: Tirrell Isaac M. , Wall Jennifer L. , Daley Christopher J. , Denial Sarah J. , Tennis Frances G. , Galens Kevin G. , O'Handley Suzanne F.
Published: March 2006

YZGD from Paenibacillus thiaminolyticus is a novel bifunctional enzyme with both PLPase (pyridoxal phosphatase) and Nudix (nucleoside diphosphate x) hydrolase activities. The PLPase activity is catalysed by the HAD (haloacid dehalogenase) superfamily motif of the enzyme, and the Nudix hydrolase activity is catalysed by the conserved Nudix signature sequence within a separate portion of the enzyme, as confirmed by site-directed mutagenesis. YZGD's phosphatase activity is very specific, with pyridoxal phosphate being the only natural substrate, while YZGD's Nudix activity is just the opposite, with YZGD being the most versatile Nudix hydrolase characterized to date. YZGD's Nudix substrates include the CDP-alcohols (CDP-ethanol, CDP-choline and CDP-glycerol), the ADP-coenzymes (NADH, NAD and FAD), ADP-sugars, TDP-glucose and, to a lesser extent, UDP- and GDP-sugars. Regardless of the Nudix substrate, one of the products is always a nucleoside monophosphate, suggesting a role in nucleotide salvage. Both the PLPase and Nudix hydrolase activities require a bivalent metal cation, but while PLPase activity is supported by Co2+, Mg2+, Zn2+ and Mn2+, the Nudix hydrolase activity is Mn2+-specific. YZGD's phosphatase activity is optimal at an acidic pH (pH 5), while YZGD's Nudix activities are optimal at an alkaline pH (pH 8.5). YZGD is the first enzyme reported to be a member of both the HAD and Nudix hydrolase superfamilies, the first PLPase to be recognized as a member of the HAD superfamily and the first Nudix hydrolase capable of hydrolysing ADP-x, CDP-x and TDP-x substrates with comparable substrate specificity.
Effects of insulin and cytosolic redox state on glucose production pathways in the isolated perfused mouse liver measured by integrated 2H and 13C NMR
Authors: Hausler Natasha , Browning Jeffrey , Merritt Matthew , Storey Charles , Milde Angela , Jeffrey F. Mark H. , Sherry A. Dean , Malloy Craig R. , Burgess Shawn C.
Published: March 2006

A great deal is known about hepatic glucose production and its response to a variety of factors such as redox state, substrate supply and hormonal control, but the effects of these parameters on the flux through biochemical pathways which integrate to control glucose production are less clear. A combination of 13C and [2H]water tracers and NMR isotopomer analysis were used to investigate metabolic fluxes in response to altered cytosolic redox state and insulin. In livers isolated from fed mice and perfused with a mixture of substrates including lactate/pyruvate (10:1, w/w), hepatic glucose production had substantial contributions from glycogen, PEP (phosphoenolpyruvate) and glycerol. Inversion of the lactate/pyruvate ratio (1:10, w/w) resulted in a surprising decrease in the contribution from glycogen and an increase in that from PEP to glucose production. A change in the lactate/pyruvate ratio from 10:1 to 1:10 also stimulated flux through the tricarboxylic acid cycle (2-fold), while leaving oxygen consumption and overall glucose output unchanged. When lactate and pyruvate were eliminated from the perfusion medium, both gluconeogenesis and tricarboxylic-acid-cycle flux were dramatically lower. Insulin lowered glucose production by inhibiting glycogenolysis at both low and high doses, but only at high levels of insulin did gluconeogenesis or tricarboxylic-acid-cycle flux tend towards lower values (P<0.1). Our data demonstrate that, in the isolated mouse liver, substrate availability and cellular redox state have a dramatic impact on liver metabolism in both the tricarboxylic acid cycle and gluconeogenesis. The tight correlation of these two pathways under multiple conditions suggest that interventions which increase or decrease hepatic tricarboxylic-acid-cycle flux will have a concomitant effect on gluconeogenesis and vice versa.
Homologous versus heterologous interactions in the bicomponent staphylococcal γ-haemolysin pore1
Authors: Viero Gabriella , Cunaccia Romina , Prévost Gilles , Werner Sandra , Monteil Henri , Keller Daniel , Joubert Olivier , Menestrina Gianfranco , Dalla Serra Mauro
Published: March 2006

Staphylococcal γ-haemolysin HlgA–HlgB forms a β-barrel transmembrane pore in cells and in model membranes. The pore is formed by the oligomerization of two different proteins and a still debated number of monomers. To clarify the topology of the pore, we have mutated single residues – placed near the right and left interfaces of each monomer into cysteine. The mutants were labelled with fluorescent probes, forming a donor–acceptor pair for FRET (fluorescence resonance energy transfer). Heterologous couples (labelled on complementary left and right interfaces) displayed a marked FRET, suggesting extensive HlgA–HlgB or HlgB–HlgA contacts. Heterologous control couples (with both components labelled on the same side) showed absent or low FRET. We found the same result for the homologous couple formed by HlgA [i.e. HlgA–HlgA in the presence of wt (wild-type) HlgB]. The homologous HlgB couple (HlgB–HlgB labelled on left and right interfaces and in the presence of wt HlgA) displayed a transient, declining FRET, which may indicate fast formation of an intermediate that is consumed during pore formation. We conclude that bicomponent pores are assembled by alternating heterologous monomers.
Minimal features of paxillin that are required for the tyrosine phosphorylation of focal adhesion kinase
Authors: Wade Ramon , Vande Pol Scott
Published: March 2006

Tyrosine phosphorylation of FAK (focal adhesion kinase) regulates signalling that results from the interaction of integrins with extracellular matrix and growth factor receptors. A critical step in this process is the phosphorylation of Tyr397 of FAK, which creates a binding site for Src family kinases, PI3K (phosphoinositide 3-kinase) and Shc (Src homology and collagen homology). An intact Tyr397 site is required for FAK-mediated regulation of cell migration, survival signals and full responsiveness to soluble growth factors. We showed previously that the adaptor protein paxillin is required for the overall tyrosine phosphorylation of FAK in embryonic stem cells [Wade, Bohl and Vande Pol (2002) Oncogene 21, 96–107]. In the present paper, we identify the minimal structural features of paxillin that are required to support overall FAK tyrosine phosphorylation and Tyr397 phosphorylation. Paxillin contains N-terminal leucine-rich LD motifs that bind directly to FAK and four LIM (Lin-11, Isl-1 and Mec-3) domains in the C-terminus. We show that paxillin LIM domains 1, 2 and 3 are each required for FAK tyrosine phosphorylation, while LIM4 is dispensable. In addition to paxillin LIM domains 1, 2 and 3, a single LD motif on paxillin is required to support FAK tyrosine phosphorylation in embryonic stem cells. Both sequence and spatial requirements exist for LD motifs to support FAK tyrosine phosphorylation. Interestingly, synthetic LD motifs that fail to bind FAK in vitro are able to fully support FAK tyrosine phosphorylation, indicating that minimal interactions of LD motifs with FAK suffice. Our results demonstrate at least four distinct structural domains of paxillin support at least three distinct functions that are each required for FAK tyrosine phosphorylation.
Identification of a mitochondrial transporter for pyrimidine nucleotides in Saccharomyces cerevisiae: bacterial expression, reconstitution and functional characterization
Authors: Marobbio Carlo Marya Thomas , Di Noia Maria Antonietta , Palmieri Ferdinando
Published: March 2006

Pyrimidine (deoxy)nucleoside triphosphates are required in mitochondria for the synthesis of DNA and the various types of RNA present in these organelles. In Saccharomyces cerevisiae, these nucleotides are synthesized outside the mitochondrial matrix and must therefore be transported across the permeability barrier of the mitochondrial inner membrane. However, no protein has ever been found to be associated with this transport activity. In the present study, Rim2p has been identified as a yeast mitochondrial pyrimidine nucleotide transporter. Rim2p (replication in mitochondria 2p) is a member of the mitochondrial carrier protein family having some special features. The RIM2 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes and its transport properties and kinetic parameters were characterized. It transported the pyrimidine (deoxy)nucleoside tri- and di-phosphates and, to a lesser extent, pyrimidine (deoxy)nucleoside monophosphates, by a counter-exchange mechanism. Transport was saturable, with an apparent Km of 207 μM for TTP, 404 μM for UTP and 435 μM for CTP. Rim2p was strongly inhibited by mercurials, bathophenanthroline, tannic acid and Bromocresol Purple, and partially inhibited by bongkrekic acid. Furthermore, the Rim2p-mediated heteroexchanges, TTP/TMP and TTP/TDP, are electroneutral and probably H+-compensated. The main physiological role of Rim2p is proposed to be to transport (deoxy)pyrimidine nucleoside triphosphates into mitochondria in exchange for intramitochondrially generated (deoxy)pyrimidine nucleoside monophosphates.
Induction of spermidine/spermine N1-acetyltransferase (SSAT) by aspirin in Caco-2 colon cancer cells
Authors: Babbar Naveen , Gerner Eugene W. , Casero Robert A.
Published: March 2006

Epidemiological, experimental and clinical results suggest that aspirin and other NSAIDs (non-steroidal anti-inflammatory drugs) inhibit the development of colon cancer. It has been shown that the NSAID sulindac induces apoptosis and suppresses carcinogenesis, in part, by a mechanism leading to the transcriptional activation of the gene encoding SSAT (spermidine/spermine N1-acetyltransferase), a rate-limiting enzyme in polyamine catabolism. In the present study, we show that a variety of NSAIDs, including aspirin, sulindac, ibuprofen and indomethacin, can induce SSAT gene expression in Caco-2 cells. Aspirin, at physiological concentrations, can induce SSAT mRNA via transcriptional initiation mechanisms. This induction leads to increased SSAT protein levels and enzyme activity. Promoter deletion analysis of the 5′ SSAT promoter-flanking region led to the identification of two NF-κB (nuclear factor κB) response elements. Electrophoretic mobility-shift assays showed binding of NF-κB complexes at these sequences after aspirin treatment. Aspirin treatment led to the activation of NF-κB signalling and increased binding at these NF-κB sites in the SSAT promoter, hence providing a potential mechanism for the induction of SSAT by aspirin in these cells. Aspirininduced SSAT ultimately leads to a decrease in cellular polyamine content, which has been associated with decreased carcinogenesis. These results suggest that activation of SSAT by aspirin and different NSAIDs may be a common property of NSAIDs that plays an important role in their chemopreventive actions in colorectal cancer.
Myogenic differentiation induces taurine transporter in association with taurine-mediated cytoprotection in skeletal muscles
Authors: Uozumi Yoriko , Ito Takashi , Hoshino Yuki , Mohri Tomomi , Maeda Makiko , Takahashi Kyoko , Fujio Yasushi , Azuma Junichi
Published: March 2006

Skeletal muscle homoeostasis is maintained by a variety of cytoprotective mechanisms. Since ablation of the TauT (taurine transporter) gene results in susceptibility to exercise-induced muscle weakness in vivo, it has been suggested that TauT is essential for skeletal muscle function. However, the regulatory mechanisms of TauT expression remain to be elucidated. In the present study, we demonstrated that TauT was up-regulated during myogenesis in C2C12 cells. Treatment with bFGF (basic fibroblast growth factor), which inhibited muscle differentiation, abrogated myogenic induction of TauT. The promoter activities of TauT were up-regulated during muscle differentiation in C2C12 cells. Database analyses identified an MEF2 (myocyte enhancer binding factor 2) consensus sequence at −844 in the rat TauT gene. Truncation of the promoter region containing the MEF2 site significantly reduced the promoter activity, demonstrating the functional importance of the MEF2 site. Electrophoretic mobility-shift assays confirmed that MEF2 bound to the MEF2 consensus sequence and that DNA–protein complex levels were increased during differentiation. Promoter analyses using mutated promoter-reporter plasmids demonstrated that this site was functional. Importantly, transfection with a MyoD expression vector markedly enhanced TauT promoter activity in the (non-myogenic) 10T1/2 cells. Moreover, co-transfection with an MEF2 expression vector augmented MyoD-induced TauT promoter activity, suggesting that MEF2 is required for full activation of TauT expression. Finally, we examined the effects of taurine on myotube atrophy to clarify the biological significance of the up-regulation of TauT, and demonstrated that taurine attenuated muscle atrophy induced by dexamethasone. TauT expression is regulated under the control of the myogenic programme, and we propose that this is the mechanism for taurine-mediated resistance to muscle atrophy.
Herpes simplex virus type 1-encoded glycoprotein C contributes to direct coagulation Factor X–virus binding
Authors: Livingston Joel R. , Sutherland Michael R. , Friedman Harvey M. , Pryzdial Edward L. G.
Published: March 2006

The HSV1 (herpes simplex virus type 1) surface has been shown recently to initiate blood coagulation by FVIIa (activated Factor VII)-dependent proteolytic activation of FX (Factor X). At least two types of direct FX–HSV1 interactions were suggested by observing that host cell-encoded tissue factor and virus-encoded gC (glycoprotein C) independently enhance FVIIa function on the virus. Using differential sedimentation to separate bound from free 125I-ligand, we report in the present study that, in the presence of Ca2+, FX binds directly to purified wild-type HSV1 with an apparent dissociation constant (Kd) of 1.5±0.4 μM and 206±24 sites per virus at saturation. The number of FX-binding sites on gC-deficient virus was reduced to 43±5, and the remaining binding had a lower Kd (0.7±0.2 μM), demonstrating an involvement of gC. Engineering gC back into the deficient strain or addition of a truncated soluble recombinant form of gC (sgC), increased the Kd and the number of binding sites. Consistent with a gC/FX stoichiometry of approximately 1:1, 121±6 125I-sgC molecules were found to bind per wild-type HSV1. In the absence of Ca2+, the number of FX-binding sites on the wild-type virus was similar to the gC-deficient strain in the presence of Ca2+. Furthermore, in the absence of Ca2+, direct sgC binding to HSV1 was insignificant, although sgC was observed to inhibit the FX–virus association, suggesting a Ca2+-independent solution-phase FX–sgC interaction. Cumulatively, these data demonstrate that gC constitutes one type of direct FX–HSV1 interaction, possibly providing a molecular basis for clinical correlations between recurrent infection and vascular pathology.
Gene expression patterns and catalytic properties of UDP-D-glucose 4-epimerases from barley (Hordeum vulgare L.)
Authors: Zhang Qisen , Hrmova Maria , Shirley Neil J. , Lahnstein Jelle , Fincher Geoffrey B.
Published: March 2006

UGE (UDP-Glc 4-epimerase or UDP-Gal 4-epimerase; EC 5.1.3.2) catalyses the interconversion of UDP-Gal and UDP-Glc. Both nucleotide sugars act as activated sugar donors for the biosynthesis of cell wall polysaccharides such as cellulose, xyloglucans, (1,3;1,4)-β-D-glucan and pectins, together with other biologically significant compounds including glycoproteins and glycolipids. Three members of the HvUGE (barley UGE) gene family, designated HvUGE1, HvUGE2 and HvUGE3, have been characterized. Q-PCR (quantitative real-time PCR) showed that HvUGE1 mRNA was most abundant in leaf tips and mature roots, but its expression levels were relatively low in basal leaves and root tips. The HvUGE2 gene was transcribed at significant levels in all organs examined, while HvUGE3 mRNA levels were very low in all the organs. Heterologous expression of a near full-length cDNA confirmed that HvUGE1 encodes a functional UGE. A non-covalently bound NAD+ was released from the enzyme after denaturing with aqueous ethanol and was identified by its spectrophotometric properties and by electrospray ionization MS. The Km values were 40 μM for UDP-Gal and 55 μM for UDP-Glc. HvUGE also catalyses the interconversion of UDP-GalNAc and UDP-GlcNAc, although it is not known if this has any biological significance. A three-dimensional model of the HvUGE revealed that its overall structural fold is highly conserved compared with the human UGE and provides a structural rationale for its ability to bind UDP-GlcNAc.
Characterization of an Arabidopsis inositol 1,3,4,5,6-pentakisphosphate 2-kinase (AtIPK1)
Authors: Sweetman Dylan , Johnson Sue , Caddick Samuel E. K. , Hanke David E. , Brearley Charles A.
Published: March 2006

The metabolic pathway(s) by which plants synthesize InsP6 (inositol 1,2,3,4,5,6-hexakisphosphate) remains largely undefined [Shears (1998) Biochim. Biophys. Acta 1436, 49–67], while the identities of the genes that encode enzymes catalysing individual steps in these pathways are, with the notable exception of myo-inositol phosphate synthase and ZmIpk [Shi, Wang, Wu, Hazebroek, Meeley and Ertl (2003) Plant Physiol. 131, 507–515], unidentified. A yeast enzyme, ScIPK1, catalyses the synthesis of InsP6 by 2-phosphorylation of Ins(1,3,4,5,6)P5 (inositol 1,3,4,5,6-pentakisphosphate). A human orthologue, HsIPK1, is able to substitute for yeast ScIPK1, restoring InsP6 production in a Saccharomyces cerevisiae mutant strain lacking the ScIPK1 open reading frame (ScIpk1Δ). We have identified an Arabidopsis genomic sequence, AtIPK1, encoding an Ins(1,3,4,5,6)P5 2-kinase. Inclusion of the AtIPK1 protein in alignments of amino acid sequences reveals that human and Arabidopis kinases are more similar to each other than to the S. cerevisiae enzyme, and further identifies an additional motif. Recombinant AtIPK1 protein expressed in Escherichia coli catalysed the synthesis of InsP6 from Ins(1,3,4,5,6)P5. The enzyme obeyed Michaelis–Menten kinetics with an apparent Vmax of 35 nmol·min−1·(mg of protein)−1 and a Km for Ins(1,3,4,5,6)P5 of 22 μM at 0.4 mM ATP. RT (reverse transcriptase)–PCR analysis of AtIPK1 transcripts revealed that AtIPK1 is expressed in siliques, leaves and cauline leaves. In situ hybridization experiments further revealed strong expression of AtIPK1 in male and female organs of flower buds. Expression of AtIPK1 protein in an ScIpk1Δ mutant strain restored InsP6 production and rescued the temperature-sensitive growth phenotype of the yeast.
Rescue of cell growth by sphingosine with disruption of lipid microdomain formation in Saccharomyces cerevisiae deficient in sphingolipid biosynthesis
Authors: Tani Motohiro , Kihara Akio , Igarashi Yasuyuki
Published: March 2006

In the yeast Saccharomyces cerevisiae, sphingolipids are essential for cell growth. Inactivation of sphingolipid biosynthesis, such as by disrupting the serine palmitoyltransferase gene (LCB2), is lethal, but cells can be rescued by supplying an exogenous LCB (long-chain base) like PHS (phytosphingosine) or DHS (dihydrosphingosine). In the present study, supplying SPH (sphingosine), an unnatural LCB for yeast, similarly rescued the Δlcb2 cells, but only when SPH 1-phosphate production was inhibited by deleting the LCB kinase gene LCB4. Exogenously added SPH was adequately converted into phosphoinositol-containing complex sphingolipids. Interestingly, cells carrying SPH-based sphingolipids exhibited a defect in the association of Pma1p with Triton X-100-insoluble membrane fractions, and displayed sensitivities to both Ca2+ and hygromycin B. These results suggest that the SPH-based sphingolipids in these cells have properties that differ from those of the PHS- or DHS-based sphingolipids in regard to lipid microdomain formation, leading to abnormal sensitivities towards certain environmental stresses. The present paper is the first report showing that in sphingolipid-deficient S. cerevisiae, the requirement for LCB can be fulfilled by exogenous SPH, although this supplement results in failure of lipid microdomain formation.
Transcription of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase type 3 gene, ATP2A3, is regulated by the calcineurin/NFAT pathway in endothelial cells
Authors: Hadri Lahouaria , Pavoine Catherine , Lipskaia Larissa , Yacoubi Sabrina , Lompré Anne-Marie
Published: March 2006

Histamine, known to induce Ca2+ oscillations in endothelial cells, was used to alter Ca2+ cycling. Treatment of HUVEC (human umbilical-vein endothelial cell)-derived EA.hy926 cells with histamine for 1–3 days increased the levels of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 3, but not of SERCA 2b, transcripts and proteins. Promoter-reporter gene assays demonstrated that this increase in expression was due to activation of SERCA 3 gene transcription. The effect of histamine was abolished by mepyramine, but not by cimetidine, indicating that the H1 receptor, but not the H2 receptor, was involved. The histamine-induced up-regulation of SERCA 3 was abolished by cyclosporin A and by VIVIT, a peptide that prevents calcineurin and NFAT (nuclear factor of activated T-cells) from interacting, indicating involvement of the calcineurin/NFAT pathway. Histamine also induced the nuclear translocation of NFAT. NFAT did not directly bind to the SERCA 3 promoter, but activated Ets-1 (E twenty-six-1), which drives the expression of the SERCA 3 gene. Finally, cells treated with histamine and loaded with fura 2 exhibited an improved capacity in eliminating high cytosolic Ca2+ concentrations, in accordance with an increase in activity of a low-affinity Ca2+-ATPase, like SERCA 3. Thus chronic treatment of endothelial cells with histamine up-regulates SERCA 3 transcription. The effect of histamine is mediated by the H1R (histamine 1 receptor) and involves activation of the calcineurin/NFAT pathway. By increasing the rate of Ca2+ sequestration, up-regulation of SERCA 3 counteracts the cytosolic increase in Ca2+ concentration.
Identification of a short form of ubiquitin-specific protease 3 that is a repressor of rat glutathione S-transferase gene expression
Authors: Whalen Richard , Liu Xiangdang , Boyer Thomas D.
Published: March 2006

The transcription rate and protein expression from both GSTA2 (glutathione S-transferase A2) and albumin genes decrease in rat liver after IL-6 (interleukin 6) plus DEX (dexamethasone) treatment of primary hepatocytes or after LPS (lipopolysaccharide)-induced acute-phase response in animals. The down-regulation is associated with the induced expression of a nuclear protein (termed IL6DEX-NP for IL-6/DEX-induced nuclear protein) that binds to a specific site on the promoter of GSTA2, leading to a decrease in transcriptional activity. IL6DEX-NP is not similar to other transcription factors, and, for identification, we functionally cloned it from a rat liver library using a yeast one-hybrid screen based on DNA-binding activity. The cloned sequence was a truncated form of USP3 (ubiquitin-specific protease 3) and the truncated USP3 protein in a yeast extract bound to DNA containing the IL6DEX-NP recognition sequence. Using 5′- and 3′-RACE (rapid amplification of cDNA ends), the complete sequence of USP3 was found in liver from LPS-treated rats. However, using Western blot analysis, only truncated forms of USP3 could be identified in nuclear extracts from LPS-treated rat livers. A GSTA2 promoter–reporter gene plasmid and USP3-expressing plasmids were transfected into rat hepatoma cells. Expression of the short form of USP3, but not the full-length protein, abolished expression from the reporter gene. Chromatin immunoprecipitation localized USP3 to the GSTA2 promoter in rat hepatocytes in vivo. We believe that the short form of USP3 is IL6DEX-NP and that it may play an important role in the negative regulation of proteins during the acute-phase response.
AToxoplasma gondiiphosphoinositide phospholipase C (TgPI-PLC) with high affinity for phosphatidylinositol
Authors: Fang Jianmin , Marchesini Norma , Moreno Silvia N. J.
Published: March 2006

The Toxoplasma gondii phosphoinositide-specific phospholipase C gene (TgPI-PLC) was cloned, sequenced and expressed in Escherichia coli and its enzymatic characteristics were investigated. TgPI-PLC is present in the genome as a single-copy gene consisting of 22 exons interrupted by 21 introns, and encodes a polypeptide of 1097 amino acids with a predicted molecular mass of 121 kDa. In addition to the conserved catalytic X and Y domains, TgPI-PLC contains an apparent N-terminal PH domain, an EF hand motif and a C-terminal C2 domain. When compared with mammalian δ-type PI-PLC, TgPI-PLC has an additional extended N-terminus and two insertions in the region between the X and Y domains, with a 31–35% identity over the whole sequence. Recombinant TgPI-PLC, as well as the native enzyme obtained from crude membrane extracts of the parasite, was more active with phosphatidylinositol than with phosphatidylinositol 4,5-bisphosphate as substrate. Indirect immunofluorescence analysis using an affinity-purified antibody against TgPI-PLC revealed that this enzyme localizes in the plasma membrane of the parasites.
Human acyl-CoA:cholesterol acyltransferase 2 gene expression in intestinal Caco-2 cells and in hepatocellular carcinoma
Authors: Song Bao-Liang , Wang Can-Hua , Yao Xiao-Min , Yang Li , Zhang Wen-Jing , Wang Zhen-Zhen , Zhao Xiao-Nan , Yang Jin-Bo , Qi Wei , Yang Xin-Ying , Inoue Kenji , Lin Zhi-Xin , Zhang Hui-Zhan , Kodama Tatsuhiko , Chang Catherine C. Y. , Liu Yin-Kun , Chang Ta-Yuan , Li Bo-Liang
Published: March 2006

Humans express two ACAT (acyl-CoA:cholesterol acyltransferase) genes, ACAT1 and ACAT2. ACAT1 is ubiquitously expressed, whereas ACAT2 is primarily expressed in intestinal mucosa and plays an important role in intestinal cholesterol absorption. To investigate the molecular mechanism(s) responsible for the tissue-specific expression of ACAT2, we identified five cis-elements within the human ACAT2 promoter, four for the intestinal-specific transcription factor CDX2 (caudal type homeobox transcription factor 2), and one for the transcription factor HNF1α (hepatocyte nuclear factor 1α). Results of luciferase reporter and electrophoretic mobility shift assays show that CDX2 and HNF1α exert a synergistic effect, enhancing the ACAT2 promoter activity through binding to these cis-elements. In undifferentiated Caco-2 cells, the ACAT2 expression is increased when exogenous CDX2 and/or HNF1α are expressed by co-transfection. In differentiated Caco-2 cells, the ACAT2 expression significantly decreases when the endogenous CDX2 or HNF1α expression is suppressed by using RNAi (RNA interference) technology. The expression levels of CDX2, HNF1α, and ACAT2 are all greatly increased when the Caco-2 cells differentiate to become intestinal-like cells. These results provide a molecular mechanism for the tissue-specific expression of ACAT2 in intestine. In normal adult human liver, CDX2 expression is not detectable and the ACAT2 expression is very low. In the hepatoma cell line HepG2 the CDX2 expression is elevated, accounting for its elevated ACAT2 expression. A high percentage (seven of fourteen) of liver samples from patients affected with hepatocellular carcinoma exhibited elevated ACAT2 expression. Thus, the elevated ACAT2 expression may serve as a new biomarker for certain form(s) of hepatocellular carcinoma.
Direct evidence for S-nitrosation of mitochondrial complex I
Authors: Burwell Lindsay S. , Nadtochiy Sergiy M. , Tompkins Andrew J. , Young Sara , Brookes Paul S.
Published: March 2006

NO• (nitric oxide) is a pleiotropic signalling molecule, with many of its effects on cell function being elicited at the level of the mitochondrion. In addition to the well-characterized binding of NO• to the CuB/haem-a3 site in mitochondrial complex IV, it has been proposed by several laboratories that complex I can be inhibited by S-nitrosation of a cysteine. However, direct molecular evidence for this is lacking. In this investigation we have combined separation techniques for complex I (blue-native gel electrophoresis, Superose 6 column chromatography) with sensitive detection methods for S-nitrosothiols (chemiluminescence, biotin-switch assay), to show that the 75 kDa subunit of complex I is S-nitrosated in mitochondria treated with S-nitrosoglutathione (10 μM–1 mM). The stoichiometry of S-nitrosation was 7:1 (i.e. 7 mol of S-nitrosothiols per mol of complex I) and this resulted in significant inhibition of the complex. Furthermore, S-nitrosothiols were detected in mitochondria isolated from hearts subjected to ischaemic preconditioning. The implications of these results for the physiological regulation of respiration, for reactive oxygen species generation and for a potential role of S-nitrosation in cardioprotection are discussed.
Two exo-β-D-glucosaminidases/exochitosanases from actinomycetes define a new subfamily within family 2 of glycoside hydrolases
Authors: Côté Nathalie , Fleury Alain , Dumont-Blanchette Émilie , Fukamizo Tamo , Mitsutomi Masaru , Brzezinski Ryszard
Published: March 2006

A GlcNase (exo-β-D-glucosaminidase) was purified from culture supernatant of Amycolatopsis orientalis subsp. orientalis grown in medium with chitosan. The enzyme hydrolysed the terminal GlcN (glucosamine) residues in oligomers of GlcN with transglycosylation observed at late reaction stages. 1H-NMR spectroscopy revealed that the enzyme is a retaining glycoside hydrolase. The GlcNase also behaved as an exochitosanase against high-molecular-mass chitosan with Km and kcat values of 0.16 mg/ml and 2832 min−1. On the basis of partial amino acid sequences, PCR primers were designed and used to amplify a DNA fragment which then allowed the cloning of the GlcNase gene (csxA) associated with an open reading frame of 1032 residues. The GlcNase has been classified as a member of glycoside hydrolase family 2 (GH2). Sequence alignments identified a group of CsxA-related protein sequences forming a distinct GH2 subfamily. Most of them have been annotated in databases as putative β-mannosidases. Among these, the SAV1223 protein from Streptomyces avermitilis has been purified following gene cloning and expression in a heterologous host and shown to be a GlcNase with no detectable β-mannosidase activity. In CsxA and all relatives, a serine-aspartate doublet replaces an asparagine residue and a glutamate residue, which were strictly conserved in previously studied GH2 members with β-galactosidase, β-glucuronidase or β-mannosidase activity and shown to be directly involved in various steps of the catalytic mechanism. Alignments of several other GH2 members allowed the identification of yet another putative subfamily, characterized by a novel, serine-glutamate doublet at these positions.
Binding of the C-terminal domain of the HIV-1 capsid protein to lipid membranes: a biophysical characterization
Authors: Barrera Francisco N. , Hurtado-Gómez Estefanía , Lidón-Moya María C. , Neira José L.
Published: March 2006

The capsid protein, CA, of HIV-1 forms a capsid that surrounds the viral genome. However, recent studies have shown that an important proportion of the CA molecule does not form part of this capsid, and its location and function are still unknown. In the present work we show, by using fluorescence, differential scanning calorimetry and Fourier-transform infrared spectroscopy, that the C-terminal region of CA, CA-C, is able to bind lipid vesicles in vitro in a peripheral fashion. CA-C had a greater affinity for negatively charged lipids (phosphatidic acid and phosphatidylserine) than for zwitterionic lipids [PC/Cho/SM (equimolar mixture of phosphatidylcholine, cholesterol and sphingomyelin) and phosphatidylcholine]. The interaction of CA-C with lipid membranes was supported by theoretical studies, which predicted that different regions, occurring close in the three-dimensional CA-C structure, were responsible for the binding. These results show the flexibility of CA-C to undergo conformational rearrangements in the presence of different binding partners. We hypothesize that the CA molecules that do not form part of the mature capsid might be involved in lipid-binding interactions in the inner leaflet of the virion envelope.
A novel role for a Drosophila homologue of cGMP-specific phosphodiesterase in the active transport of cGMP
Authors: Day Jonathan P. , Houslay Miles D. , Davies Shireen-A.
Published: March 2006

cGMP was first discovered in urine, demonstrating that kidney cells extrude this cyclic nucleotide. Drosophila Malpighian tubules provide a model renal system in which a homologue of mammalian PDE (phosphodiesterase) 6 is expressed. In humans, this cG-PDE (cGMP-specific PDE) is specifically expressed in the retinal system, where it controls visual signal transduction. In order to gain insight into the functional role of DmPDE6 (Drosophila PDE6-like enzyme) in epithelial function, we generated transgenic animals with targeted expression of DmPDE6 to tubule Type I (principal) cells. This revealed localization of DmPDE6 primarily at the apical membranes. As expected, overexpression of DmPDE6 resulted in elevated cG-PDE activity and decreased tubule cGMP content. However, such targeted overexpression of DmPDE6 creates a novel phenotype that manifests itself in inhibition of the active transport and efflux of cGMP by tubules. This effect is specific to DmPDE6 action, as no effect on cGMP transport is observed in tubules from a bovine PDE5 transgenic line which display reduced rates of fluid secretion, an effect not seen in DmPDE6 transgenic animals. Specific ablation of DmPDE6 in tubule principal cells, via expression of a targeted DmPDE6 RNAi (RNA interference) transgene, conferred increased active transport of cGMP, confirming a direct role for DmPDE6 in regulating cGMP transport in tubule principal cells. Pharmacological inhibition of DmPDE6 in wild-type tubules using the cG-PDE inhibitor, zaprinast, similarly results in stimulated cGMP transport. We provide the first demonstration of a novel role for a cG-PDE in modulating cGMP transport and efflux.
Phosphodiesterase-4 influences the PKA phosphorylation status and membrane translocation of G-protein receptor kinase 2 (GRK2) in HEK-293β2 cells and cardiac myocytes
Authors: Li Xiang , Huston Elaine , Lynch Martin J. , Houslay Miles D. , Baillie George S.
Published: March 2006

Membrane-recruitment of GRK2 (G-protein receptor kinase 2) provides a fundamental step in the desensitization process controlling GPCRs (G-protein-coupled receptors), such as the β2AR (β2-adrenergic receptor). In the present paper, we show that challenge of HEK-293β2 [human embryonic kidney cells stably overexpressing the FLAG-tagged β2AR–GFP (green fluorescent protein)] cells with the β-adrenoceptor agonist, isoprenaline, causes GRK2 to become phosphorylated by PKA (cAMP-dependent protein kinase). This action is facilitated when cAMP-specific PDE4 (phosphodiesterase-4) activity is selectively inactivated, either chemically with rolipram or by siRNA (small interfering RNA)-mediated knockdown of PDE4B and PDE4D. PDE4-selective inhibition by rolipram facilitates the isoprenaline-induced membrane translocation of GRK2, phosphorylation of the β2AR by GRK2, membrane translocation of β-arrestin and internalization of β2ARs. PDE4-selective inhibition also enhances the ability of isoprenaline to trigger the PKA phosphorylation of GRK2 in cardiac myocytes. In the absence of isoprenaline, rolipram-induced inhibition of PDE4 activity in HEK-293β2 cells acts to stimulate PKA phosphorylation of GRK2, with consequential effects on GRK2 membrane recruitment and GRK2-mediated phosphorylation of the β2AR. We propose that a key role for PDE4 enzymes is: (i) to gate the action of PKA on GRK2, influencing the rate of GRK2 phosphorylation of the β2AR and consequential recruitment of β-arrestin subsequent to β-adrenoceptor agonist challenge, and (ii) to protect GRK2 from inappropriate membrane recruitment in unstimulated cells through its phosphorylation by PKA in response to fluctuations in basal levels of cAMP.
Spectroscopic and kinetic characterization of the light-dependent enzyme protochlorophyllide oxidoreductase (POR) using monovinyl and divinyl substrates
Authors: Heyes Derren J. , Kruk Jerzy , Hunter C. Neil
Published: March 2006

The enzyme POR [Pchlide (protochlorophyllide) oxidoreductase] catalyses the reduction of Pchlide to chlorophyllide, which is a key step in the chlorophyll biosynthesis pathway. This light-dependent reaction has previously been studied in great detail but recent reports suggest that a mixture of MV (monovinyl) and DV (divinyl) Pchlides may have influenced some of these properties of the reaction. Low-temperature absorbance and fluorescence spectroscopy have revealed several spectral differences between MV and DV Pchlides, which were purified from a Rhodobacter capsulatus strain that was shown to contain a mixture of the two pigments. A thorough steady-state kinetic characterization using both Pchlide forms demonstrates that neither pigment appears to affect the kinetic properties of the enzyme. The reaction has also been monitored following illumination at low temperatures and was shown to consist of an initial photochemical step followed by four ‘dark’ steps for both pigments. However, minor differences were observed in the spectral properties of some of the intermediates, although the temperature dependency of each step was nearly identical for the two pigments. This work provides the first detailed kinetic and spectroscopic study of this unique enzyme using biologically important MV and DV substrate analogues. It also has significant implications for the DV reductase enzyme, which is responsible for converting DV pigments into their MV counterparts, and its position in the sequence of reactions that comprise the chlorophyll biosynthesis pathway.
Electrohydrodynamic jetting of mouse neuronal cells
Authors: Eagles Peter A. M. , Qureshi Amer N. , Jayasinghe Suwan N.
Published: March 2006

CAD (Cath.a-differentiated) cells, a mouse neuronal cell line, were subjected to electrohydrodynamic jetting at a field strength of 0.47–0.67 kV/mm, corresponding to an applied voltage of 7–10 kV. After jetting, the cells appeared normal and continued to divide at rates similar to those shown by control samples. Jetted cells, when placed in serum-free medium, underwent differentiation that was sustained for at least 1 month. Some of the droplets produced by jetting contained only a few cells. These results indicate that the process of jetting does not significantly perturb neuronal cells and that this novel approach might in the future be a useful way to deposit small numbers of living nerve cells on to surfaces.
Life cycle of connexins in health and disease
Authors: Laird Dale W.
Published: March 2006

Evaluation of the human genome suggests that all members of the connexin family of gap-junction proteins have now been successfully identified. This large and diverse family of proteins facilitates a number of vital cellular functions coupled with their roles, which range from the intercellular propagation of electrical signals to the selective intercellular passage of small regulatory molecules. Importantly, the extent of gap-junctional intercellular communication is under the direct control of regulatory events associated with channel assembly and turnover, as the vast majority of connexins have remarkably short half-lives of only a few hours. Since most cell types express multiple members of the connexin family, compensatory mechanisms exist to salvage tissue function in cases when one connexin is mutated or lost. However, numerous studies of the last decade have revealed that mutations in connexin genes can also lead to severe and debilitating diseases. In many cases, single point mutations lead to dramatic effects on connexin trafficking, assembly and channel function. This review will assess the current understanding of wild-type and selected disease-linked mutant connexin transport through the secretory pathway, gap-junction assembly at the cell surface, internalization and degradation.
Role of HNF4α in the superinduction of the IL-1β-activated iNOS gene by oxidative stress
Authors: Gonzalez Frank J.
Published: March 2006

IL-1β (interleukin-1β) treatment of hepatocytes results in an NF-κB (nuclear factor-κB)-mediated activation of the iNOS (induced nitric oxide synthase) gene, and this increase in gene expression is further augmented by oxidative stress. Oxidative stress alone has no influence on the iNOS promoter, therefore indicating that the promoter needs to be primed by NF-κB. In this issue of the Biochemical Journal, Guo et al. extend their earlier work, showing that HNF4α (hepatocyte nuclear factor 4α) mediates the superinduction of iNOS observed by co-treating cells with IL-1β plus H2O2. A specific phosphorylation by p38 kinase at Ser-158 of HNF4α results in increased binding of HNF4α to the iNOS promoter, leading to enhanced transcription. The study by Guo et al. is the first to show definitively that HNF4α can be modulated to differentially activate specific genes. However, issues remain to determine the functional significance in vivo of the elevated iNOS activity, and the mechanism that governs the specificity of HNF4α towards the iNOS promoter element as compared with many other HNF4α target genes in the hepatocyte.