A gold-containing drug against parasitic polyamine metabolism: the X-ray structure of trypanothione reductase from Leishmania infantum in complex with auranofin reveals a dual mechanism of enzyme inhibition

Auranofin is a gold(I)-containing drug in clinical use as an antiarthritic agent. Recent studies showed that auranofin manifests interesting antiparasitic actions very likely arising from inhibition of parasitic enzymes involved in the control of the redox metabolism. Trypanothione reductase is a key enzyme of Leishmania infantum polyamine-dependent redox metabolism, and a validated target for antileishmanial drugs. As trypanothione reductase contains a dithiol motif at its active site and gold(I) compounds are known to be highly thiophilic, we explored whether auranofin might behave as an effective enzyme inhibitor and as a potential antileishmanial agent. Notably, enzymatic assays revealed that auranofin causes indeed a pronounced enzyme inhibition. To gain a deeper insight into the molecular basis of enzyme inhibition, crystals of the auranofin-bound enzyme, in the presence of NADPH, were prepared, and the X-ray crystal structure of the auranofin–trypanothione reductase–NADPH complex was solved at 3.5 Å resolution. In spite of the rather low resolution, these data were of sufficient quality as to identify the presence of the gold center and of the thiosugar of auranofin, and to locate them within the overall protein structure. Gold binds to the two active site cysteine residues of TR, i.e. Cys52 and Cys57, while the thiosugar moiety of auranofin binds to the trypanothione binding site; thus auranofin appears to inhibit TR through a dual mechanism. Auranofin kills the promastigote stage of L. infantum at micromolar concentration; these findings will contribute to the design of new drugs against leishmaniasis

Cell Death by SecTRAPs: Thioredoxin Reductase as a Prooxidant Killer of Cells

BACKGROUND: SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins) can be formed from the selenoprotein thioredoxin reductase (TrxR) by targeting of its selenocysteine (Sec) residue with electrophiles, or by its removal through C-terminal truncation. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured cancer cell lines by a gain of function. PRINCIPAL FINDINGS: Both human and rat SecTRAPs killed human A549 and HeLa cells. The cell death displayed both apoptotic and necrotic features. It did not require novel protein synthesis nor did it show extensive nuclear fragmentation, but it was attenuated by use of caspase inhibitors. The redox active disulfide/dithiol motif in the N-terminal domain of TrxR had to be maintained for manifestation of SecTRAP cytotoxicity. Stopped-flow kinetics showed that NADPH can reduce the FAD moiety in SecTRAPs at similar rates as in native TrxR and purified SecTRAPs could maintain NADPH oxidase activity, which was accelerated by low molecular weight substrates such as juglone. In a cellular context, SecTRAPs triggered extensive formation of reactive oxygen species (ROS) and consequently antioxidants could protect against the cell killing by SecTRAPs. CONCLUSIONS: We conclude that formation of SecTRAPs could contribute to the cytotoxicity seen upon exposure of cells to electrophilic agents targeting TrxR. SecTRAPs are prooxidant killers of cells, triggering mechanisms beyond those of a mere loss of thioredoxin reductase activity

A transcriptomic analysis of Echinococcus granulosus larval stages:implications for parasite biology and host adaptation

The cestode Echinococcus granulosus--the agent of cystic echinococcosis, a zoonosis affecting humans and domestic animals worldwide--is an excellent model for the study of host-parasite cross-talk that interfaces with two mammalian hosts. To develop the molecular analysis of these interactions, we carried out an EST survey of E. granulosus larval stages. We report the salient features of this study with a focus on genes reflecting physiological adaptations of different parasite stages.We generated ~10,000 ESTs from two sets of full-length enriched libraries (derived from oligo-capped and trans-spliced cDNAs) prepared with three parasite materials: hydatid cyst wall, larval worms (protoscoleces), and pepsin/H(+)-activated protoscoleces. The ESTs were clustered into 2700 distinct gene products. In the context of the biology of E. granulosus, our analyses reveal: (i) a diverse group of abundant long non-protein coding transcripts showing homology to a middle repetitive element (EgBRep) that could either be active molecular species or represent precursors of small RNAs (like piRNAs); (ii) an up-regulation of fermentative pathways in the tissue of the cyst wall; (iii) highly expressed thiol- and selenol-dependent antioxidant enzyme targets of thioredoxin glutathione reductase, the functional hub of redox metabolism in parasitic flatworms; (iv) candidate apomucins for the external layer of the tissue-dwelling hydatid cyst, a mucin-rich structure that is critical for survival in the intermediate host; (v) a set of tetraspanins, a protein family that appears to have expanded in the cestode lineage; and (vi) a set of platyhelminth-specific gene products that may offer targets for novel pan-platyhelminth drug development.This survey has greatly increased the quality and the quantity of the molecular information on E. granulosus and constitutes a valuable resource for gene prediction on the parasite genome and for further genomic and proteomic analyses focused on cestodes and platyhelminths

Isotopic techniques to measure N2O, N2 and their sources

GHG emissions are usually the result of several simultaneous processes. Furthermore, some gases such as N2 are very difficult to quantify and require special techniques. Therefore, in this chapter, the focus is on stable isotope methods. Both natural abundance techniques and enrichment techniques are used. Especially in the last decade, a number of methodological advances have been made. Thus, this chapter provides an overview and description of a number of current state-of-theart techniques, especially techniques using the stable isotope 15N. Basic principles and recent advances of the 15N gas flux method are presented to quantify N2 fluxes, but also the latest isotopologue and isotopomer methods to identify pathways for N2O production. The second part of the chapter is devoted to 15N tracing techniques, the theoretical background and recent methodological advances. A range of different methods is presented from analytical to numerical tools to identify and quantify pathway-specific N2O emissions. While this chapter is chiefly concerned with gaseous N emissions, a lot of the techniques can also be applied to other gases such as methane (CH4), as outlined in Sect. 5.3

Microbe-host interplay in atopic dermatitis and psoriasis

Despite recent advances in understanding microbial diversity in skin homeostasis, the relevance of microbial dysbiosis in inflammatory disease is poorly understood. Here we perform a comparative analysis of skin microbial communities coupled to global patterns of cutaneous gene expression in patients with atopic dermatitis or psoriasis. The skin microbiota is analysed by 16S amplicon or whole genome sequencing and the skin transcriptome by microarrays, followed by integration of the data layers. We find that atopic dermatitis and psoriasis can be classified by distinct microbes, which differ from healthy volunteers microbiome composition. Atopic dermatitis is dominated by a single microbe (Staphylococcus aureus), and associated with a disease relevant host transcriptomic signature enriched for skin barrier function, tryptophan metabolism and immune activation. In contrast, psoriasis is characterized by co-occurring communities of microbes with weak associations with disease related gene expression. Our work provides a basis for biomarker discovery and targeted therapies in skin dysbiosis.Peer reviewe

Exploring thioredoxin reductase as an anticancer drug target

Mammalian thioredoxin reductase (TrxR; EC 1.8.1.9) is a homodimeric NADPH-dependent selenium-containing flavoenzyme with disulfide oxidoreductase activity. The mammalian thioredoxin (Trx) system exerts an impressive spectrum of functions either directly through reactions catalyzed by TrxR or by its prime substrate Trx. It is involved in redox regulation, antioxidant defense, cell growth, replication and regulation of transcription factors. TrxR was found to be overexpressed in a variety of tumor cells and may be involved in several, if not all, steps of carcinogenesis. Therefore, TrxR has been proposed as a potential target for anticancer therapy. In fact, inhibition of TrxR has been shown by a number of chemotherapeutic drugs as part of their mechanism of action. The rather unique biochemistry of TrxR indeed supports efficient targeting by electrophiles. TrxR has an N-terminal active site with two cysteines (Cys) and a Cterminal active site with a Cys and a selenocysteine (Sec). Both active sites are necessary for native TrxR function. Sec is a rare, naturally occurring amino acid encoded by a UGA codon. As this codon normally signals a termination of translation, a recoding signal, the Sec insertion sequence is present in the 3´ untranslated region of the mRNA. A specific translation machinery then guides the insertion of the Sec into the polypeptide chain. Sec is a highly reactive Cys analog with a selenium atom in place of the sulfur, resulting in a significantly lower pKa value. Therefore, Sec is mainly deprotonated at physiological pH and thus highly reactive towards alkylation by electrophilic agents. This results in abrogation of redox activity of the enzyme´s C-terminal active site that is essential for reduction of Trx and most other substrates. However, specific targeting of the Sec does not render a completely inactive protein. With an intact N-terminal active site, inhibited TrxR may still induce a rapid cell death in cancer cells presumably by redox cycling with an endogenous substrate inducing oxidative stress as shown in this thesis. We named this cytotoxic form of TrxR SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins). Introducing SecTRAPs directly into A549 (lung carcinoma) or HeLa (cervical adenocarcinoma) cells using the protein delivery reagent BioPORTER® we observed DNA condensation, phosphatidylserine exposure prior to loss of membrane integrity and production of reactive oxygen species. Cell death was caspase-dependent but independent of novel protein synthesis. Cisplatin was shown to inhibit TrxR and form SecTRAPs in vitro. We investigated the elemental differences in TrxR inhibition of simple Pt, Pd and Au salts, showing Pd and Au to be superior in this aspect. All salts seemingly target the C-terminal active site of TrxR in a specific manner, potentially forming SecTRAPs. Inhibition of cellular TrxR was shown to correlate with cytotoxicity of nitroaromatic and quinone compounds involving caspase activation. However, the extent of cell death was in some cases, but not all, dependent on TrxR1 levels. Using siRNA constructs to specifically knock down TrxR1 by 90% in A549 cells resulted in increased susceptibility towards DNCB and menadione, and decreased sensitivity to cisplatin. This may indicate that different TrxR inhibition mechanisms can lead to different treatment outcomes and that cisplatin may indeed form toxic SecTRAPs within cells. This occurs possibly at a greater extent in tumors where high expression of TrxR is prevalent. The potential of TrxR as an anticancer drug target is discussed in this thesis with special focus on drugs likely to form SecTRAPs and possibly to induce selective tumor killing

Fecal transfers from children on the ketogenic diet mimic the anti-seizure effect in mice

The ketogenic diet (KD) mediates its anti-seizure effect through the gut microbiota in epilepsy mouse models.1 Lum et al.2 demonstrated that fecal microbiota from children with epilepsy treated with the KD decreases seizure susceptibility in mice after transfer