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M P Barrett

Publications and source records attributed to M P Barrett.

At least 37 records · Page 2Linked to original sources
PubMed1999

The biochemical basis of arsenical-diamidine crossresistance in African trypanosomes.

Resistance to currently used drugs is a serious problem in most fields of antimicrobial chemotherapy. Crossresistance between two of the major classes of drug used in the treatment of African trypanosomiasis, the melaminophenyl arsenicals and diamidines is easily selected in the laboratory. Here, Mike Barrett and Alan Fairlamb outline the mechanism underlying this crossresistance, which appears to arise as a result of alterations in an unusual adenosine transporter involved in the uptake of these drugs.

Animals↗
PubMed1999

Structure and function of facilitative sugar transporters.

Sugar transporters from one group of the major facilitator superfamily of membrane transporters. A conserved common central pore structure lies at the heart of these transporters and diverse functionality is brought about by alterations to this pore or regions associated with it. Recent mutagenesis studies of sugar transporters within the framework of tenable models for the distantly related lactose permease argue that this model is a good paradigm for other members of the major facilitator superfamily.

Amino Acid Sequence↗
PubMed1999

Recent advances in identifying and validating drug targets in trypanosomes and leishmanias.

The unique aspects of the biochemistry of trypanosomatids make rational drug design an attractive approach, but targets must be selected carefully. Genetic manipulation provides a valuable means of mimicking loss of function attributable to therapeutic intervention, but caution must be exercised when interpreting such data with respect to target validation.

Animals↗
PubMed1999

Affinity chromatography using trypanocidal arsenical drugs identifies a specific interaction between glycerol-3-phosphate dehydrogenase from Trypanosoma brucei and Cymelarsan.

A 36-kDa protein was isolated by affinity chromatography using Cymelarsan, an arsenical drug currently used in African trypanosomiasis treatment, as ligand. This protein was identified as glycerol-3-phosphate dehydrogenase. Trypanosomal glycerol-3-phosphate was bound covalently, whereas its counterpart from rabbit muscle bound by ionic interaction. Arsenical drugs inhibit the enzyme in a dose-dependent manner. Oxidation of cysteine residues protects against inactivation without significantly diminishing enzymic activity. Drug concentrations giving 50% inhibition of the dehydrogenase activity were determined for the enzyme from both Trypanosoma brucei and rabbit and indicate a higher sensitivity of the trypanosomal enzyme to arsenical drugs and thiol reagents. MS was used to identify residues of glycerol-3-phosphate dehydrogenase bound by Cymelarsan; they are not conserved in the mammalian enzyme.

Animals↗
PubMed1998

6-Phosphogluconate dehydrogenase: the mechanism of action investigated by a comparison of the enzyme from different species.

The mechanism of action of 6-phosphogluconate dehydrogenase with the alternative substrate 2-deoxy 6-phosphogluconate was investigated using enzymes from sheep liver, human erythrocytes and Trypanosoma brucei. The three enzymes oxidize 2-deoxy 6-phosphogluconate, but only the sheep liver enzyme releases the intermediate 2-deoxy,3-keto 6-phosphogluconate. Kinetic comparison showed that an increase in the rate of NADP+ reduction at high pH is due to increased release of the intermediate, rather than an increase in the overall reaction rate. 2-Deoxy,3-keto 6-phosphogluconate is decarboxylated by the erythrocyte and trypanosome enzymes but not the liver one in the absence of either NADPH or 6-phosphogluconate, which act as activators. The pH dependence of decarboxylation and the degree of activation suggest that 6-phosphogluconate is the activator which operates under normal assay conditions, while NADPH acts mainly by increasing the binding of the intermediate. The data suggest that the activity of 6PGDH is subjected to a two-way regulation: NADPH, which regulates the pentose phosphate pathway, inhibits the enzyme, while 6-phosphogluconate, levels of which rise when NADPH inhibition is removed, acts as an activator ensuring that 6-phosphogluconate is rapidly removed.

Allosteric Regulation↗
PubMed1998

Crystallization and preliminary X-ray diffraction studies of 6-phosphogluconate dehydrogenase from Lactococcus lactis.

6-Phosphogluconate dehydrogenase is one of the seven enzymes involved in the pentose phosphate pathway. Crystals of a mammalian and a protozoan enzyme have been obtained previously and structures determined. It is reported here that a bacterial 6-phosphogluconate dehydrogenase, from Lactococcus lactis, has been purified and used in crystallization trials. Large prisms suitable for a detailed structural analysis have been obtained and characterized as orthorhombic, space group F222, with a = 70.4, b = 105.7, c = 474.6 A. Diffraction has been observed to 2.2 A resolution using synchrotron radiation. Structural analysis, in combination with ongoing biochemical characterization, will assist the elucidation of the structure-activity relationships of this enzyme.

Bacterial Proteins↗
PubMed1998

A 2.8 A resolution structure of 6-phosphogluconate dehydrogenase from the protozoan parasite Trypanosoma brucei: comparison with the sheep enzyme accounts for differences in activity with coenzyme and substrate analogues.

The three-dimensional structure of 6-phosphogluconate dehydrogenase (6PGDH) from the parasitic protozoan Trypanosoma brucei has been solved at 2.8 A resolution. This pentose phosphate pathway enzyme is NADP-dependent; NADPH generated in the reaction protects against oxidative stress. The enzyme crystallises in the space-group P3121 with a dimer in the asymmetric unit and cell dimensions a=b=135.13 A, c=116.74 A, alpha=beta=90 degrees, gamma=120 degrees. The structure has refined to R=18.6% (Rfree=27.3%) with good geometry. The amino acid sequence of T. brucei 6PGDH is only 35% identical to that of the sheep liver enzyme and significant activity differences have been observed. The active dimer assembles with the C-terminal tail of one subunit threaded through the other, forming part of the substrate binding site. The tail of T. brucei 6PGDH is shorter than that of the sheep enzyme and its terminal residues associate tightly with the second monomer. The three-dimensional structure shows this generates additional interactions between the subunits close to the active site; the coenzyme binding domain is thereby associated more tightly with the helical domain. Three residues, conserved in all other known sequences, are important in creating a salt bridge between monomers close to the substrate binding site. The differences could explain the 200-fold enhanced affinity observed for the substrate analogue 6-phospho-2-deoxy-D-gluconate and suggest targets for anti-parasite drug design. The coenzyme binding domain of 6PGDH has a beta-alpha-beta fold; while in most species the "fingerprint" sequence is GxAxxG, in the T. brucei enzyme it is GxGxxG. Additional interactions between the enzyme and the coenzyme bis-phosphate are likely in the parasite 6PGDH, accounting for greater inhibition (40-fold) of 2'5'-ADP. While the core of the T. brucei dimer was restrained during refinement, several conformational differences have been found between the monomers; those at the coenzyme binding site suggest the molecule could be asymmetric during the enzyme reaction.

Amino Acid Sequence↗
PubMed1998

Cloning and characterization of the two enzymes responsible for trypanothione biosynthesis in Crithidia fasciculata.

Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (gamma-Glu-Cys-Gly) and spermidine to form trypanothione (N1,N8-bis(glutathionyl)spermidine), which is involved in maintaining intracellular thiol redox and in defense against oxidants. In this study, the genes from Crithidia fasciculata, Cf-GSS and Cf-TRS, which encode, respectively, glutathionylspermidine synthetase (EC 6.3.1.8) and trypanothione synthetase (EC 6.3.1.9) have been cloned and expressed. The deduced amino acid sequence of both Cf-GSS and Cf-TRS share 50% sequence similarity with the Escherichia coli glutathionylspermidine synthetase/amidase. Both genes are present as single copies in the C. fasciculata genome. When expressed in E. coli and Saccharomyces cerevisiae, neither protein was present in an active soluble form. However, thiol analysis of S. cerevisiae demonstrated that cells transformed with the Cf-GSS gene contained substantial amounts of glutathionylspermidine, whereas cells expressing both the Cf-GSS and Cf-TRS genes contained glutathionylspermidine and trypanothione, confirming that these genes encode the functional glutathionylspermidine and trypanothione synthetases from C. fasciculata. The translation products of Cf-GSS and Cf-TRS show significant homology to the amidase domain present in E. coli glutathionylspermidine synthetase, which can catalyze both synthesis and degradation of glutathionylspermidine. Glutathionylspermidine synthetase isolated from C. fasciculata was found to possess a similar amidase activity.

Amide Synthases↗
PubMed1998

An approach to use an unusual adenosine transporter to selectively deliver polyamine analogues to trypanosomes.

In this paper we describe an approach to selectively deliver compounds to trypanosomes using an adenosine transporter which is unique to the trypanosome. Various polyamine analogues have been attached to known substrates of this adenosine transporter. The compounds prepared interact specifically with the adenosine transporter, some with a similar efficiency to berenil, a known substrate.

Adenosine↗
PubMed1998

Sugar transporters from bacteria, parasites and mammals: structure-activity relationships.

Sugar transport across the plasma membrane is one of the most important transport processes. The cloning and expression of cDNAs from a superfamily of related sugar transporters that all adopt a 12-membrane-spanning-domain structure has opened new avenues of investigation, including presteady-state kinetic analysis. Structure-function analyses of mammalian and bacterial sugar transporters, and comparisons of these transporters with those of parasitic trypanosomatids, indicate that different environmental pressures have tailored the evolution of the various members of the sugar-transporter superfamily. Subtle distinctions in the function of these proteins can be related to particular amino acid residue substitutions.

Amino Acid Sequence↗
PubMed1997

Kinetoplastid glucose transporters.

Protozoa of the order kinetoplastida have colonized many habitats, and several species are important parasites of humans. Adaptation to different environments requires an associated adaptation at a cell's interface with its environment, i.e. the plasma membrane. Sugar transport by the kinetoplastida as a phylogenetically related group of organisms offers an exceptional model in which to study the ways by which the carrier proteins involved in this process may evolve to meet differing environmental challenges. Seven genes encoding proteins involved in glucose transport have been cloned from several kinetoplastid species. The transporters all belong to the glucose transporter superfamily exemplified by the mammalian erythrocyte transporter GLUT1. Some species, such as the African trypanosome Trypanosoma brucei, which undergo a life cycle where the parasites are exposed to very different glucose concentrations in the mammalian bloodstream and tsetse-fly midgut, have evolved two different transporters to deal with this fluctuation. Other species, such as the South American trypanosome Trypanosoma cruzi, multiply predominantly in conditions of relative glucose deprivation (intracellularly in the mammalian host, or within the reduviid bug midgut) and have a single, relatively high-affinity type, transporter. All of the kinetoplastid transporters can also transport d-fructose, and are relatively insensitive to the classical inhibitors of GLUT1 transport cytochalasin B and phloretin.

Amino Acid Sequence↗
PubMed1997

The pentose phosphate pathway and parasitic protozoa.

The pentose phosphate pathway plays a crucial role in the host-parasite relationship. It maintains a pool of NADPH, which serves to protect against oxidant stress and which generates carbohydrate intermediates used in nucleotide and other biosynthetic pathways. Deficiency in the first enzyme of the pathway, glucose-6-phosphate dehydrogenase, protects human erythrocytes from infection with Plasmodium falciparum for reasons that remain obscure. Loss of the third enzyme of the pathway, 6-phosphogluconate de-hydrogenase, is toxic, suggesting this enzyme might be a target for chemotherapy. Mike Barrett here summarizes the roles of the pentose phosphate pathway in various parasitic protozoa.

Journal Article↗
PubMed1997

The cross-linking by o-phthalaldehyde of two amino acid residues at the active site of 6-phosphogluconate dehydrogenase.

o-phthalaldehyde inactivates homodimeric, NADP+ dependent, 6-phosphogluconate dehydrogenase from sheep liver, upon formation of a single isoindole derivative per enzyme subunit. This indicates that the thiol group of a cysteine residue or the epsilon-amino group of a lysine residue located within 3 A and crosslinked by the reagent is essential for catalysis. Fluorescence analyses of the modified enzyme suggest that the isoindole derivative forms at the binding site of the nicotinamide moiety of NADP+. The enzymes from Trypanosoma brucei and Lactococcus lactis are also inactivated suggesting a similar three-dimensional structure in this domain. The isoindole derivative does not form with two mutants of the T. brucei enzyme (Lys185His and Lys185Leu), this allowing to identify not only the lysine but also the cysteine involved in the cross-linking. The formation of the isoindole derivative inactivates not only the oxidative decarboxylation, but also two partial reactions catalysed by the enzyme.

Animals↗
PubMed1996

6-Phosphogluconate dehydrogenase from Trypanosoma brucei. Kinetic analysis and inhibition by trypanocidal drugs.

The kinetics of 6-phosphogluconate dehydrogenase from Trypanosoma brucei was examined and compared to those of the same enzyme from lamb's liver. Variation of kinetic parameters as a function of pH suggests a chemical mechanism similar to other 6-phosphogluconate dehydrogenases. The comparison extended to a detailed analysis of the effect on enzyme activity by several inhibitors including the trypanocidal drugs suramin, melarsoprol and analogues of these compounds. The T. brucei enzyme differs significantly from its mammalian counterpart with respect to several inhibitors, particularly the substrate analogue 6-phospho-2-deoxygluconate and the coenzyme analogue adenosine 2',5'-bisphosphate which have respectively 170-fold and 40-fold higher affinity for the parasite enzyme.

Animals↗
PubMed1996

Hexose uptake in Trypanosoma cruzi: structure-activity relationship between substrate and transporter.

The gene encoding a hexose transporter, TcrHt1, from Trypanosoma cruzi has been functionally expressed in mammalian Chinese hamster ovary cells. Kinetic parameters of the heterologously expressed protein are very similar to those of the transporter identified in T. cruzi epimastigotes, confirming that TcrHT1 is the major transporter functioning in these parasites. A detailed analysis of substrate recognition using analogues of D-glucose substituted at each carbon position has been performed. The glucose transporter of T. cruzi does not recognize C-3 or C-6 analogues of D-glucose, whereas these analogues were recognized by the glucose transporter of bloodstream-form T. brucei. As for other kinetoplastid transporters, but in stark contrast to the mammalian GLUT family, TcrHT1 can also transport D-fructose, with relatively high affinity (Km = 0.682 +/- 0.003 mM). Amino acid side-chain-modifying reagents were also used to identify residues of the transporter present at the substrate-binding site. While specific modifiers of cysteine, histidine and arginine all inhibited catalytic activity, protection using substrate was only observed using the arginine-specific reagent, phenylglyoxal. Reagents which modify lysine residues had no effect on transport.

Amino Acids↗