Identification of Proteins Targeted by the Thioredoxin Superfamily in Plasmodium falciparum

The malarial parasite Plasmodium falciparum possesses a functional thioredoxin and glutathione system comprising the dithiol-containing redox proteins thioredoxin (Trx) and glutaredoxin (Grx), as well as plasmoredoxin (Plrx), which is exclusively found in Plasmodium species. All three proteins belong to the thioredoxin superfamily and share a conserved Cys-X-X-Cys motif at the active site. Only a few of their target proteins, which are likely to be involved in redox reactions, are currently known. The aim of the present study was to extend our knowledge of the Trx-, Grx-, and Plrx-interactome in Plasmodium. Based on the reaction mechanism, we generated active site mutants of Trx and Grx lacking the resolving cysteine residue. These mutants were bound to affinity columns to trap target proteins from P. falciparum cell extracts after formation of intermolecular disulfide bonds. Covalently linked proteins were eluted with dithiothreitol and analyzed by mass spectrometry. For Trx and Grx, we were able to isolate 17 putatively redox-regulated proteins each. Furthermore, the approach was successfully established for Plrx, leading to the identification of 21 potential target proteins. In addition to confirming known interaction partners, we captured potential target proteins involved in various processes including protein biosynthesis, energy metabolism, and signal transduction. The identification of three enzymes involved in S-adenosylmethionine (SAM) metabolism furthermore suggests that redox control is required to balance the metabolic fluxes of SAM between methyl-group transfer reactions and polyamine synthesis. To substantiate our data, the binding of the redoxins to S-adenosyl-L-homocysteine hydrolase and ornithine aminotransferase (OAT) were verified using BIAcore surface plasmon resonance. In enzymatic assays, Trx was furthermore shown to enhance the activity of OAT. Our approach led to the discovery of several putatively redox-regulated proteins, thereby contributing to our understanding of the redox interactome in malarial parasites

Arylmethylamino steroids as antiparasitic agents

In search of antiparasitic agents, we here identify arylmethylamino steroids as potent compounds and characterize more than 60 derivatives. The lead compound 1o is fast acting and highly active against intraerythrocytic stages of chloroquine-sensitive and resistant Plasmodium falciparum parasites (IC50 1–5?nM) as well as against gametocytes. In P. berghei-infected mice, oral administration of 1o drastically reduces parasitaemia and cures the animals. Furthermore, 1o efficiently blocks parasite transmission from mice to mosquitoes. The steroid compounds show low cytotoxicity in mammalian cells and do not induce acute toxicity symptoms in mice. Moreover, 1o has a remarkable activity against the blood-feeding trematode parasite Schistosoma mansoni. The steroid and the hydroxyarylmethylamino moieties are essential for antimalarial activity supporting a chelate-based quinone methide mechanism involving metal or haem bioactivation. This study identifies chemical scaffolds that are rapidly internalized into blood-feeding parasites

Identification of Proteins Targeted by the Thioredoxin Superfamily in Plasmodium falciparum

The malarial parasite Plasmodium falciparum possesses a functional thioredoxin and glutathione system comprising the dithiol-containing redox proteins thioredoxin (Trx) and glutaredoxin (Grx), as well as plasmoredoxin (Plrx), which is exclusively found in Plasmodium species. All three proteins belong to the thioredoxin superfamily and share a conserved Cys-X-X-Cys motif at the active site. Only a few of their target proteins, which are likely to be involved in redox reactions, are currently known. The aim of the present study was to extend our knowledge of the Trx-, Grx-, and Plrx-interactome in Plasmodium. Based on the reaction mechanism, we generated active site mutants of Trx and Grx lacking the resolving cysteine residue. These mutants were bound to affinity columns to trap target proteins from P. falciparum cell extracts after formation of intermolecular disulfide bonds. Covalently linked proteins were eluted with dithiothreitol and analyzed by mass spectrometry. For Trx and Grx, we were able to isolate 17 putatively redox-regulated proteins each. Furthermore, the approach was successfully established for Plrx, leading to the identification of 21 potential target proteins. In addition to confirming known interaction partners, we captured potential target proteins involved in various processes including protein biosynthesis, energy metabolism, and signal transduction. The identification of three enzymes involved in S-adenosylmethionine (SAM) metabolism furthermore suggests that redox control is required to balance the metabolic fluxes of SAM between methyl-group transfer reactions and polyamine synthesis. To substantiate our data, the binding of the redoxins to S-adenosyl-L-homocysteine hydrolase and ornithine aminotransferase (OAT) were verified using BIAcore surface plasmon resonance. In enzymatic assays, Trx was furthermore shown to enhance the activity of OAT. Our approach led to the discovery of several putatively redox-regulated proteins, thereby contributing to our understanding of the redox interactome in malarial parasites

Redox Regulation of Plasmodium falciparum Ornithine δ−Aminotransferase

Ornithine δ−aminotransferase (OAT) of the malaria parasite Plasmodium falciparum catalyzes the reversible conversion of ornithine into glutamate−5−semialdehyde and glutamate and is−in contrast to its human counterpart−activated by thioredoxin (Trx) by a factor of 10. Trx, glutaredoxin, and plasmoredoxin are redox−active proteins that play a crucial role in the maintenance and control of redox reactions, and were shown to interact with P. falciparum OAT. OAT, which is involved in ornithine homeostasis and proline biosynthesis, is essential for mitotic cell division in rapidly growing cells, thus representing a potential target for chemotherapeutic intervention. Here we report the three−dimensional crystal structure of P. falciparum OAT at 2.3 Å resolution. The overall structure is very similar to that of the human OAT. However, in plasmodial OAT, the loop involved in substrate binding contains two cysteine residues, which are lacking in human OAT. Site−directed mutagenesis of these cysteines and functional analysis demonstrated that Cys154 and Cys163 mediate the interaction with Trx. Interestingly, the Cys154 ? Ser mutant has a strongly reduced specific activity, most likely due to impaired binding of ornithine. Cys154 and Cys163 are highly conserved in Plasmodium but do not exist in other organisms, suggesting that redox regulation of OAT by Trx is specific for malaria parasites. Plasmodium might require a tight Trx−mediated control of OAT activity for coordinating ornithine homeostasis, polyamine synthesis, proline synthesis, and mitotic cell division

H₂O₂ dynamics in the malaria parasite <i>Plasmodium falciparum</i>

<div><p>Hydrogen peroxide is an important antimicrobial agent but is also crucially involved in redox signaling and pathogen-host cell interactions. As a basis for systematically investigating intracellular H₂O₂ dynamics and regulation in living malaria parasites, we established the genetically encoded fluorescent H₂O₂ sensors roGFP2-Orp1 and HyPer-3 in <i>Plasmodium falciparum</i>. Both ratiometric redox probes as well as the pH control SypHer were expressed in the cytosol of blood-stage parasites. Both redox sensors showed reproducible sensitivity towards H₂O₂ in the lower micromolar range <i>in vitro</i> and in the parasites. Due to the pH sensitivity of HyPer-3, we used parasites expressing roGFP2-Orp1 for evaluation of short-, medium-, and long-term effects of antimalarial drugs on H₂O₂ levels and detoxification in <i>Plasmodium</i>. None of the quinolines or artemisinins tested had detectable direct effects on the H₂O₂ homeostasis at pharmacologically relevant concentrations. However, pre-treatment of the cells with antimalarial drugs or heat shock led to a higher tolerance towards exogenous H₂O₂. The systematic evaluation and comparison of the two genetically encoded cytosolic H₂O₂ probes in malaria parasites provides a basis for studying parasite-host cell interactions or drug effects with spatio-temporal resolution while preserving cell integrity.</p></div

Redox status of patients before cardiac surgery

Objectives: Redox regulation plays a crucial role in balancing the cardiovascular system. In this prospective study we aimed to identify currently unknown correlations valuable to cardiovascular research and patient management. Methods: Blood samples from 500 patients were collected directly before cardiosurgical interventions (Ethics Committee reference number 85/11). Four central redox parameters were determined together with about 30 clinical, anthropometric, and metabolic parameters. Results: Creatinine levels and pulmonary hypertension were significant predictors of the total antioxidant status (TAOS) in the patients; total glutathione levels were linked to C-peptide, and creatinine, gender, and ventricular arrhythmia influenced nitrate/nitrite levels. Notably, significant interactions were found between medication and redox parameters. Calcium channel blockers (CCBs) were positive predictors of total glutathione levels, whereas angiotensin-converting enzyme inhibitors and CCBs were negative predictors of NOx levels. Age showed the highest correlation with the duration of the intensive care stay, followed by NOx levels, creatinine, TAOS, and C-reactive protein. Discussion: In this prospective study we determined multiple correlations between redox markers and parameters linked to cardiovascular diseases. The data point towards so far unknown interdependencies, particularly between antihypertensive drugs and redox metabolism. A thorough follow-up to these data has the potential to improve patient management. Abbreviations: A: absorption; ΔA: absorption difference; ABTS: 2,2′-azino-di(3-ethylbenzothiazoline sulfonate); ACE: angiotensin-converting enzyme; AO: antioxidant; ARB: angiotensin receptor blocker; BMI: body mass index; CAD: coronary artery disease; CCB: calcium channel blocker; CDC: coronary heart diseases; COPD: chronic obstructive pulmonary disease; CRP: C-reactive protein; CVD: cardiovascular diseases; Cu-OOH: cumene hydroperoxide; D: dilution factor; DAN: 2,3-diaminonaphtalene; DMSO: dimethylsulfoxide; DNA: deoxyribonucleic acid; DTNB: 5,5-dithiobis(2-nitrobenzoate); ε: extinction coefficient; EDRF: endothelium-derived relaxing factor; fc: final concentration; GPx: glutathione peroxidases; (h)GR: (human) glutathione reductase; GSH: (reduced) glutathione; GSSG: glutathione disulfide; GST: glutathione-S-transferase; Hb: hemoglobin; HDL: high-density lipoprotein; Hk: hematocrit; H2O2: hydrogen peroxide; ICS: intensive care stay; LDH: lactate dehydrogenase; LDL: low-density lipoprotein; MI: myocardial infarction; NED: N-(1-naphthyl)-ethylendiamine-dihydrochloride; NOS: nitric oxide synthase; NOx: nitrate/nitrite; NR: nitrate reductase; PBS: phosphate buffered saline; PCA: principle component analysis; PH: pulmonary hypertension; ROS: reactive oxygen species; RNS: reactive nitrogen species; RT: room temperature (25°C); SA: sulfanilamide; SOD: superoxide dismutase; SSA: sulfosalicylic acid; TAC: total antioxidant capacity; TAOS: total antioxidant status; TEAC: trolox equivalent antioxidative capacity; TG: triglycerides; tGSH: total glutathione; TNB-: 2-nitro-5-thiobenzoate; U: unit; UV: ultraviolet; VA: volume activity; Wc: working concentration; WHR: waist-hip ratio

Short-term effects of antimalarial drugs on the redox ratio of <i>P</i>. <i>falciparum</i> 3D7^{[roGFP2-Orp1]}-transfected parasites.

<p>After 15 s baseline monitoring, the parasites were exposed to 100 <b>μ</b>M antimalarial drugs and monitored for 3 min at the CLSM. Upon addition of all drugs, an increase in the 405/488 nm ratio was detected which was more pronounced for the quinolines, particularly MQ. Each data point (mean ± SEM) is composed of values from nine trophozoites analyzed in three independent experiments.</p

Effect of H₂O₂ on <i>P</i>. <i>falciparum</i> 3D7^{[roGFP2-Orp1]}- and 3D7^[HyPer-3]-transfected parasites within intact RBCs and after RBC lysis_.

<p>After 15 s baseline monitoring, 3D7^{[roGFP2-Orp1]} (<b>A</b>) and 3D7^[HyPer-3] parasites (<b>C</b>) in intact RBCs as well as 3D7^{[roGFP2-Orp1]} (<b>B</b>) and 3D7^[HyPer-3] parasites (<b>D</b>) after saponin lysis of their host cells were exposed to H₂O₂ (20 <b>μ</b>M to 1 mM) and monitored for 3 min at the CLSM. Non-treated parasites served as controls. In both setups, 3D7^[HyPer-3] showed a higher sensitivity to H₂O₂ than 3D7^{[roGFP2-Orp1]}. Moreover, the H₂O₂ response of 3D7^[HyPer-3] increased by a factor of 1.6 after lysis of the host cell (<b>D</b>). The initial response of 3D7^{[roGFP2-Orp1]} in intact RBCs (<b>A</b>) and after saponin-lysis of the host cells (<b>B</b>) was comparable; however, in (<b>B</b>) the constant decrease of the fluorescence ratio in treated parasites and controls indicates bleaching of the sensor. For each H₂O₂ concentration, data from nine trophozoites in total, examined in three independent experiments, were analyzed per data point. Mean and standard error of the mean (SEM) are shown.</p