title: Cysteine-protease activity elicited by Ca2+ stimulus in Plasmodium.
abstract: Bloodstage malaria parasites require proteolytic activity for key processes as invasion, hemoglobin degradation and merozoite escape from red blood cells (RBCs). We investigated by confocal microscopy the presence of cysteine-protease activity elicited by calcium stimulus in Plasmodium chabaudi and Plasmodium falciparum in free trophozoites or for the later parasite within RBC using fluorescence resonance energy transfer (FRET) peptides. Peptide probes access, to either free or intraerythrocytic parasites, was also tested by selecting a range of fluorescent peptides (653-3146 Da molecular mass) labeled with Abz or FITC. In the present work we show that Ca2+ stimulus elicited by treatment with either melatonin, thapsigargin, ionomicin or nigericin, promotes an increase of substrate hydrolysis, which was blocked by the specific cysteine-protease inhibitor E-64 and the intracellular Ca2+ chelator, BAPTA. When parasites were treated with cytoplasmic Ca2+ releasing compounds, a cysteine-protease was labeled in the parasite cytoplasm by the fluorescent specific irreversible inhibitor, Ethyl-Eps-Leu-Tyr-Cap-Lys(Abz)-NH2, where Ethyl-Eps is Ethyl-(2S,3S)-oxirane-2,3-dicarboxylate. In summary, we demonstrate that P. chabaudi and P. falciparum have a cytoplasmic dependent cysteine-protease activity elicited by Ca2+.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, SP, Brazil.
authors.forename: Shirley L
authors.initials: SL
authors.lastname: Farias
authors.forename: Marcos L
authors.initials: ML
authors.lastname: Gazarini
authors.forename: Robson L
authors.initials: RL
authors.lastname: Melo
authors.forename: Izaura Y
authors.initials: IY
authors.lastname: Hirata
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Célia R S
authors.initials: CR
authors.lastname: Garcia
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism / Q000469:parasitology*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008550:Melatonin / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009550:Nigericin / Q000494:pharmacology; D016788:Plasmodium chabaudi / Q000187:drug effects / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000187:drug effects / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007476:Ionophores; D015759:Ionomycin; D019284:Thapsigargin; D003546:Cysteine Endopeptidases; D008550:Melatonin; D009550:Nigericin; D002118:Calcium