Characterization of the Phytochelatin Synthase of Schistosoma mansoni

Treatment for schistosomiasis, which is responsible for more than 280,000 deaths annually, depends exclusively on the use of praziquantel. Millions of people are treated annually with praziquantel and drug resistant parasites are likely to evolve. In order to identify novel drug targets the Schistosoma mansoni sequence databases were queried for proteins involved in glutathione metabolism. One potential target identified was phytochelatin synthase (PCS). Phytochelatins are oligopeptides synthesized enzymatically from glutathione by PCS that sequester toxic heavy metals in many organisms. However, humans do not have a PCS gene and do not synthesize phytochelatins. In this study we have characterized the PCS of S. mansoni (SmPCS). The conserved catalytic triad of cysteine-histidine-aspartate found in PCS proteins and cysteine proteases is also found in SmPCS, as are several cysteine residues thought to be involved in heavy metal binding and enzyme activation. The SmPCS open reading frame is considerably extended at both the N- and C-termini compared to PCS from other organisms. Multiple PCS transcripts are produced from the single encoded gene by alternative splicing, resulting in both mitochondrial and cytoplasmic protein variants. Expression of SmPCS in yeast increased cadmium tolerance from less than 50 µM to more than 1,000 µM. We confirmed the function of SmPCS by identifying PCs in yeast cell extracts using HPLC-mass spectrometry. SmPCS was found to be expressed in all mammalian stages of worm development investigated. Increases in SmPCS expression were seen in ex vivo worms cultured in the presence of iron, copper, cadmium, or zinc. Collectively, these results indicate that SmPCS plays an important role in schistosome response to heavy metals and that PCS is a potential drug target for schistosomiasis treatment. This is the first characterization of a PCS from a parasitic organism

a common highly conserved cadmium detoxification mechanism from bacteria to humans HEAVY METAL TOLERANCE CONFERRED BY THE ATP-BINDING CASSETTE (ABC)TRANSPORTER SpHMT1 REQUIRES GLUTATHIONE BUT NON METAL-CHELATING PHYTOCHELATIN PEPTIDES

International audienceCadmium poses a significant threat to human health due to its toxicity. In mammals and in bakers'yeast, cadmium is detoxified by ATP-binding cassette transporters after conjugation to glutathione. In fission yeast, phytochelatins constitute the co-substrate with cadmium for the transporter SpHMT1. In plants, a detoxification mechanism similar to the one in fission yeast is supposed, but the molecular nature of the transporter is still lacking. To investigate further the relationship between SpHMT1 and its co-substrate, we overexpressed the transporter in a Schizosaccharomyces pombe strain deleted for the phytochelatin synthase gene and heterologously in Saccharomyces cerevisiae and in Escherichia coli. In all organisms, overexpression of SpHMT1 conferred a markedly enhanced tolerance to cadmium but not to Sb(III), AgNO3, As(III), As(V), CuSO4, or HgCl2. Abolishment of the catalytic activity by expression of SpHMT1K623M mutant suppressed the cadmium tolerance phenotype independently of the presence of phytochelatins. Depletion of the glutathione pool inhibited the SpHMT1 activity but not that of AtHNIA4, a P-type ATPase, indicating that GSH is necessary for the SpHMT1-mediated cadmium resistance. In E. coli, SpHMT1 was targeted to the periplasmic membrane and led to an increased amount of cadmium in the periplasm. These results demonstrate that SpHMT1 confers cadmium tolerance in the absence of phytochelatins but depending on the presence of GSH and ATP. Our results challenge the dogma of the two separate cadmium detoxification pathways and demonstrate that a common highly conserved mechanism has been selected during the evolution from bacteria to humans. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc

Glutathione in Synechocystis 6803: A closer look into the physiology of a ΔgshB mutant

Glutathione (GSH) is a low molecular weight thiol compound that plays many roles in photosynthetic organisms. We utilized a ΔgshB (glutathione synthetase) mutant strain as a tool to evaluate the role of GSH in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803), a model photosynthetic organism. The ΔgshB mutant does not synthesize glutathione, but instead accumulates the GSH precursor, γ-glutamylcysteine (γ-EC), to millimolar levels. We found that γ-EC was sufficient to permit cellular proliferation during optimal conditions, but not when cells were exposed to conditions promoting oxidative stress. Furthermore, we found that many factors affecting growth rate and photosynthetic activities strongly influenced cellular thiol content. Here, we are providing some additional insights into the role of GSH and γ-EC in Synechocystis 6803 during conditions promoting oxidative stress