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In eukaryotes the roles of protein kinases (PKs) regulating important biological processes such as growth and differentiation are well known. Molecular, biochemical, and physiological analyses trying to unravel principles of schistosome development have substantiated the importance for PKs also in this parasite. Amongst others the role of SmTK3 was studied, one of the first cellular PKs characterized from Schistosoma mansoni. Its function was demonstrated in mitogenic and differentiation processes in the gonads. Furthermore, first insights were obtained for the downstream part of a signal transduction cascade SmTK3 is involved in, which includes the diaphanous homolog SmDia. Here we attempted to further unravel the SmTK3 signaling cascade by searching for upstream interaction partners. Using yeast three-hybrid (Y3H) analyses we detected the epidermal growth factor receptor (EGFR) pathway substrate 8 of S. mansoni (SmEps8) as the most interesting candidate. By detailed interaction analyses we showed a contribution of the Src homology (SH) domains SH2 and SH3 of SmTK3 to binding, with a clear bias towards SH2. Compared to full-length SmEps8, binding was enhanced when only its 5' part including the phosphotyrosine binding domain (PTB) was used for interaction analyses including the SH2 domain of SmTK3, although phosphorylation seemed not to play a decisive role for binding. RT-PCR analyses and in situ hybridization experiments demonstrated similar transcription patterns of SmTK3 and SmEPS8, which co-localize in the reproductive organs. Furthermore, first evidence was obtained for SmEps8 interaction and colocalization with SER, one of the epidermal growth factor receptor (EGFR) homologs detected in S. mansoni. The results of this study provide first evidence for a SER-SmEps8-SmTK3-SmDia signal transduction pathway controlling differentiation processes in the gonads of S. mansoni.

SUMMARYAmong the topics of considerable interest concerning our understanding of the unusual biology of schistosomes is the sexual maturation of the female. The identification of genes coding for signal transduction proteins controlling essential steps of the pairing-dependent differentiation of the reproductive organs, vitellarium and ovary will help to substantiate our knowledge about this unique parasite. Furthermore, such signalling proteins could be potential targets to interfere with the development of this parasite to combat schistosomiasis since its pathology is caused by the eggs. This review summarises first post-genomic steps to elucidate the function of gonad-specific signalling molecules which were identified by homology-based cloning strategies, byin silicoidentification or by yeast two-hybrid interaction analyses, using a combination of novel techniques. These include thein vitroculture of adult schistosomes, their treatment with chemical inhibitors to block enzyme activity, the use of RNAi to silence gene function post-transcriptionally, and confocal laser scanning microscopy to study the morphological consequences of these experimental approaches. Finally, we propose a first model of protein networks that are active in the ovary regulating mitogenic activity and differentiation. Some of these molecules are also active in the testes of males, probably fulfilling similar roles as in the ovary.

Onchocerciasis or river blindness, caused by the filarial nematode Onchocerca volvulus, is the second leading cause of blindness due to infectious diseases. The protective role of the omega-class glutathione transferase 3 from O. volvulus (OvGST3) against intracellular and environmental reactive oxygen species has been described previously. In the present study, we continue our investigation of the highly stress-responsive OvGST3. Alternative splicing of two exons and one intron retention generates five different transcript isoforms that possess a spliced leader at their 5'-end, indicating that the mechanism of mature mRNA production involves alternative-, cis- and trans-splicing processes. Interestingly, the first two exons of the ovgst3 gene encode a signal peptide before sequence identity to other omega-class glutathione transferases begins. Only the recombinant expression of the isoform that encodes the longest deduced amino acid sequence (OvGST3/5) was successful, with the purified enzyme displaying modest thiol oxidoreductase activity. Significant IgG1 and IgG4 responses against recombinantly expressed OvGST3/5 were detected in sera from patients with the generalized as well as the chronic hyperreactive form of onchocerciasis, indicating exposure of the secreted protein to the human host's immune system and its immunogenicity. Immunohistological localization studies performed at light and electron microscopy levels support the extracellular localization of the protein. Intensive labeling of the OvGST3 was observed in the egg shell at the morula stage of the embryo, indicating extremely defined, stage-specific expression for a short transient period only.

Onchocerciasis or river blindness, caused by the filarial worm Onchocerca volvulus, is the world's second leading infectious cause of blindness. In order to chronically infect the host, O. volvulus has evolved molecular strategies that influence and direct immune responses away from the modes most damaging to it. The O. volvulus GST1 (OvGST1) is a unique glutathione S-transferase (GST) in that it is a glycoprotein and possesses a signal peptide that is cleaved off in the process of maturation. The mature protein starts with a 25-amino-acid extension not present in other GSTs. In all life stages of the filarial worm, it is located directly at the parasite-host interface. Here, the OvGST1 functions as a highly specific glutathione-dependent prostaglandin D synthase (PGDS). The enzyme therefore has the potential to participate in the modulation of immune responses by contributing to the production of parasite-derived prostanoids and restraining the host's effector responses, making it a tempting target for chemotherapy and vaccine development. Here, we report the crystal structure of the OvGST1 bound to its cofactor glutathione at 2.0 A resolution. The structure reveals an overall structural homology to the haematopoietic PGDS from vertebrates but, surprisingly, also a large conformational change in the prostaglandin binding pocket. The observed differences reveal a different vicinity of the prostaglandin H(2) binding pocket that demands another prostaglandin H(2) binding mode to that proposed for the vertebrate PGDS. Finally, a putative substrate binding mode for prostaglandin H(2) is postulated based on the observed structural insights.

Drug-induced suppression of female schistosome sexual maturation is an auspicious strategy to combat schistosomiasis since the eggs are the causative agent. The establishment of drug targets requires knowledge about the molecular mechanisms that regulate the development of the female reproductive organs, which include vitellarium and ovary. This review summarizes recent studies suggesting tyrosine kinases as important factors for the regulation of female gonad development. In this context, especially cytoplasmatic tyrosine kinases of the Src class seem to play dominant roles. Moreover, experimental data and theoretical concepts are provided supporting a crosstalk between tyrosine kinase and TGFbeta signaling in the production of vitellocytes. Finally, we take advantage from the schistosome genome project to propose a model for the regulation of vitelline-cell production and differentiation.

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 threedimensional 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.

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.

Glutathione S-transferases (GSTs) belong to a family of detoxification enzymes that conjugate glutathione to various xenobiotics, thus facilitating their expulsion from the cells. For high-resolution crystallographic investigations, GST from the human malarial parasite Plasmodium falciparum was overexpressed in bacterial cells and crystallized using hanging-drop vapour diffusion. X-ray intensity data to 2.8 A resolution were collected from an orthorhombic crystal form with unit-cell parameters a = 62.2, b = 88.3, c = 75.3 A. A search for heavy-atom derivatives has been initiated, along with phase-determination efforts by molecular replacement.

Besides their great medical importance as causative agents of schistosomiasis, an infectious disease affecting humans and animals in tropical and subtropical regions worldwide, schistosomes exhibit distinctive biological features. Living in the blood vessels of infected hosts, these blood flukes survive permanent attacks of the immune system over many years. Furthermore, schistosomes represent the only genus of the class trematoda which live doeciously. Their most remarkable attribute, however, is the continuous pairing-contact which is both obligatory for the development of the female reproductive organs and prerequisite for egg production

Onchocerciasis or river blindness, caused by the filarial nematode Onchocerca volvulus, is the second leading cause of blindness due to infectious diseases. The protective role of the omega-class glutathione transferase 3 from O. volvulus (OvGST3) against intracellular and environmental reactive oxygen species has been described previously. In the present study, we continue our investigation of the highly stress-responsive OvGST3. Alternative splicing of two exons and one intron retention generates five different transcript isoforms that possess a spliced leader at their 5'-end, indicating that the mechanism of mature mRNA production involves alternative-, cis- and trans-splicing processes. Interestingly, the first two exons of the ovgst3 gene encode a signal peptide before sequence identity to other omega-class glutathione transferases begins. Only the recombinant expression of the isoform that encodes the longest deduced amino acid sequence (OvGST3/5) was successful, with the purified enzyme displaying modest thiol oxidoreductase activity. Significant IgG1 and IgG4 responses against recombinantly expressed OvGST3/5 were detected in sera from patients with the generalized as well as the chronic hyperreactive form of onchocerciasis, indicating exposure of the secreted protein to the human host's immune system and its immunogenicity. Immunohistological localization studies performed at light and electron microscopy levels support the extracellular localization of the protein. Intensive labeling of the OvGST3 was observed in the egg shell at the morula stage of the embryo, indicating extremely defined, stage-specific expression for a short transient period only.

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 approximately 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.

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 parasite Plasmodium falciparum causes malaria tropica, the most prevailing parasitic disease worldwide, with 300-500 million infections and 1.5-2.7 million deaths/year. The emergence of strains resistant to drugs used for prophylaxis and treatment and no vaccine available makes the structural analysis of potential drug targets essential. For that reason, we analyzed the three-dimensional structure of the glutathione S-transferase from P. falciparum (Pf-GST1) in the apoform and in complex with its inhibitor S-hexyl-glutathione. The structures have been analyzed to 2.6 and 2.2 A, respectively. Pf-GST1 shares several structural features with the Mu-type GSTs and is therefore closely related to this class, even though alignments with its members display low sequence identities in the range of 20-33%. Upon S-hexyl-glutathione binding, the overall structure and the glutathione-binding site (G-site) remain almost unchanged with the exception of the flexible C terminus. The detailed comparison of the parasitic enzyme with the human host Mu-class enzyme reveals that, although the overall structure is homologue, the shape of the hydrophobic binding pocket (H-site) differs substantially. In the human enzyme, it is shielded from one side by the large Mu-loop, whereas in Pf-GST1 the Mu-loop is truncated and the space to recognize and bind voluminous substrates is extended. This structural feature can be exploited to support the design of specific and parasite-selective inhibitors.