Kai Lüersen

Oxidative stress in Caenorhabditis elegans: protective effects of the Omega class glutathione transferase (GSTO-1)

To elucidate the function of Omega class glutathione transferases (GSTs) (EC 2.5.1.18) in multicellular organisms, the GSTO‐1 from Caenorhabditis elegans (GSTO‐1; C29E4.7) was investigated. Disc diffusion assays using Escherichia coli overexpressing GSTO‐1 provided a test of resistance to long‐term exposure under oxidative stress. After affinity purification, the recombinant GSTO‐1 had minimal catalytic activity toward classic GST substrates but displayed significant thiol oxidoreductase and dehydroascorbate reductase activity. Microinjection of the GSTO‐1‐promoter green fluorescent protein construct and immunolocalization by electron microscopy localized the protein exclusively in the intestine of all postembryonic stages of C. elegans. Deletion analysis identified an ~300‐nucleotide sequence upstream of the ATG start site necessary for GSTO‐1 expression. Site‐specific mutagenesis of a GATA transcription factor binding motif in the minimal promoter led to the loss of reporter expression. Similarly, RNA interference (RNAi) of Elt‐2 indicated the involvement of this gut‐specific transcription factor in GSTO‐1 expression. Transcriptional up‐regulation under stress conditions of GSTO‐1 was confirmed by analyzing promoter‐reporter constructs in transgenic C. elegans strains. To investigate the function of GSTO‐1 in vivo, transgenic animals overexpressing GSTO‐1 were generated exhibiting an increased resistance to juglone‐, paraquat‐, and cumene hydroperoxide‐induced oxidative stress. Specific silencing of the GSTO‐1 by RNAi created worms with an increased sensitivity to several prooxidants, arsenite, and heat shock. We conclude that the stress‐responsive GSTO‐1 plays a key role in counteracting environmental stress.—Burmeister, C., Lüersen, K., Heinick, A., Hussein, A., Domagalski, M., Walter, R. D., Liebau, E. Oxidative stress in Caenorhabditis elegans: protective effects of the Omega class glutathione transferase (GSTO‐1). FASEB J. 22, 343–354 (2008)

3-Aminooxy-1-aminopropane and derivatives have an antiproliferative effect on cultured Plasmodium falciparum by decreasing intracellular polyamine concentrations.

ABSTRACT The intraerythrocytic development of Plasmodium falciparum correlates with increasing levels of the polyamines putrescine, spermidine, and spermine in the infected red blood cells; and compartmental analyses revealed that the majority is associated with the parasite. Since depletion of cellular polyamines is a promising strategy for inhibition of parasite proliferation, new inhibitors of polyamine biosynthesis were tested for their antimalarial activities. The ornithine decarboxylase (ODC) inhibitor 3-aminooxy-1-aminopropane (APA) and its derivatives CGP 52622A and CGP 54169A as well as the S-adenosylmethionine decarboxlyase (AdoMetDC) inhibitors CGP 40215A and CGP 48664A potently affected the bifunctional P. falciparum ODC-AdoMetDC, with Ki values in the low nanomolar and low micromolar ranges, respectively. Furthermore, the agents were examined for their in vitro plasmodicidal activities in 48-h incubation assays. APA, CGP 52622A, CGP 54169A, and CGP 40215A were the most effective, with 50% inhibitory concentrations below 3 μM. While the effects of the ODC inhibitors were completely abolished by the addition of putrescine, growth inhibition by the AdoMetDC inhibitor CGP 40215A could not be antagonized by putrescine or spermidine. Moreover, CGP 40215A did not affect the cellular polyamine levels, indicating a mechanism of action against P. falciparum independent of polyamine synthesis. In contrast, the ODC inhibitors led to decreased cellular putrescine and spermidine levels in P. falciparum, supporting the fact that they exert their antimalarial activities by inhibition of the bifunctional ODC-AdoMetDC.

The glutathione S-transferase from Plasmodium falciparum.

Liebau, E., Bergmann, B., Campbell, A. M., Teesdale-Spittle, P., Brophy, P. M., Luersen, K., Walter, R. D. (2002). The glutathione S-transferase from Plasmodium falciparum. Molecular and Biochemical Parasitology, 124, (1-2), 85-90

Assessing the polyamine metabolism of Plasmodium falciparum as chemotherapeutic target

More than 30 years ago the potent ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) was designed as new anticancer drug. Its efficacy was not as expected since the polyamine metabolism in mammalian cells seemed to be far more complex. However when DFMO was applied to African trypanosomes its effect on this protozoan parasite was highly convincing. Thenceforward many researchers tested DFMO and also other polyamine synthesis inhibitors against different parasites among them the causative agent of malaria Plasmodium. This review recapitulates the different attempts to interfere chemically with the plasmodial polyamine metabolism, the impact on the disease as well as its biochemical and molecular background. It will show that this fast proliferating organism depends for growth on high amounts of polyamines and that Plasmodium has its own and unique polyamine synthesis, differing highly from the mammalian one mainly in the arrangement of the key enzymes, S-adenosylmethionine decarboxylase and ornithine decarboxylase (AdoMetDC/ODC), on a bifunctional protein.

Functional analysis of the methylmalonyl-CoA epimerase from Caenorhabditis elegans.

Methylmalonyl‐CoA epimerase (MCE) is an enzyme involved in the propionyl‐CoA metabolism that is responsible for the degradation of branched amino acids and odd‐chain fatty acids. This pathway typically functions in the reversible conversion of propionyl‐CoA to succinyl‐CoA. The Caenorhabditis elegans genome contains a single gene encoding MCE (mce‐1) corresponding to a 15 kDa protein. This was expressed in Escherichia coli and the enzymatic activity was determined. Analysis of the protein expression pattern at both the tissue and subcellular level by microinjection of green fluorescent protein constructs revealed expression in the pharynx, hypodermis and, most prominently in body wall muscles. The subcellular pattern agrees with predictions of mitochondrial localization. The sequence similarity to an MCE of known structure was high enough to permit a three‐dimensional model to be built, suggesting conservation of ligand and metal binding sites. Comparison with corresponding sequences from a variety of organisms shows more than 1/6 of the sequence is completely conserved. Mutants allelic to mce‐1 showed no obvious phenotypic alterations, demonstrating that the enzyme is not essential for normal worm development under laboratory conditions. However, survival of the knockout mutants was altered when exposed to stress conditions, with mutants surprisingly showing an increased resistance to oxidative stress.

The putative γ-glutamylcysteine synthetase from Plasmodium falciparum contains large insertions and a variable tandem repeat☆

The tripeptide glutathione plays a pivotal role in the maintenance of the thiol redox state of the cell and for the detoxification of reactive oxygen species. Glutathione is synthesized in two consecutive reactions by y-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase, respectively. The former enzyme represents the rate limiting step of the synthetic pathway. We have cloned the cDNA and gene of a putative gamma-GCS from Plasmodium falciparum. The contiguous cDNA sequences obtained from various cDNA libraries of P. falciparum K1 and 3D7 encompass 4206 bp or 4038 bp and encode polypeptides of 1119 and 1063 amino acids, respectively. The deduced amino acid sequences show four regions of homology (identity: 31.3-43.9%) to human and Trypanosoma brucei gamma-GCS. These regions are interrupted by three large insertions between 94 and 239 amino acids. Within the first insert a variable repetitive motif was identified, which is responsible for the differing sizes of the sequences. We have analysed this phenomenon in five additional P. falciparum strains and found a high degree of variability in the number of the repeated octamer (Y/C)S(N/D)LQQ(Q/R). Therefore the predicted molecular mass of the proteins from different P. falciparum strains ranges from 124.4 to 133.2 kDa, which is almost twice that of the catalytic subunit of the human host enzyme. Isolation of three genomic clones revealed that the gene does not contain introns. P. falciparum gamma-GCS transcription peaks in trophozoites (24-30 h) suggesting that the antioxidant glutathione is predominantly produced at a time where hemoglobin degradation and the simultaneous formation of reactive oxygen species is maximal.

Thioredoxin and glutathione system of malaria parasite Plasmodium falciparum.

SummaryPlasmodium falciparum is the causative agent of malaria tropica. Due to the increasing resistance towards the commonly used plasmodicidal drugs there is an urgent need to identify and assess new targets for the chemotherapeutic intervention of parasite development in the human host. It is established thatP. falciparum-infected erythrocytes are vulnerable to oxidative stress, and therefore efficient antioxidative systems are required to ensure parasite development within the host cell. The thioredoxin and glutathione redox systems represent two powerful means to detoxify reactive oxygen species and this article summarizes some of the recent work which has led to a better understanding of these systems in the parasite and will help to assess them as potential targets for the development of new chemotherapeutics of malaria.

Leishmania major thialysine Nε-acetyltransferase: Identification of amino acid residues crucial for substrate binding

Thialysine N ε‐acetyltransferases and spermidine/spermine N‐acetyltransferases (SSAT) are closely related members of the GCN5‐related N‐acetyltransferase superfamily. Accordingly, a putative orthologue from the human protozoan parasite Leishmania major exhibits an almost equal similarity to human SSAT and thialysine N ε‐acetyltransferase. Characterisation of the recombinantly expressed L. major protein indicated that it represents a thialysine N ε‐acetyltransferase, preferring thialysine (S‐aminoethyl‐l‐cysteine) and structurally related amino acids as acceptor molecules. The known thialysine N ε‐acetyltransferases contain five conserved amino acid residues that are replaced in SSAT sequences. Kinetic analyses of the respective recombinant mutant proteins suggest that Ser82 and Thr83 of L. major thialysine N ε‐acetyltransferase are key residues for acceptor binding. In addition, the conserved Leu130 is tentatively involved in specific interaction with the sulphur‐containing side chain of thialysine. The presence of these three amino acid residues is suggested to be a means by which thialysine N ε‐acetyltransferases can be distinguished from SSAT sequences.

The ornithine decarboxylase domain of the bifunctional ornithine decarboxylase/S-adenosylmethionine decarboxylase of Plasmodium falciparum: recombinant expression and catalytic properties of two different constructs.

The polyamines putrescine, spermidine and spermine play an essential role in cell differentiation and proliferation. Inhibition of the rate-limiting enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), has been proposed as a therapeutic strategy against cancer and parasitic infections. In the case of Plasmodium falciparum, the causative agent of malaria tropica, this approach is especially interesting, because here both key enzymes, ODC and AdoMetDC, are combined in a bifunctional protein, ODC/AdoMetDC. This arrangement has not been found in any other organism investigated so far. We report the cloning and recombinant expression of the ODC domain of P. falciparum in Escherichia coli. First, we expressed the mere recombinant ODC domain (rPfODC). Secondly, we expressed the recombinant ODC domain in conjunction with the preceding part of the hinge region of the bifunctional ODC/AdoMetDC (rPfHinge-ODC). K(m) values for L-ornithine were 47.3 microM for the rPfHinge-ODC and 161. 5 microM for the rPfODC. Both recombinant enzymes were inhibited by putrescine, but the K(i) value for the rPfHinge-ODC was 50.4 microM (IC(50)=157 microM), whereas the IC(50) for the rPfODC was 500 microM. Spermidine was a weak inhibitor in both cases. alpha-Difluoromethylornithine inhibited the rPfHinge-ODC with a K(i) value of 87.6 microM. For two novel ODC inhibitors, CGP52622A and CGP54619A, the K(i) values of the rPfHinge-ODC were in the nanomolar range.

Detection of a chemotactic factor for neutrophils in extracts of female Onchocerca volvulus

Neutrophilic granulocytes and macrophages are the dominant inflammatory cell types observed in the vicinity of and attached to adult Onchocerca volvulus in the subcutaneous nodules. Crude extract from female O. volvulus was examined for chemotactic activity for peripheral neutrophils from healthy individuals by use of an endogenous component chemotactic assay in Boyden chambers. Significant chemotactic responses of neutrophils were detected using O. volvulus extracts at > or = 15 microg/ml in a dose-dependent manner. Checkerboard analysis demonstrated low chemokinetic in addition to chemotactic activity. Neutrophil migration was also elicited by excretory-secretory products of vital females. Fractionation of the female worm extract by FPLC revealed two components with chemotactic activity, one with a molecular mass less than 12 kDa and another with a molecular mass of > 200 kDa. Immunohistological examination of onchocercomas containing only one adult alive filarial worm demonstrated that neutrophils were accumulated near and attached to the cuticle of immature females, females producing microfilariae and males.