Biochimica Et Biophysica Acta - Biomembranes, Aug 1, 2001
The glucose transporter of Trypanosoma brucei was reconstituted by incorporating Escherichia coli... more The glucose transporter of Trypanosoma brucei was reconstituted by incorporating Escherichia coli phospholipid liposomes into detergent-solubilised trypanosome membranes. Proteoliposome vesicles were formed by detergent dilution and used in glucose-uptake assays. The minima for functional reconstitution of the glucose transporter were established and used to probe the mechanism of glucose transport. The uptake pattern of radiolabelled glucose showed a counterflow transient at about 3 s, after which the sugar equilibrated across the proteoliposomal membrane. This observation is consistent with a facilitated transporter. There was a six-fold increase in the initial rate of glucose uptake compared to non-reconstituted or native membranes. In addition, the transporter exhibited stereospecificity to D-glucose but poorly transported L-glucose. Directionality, stereoselectivity or substrate specificity and cis-inhibition by phloridzin were therefore the main criteria for validation of glucose transport. The observed counterflow transient also provided further evidence for a facilitated glucose transporter within the trypanosome plasma membrane, and was the single most important criterion for this assertion. A stoichiometry of 0.78 mol of glucose per mol of transporter was estimated.
Biochimica Et Biophysica Acta - General Subjects, Mar 1, 2010
Background: Factor VIII is the cofactor for Factor X activation by Factor IXa. Activated Factor X... more Background: Factor VIII is the cofactor for Factor X activation by Factor IXa. Activated Factor X, Factor Xa, in turn activates prothrombin in a sequence that leads to fibrin clot formation at the site of vascular injury. Although the biochemistry of the cascade has been well studied, the molecular mechanism underlying the cofactor role of Factor VIII is not understood. Methods: We screened a bacterial peptide display library with Factor IXa and Factor X co-immobilized on tosylactivated Dynabeads which were then used as platelet surrogates. Validation of peptide selection procedure and comparison of Factor VIII-like cofactor activity of oxidoreductases was performed using COATEST assays. Determination of Factor VIII as a folding catalyst with potential disulphide isomerase activity was determined using the RNase A renaturation assay. Results: We set out to identify the cofactor requirements of the Factor IXa/Factor X procoagulant complex by random peptide display, and isolated a peptide with the active-site sequence, CGPC, of thioredoxin. This peptide was able to activate Factor X in a Factor IXa-dependent manner. Redox catalysts or oxidoreductases with homologous active-site vicinal cysteines such as PDI and DsbA also mimicked Factor VIII in their requirement of Factor IXa in Factor X activation. However, the cofactor activity of these peptides was up to a 1000-fold lower than that of Factor VIII and they were therefore unable to catalyse blood coagulation. Factor X activation by PDI and by Factor VIII was abolished by oxidation in an isolated system, which implies a possible role for thiol-disulphide exchange in the activity of the tenase complex. Using scrambled RNase A as a surrogate substrate, we also found that Factor VIII could renature this enzyme. Conclusion: Our findings suggest that Factor VIII may be a specialized folding catalyst with disulphide isomerase activity. We suggest that it is this activity that may underlie its cofactor function in Factor X activation, and that this function is interchangeable with classical oxidoreductases. General significance: The possible involvement of thiol-disulphide interchange as a mechanism underlying Factor VIII cofactor activity may provide some insight into the biochemistry of the intrinsic tenase complex.
Glucose transport in Trypanosoma brucei is facilitated by a transporter that is kinetically marke... more Glucose transport in Trypanosoma brucei is facilitated by a transporter that is kinetically markedly different from its mammalian homologue. In this regard, the trypanosomal transporter may be selectively targeted. We investigated the potential of a series of triazinyl derivatives as inhibitors of glucose transport in T. brucei. A graded response of glucose transporter inhibition by these compounds was observed, with Cibacron blue 3GA, CiB, being the most potent. This inhibited transport by up to 90% in a concentration-dependent manner, with an IC50 of about 19.4 microM. A Dixon plot of different concentrations of this triazine and the rate of transport suggested that inhibition may be simple and competitive. The inhibition constant Ki was 14.8 microM. Although cytochalasin B has been widely reported to inhibit glucose uptake by mammalian and other eukaryotic glucose transporters, it had no effect at all on the trypanosome transporter at concentrations equivalent to those of the triazines. This may suggest structural differences between the trypanosome and mammalian glucose transporters and also suggests that the triazine moiety may serve as a template for the design potent trypanocides targeted at the glucose transporter.
The African trypanosome Trypanosoma brucei has a digenetic life cycle that involves the insect ve... more The African trypanosome Trypanosoma brucei has a digenetic life cycle that involves the insect vector and the mammalian host. This is underscored by biochemical switches in its nutritional requirements. In the insect vector, the parasite relies on amino acid catabolism, but in the mammalian host, it derives its energy exclusively from blood glucose. Glucose transport is facilitated, and constitutes the rate-limiting step in ATP synthesis. Here, we report the cloning of a novel glucose transporter-related gene by heterologous screening of a EMBL4 genomic library of T. brucei EATRO 164 using a rat liver glucose transporter cDNA clone. Genomic analysis shows that the gene is present as a single copy within the parasite genome. The gene encodes a protein with an estimated molecular mass of 55.9 kDa, which shares only segmental homology with members of the glucose transporter superfamily. Several potential post-translational modification sites including phosphorylation, N-glycosylation, and cotranslational myristoylation sites also punctuate the sequence. It is distinguished from classical transporter proteins by the absence of putative hydrophobic membrane-spanning domains. However, this protein was capable of complementing Schizosaccharomyces pombe glucose transporter mutants. The rescued phenotype conferred the ability of the cells to grow on a broad range of sugars, both monosaccharides and disaccharides. The kinetics of glucose uptake reflected those in T. brucei. In addition to complementation in yeast, we also showed that the gene enhanced glucose uptake in cultured mammalian cells. The bloodstream forms of Trypanosoma brucei are absolutely dependent on peripheral glucose supply and derive their energy by glycolysis (1, 2). The glycosome is the major site for glycolysis, and most glycolytic enzymes are compartmentalized here (3). There is evidence suggesting the differential expression of glycolytic enzymes in the bloodstream forms (4). This may be indicative of a differentially expressed glucose transporter, assuming that the two systems operate in tandem. In order to be shunted into the glycolytic pathway, glucose must therefore cross the plasma membrane and then pass through the glycosomal membrane. It might appear that both compartments are equipped with glucose transporters, although it is suggested that the flagellar pocket may serve in solute uptake by endocytosis (5). Glucose uptake by bloodstream forms is mediated by a facilitated glucose transporter (6-8). Kinetic measurements indicate that there are substantial differences between this transporter and that of the mammalian host. Some of these differences include a 20-50-fold higher rate of glucose metabolism than the mammalian host cells (9); insensitivity to cytochalasin B (unpublished observations), and the ability to transport fructose (10). Some work has shown that several glucose transporters are present in the kinetoplastids, which differ largely in the stage specificity of expression (11-14). These transporters showed apparent homology to members of the facilitative glucose transporters including the presence of putative transmembrane segments. The kinetics of glucose uptake and sensitivity to known inhibitors of transport were also similar in many respects. However, because those transporters were identified either with variant surface glycoprotein gene probes (11) or based on developmental expression (13), we reasoned that other unidentified transporters still exist in T. brucei. By heterologous probing with a rat liver glucose transporter cDNA, we isolated and cloned a trypanosome protein that is distinct from any previously reported. Although it has only residual homology to the classical glucose transporters, it was able to rescue fission yeast glucose transporter mutants by supporting growth on sugars, both disaccharides and monosaccharides. This protein may therefore belong to a new class of transporters, or it may be tightly associated with glucose uptake and metabolism.
SIRT1 protects against several complex metabolic and ageing-related diseases (MARDs), and is ther... more SIRT1 protects against several complex metabolic and ageing-related diseases (MARDs), and is therefore considered a polypill target to improve healthy ageing. Although dietary sirtuinactivating compounds (dSTACs) including resveratrol are promising drug candidates, their clinical application has been frustrated by an imprecise understanding of how their signals are transduced into increased healthspan. Recent work indicates that SIRT1 and orthologous sirtuins coactivate the oestrogen receptor/ER and the worm steroid receptor DAF-12. Here they are further shown to ligand-independently transduce dStAcs signals through these receptors. While some dStAcs elicit eR subtype-selectivity in the presence of hormone, most synergize with 17β-oestradiol and dafachronic acid respectively to increase ER and DAF-12 coactivation by the sirtuins. These data suggest that dStAcs functionally mimic gonadal steroid hormones, enabling sirtuins to transduce the cognate signals through a conserved endocrine pathway. Interestingly, resveratrol non-monotonically modulates sirtuin signalling, suggesting that it may induce hormesis, i.e. "less is more". together, the findings suggest that dSTACs may be informational molecules that use exploitative mimicry to modulate sirtuin signalling through steroid receptors. Hence dStAcs' intrinsic oestrogenicity may underlie their proven ability to impart the health benefits of oestradiol, and also provides a mechanistic insight into how they extend healthspan or protect against MARDs. Among the seven human sirtuins, SIRT1 (silent information regulator 2 homologue 1) has received the most attention because of its many roles including gene regulation, genomic stability and energy metabolism 1,2. SIRT1 is also of enormous interest as a viable drug target because it protects against several conditions including obesity, type 2 diabetes, cancer and cardiovascular and neurodegenerative diseases 3,4. However, deciphering how this protein is regulated is complicated by the fact that it appears to be a hub for multiple networks while also participating in several reciprocal interactions and autoregulatory loops 5. This further frustrates our understanding of how it transduces signals from interacting partners but especially from sirtuin activating compounds (STACs) such as resveratrol. These molecules are thought to allosterically activate SIRT1 by directly binding to its N-terminal STACs-activation domain, STACs-AD. Although this proposed mechanism has been intensely controversial 6-8 , evidence suggests that a single residue (E230) within the STACs-AD may be responsible for allosteric activation 9. Furthermore, co-crystal structures of STACs and SIRT1 support direct binding consistent with an allosteric mechanism 10,11. Alternative mechanisms propose that AMP-activated protein kinase may transduce STACs signals but the pathway leading to SIRT1 activation is unclear 12,13. Hence there is no consensus on how SIRT1 and orthologous sirtuins translate the beneficial effects of resveratrol and related dietary STACs (dSTACs) to extend healthspan in diverse organisms including humans. Since dSTACs can allosterically increase sirtuin deacetylase activities, they portend pharmacological interventions for MARDs 8,14-16. However this has been slowed by an imprecise understanding of how the cognate allosteric signals are transduced by the sirtuins, partly because dSTACs are functionally promiscuous 8,17. A clue to how they work in vivo may be in the fact that with the exception of SRT1720 and its relatives, all are phytoestrogens 8,17 that are structurally similar to the oestrogen receptor (ER) steroidal ligand 17β-oestradiol (hereafter referred to as E2 or oestrogen), and have been shown to compete with this hormone for ER binding 18-21. In what appears to hint at how dSTACs may modulate sirtuin signalling, resveratrol in particular has been shown to activate the ER and to competitively inhibit oestradiol binding to the receptor's ligand binding domain (LBD) 22. Recent work 23 described SIRT1 and the orthologous sirtuins Sir2 (Saccharomyces cerevisiae) and Sir-2.1 (Caenorhabditis elegans) as nuclear receptor coregulators that coactivate the ER and DAF-12, the steroid receptor that regulates nematode lifespan and reproductive development 24-26. The data reported herein build on those findings to show that dSTACs ligand-independently enhance sirtuin signalling through these receptors. The results also suggest
Rational drug discovery relies on pathognomonic molecular reporters of disease or biomarkers. The... more Rational drug discovery relies on pathognomonic molecular reporters of disease or biomarkers. Therefore biomarkers contain relational or contextual information about disease pathophysiology. Two broad pathways can be taken to identify biomarkers: a 'top-down', holistic approach that makes no assumptions about biomarker type, or the 'bottom-up' approach, which is hypothesis driven and relies on a priori information. Both approaches involve parallel or sequential methods that include genomic and proteomic profiling. Biomarker discovery and translational medicine owe much to isotopic techniques because these provide near-real-time information about disease status as diagnostics, in drug delivery and for monitoring treatment. Here, we provide an overview of recent developments and some insight into the future role of isotopes in biomarker discovery and disease therapy. Reviews POST SCREEN
RNA polymerase II (pol II) promoters are rare in the African trypanosome Trypanosoma brucei becau... more RNA polymerase II (pol II) promoters are rare in the African trypanosome Trypanosoma brucei because gene regulation in the parasite is complex and polycistronic. Here, we describe a putative pol II promoter and its structure-function relationship. The promoter has features of an archetypal eukaryotic pol II promoter including putative canonical CCAAT and TATA boxes, and an initiator element. However, the spatial arrangement of these elements is only similar to yeast pol II promoters. Deletion mapping and transcription assays enabled delineation of a minimal promoter that could drive orientation-independent reporter gene expression suggesting that it may be a bidirectional promoter. In vitro transcription in a heterologous nuclear extract revealed that the promoter can be recognized by the basal eukaryotic transcription complex. This suggests that the transcription machinery in the parasite may be very similar to those of other eukaryotes.
Available from British Library Document Supply Centre- DSC:DX97425 / BLDSC - British Library Docu... more Available from British Library Document Supply Centre- DSC:DX97425 / BLDSC - British Library Document Supply CentreSIGLEGBUnited Kingdo
Des dendrimeres de lipides cationiques de diametre bien defini et de groupement amines terminaux ... more Des dendrimeres de lipides cationiques de diametre bien defini et de groupement amines terminaux en nombre precis (de huit a trente-deux) ont ete synthetises sur un support solide. L'utilisation de dendrimeres de geometries variees a ete envisagee pour l'administration de genes par evaluation de l'efficacite de transfection des membres d'une serie variant par la longueur des ramifications, le niveau de liaisons des lipides, la presence de sucre et la presence d'un peptide a localisation nucleaire (nuclear localization signal'). L'activite de transfection des produits a ete etudiee in vitro sur des cellules cos 7 (fibroblastes). Les dendrimeres (1a et 1b) presentent de fortes activites de transfection. Les resultats montrent que la presence d'un plus grand nombre de groupements amino a la surface des dendrimeres peut ameliorer l'administration des genes. Une premiere caracterisation physicochimique des complexes ADN/lipide a permis de demontrer la quantite minimale de dendrimeres necessaires pour la transfection de 2,5 μg de plasmide (10 μg/ml pour les dendrimeres a huit groupes amino libres terminaux et 5 et 2,5 μg/ml pour les dendrimeres avec respectivement seize et trente-deux groupes amino terminaux).
Resveratrol has been widely investigated for its potential health properties, although little is ... more Resveratrol has been widely investigated for its potential health properties, although little is known about its metabolism in vivo. Here we investigated the distribution of metabolic products of [ 3 H]trans-resveratrol, following gastric administration. At 2 h, plasma concentrations reached 1•7 % of the administered dose, whilst liver and kidney concentrations achieved 1•0 and 0•6 %, respectively. Concentrations detected at 18 h were lower, being only 0•5 % in plasma and a total of 0•35 % in tissues. Furthermore, whilst kidney and liver concentrations fell to 10 and 25 %, respectively, of concentrations at 2 h, the brain retained 43 % of that measured at 2 h. Resveratrol-glucuronide was identified as the major metabolite, reaching 7 mM in plasma at 2 h. However, at 18 h the main form identified in liver, heart, lung and brain was native resveratrol aglycone, indicating that it is the main form retained in the tissues. No phenolic degradation products were detected in urine or tissues, indicating that, unlike flavonoids, resveratrol does not appear to serve as a substrate for colonic microflora. The present study provides additional information about the nature of resveratrol metabolites and which forms might be responsible for its in vivo biological effects.
Biochemical and Biophysical Research Communications, 2016
The liver expresses batteries of cytoprotective genes that confer cellular resistance to oxidativ... more The liver expresses batteries of cytoprotective genes that confer cellular resistance to oxidative stress and xenobiotic toxins, and protection against cancer and other stress-related diseases. These genes are mainly regulated by Nrf2, making this transcription factor a target for small molecule discovery to treat such diseases. In this report, we identified dietary polyphenolic antioxidants that not only activated these genes but also relieved Nrf2 repression by Keap1, a Cul3dependent ubiquitin ligase adaptor protein that mediates its degradation. Analysis of postprandial liver RNA revealed a marked activation of both genes by all test polyphenols compared with controls. Nrf2 inhibition by RNA interference reduced polyphenol effects on its target gene expression. Our data suggest that polyphenols may induce cellular defense genes by derepressing Nrf2 inhibition by Keap1. We posit that this ability to derepress Nrf2 and reactivate its target genes may underlie the protection conferred by polyphenols against oxidative stress-related diseases.
The 235 kDa rhoptry protein Py235 of Plasmodium yoelii, has been implicated in erythrocyte invasi... more The 235 kDa rhoptry protein Py235 of Plasmodium yoelii, has been implicated in erythrocyte invasion by the merozoite forms of the parasite. Py235 is encoded by a large, highly polymorphic gene family, members of which appear to be differentially transcribed. However, it is not clear how many variants are expressed at the protein level during an infection cycle and whether or not these variants are expressed selectively or combinatorially. Certain monoclonal antibodies to Py235 have been shown to attenuate parasite virulence upon passive transfer into mice, suggesting that this antigen or its derivatives may be useful vaccine candidates. To provide a basis for this, we sought to identify those variants that are recognised by the host immune system, and to establish the pattern of expression of the antigen in mice during infection. Using Py235 monoclonal antibodies as probes, we isolated distinct antigenic variants from an expression library, suggesting that the antigen repertoire is potentially large and that different Py235 variants may be produced during infection. The implications of these observations are discussed with respect to the ability of a cloned parasite line to express distinct antigenic variants in vivo.
Fems Immunology and Medical Microbiology, Aug 1, 2007
The 235-kDa antigenic rhoptry protein Py235 of Plasmodium yoelii is encoded by a large, highly po... more The 235-kDa antigenic rhoptry protein Py235 of Plasmodium yoelii is encoded by a large, highly polymorphic gene family. Monoclonal antibodies to some of these antigens have been shown to attenuate the virulence of the lethal YM strain of the parasite, converting a potentially fatal YM infection to a fulminating one typical of the nonlethal 17X strain, by inducing a switch in target cell preference from mature red blood cells to reticulocytes. The reason for this is not known but would suggest that antigenic determinants of Py235 may be useful in or as subunit vaccines. To identify such determinants, we constructed an epitope expression library of one Py235 variant and screened the library with the antibodies. Thus, we mapped 5-and 12-amino acid epitopes to the C-terminus of the antigen. Both epitopes were more reactive with protective than with nonprotective monoclonal antibodies. This may explain the differential protection conferred by these antibodies upon their passive transfer into mice.
Introducción: La transferrina (Tf) ejerce una función crucial en el mantenimiento de la homeostas... more Introducción: La transferrina (Tf) ejerce una función crucial en el mantenimiento de la homeostasis sistémica del hierro. La expresión del gen de la transferrina es controlada a nivel transcripcional, aunque la posible influencia de factores genéticos todavía se desconoce. Objetivo: Estudiar el papel del rs3811647 en la expresión de la transferrina mediante un ensayo in-vitro en células de hepatoma. Diseño y métodos: Células Hep3B fueron co-transfectadas con vectores que contenían las variantes A (VarA-Tfluc) y G (VarG-Tf-luc) del rs3811647, utilizandose la luciferasa como marcador de la expresión del gen Tf. Resultados: Los ensayos con la luciferasa mostraron un mayor aumento de la expresión del gen Tf en presencia de la variante A comparada con la G (p < 0,05). El análisis in silico del SNP rs3811647 mostró que la presencia del alelo A puede constituir un sitio de unión del receptor de glucocorticoides (GR). Conclusión: El alelo A del SNP rs3811647 incrementa la expresión del gen Tf de modo que podría modular la variación interindividual en los niveles de transferrina sérica observados en diferentes poblaciones.
Hepcidin is a small acute phase peptide that regulates iron absorption. It is induced by inflamma... more Hepcidin is a small acute phase peptide that regulates iron absorption. It is induced by inflammation and infection, but is repressed by anaemia and hypoxia. Here we further reveal that hepcidin transcription also involves interactions between functional metal response elements (MREs) in its promoter, and the MRE-binding transcription factor-1. Analysis of hepcidin mRNA and protein levels in hepatoma cells suggests that its expression may be regulated by divalent metal ions, with zinc inducing maximal effects on hepcidin levels. These data suggest that this peptide may be a pleiotropic sensor of divalent metals, some of which are xenobiotic environmental toxins.
Ferroportin (Fpn/IREG1/MTP1) is the only known transporter mediating iron efflux from epithelial ... more Ferroportin (Fpn/IREG1/MTP1) is the only known transporter mediating iron efflux from epithelial cells and macrophages, and thus regulates how much iron is released into the circulation. Consequently, Fpn mutations are associated with haemochromatosis. Fpn itself is post-translationally regulated by hepcidin (Hepc) which induces its redistribution and degradation in a ubiquitin-dependent process. Together, the two proteins appear to be the nexus for iron homeostasis. Here we show that a rare gain-of-function mutation (K240E) that is associated with iron overload, impedes Fpn binding and subcellular trafficking by the small ubiquitin-like modifier (SUMO). Whereas wild-type Fpn is ensconced within vesicular bodies, the FpnK240E mutant appeared diffused within the cell when co-expressed with SUMO. Furthermore, compared with wild type Fpn, the sumoylation-defective mutant was constitutively-active, resulting in a lower intracellular labile iron pool than the former. These findings suggest that SUMO may regulate iron homeostasis by controlling Fpn trafficking.
Introduction: Transferrin (Tf) exerts a crucial function in the maintenance of systemic iron home... more Introduction: Transferrin (Tf) exerts a crucial function in the maintenance of systemic iron homeostasis. The expression of the Tf gene is controlled by transcriptional mechanism, although little is known about genetic factors influence. Objective: To study the role of rs3811647 in Tf expression using an in-vitro assay on hepatoma cells. Design and methods: Hep3B cells were co-transfected with constructs containing A (VarA-Tf-luc) and G (VarG-Tf-luc) variants of rs3811647, using luciferase as a surrogate reporter of Tf expression. Results: Luciferase assays showed a higher intrinsic enhancer activity (p < 0.05) in the A compared with the G variant. In silico analysis of SNP rs3811647 showed that the A allele might constitute a binding site for the transcription factor glucocorticoid receptor (GR). Conclusion: The A allele of SNP rs3811647 increases Tf expression in a manner that might underlie inter-individual variation in serum transferrin levels observed in different population groups.
Biochimica Et Biophysica Acta - Biomembranes, Aug 1, 2001
The glucose transporter of Trypanosoma brucei was reconstituted by incorporating Escherichia coli... more The glucose transporter of Trypanosoma brucei was reconstituted by incorporating Escherichia coli phospholipid liposomes into detergent-solubilised trypanosome membranes. Proteoliposome vesicles were formed by detergent dilution and used in glucose-uptake assays. The minima for functional reconstitution of the glucose transporter were established and used to probe the mechanism of glucose transport. The uptake pattern of radiolabelled glucose showed a counterflow transient at about 3 s, after which the sugar equilibrated across the proteoliposomal membrane. This observation is consistent with a facilitated transporter. There was a six-fold increase in the initial rate of glucose uptake compared to non-reconstituted or native membranes. In addition, the transporter exhibited stereospecificity to D-glucose but poorly transported L-glucose. Directionality, stereoselectivity or substrate specificity and cis-inhibition by phloridzin were therefore the main criteria for validation of glucose transport. The observed counterflow transient also provided further evidence for a facilitated glucose transporter within the trypanosome plasma membrane, and was the single most important criterion for this assertion. A stoichiometry of 0.78 mol of glucose per mol of transporter was estimated.
Biochimica Et Biophysica Acta - General Subjects, Mar 1, 2010
Background: Factor VIII is the cofactor for Factor X activation by Factor IXa. Activated Factor X... more Background: Factor VIII is the cofactor for Factor X activation by Factor IXa. Activated Factor X, Factor Xa, in turn activates prothrombin in a sequence that leads to fibrin clot formation at the site of vascular injury. Although the biochemistry of the cascade has been well studied, the molecular mechanism underlying the cofactor role of Factor VIII is not understood. Methods: We screened a bacterial peptide display library with Factor IXa and Factor X co-immobilized on tosylactivated Dynabeads which were then used as platelet surrogates. Validation of peptide selection procedure and comparison of Factor VIII-like cofactor activity of oxidoreductases was performed using COATEST assays. Determination of Factor VIII as a folding catalyst with potential disulphide isomerase activity was determined using the RNase A renaturation assay. Results: We set out to identify the cofactor requirements of the Factor IXa/Factor X procoagulant complex by random peptide display, and isolated a peptide with the active-site sequence, CGPC, of thioredoxin. This peptide was able to activate Factor X in a Factor IXa-dependent manner. Redox catalysts or oxidoreductases with homologous active-site vicinal cysteines such as PDI and DsbA also mimicked Factor VIII in their requirement of Factor IXa in Factor X activation. However, the cofactor activity of these peptides was up to a 1000-fold lower than that of Factor VIII and they were therefore unable to catalyse blood coagulation. Factor X activation by PDI and by Factor VIII was abolished by oxidation in an isolated system, which implies a possible role for thiol-disulphide exchange in the activity of the tenase complex. Using scrambled RNase A as a surrogate substrate, we also found that Factor VIII could renature this enzyme. Conclusion: Our findings suggest that Factor VIII may be a specialized folding catalyst with disulphide isomerase activity. We suggest that it is this activity that may underlie its cofactor function in Factor X activation, and that this function is interchangeable with classical oxidoreductases. General significance: The possible involvement of thiol-disulphide interchange as a mechanism underlying Factor VIII cofactor activity may provide some insight into the biochemistry of the intrinsic tenase complex.
Glucose transport in Trypanosoma brucei is facilitated by a transporter that is kinetically marke... more Glucose transport in Trypanosoma brucei is facilitated by a transporter that is kinetically markedly different from its mammalian homologue. In this regard, the trypanosomal transporter may be selectively targeted. We investigated the potential of a series of triazinyl derivatives as inhibitors of glucose transport in T. brucei. A graded response of glucose transporter inhibition by these compounds was observed, with Cibacron blue 3GA, CiB, being the most potent. This inhibited transport by up to 90% in a concentration-dependent manner, with an IC50 of about 19.4 microM. A Dixon plot of different concentrations of this triazine and the rate of transport suggested that inhibition may be simple and competitive. The inhibition constant Ki was 14.8 microM. Although cytochalasin B has been widely reported to inhibit glucose uptake by mammalian and other eukaryotic glucose transporters, it had no effect at all on the trypanosome transporter at concentrations equivalent to those of the triazines. This may suggest structural differences between the trypanosome and mammalian glucose transporters and also suggests that the triazine moiety may serve as a template for the design potent trypanocides targeted at the glucose transporter.
The African trypanosome Trypanosoma brucei has a digenetic life cycle that involves the insect ve... more The African trypanosome Trypanosoma brucei has a digenetic life cycle that involves the insect vector and the mammalian host. This is underscored by biochemical switches in its nutritional requirements. In the insect vector, the parasite relies on amino acid catabolism, but in the mammalian host, it derives its energy exclusively from blood glucose. Glucose transport is facilitated, and constitutes the rate-limiting step in ATP synthesis. Here, we report the cloning of a novel glucose transporter-related gene by heterologous screening of a EMBL4 genomic library of T. brucei EATRO 164 using a rat liver glucose transporter cDNA clone. Genomic analysis shows that the gene is present as a single copy within the parasite genome. The gene encodes a protein with an estimated molecular mass of 55.9 kDa, which shares only segmental homology with members of the glucose transporter superfamily. Several potential post-translational modification sites including phosphorylation, N-glycosylation, and cotranslational myristoylation sites also punctuate the sequence. It is distinguished from classical transporter proteins by the absence of putative hydrophobic membrane-spanning domains. However, this protein was capable of complementing Schizosaccharomyces pombe glucose transporter mutants. The rescued phenotype conferred the ability of the cells to grow on a broad range of sugars, both monosaccharides and disaccharides. The kinetics of glucose uptake reflected those in T. brucei. In addition to complementation in yeast, we also showed that the gene enhanced glucose uptake in cultured mammalian cells. The bloodstream forms of Trypanosoma brucei are absolutely dependent on peripheral glucose supply and derive their energy by glycolysis (1, 2). The glycosome is the major site for glycolysis, and most glycolytic enzymes are compartmentalized here (3). There is evidence suggesting the differential expression of glycolytic enzymes in the bloodstream forms (4). This may be indicative of a differentially expressed glucose transporter, assuming that the two systems operate in tandem. In order to be shunted into the glycolytic pathway, glucose must therefore cross the plasma membrane and then pass through the glycosomal membrane. It might appear that both compartments are equipped with glucose transporters, although it is suggested that the flagellar pocket may serve in solute uptake by endocytosis (5). Glucose uptake by bloodstream forms is mediated by a facilitated glucose transporter (6-8). Kinetic measurements indicate that there are substantial differences between this transporter and that of the mammalian host. Some of these differences include a 20-50-fold higher rate of glucose metabolism than the mammalian host cells (9); insensitivity to cytochalasin B (unpublished observations), and the ability to transport fructose (10). Some work has shown that several glucose transporters are present in the kinetoplastids, which differ largely in the stage specificity of expression (11-14). These transporters showed apparent homology to members of the facilitative glucose transporters including the presence of putative transmembrane segments. The kinetics of glucose uptake and sensitivity to known inhibitors of transport were also similar in many respects. However, because those transporters were identified either with variant surface glycoprotein gene probes (11) or based on developmental expression (13), we reasoned that other unidentified transporters still exist in T. brucei. By heterologous probing with a rat liver glucose transporter cDNA, we isolated and cloned a trypanosome protein that is distinct from any previously reported. Although it has only residual homology to the classical glucose transporters, it was able to rescue fission yeast glucose transporter mutants by supporting growth on sugars, both disaccharides and monosaccharides. This protein may therefore belong to a new class of transporters, or it may be tightly associated with glucose uptake and metabolism.
SIRT1 protects against several complex metabolic and ageing-related diseases (MARDs), and is ther... more SIRT1 protects against several complex metabolic and ageing-related diseases (MARDs), and is therefore considered a polypill target to improve healthy ageing. Although dietary sirtuinactivating compounds (dSTACs) including resveratrol are promising drug candidates, their clinical application has been frustrated by an imprecise understanding of how their signals are transduced into increased healthspan. Recent work indicates that SIRT1 and orthologous sirtuins coactivate the oestrogen receptor/ER and the worm steroid receptor DAF-12. Here they are further shown to ligand-independently transduce dStAcs signals through these receptors. While some dStAcs elicit eR subtype-selectivity in the presence of hormone, most synergize with 17β-oestradiol and dafachronic acid respectively to increase ER and DAF-12 coactivation by the sirtuins. These data suggest that dStAcs functionally mimic gonadal steroid hormones, enabling sirtuins to transduce the cognate signals through a conserved endocrine pathway. Interestingly, resveratrol non-monotonically modulates sirtuin signalling, suggesting that it may induce hormesis, i.e. "less is more". together, the findings suggest that dSTACs may be informational molecules that use exploitative mimicry to modulate sirtuin signalling through steroid receptors. Hence dStAcs' intrinsic oestrogenicity may underlie their proven ability to impart the health benefits of oestradiol, and also provides a mechanistic insight into how they extend healthspan or protect against MARDs. Among the seven human sirtuins, SIRT1 (silent information regulator 2 homologue 1) has received the most attention because of its many roles including gene regulation, genomic stability and energy metabolism 1,2. SIRT1 is also of enormous interest as a viable drug target because it protects against several conditions including obesity, type 2 diabetes, cancer and cardiovascular and neurodegenerative diseases 3,4. However, deciphering how this protein is regulated is complicated by the fact that it appears to be a hub for multiple networks while also participating in several reciprocal interactions and autoregulatory loops 5. This further frustrates our understanding of how it transduces signals from interacting partners but especially from sirtuin activating compounds (STACs) such as resveratrol. These molecules are thought to allosterically activate SIRT1 by directly binding to its N-terminal STACs-activation domain, STACs-AD. Although this proposed mechanism has been intensely controversial 6-8 , evidence suggests that a single residue (E230) within the STACs-AD may be responsible for allosteric activation 9. Furthermore, co-crystal structures of STACs and SIRT1 support direct binding consistent with an allosteric mechanism 10,11. Alternative mechanisms propose that AMP-activated protein kinase may transduce STACs signals but the pathway leading to SIRT1 activation is unclear 12,13. Hence there is no consensus on how SIRT1 and orthologous sirtuins translate the beneficial effects of resveratrol and related dietary STACs (dSTACs) to extend healthspan in diverse organisms including humans. Since dSTACs can allosterically increase sirtuin deacetylase activities, they portend pharmacological interventions for MARDs 8,14-16. However this has been slowed by an imprecise understanding of how the cognate allosteric signals are transduced by the sirtuins, partly because dSTACs are functionally promiscuous 8,17. A clue to how they work in vivo may be in the fact that with the exception of SRT1720 and its relatives, all are phytoestrogens 8,17 that are structurally similar to the oestrogen receptor (ER) steroidal ligand 17β-oestradiol (hereafter referred to as E2 or oestrogen), and have been shown to compete with this hormone for ER binding 18-21. In what appears to hint at how dSTACs may modulate sirtuin signalling, resveratrol in particular has been shown to activate the ER and to competitively inhibit oestradiol binding to the receptor's ligand binding domain (LBD) 22. Recent work 23 described SIRT1 and the orthologous sirtuins Sir2 (Saccharomyces cerevisiae) and Sir-2.1 (Caenorhabditis elegans) as nuclear receptor coregulators that coactivate the ER and DAF-12, the steroid receptor that regulates nematode lifespan and reproductive development 24-26. The data reported herein build on those findings to show that dSTACs ligand-independently enhance sirtuin signalling through these receptors. The results also suggest
Rational drug discovery relies on pathognomonic molecular reporters of disease or biomarkers. The... more Rational drug discovery relies on pathognomonic molecular reporters of disease or biomarkers. Therefore biomarkers contain relational or contextual information about disease pathophysiology. Two broad pathways can be taken to identify biomarkers: a 'top-down', holistic approach that makes no assumptions about biomarker type, or the 'bottom-up' approach, which is hypothesis driven and relies on a priori information. Both approaches involve parallel or sequential methods that include genomic and proteomic profiling. Biomarker discovery and translational medicine owe much to isotopic techniques because these provide near-real-time information about disease status as diagnostics, in drug delivery and for monitoring treatment. Here, we provide an overview of recent developments and some insight into the future role of isotopes in biomarker discovery and disease therapy. Reviews POST SCREEN
RNA polymerase II (pol II) promoters are rare in the African trypanosome Trypanosoma brucei becau... more RNA polymerase II (pol II) promoters are rare in the African trypanosome Trypanosoma brucei because gene regulation in the parasite is complex and polycistronic. Here, we describe a putative pol II promoter and its structure-function relationship. The promoter has features of an archetypal eukaryotic pol II promoter including putative canonical CCAAT and TATA boxes, and an initiator element. However, the spatial arrangement of these elements is only similar to yeast pol II promoters. Deletion mapping and transcription assays enabled delineation of a minimal promoter that could drive orientation-independent reporter gene expression suggesting that it may be a bidirectional promoter. In vitro transcription in a heterologous nuclear extract revealed that the promoter can be recognized by the basal eukaryotic transcription complex. This suggests that the transcription machinery in the parasite may be very similar to those of other eukaryotes.
Available from British Library Document Supply Centre- DSC:DX97425 / BLDSC - British Library Docu... more Available from British Library Document Supply Centre- DSC:DX97425 / BLDSC - British Library Document Supply CentreSIGLEGBUnited Kingdo
Des dendrimeres de lipides cationiques de diametre bien defini et de groupement amines terminaux ... more Des dendrimeres de lipides cationiques de diametre bien defini et de groupement amines terminaux en nombre precis (de huit a trente-deux) ont ete synthetises sur un support solide. L'utilisation de dendrimeres de geometries variees a ete envisagee pour l'administration de genes par evaluation de l'efficacite de transfection des membres d'une serie variant par la longueur des ramifications, le niveau de liaisons des lipides, la presence de sucre et la presence d'un peptide a localisation nucleaire (nuclear localization signal'). L'activite de transfection des produits a ete etudiee in vitro sur des cellules cos 7 (fibroblastes). Les dendrimeres (1a et 1b) presentent de fortes activites de transfection. Les resultats montrent que la presence d'un plus grand nombre de groupements amino a la surface des dendrimeres peut ameliorer l'administration des genes. Une premiere caracterisation physicochimique des complexes ADN/lipide a permis de demontrer la quantite minimale de dendrimeres necessaires pour la transfection de 2,5 μg de plasmide (10 μg/ml pour les dendrimeres a huit groupes amino libres terminaux et 5 et 2,5 μg/ml pour les dendrimeres avec respectivement seize et trente-deux groupes amino terminaux).
Resveratrol has been widely investigated for its potential health properties, although little is ... more Resveratrol has been widely investigated for its potential health properties, although little is known about its metabolism in vivo. Here we investigated the distribution of metabolic products of [ 3 H]trans-resveratrol, following gastric administration. At 2 h, plasma concentrations reached 1•7 % of the administered dose, whilst liver and kidney concentrations achieved 1•0 and 0•6 %, respectively. Concentrations detected at 18 h were lower, being only 0•5 % in plasma and a total of 0•35 % in tissues. Furthermore, whilst kidney and liver concentrations fell to 10 and 25 %, respectively, of concentrations at 2 h, the brain retained 43 % of that measured at 2 h. Resveratrol-glucuronide was identified as the major metabolite, reaching 7 mM in plasma at 2 h. However, at 18 h the main form identified in liver, heart, lung and brain was native resveratrol aglycone, indicating that it is the main form retained in the tissues. No phenolic degradation products were detected in urine or tissues, indicating that, unlike flavonoids, resveratrol does not appear to serve as a substrate for colonic microflora. The present study provides additional information about the nature of resveratrol metabolites and which forms might be responsible for its in vivo biological effects.
Biochemical and Biophysical Research Communications, 2016
The liver expresses batteries of cytoprotective genes that confer cellular resistance to oxidativ... more The liver expresses batteries of cytoprotective genes that confer cellular resistance to oxidative stress and xenobiotic toxins, and protection against cancer and other stress-related diseases. These genes are mainly regulated by Nrf2, making this transcription factor a target for small molecule discovery to treat such diseases. In this report, we identified dietary polyphenolic antioxidants that not only activated these genes but also relieved Nrf2 repression by Keap1, a Cul3dependent ubiquitin ligase adaptor protein that mediates its degradation. Analysis of postprandial liver RNA revealed a marked activation of both genes by all test polyphenols compared with controls. Nrf2 inhibition by RNA interference reduced polyphenol effects on its target gene expression. Our data suggest that polyphenols may induce cellular defense genes by derepressing Nrf2 inhibition by Keap1. We posit that this ability to derepress Nrf2 and reactivate its target genes may underlie the protection conferred by polyphenols against oxidative stress-related diseases.
The 235 kDa rhoptry protein Py235 of Plasmodium yoelii, has been implicated in erythrocyte invasi... more The 235 kDa rhoptry protein Py235 of Plasmodium yoelii, has been implicated in erythrocyte invasion by the merozoite forms of the parasite. Py235 is encoded by a large, highly polymorphic gene family, members of which appear to be differentially transcribed. However, it is not clear how many variants are expressed at the protein level during an infection cycle and whether or not these variants are expressed selectively or combinatorially. Certain monoclonal antibodies to Py235 have been shown to attenuate parasite virulence upon passive transfer into mice, suggesting that this antigen or its derivatives may be useful vaccine candidates. To provide a basis for this, we sought to identify those variants that are recognised by the host immune system, and to establish the pattern of expression of the antigen in mice during infection. Using Py235 monoclonal antibodies as probes, we isolated distinct antigenic variants from an expression library, suggesting that the antigen repertoire is potentially large and that different Py235 variants may be produced during infection. The implications of these observations are discussed with respect to the ability of a cloned parasite line to express distinct antigenic variants in vivo.
Fems Immunology and Medical Microbiology, Aug 1, 2007
The 235-kDa antigenic rhoptry protein Py235 of Plasmodium yoelii is encoded by a large, highly po... more The 235-kDa antigenic rhoptry protein Py235 of Plasmodium yoelii is encoded by a large, highly polymorphic gene family. Monoclonal antibodies to some of these antigens have been shown to attenuate the virulence of the lethal YM strain of the parasite, converting a potentially fatal YM infection to a fulminating one typical of the nonlethal 17X strain, by inducing a switch in target cell preference from mature red blood cells to reticulocytes. The reason for this is not known but would suggest that antigenic determinants of Py235 may be useful in or as subunit vaccines. To identify such determinants, we constructed an epitope expression library of one Py235 variant and screened the library with the antibodies. Thus, we mapped 5-and 12-amino acid epitopes to the C-terminus of the antigen. Both epitopes were more reactive with protective than with nonprotective monoclonal antibodies. This may explain the differential protection conferred by these antibodies upon their passive transfer into mice.
Introducción: La transferrina (Tf) ejerce una función crucial en el mantenimiento de la homeostas... more Introducción: La transferrina (Tf) ejerce una función crucial en el mantenimiento de la homeostasis sistémica del hierro. La expresión del gen de la transferrina es controlada a nivel transcripcional, aunque la posible influencia de factores genéticos todavía se desconoce. Objetivo: Estudiar el papel del rs3811647 en la expresión de la transferrina mediante un ensayo in-vitro en células de hepatoma. Diseño y métodos: Células Hep3B fueron co-transfectadas con vectores que contenían las variantes A (VarA-Tfluc) y G (VarG-Tf-luc) del rs3811647, utilizandose la luciferasa como marcador de la expresión del gen Tf. Resultados: Los ensayos con la luciferasa mostraron un mayor aumento de la expresión del gen Tf en presencia de la variante A comparada con la G (p < 0,05). El análisis in silico del SNP rs3811647 mostró que la presencia del alelo A puede constituir un sitio de unión del receptor de glucocorticoides (GR). Conclusión: El alelo A del SNP rs3811647 incrementa la expresión del gen Tf de modo que podría modular la variación interindividual en los niveles de transferrina sérica observados en diferentes poblaciones.
Hepcidin is a small acute phase peptide that regulates iron absorption. It is induced by inflamma... more Hepcidin is a small acute phase peptide that regulates iron absorption. It is induced by inflammation and infection, but is repressed by anaemia and hypoxia. Here we further reveal that hepcidin transcription also involves interactions between functional metal response elements (MREs) in its promoter, and the MRE-binding transcription factor-1. Analysis of hepcidin mRNA and protein levels in hepatoma cells suggests that its expression may be regulated by divalent metal ions, with zinc inducing maximal effects on hepcidin levels. These data suggest that this peptide may be a pleiotropic sensor of divalent metals, some of which are xenobiotic environmental toxins.
Ferroportin (Fpn/IREG1/MTP1) is the only known transporter mediating iron efflux from epithelial ... more Ferroportin (Fpn/IREG1/MTP1) is the only known transporter mediating iron efflux from epithelial cells and macrophages, and thus regulates how much iron is released into the circulation. Consequently, Fpn mutations are associated with haemochromatosis. Fpn itself is post-translationally regulated by hepcidin (Hepc) which induces its redistribution and degradation in a ubiquitin-dependent process. Together, the two proteins appear to be the nexus for iron homeostasis. Here we show that a rare gain-of-function mutation (K240E) that is associated with iron overload, impedes Fpn binding and subcellular trafficking by the small ubiquitin-like modifier (SUMO). Whereas wild-type Fpn is ensconced within vesicular bodies, the FpnK240E mutant appeared diffused within the cell when co-expressed with SUMO. Furthermore, compared with wild type Fpn, the sumoylation-defective mutant was constitutively-active, resulting in a lower intracellular labile iron pool than the former. These findings suggest that SUMO may regulate iron homeostasis by controlling Fpn trafficking.
Introduction: Transferrin (Tf) exerts a crucial function in the maintenance of systemic iron home... more Introduction: Transferrin (Tf) exerts a crucial function in the maintenance of systemic iron homeostasis. The expression of the Tf gene is controlled by transcriptional mechanism, although little is known about genetic factors influence. Objective: To study the role of rs3811647 in Tf expression using an in-vitro assay on hepatoma cells. Design and methods: Hep3B cells were co-transfected with constructs containing A (VarA-Tf-luc) and G (VarG-Tf-luc) variants of rs3811647, using luciferase as a surrogate reporter of Tf expression. Results: Luciferase assays showed a higher intrinsic enhancer activity (p < 0.05) in the A compared with the G variant. In silico analysis of SNP rs3811647 showed that the A allele might constitute a binding site for the transcription factor glucocorticoid receptor (GR). Conclusion: The A allele of SNP rs3811647 increases Tf expression in a manner that might underlie inter-individual variation in serum transferrin levels observed in different population groups.
Uploads
Papers by Henry Bayele