title: Cathepsin B-like and cell death in the unicellular human pathogen Leishmania.
abstract: In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H(2)O(2)), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.
authors.affiliation: Department of Biochemistry, Faculty of Biology and Medicine, University of Lausanne, Epalinges, Switzerland.
authors.forename: A K
authors.initials: AK
authors.lastname: El-Fadili
authors.forename: H
authors.initials: H
authors.lastname: Zangger
authors.forename: C
authors.initials: C
authors.lastname: Desponds
authors.forename: I J
authors.initials: IJ
authors.lastname: Gonzalez
authors.forename: H
authors.initials: H
authors.lastname: Zalila
authors.forename: C
authors.initials: C
authors.lastname: Schaff
authors.forename: A
authors.initials: A
authors.lastname: Ives
authors.forename: S
authors.initials: S
authors.lastname: Masina
authors.forename: J C
authors.initials: JC
authors.lastname: Mottram
authors.forename: N
authors.initials: N
authors.lastname: Fasel
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis; D002401:Cathepsin B / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007891:Leishmania / Q000201:enzymology*; D011485:Protein Binding; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D015800:Protozoan Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D006861:Hydrogen Peroxide; D002401:Cathepsin B