Biophysical characterization of the complex between the iron-responsive transcription factor Fep1 and DNA.

Fep1 is an iron-responsive GATA-type transcriptional repressor present in numerous fungi. The DNA-binding domain of this protein is characterized by the presence of two zinc fingers of the Cys2-Cys2 type and a Cys-X5-Cys-X8-Cys-X2-Cys motif located between the two zinc fingers, that is involved in binding of a [2Fe-2S] cluster. In this work, biophysical characterization of the DNA-binding domain of Pichia pastoris Fep1 and of the complex of the protein with cognate DNA has been undertaken. The results obtained by analytical ultracentrifugation sedimentation velocity, small-angle X-ray scattering and differential scanning calorimetry indicate that Fep1 is a natively unstructured protein that is able to bind DNA forming 1:1 and 2:1 complexes more compact than the individual partners. Complex formation takes place independently of the presence of a stoichiometric [2Fe-2S] cluster, suggesting that the cluster may play a role in recruiting other protein(s) required for regulation of transcription in response to changes in intracellular iron levels.

Omic approaches to decipher the molecular mechanisms of fibrosis, and design new anti-fibrotic strategies.

We review here omics approaches including transcriptomics, proteomics, glycomics, metabolomics and interactomics, databases and computational tools for omic and multi-omic investigations of fibrosis to understand the molecular mechanisms underlying fibrogenesis and fibrosis, to identify biomarkers of diagnosis, prognosis or disease progression, and new therapeutic targets and to design new anti-fibrotic drugs. We also provide perspectives for future studies including lipid and glycosaminoglycan profiling, and the design of virtual patient models as a basis for personalised medicine and virtualisation of drug development.

A comparative analysis of secreted protein disulfide isomerases from the tropical co-endemic parasites Schistosoma mansoni and Leishmania major.

The human parasites Schistosoma mansoni and Leishmania major are co-endemic and a major threat to human health. Though displaying different tissue tropisms, they excrete/secrete similar subsets of intracellular proteins that, interacting with the host extracellular matrix (ECM), help the parasites invading the host. We selected one of the most abundant proteins found in the secretomes of both parasites, protein disulfide isomerase (PDI), and performed a comparative screening with surface plasmon resonance imaging (SPRi), looking for ECM binding partners. Both PDIs bind heparan sulfate; none of them binds collagens; each of them binds further ECM components, possibly linked to the different tropisms. We investigated by small-angle X-ray scattering both PDIs structures and those of a few complexes with host partners, in order to better understand the differences within this conserved family fold. Furthermore, we highlighted a previously undisclosed moonlighting behaviour of both PDIs, namely a concentration-dependent switch of function from thiol-oxidoreductase to holdase. Finally, we have tried to exploit the differences to look for possible compounds able to interfere with the redox activity of both PDI.

Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners.

Lysyl oxidase (LOX) catalyzes the oxidative deamination of lysine and hydroxylysine residues in collagens and elastin, which is the first step of the cross-linking of these extracellular matrix proteins. It is secreted as a proenzyme activated by bone morphogenetic protein-1, which releases the LOX catalytic domain and its bioactive N-terminal propeptide. We characterized the recombinant human propeptide by circular dichroism, dynamic light scattering, and small-angle X-ray scattering (SAXS), and showed that it is elongated, monomeric, disordered and flexible (Dmax: 11.7 nm, Rg: 3.7 nm). We generated 3D models of the propeptide by coarse-grained molecular dynamics simulations restrained by SAXS data, which were used for docking experiments. Furthermore, we have identified 17 new binding partners of the propeptide by label-free assays. They include four glycosaminoglycans (hyaluronan, chondroitin, dermatan and heparan sulfate), collagen I, cross-linking and proteolytic enzymes (lysyl oxidase-like 2, transglutaminase-2, matrix metalloproteinase-2), a proteoglycan (fibromodulin), one growth factor (Epidermal Growth Factor, EGF), and one membrane protein (tumor endothelial marker-8). This suggests new roles for the propeptide in EGF signaling pathway.

Fragment-Based Discovery of a Regulatory Site in Thioredoxin Glutathione Reductase Acting as "Doorstop" for NADPH Entry.

Members of the FAD/NAD-linked reductase family are recognized as crucial targets in drug development for cancers, inflammatory disorders, and infectious diseases. However, individual FAD/NAD reductases are difficult to inhibit in a selective manner with off-target inhibition reducing usefulness of identified compounds. Thioredoxin glutathione reductase (TGR), a high molecular weight thioredoxin reductase-like enzyme, has emerged as a promising drug target for the treatment of schistosomiasis, a parasitosis afflicting more than 200 million people. Taking advantage of small molecules selected from a high-throughput screen and using X-ray crystallography, functional assays, and docking studies, we identify a critical secondary site of the enzyme. Compounds binding at this site interfere with well-known and conserved conformational changes associated with NADPH reduction, acting as a doorstop for cofactor entry. They selectively inhibit TGR from Schistosoma mansoni and are active against parasites in culture. Since many members of the FAD/NAD-linked reductase family have similar catalytic mechanisms, the unique mechanism of inhibition identified in this study for TGR broadly opens new routes to selectively inhibit homologous enzymes of central importance in numerous diseases.

Pichia pastoris Fep1 is a [2Fe-2S] protein with a Zn finger that displays an unusual oxygen-dependent role in cluster binding.

Fep1, the iron-responsive GATA factor from the methylotrophic yeast Pichia pastoris, has been characterised both in vivo and in vitro. This protein has two Cys2-Cys2 type zinc fingers and a set of four conserved cysteines arranged in a Cys-X5-Cys-X8-Cys-X2-Cys motif located between the two zinc fingers. Electronic absorption and resonance Raman spectroscopic analyses in anaerobic and aerobic conditions indicate that Fep1 binds iron in the form of a [2Fe-2S] cluster. Site-directed mutagenesis shows that replacement of the four cysteines with serine inactivates this transcriptional repressor. Unexpectedly, the inactive mutant is still able to bind a [2Fe-2S] cluster, employing two cysteine residues belonging to the first zinc finger. These two cysteine residues can act as alternative cluster ligands selectively in aerobically purified Fep1 wild type, suggesting that oxygen could play a role in Fep1 function by causing differential localization of the [Fe-S] cluster.

Uncovering new structural insights for antimalarial activity from cost-effective aculeatin-like derivatives.

A series of new aculeatin-like analogues were synthesized in two steps by combining two sets of building blocks. Many compounds showed inhibitory activities in vitro against Plasmodium falciparum and have helped to gain more insight into structure-activity relationships around the spirocyclohexadienone pharmacophoric scaffold. Plasmodium falciparum thioredoxin reductase (PfTrxR) has been investigated as a putative cellular target. Moreover, a new aculeatin-like scaffold without Michael acceptor properties, efficient at 0.86 µM against P. falciparum 3D7, was identified and raises the prospect of developing a new antimalarial agent.

Selenocysteine robustness versus cysteine versatility: a hypothesis on the evolution of the moonlighting behaviour of peroxiredoxins.

Peroxiredoxins (Prxs) and glutathione peroxidases (Gpxs) provide the majority of peroxides reducing activity in the cytoplasm. Both are peroxidases but differences in the chemical mechanism of reduction of oxidative agents, as well as in the reactivity of the catalytically active residues, confer peculiar features on them. Ultimately, Gpx should be regarded as an efficient peroxides scavenger having a high-reactive selenocysteine (Sec) residue. Prx, by having a low pKa cysteine, is less efficient than Gpx in reduction of peroxides under physiological conditions, but the chemistry of the sulfur together with the peculiar structural arrangement of the active site, in typical Prxs, make it suitable to sense a redox environment and to switch-in-function so as to exert holdase activity under redox-stress conditions. The complex macromolecular assembly would have evolved the chaperone holdase function and the moonlighting behaviour typical of many Prxs.

Thioredoxin reductase and its inhibitors.

Thioredoxin plays a crucial role in a wide number of physiological processes, which span from reduction of nucleotides to deoxyriboucleotides to the detoxification from xenobiotics, oxidants and radicals. The redox function of Thioredoxin is critically dependent on the enzyme Thioredoxin NADPH Reductase (TrxR). In view of its indirect involvement in the above mentioned physio/pathological processes, inhibition of TrxR is an important clinical goal. As a general rule, the affinities and mechanisms of binding of TrxR inhibitors to the target enzyme are known with scarce precision and conflicting results abound in the literature. A relevant analysis of published results as well as the experimental procedures is therefore needed, also in view of the critical interest of TrxR inhibitors. We review the inhibitors of TrxR and related flavoreductases and the classical treatment of reversible, competitive, non competitive and uncompetitive inhibition with respect to TrxR, and in some cases we are able to reconcile contradictory results generated by oversimplified data analysis.

Crystal structure of Plasmodium falciparum thioredoxin reductase, a validated drug target.

Plasmodium falciparum is the vector of the most prevalent and deadly form of malaria, and, among the Plasmodium species, it is the one with the highest rate of drug resistance. At the basis of a rational drug design project there is the selection and characterization of suitable target(s). Thioredoxin reductase, the first protection against reactive oxygen species in the erythrocytic phase of the parasite, is essential for its survival. Hence it represents a good target for the design of new anti-malarial active compounds. In this paper we present the first crystal structure of recombinant P. falciparum thioredoxin reductase (PfTrxR) at 2.9Å and discuss its differences with respect to the human orthologue. The most important one resides in the dimer interface, which offers a good binding site for selective non competitive inhibitors. The striking conservation of this feature among the Plasmodium parasites, but not among other Apicomplexa parasites neither in mammals, boosts its exploitability.

Moonlighting by different stressors: crystal structure of the chaperone species of a 2-Cys peroxiredoxin.

2-Cys peroxiredoxins (Prxs) play two different roles depending on the physiological status of the cell. They are thioredoxin-dependent peroxidases under low oxidative stress and ATP-independent chaperones upon exposure to high peroxide concentrations. These alternative functions have been associated with changes in the oligomerization state from low-(LMW) to high-molecular-weight (HMW) species. Here we present the structures of Schistosoma mansoni PrxI in both states: the LMW decamer and the HMW 20-mer formed by two stacked decamers. The latter is the structure of a 2-Cys Prx chaperonic form. Comparison of the structures sheds light on the mechanism by which chemical stressors, such as high H(2)O(2) concentration and acidic pH, are sensed and translated into a functional switch in this protein family. We also propose a model to account for the in vivo formation of long filaments of stacked Prx rings.

On the mechanism and rate of gold incorporation into thiol-dependent flavoreductases.

NADPH-dependent flavoreductases are important drug targets. During their enzymatic cycle thiolates and selenolates that have high affinity for transition metals are generated. Auranofin (AF), a gold-containing compound, is classified by the World Health Organization as an antirheumatic agent and it is indicated as the scaffold for the development of new anticancer and antiparasitic drugs. AF inhibits selenocysteine-containing flavoreductases (thioredoxin reductase and thioredoxin glutathione reductase) more effectively than non Se-containing ones (glutathione reductase); this preference has been ascribed to the high affinity of selenium for gold. We solved the 3D structure of the Se-containing Thioredoxin Glutathione Reductase from the human parasite Schistosoma mansoni complexed with Au and our results challenge this view: we believe that the relative velocity of the reaction rather than the relative affinity, depends on the presence of Sec residues, which appear to dictate AF selectivity.

Structural and functional characterization of Schistosoma mansoni Thioredoxin.

Schistosomiasis, the human parasitosis caused by various species of the blood-fluke Schistosoma, is a debilitating disease affecting 200 million people in tropical areas. The massive administration of the only effective drug, praziquantel, leads to the appearance of less sensitive parasite strains, thus, making urgent the search for new therapeutic approaches and new suitable targets. The thiol-mediated detoxification pathway has been identified as a promising target, being essential during all the parasite developmental stages and sufficiently different from the host counterpart. As a part of a project aimed at the structural characterization of all the proteins involved in this pathway, we describe hereby the high-resolution crystal structure of Schistosoma mansoni Thioredoxin (SmTrx) in three states, namely: the wild-type oxidized adult enzyme and the oxidized and reduced forms of a juvenile isoform, carrying an N-terminal extension. SmTrx shows a typical thioredoxin fold, highly similar to the other components of the superfamily. Although probably unlikely to be a reasonable drug target given its high similarity with the human counterpart, SmTrx completes the characterization of the whole set of thiol-mediated detoxification pathway components. Moreover, it can reduce oxidized glutathione and is one of the few defence proteins expressed in mature eggs and in the hatch fluid, thus confirming an important role in the parasite. We believe its crystal structure may provide clues for the formation of granulomas and the pathogenesis of the chronic disease.

Mapping the catalytic cycle of Schistosoma mansoni thioredoxin glutathione reductase by X-ray crystallography.

Schistosomiasis is the second most widespread human parasitic disease. It is principally treated with one drug, praziquantel, that is administered to 100 million people each year; less sensitive strains of schistosomes are emerging. One of the most appealing drug targets against schistosomiasis is thioredoxin glutathione reductase (TGR). This natural chimeric enzyme is a peculiar fusion of a glutaredoxin domain with a thioredoxin selenocysteine (U)-containing reductase domain. Selenocysteine is located on a flexible C-terminal arm that is usually disordered in the available structures of the protein and is essential for the full catalytic activity of TGR. In this study, we dissect the catalytic cycle of Schistosoma mansoni TGR by structural and functional analysis of the U597C mutant. The crystallographic data presented herein include the following: the oxidized form (at 1.9 Å resolution); the NADPH- and GSH-bound forms (2.3 and 1.9 Å, respectively); and a different crystal form of the (partially) reduced enzyme (3.1 Å), showing the physiological dimer and the entire C terminus of one subunit. Whenever possible, we determined the rate constants for the interconversion between the different oxidation states of TGR by kinetic methods. By combining the crystallographic analysis with computer modeling, we were able to throw further light on the mechanism of action of S. mansoni TGR. In particular, we hereby propose the putative functionally relevant conformational change of the C terminus after the transfer of reducing equivalents from NADPH to the redox sites of the enzyme.

Inhibition of Schistosoma mansoni thioredoxin-glutathione reductase by auranofin: structural and kinetic aspects.

Schistosomiasis is a parasitic disease affecting over 200 million people currently treated with one drug, praziquantel. A possible drug target is the seleno-protein thioredoxin-glutathione reductase (TGR), a key enzyme in the pathway of the parasite for detoxification of reactive oxygen species. The enzyme is a unique fusion of a glutaredoxin domain with a thioredoxin reductase domain, which contains a selenocysteine (Sec) as the penultimate amino acid. Auranofin (AF), a gold-containing compound already in clinical use as an anti-arthritic drug, has been shown to inhibit TGR and to substantially reduce worm burden in mice. Using x-ray crystallography we solved (at 2.5 A resolution) the structure of wild type TGR incubated with AF. The electron density maps show that the actual inhibitor is gold, released from AF. Gold is bound at three different sites not directly involving the C-terminal Sec residue; however, because the C terminus in the electron density maps is disordered, we cannot exclude the possibility that gold may also bind to Sec. To investigate the possible role of Sec in the inactivation kinetics, we tested the effect of AF on a model enzyme of the same superfamily, i.e. the naturally Sec-lacking glutathione reductase, and on truncated TGR. We demonstrate that the role of selenium in the onset of inhibition by AF is catalytic and can be mimicked by an external source of selenium (benzeneselenol). Therefore, we propose that Sec mediates the transfer of gold from its ligands in AF to the redox-active Cys couples of TGR.

Investigating the structural plasticity of a cytochrome P450: three-dimensional structures of P450 EryK and binding to its physiological substrate.

Cytochrome P450s are heme-containing proteins that catalyze the oxidative metabolism of many physiological endogenous compounds. Because of their unique oxygen chemistry and their key role in drug and xenobiotic metabolism, particular attention has been devoted in elucidating their mechanism of substrate recognition. In this work, we analyzed the three-dimensional structures of a monomeric cytochrome P450 from Saccharopolyspora erythraea, commonly called EryK, and the binding kinetics to its physiological ligand, erythromycin D. Three different structures of EryK were obtained: two ligand-free forms and one in complex with its substrate. Analysis of the substrate-bound structure revealed the key structural determinants involved in substrate recognition and selectivity. Interestingly, the ligand-free structures of EryK suggested that the protein may explore an open and a closed conformation in the absence of substrate. In an effort to validate this hypothesis and to investigate the energetics between such alternative conformations, we performed stopped-flow absorbance experiments. Data demonstrated that EryK binds erythromycin D via a mechanism involving at least two steps. Contrary to previously characterized cytochrome P450s, analysis of double jump mixing experiments confirmed that this complex scenario arises from a pre-existing equilibrium between the open and closed subpopulations of EryK, rather than from an induced-fit type mechanism.

Pattern of cavities in globins: the case of human hemoglobin.

Our aim is to shed light on the conservation of potential ligand docking sites that play an important role in ligand dynamics of globins by using the technique of filling internal cavities naturally present in hemoglobin and myoglobin with xenon atoms. In particular, we present the high resolution structures of the Xe-adduct of deoxygenated wild type human hemoglobin and a quadruple mutant (L(B10)Y and H(E7)Q in alpha and beta chains). For the sake of comparison we also determined under the same experimental conditions the xenon complex of wild type sperm whale myoglobin. The analysis revealed that the number and position of Xe binding cavities are different in the alpha and beta subunits, the latter being more similar to myoglobin. Notably, no proximal Xe docking site was detected in hemoglobin, at variance with myoglobin. The pattern of internal cavities accessibility and affinity for xenon suggests a different role for the dynamics of ligand migration in the two types of hemoglobin chains as compared to myoglobin. The number and position of hydrophobic cavities in hemoglobin are briefly discussed also in comparison with the data available for other members of the globin superfamily.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of C-12 hydroxylase EryK from Saccharopolyspora erythraea.

Erythromycin A is produced by Saccharopolyspora erythraea via a secondary metabolic pathway using several steps including glycosylations and hydroxylations of the first macrolide intermediate 6-deoxyerythronolide B. Erythromycin C-12 hydroxylase (CYP113A1), the P450 cytochrome active in the final stages of erythromycin biosynthesis, was cloned and expressed in E. coli. Different crystal forms were harvested from distinct crystallization conditions: two ligand-free forms, one substrate bound and two inhibitors-bound. All crystals belong either to the monoclinc P2(1)or to the orthorhombic P2(1)2(1)2(1) space groups, and exhibit diffraction limits ranging from 2.3 to 1.6 A. The structures will be determined by molecular replacement.

Will new antischistosomal drugs finally emerge?

It has been often observed that the chemotherapeutic armamentarium against an important disease such as schistosomiasis consists of just one drug, praziquantel. Thus, development of drug resistance is an impending danger, with serious implications for the health protection of many millions of people. This rational and legitimate concern might now begin to be relieved by the recent proposal of a new class of compounds that could represent a novel source of drugs against schistosomiasis.

Glutathione reductase and thioredoxin reductase at the crossroad: the structure of Schistosoma mansoni thioredoxin glutathione reductase.

Thioredoxin glutathione reductase (TGR) is a key flavoenzyme expressed by schistosomes that bridges two detoxification pathways crucial for the parasite survival in the host's organism. In this article we report the crystal structure (at 2.2 A resolution) of TGR from Schistosoma mansoni (SmTGR), deleted in the last two residues. The structure reveals the peculiar architecture of this chimeric enzyme: the small Glutaredoxin (Grx) domain at the N-terminus is joined to the large thioredoxin reductase (TR) one via an extended complementary surface, involving residues not conserved in the Grx superfamily; the TR domain interacts with an identical partner via its C-terminal domain, forming a dimer with a twisted "W" shape. Although lacking the penultimate Selenocysteine residue (Sec), the enzyme is still able to reduce oxidized glutathione. These data update the interpretation of the interdomain communication in TGR enzymes. The possible function of this enzyme in pathogenic parasites is discussed.