Deoxyhypusine Hydroxylase From Plasmodium Vivax, the Neglected Human Malaria Parasite: Molecular Cloning, Expression and Specific Inhibition by the 5-LOX Inhibitor Zileuton.

Deoxyhypusine Hydroxylase from Plasmodium vivax, the Neglected Human Malaria Parasite: Molecular Cloning, Expression and Specific Inhibition by the 5-LOX Inhibitor Zileuton.

PLoS One. 2013; 8(3): e58318
Atemnkeng VA, Pink M, Schmitz-Spanke S, Wu XJ, Dong LL, Zhao KH, May C, Laufer S, Langer B, Kaiser A

Primaquine, an 8-aminoquinoline, is the only drug which cures the dormant hypnozoites of persistent liver stages from P. vivax. Increasing resistance needs the discovery of alternative pathways as drug targets to develop novel drug entities. Deoxyhypusine hydroxylase (DOHH) completes hypusine biosynthesis in eukaryotic initiation factor (eIF-5A) which is the only cellular protein known to contain the unusual amino acid hypusine. Modified EIF-5A is important for proliferation of the malaria parasite. Here, we present the first successful cloning and expression of DOHH from P. vivax causing tertiary malaria. The nucleic acid sequence of 1041 bp encodes an open reading frame of 346 amino acids. Histidine tagged expression of P. vivax DOHH detected a protein of 39.01 kDa in E. coli. The DOHH protein from P. vivax shares significant amino acid identity to the simian orthologues from P. knowlesi and P. yoelii strain H. In contrast to P. falciparum only four E-Z-type HEAT-like repeats are present in P. vivax DOHH with different homology to phycocyanin lyase subunits from cyanobacteria and in proteins participating in energy metabolism of Archaea and Halobacteria. However, phycocyanin lyase activity is absent in P. vivax DOHH. The dohh gene is present as a single copy gene and transcribed throughout the whole erythrocytic cycle. Specific inhibition of recombinant P. vivax DOHH is possible by complexing the ferrous iron with zileuton, an inhibitor of mammalian 5-lipoxygenase (5-LOX). Ferrous iron in the active site of 5-LOX is coordinated by three conserved histidines and the carboxylate of isoleucine(673). Zileuton inhibited the P. vivax DOHH protein with an IC50 of 12,5 nmol determined by a relative quantification by GC/MS. By contrast, the human orthologue is only less affected with an IC50 of 90 nmol suggesting a selective iron-complexing strategy for the parasitic enzyme. HubMed – drug

Analysis of the Membrane Proteome of Ciprofloxacin-Resistant Macrophages by Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC).

PLoS One. 2013; 8(3): e58285
Caceres NE, Aerts M, Marquez B, Mingeot-Leclercq MP, Tulkens PM, Devreese B, Van Bambeke F

Overexpression of multidrug transporters is a well-established mechanism of resistance to chemotherapy, but other changes may be co-selected upon exposure to drugs that contribute to resistance. Using a model of J774 macrophages made resistant to the fluoroquinolone antibiotic ciprofloxacin and comparing it with the wild-type parent cell line, we performed a quantitative proteomic analysis using the stable isotope labeling with amino acids in cell culture technology coupled with liquid chromatography electrospray ionization Fourier transform tandem mass spectrometry (LC-ESI-FT-MS/MS) on 2 samples enriched in membrane proteins (fractions F1 and F2 collected from discontinuous sucrose gradient). Nine hundred proteins were identified with at least 3 unique peptides in these 2 pooled fractions among which 61 (F1) and 69 (F2) showed a significantly modified abundance among the 2 cell lines. The multidrug resistance associated protein Abcc4, known as the ciprofloxacin efflux transporter in these cells, was the most upregulated, together with Dnajc3, a protein encoded by a gene located downstream of Abcc4. The other modulated proteins are involved in transport functions, cell adhesion and cytoskeleton organization, immune response, signal transduction, and metabolism. This indicates that the antibiotic ciprofloxacin is able to trigger a pleiotropic adaptative response in macrophages that includes the overexpression of its efflux transporter. HubMed – drug

Enhanced a?1-40 production in endothelial cells stimulated with fibrillar a?1-42.

PLoS One. 2013; 8(3): e58194
Rajadas J, Sun W, Li H, Inayathullah M, Cereghetti D, Tan A, Mello Coelho Vd, Chrest FJ, Kusiak JW, Smith WW, Taub D, Wu JC, Rifkind JM

Amyloid accumulation in the brain of Alzheimer’s patients results from altered processing of the 39- to 43-amino acid amyloid ? protein (A?). The mechanisms for the elevated amyloid (A?1-42) are considered to be over-expression of the amyloid precursor protein (APP), enhanced cleavage of APP to A?, and decreased clearance of A? from the central nervous system (CNS). We report herein studies of A? stimulated effects on endothelial cells. We observe an interesting and as yet unprecedented feedback effect involving A?1-42 fibril-induced synthesis of APP by Western blot analysis in the endothelial cell line Hep-1. We further observe an increase in the expression of A?1-40 by flow cytometry and fluorescence microscopy. This phenomenon is reproducible for cultures grown both in the presence and absence of serum. In the former case, flow cytometry reveals that A?1-40 accumulation is less pronounced than under serum-free conditions. Immunofluorescence staining further corroborates these observations. Cellular responses to fibrillar A?1-42 treatment involving eNOS upregulation and increased autophagy are also reported. HubMed – drug

Pyrimidine Biosynthesis Is Not an Essential Function for Trypanosoma brucei Bloodstream Forms.

PLoS One. 2013; 8(3): e58034
Ali JA, Tagoe DN, Munday JC, Donachie A, Morrison LJ, de Koning HP

BACKGROUND: African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host, but it is unknown whether either process is essential to the parasite. METHODOLOGYPRINCIPAL FINDINGS: Pyrimidine requirements for growth were investigated using strictly pyrimidine-free media, with or without single added pyrimidine sources. Growth rates of wild-type bloodstream form Trypanosoma brucei brucei were unchanged in pyrimidine-free medium. The essentiality of the de novo pyrimidine biosynthesis pathway was studied by knocking out the PYR6-5 locus that produces a fusion product of orotate phosphoribosyltransferase (OPRT) and Orotidine Monophosphate Decarboxylase (OMPDCase). The pyrimidine auxotroph was dependent on a suitable extracellular pyrimidine source. Pyrimidine starvation was rapidly lethal and non-reversible, causing incomplete DNA content in new cells. The phenotype could be rescued by addition of uracil; supplementation with uridine, 2’deoxyuridine, and cytidine allowed a diminished growth rate and density. PYR6-5(-)/(-) trypanosomes were more sensitive to pyrimidine antimetabolites and displayed increased uracil transport rates and uridine phosphorylase activity. Pyrimidine auxotrophs were able to infect mice although the infection developed much more slowly than infection with the parental, prototrophic trypanosome line. CONCLUSIONSSIGNIFICANCE: Pyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the pyrimidine biosynthesis pathway is not a viable drug target, although any interruption of pyrimidine supply was lethal. HubMed – drug