title: FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii.
abstract: Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca(2+) is a ubiquitous signalling ion that appears to regulate protease activity through changes in its intracellular concentration. Proteases are crucial to Plasmodium development, but the role of Ca(2+) in their activity is not fully understood. Here we investigated the role of Ca(2+) in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca(2+) from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca(2+)-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K(+)/H(+) exchanger) or monensin (a Na(+)/H(+) exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca(2+) chelators were used to investigate the role played by Ca(2+) in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis in P. berghei but decreased it in P. yoelii. Conversely, E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca(2+) between species of Plasmodium.
authors.affiliation: Department of Parasitology, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1374 Edifício Biomédicas II, CEP 05508-900, São Paulo, SP, Brazil.
authors.forename: Laura Nogueira da
authors.initials: LN
authors.lastname: Cruz
authors.forename: Eduardo
authors.initials: E
authors.lastname: Alves
authors.forename: Mônica Teixeira
authors.initials: MT
authors.lastname: Leal
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Rosenthal
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Celia R S
authors.initials: CR
authors.lastname: Garcia
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D020013:Calcium Signaling; D004789:Enzyme Activation / Q000187:drug effects*; D004912:Erythrocytes / Q000469:parasitology*; D031541:Fluorescence Resonance Energy Transfer; D008985:Monensin / Q000494:pharmacology; D009550:Nigericin / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D010962:Plasmodium berghei / Q000187:drug effects / Q000201:enzymology*; D010967:Plasmodium yoelii / Q000187:drug effects / Q000201:enzymology*; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D010455:Peptides; D019284:Thapsigargin; D008985:Monensin; D010447:Peptide Hydrolases; D009550:Nigericin; D002118:Calcium