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

Depletion of Plasmodium berghei Plasmoredoxin Reveals a Non-Essential Role for Life Cycle Progression of the Malaria Parasite

Proliferation of the pathogenic Plasmodium asexual blood stages in host erythrocytes requires an exquisite capacity to protect the malaria parasite against oxidative stress. This function is achieved by a complex antioxidant defence system composed of redox-active proteins and low MW antioxidants. Here, we disrupted the P. berghei plasmoredoxin gene that encodes a parasite-specific 22 kDa member of the thioredoxin superfamily. The successful generation of plasmoredoxin knockout mutants in the rodent model malaria parasite and phenotypic analysis during life cycle progression revealed a non-vital role in vivo. Our findings suggest that plasmoredoxin fulfils a specialized and dispensable role for Plasmodium and highlights the need for target validation to inform drug development strategies

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

Compartmentation of Redox Metabolism in Malaria Parasites

Malaria, caused by the apicomplexan parasite Plasmodium, still represents a major threat to human health and welfare and leads to about one million human deaths annually. Plasmodium is a rapidly multiplying unicellular organism undergoing a complex developmental cycle in man and mosquito – a life style that requires rapid adaptation to various environments. In order to deal with high fluxes of reactive oxygen species and maintain redox regulatory processes and pathogenicity, Plasmodium depends upon an adequate redox balance. By systematically studying the subcellular localization of the major antioxidant and redox regulatory proteins, we obtained the first complete map of redox compartmentation in Plasmodium falciparum. We demonstrate the targeting of two plasmodial peroxiredoxins and a putative glyoxalase system to the apicoplast, a non-photosynthetic plastid. We furthermore obtained a complete picture of the compartmentation of thioredoxin- and glutaredoxin-like proteins. Notably, for the two major antioxidant redox-enzymes – glutathione reductase and thioredoxin reductase – Plasmodium makes use of alternative-translation-initiation (ATI) to achieve differential targeting. Dual localization of proteins effected by ATI is likely to occur also in other Apicomplexa and might open new avenues for therapeutic intervention

The Structure, Dynamics and Electronic Structure of Liquid Ag-Se Alloys Investigated by Ab Initio Simulation

Ab initio molecular-dynamics simulations have been used to investigate the structure, dynamics and electronic properties of the liquid alloy Ag(1-x)Se(x) at 1350 K and at the three compositions x=0.33, 0.42 and 0.65. The calculations are based on density-functional theory in the local density approximation and on the pseudopotential plane-wave method. The reliability of the simulations is confirmed by detailed comparisons with very recent neutron diffraction results for the partial structure factors and radial distribution functions (RDF) of the stoichiometric liquid Ag2Se. The simulations show a dramatic change of the Se-Se RDF with increasing Se content. This change is due to the formation of Se clusters bound by covalent bonds, the Se-Se bond length being almost the same as in pure c-Se and l-Se. The clusters are predominantly chain-like, but for higher x a large fraction of 3-fold coordinated Se atoms is also found. It is shown that the equilibrium fractions of Se present as isolated atoms and in clusters can be understood on a simple charge-balance model based on an ionic interpretation. The Ag and Se diffusion coefficients both increase with Se content, in spite of the Se clustering. An analysis of the Se-Se bond dynamics reveals surprisingly short bond lifetimes of less than 1 ps. The changes in the density of states with composition arise directly from the formation of Se-Se covalent bonds. Results for the electronic conductivity obtained using the Kubo-Greenwood approximation are in adequate agreement with experiment for l-Ag2Se, but not for the high Se contents. Possible reasons for this are discussed.Comment: 14 pages, Revtex, 14 Postscript figures embedded in the tex

Poverty in the Social World of the Wise

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/68560/2/10.1177_030908928701203704.pd

Inherited Glutathione Reductase Deficiency and Plasmodium falciparum Malaria—A Case Study

In Plasmodium falciparum-infected red blood cells (RBCs), the flavoenzyme glutathione reductase (GR) regenerates reduced glutathione, which is essential for antioxidant defense. GR utilizes NADPH produced in the pentose phosphate shunt by glucose-6-phosphate dehydrogenase (G6PD). Thus, conditions affecting host G6PD or GR induce increased sensitivity to oxidants. Hereditary G6PD deficiency is frequent in malaria endemic areas and provides protection against severe malaria. Furthermore, GR deficiency resulting from insufficient saturation of the enzyme with its prosthetic group FAD is common. Based on these naturally occurring phenomena, GR of malaria parasites and their host cells represent attractive antimalarial drug targets. Recently we were given the opportunity to examine invasion, growth, and drug sensitivity of three P. falciparum strains (3D7, K1, and Palo Alto) in the RBCs from three homozygous individuals with total GR deficiency resulting from mutations in the apoprotein. Invasion or growth in the GR-deficient RBCs was not impaired for any of the parasite strains tested. Drug sensitivity to chloroquine, artemisinin, and methylene blue was comparable to parasites grown in GR-sufficient RBCs and sensitivity towards paraquat and sodium nitroprusside was only slightly enhanced. In contrast, membrane deposition of hemichromes as well as the opsonizing complement C3b fragments and phagocytosis were strongly increased in ring-infected RBCs of the GR-deficient individuals compared to ring-infected normal RBCs. Also, in one of the individuals, membrane-bound autologous IgGs were significantly enhanced. Thus, based on our in vitro data, GR deficiency and drug-induced GR inhibition may protect from malaria by inducing enhanced ring stage phagocytosis rather than by impairing parasite growth directly

Author correction: Arylmethylamino steroids as antiparasitic agents

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