Abstract
Leishmania major is a protozoan parasite from one of the most ancient phylogenic branches of unicellular eukaryotes, and containing only one giant mitochondrion. Here we report that staurosporine, that induces apoptosis in all mammalian nucleated cells, also induces in L. major a death process with several cytoplasmic and nuclear features of apoptosis, including cell shrinkage, phosphatidyl serine exposure, maintenance of plasma membrane integrity, mitochondrial transmembrane potential (ΔΨm) loss and cytochrome c release, nuclear chromatin condensation and fragmentation, and DNA degradation. Nuclear apoptosis-like features were prevented by cysteine proteinase inhibitors, and cell free assays using dying L. major cytoplasmic extracts indicated that the cysteine proteinases involved (i) also induced nuclear apoptosis-like features in isolated mammalian nuclei, and (ii) shared at least two nuclear substrates, but no cleavage site preference, with human effector caspases. Finally, isolated L. major mitochondria released cytochrome c and cysteine proteinases with nuclear pro-apoptotic activity when incubated with human recombinant Bax, even (although much less efficiently) when Bax was deleted of its transmembrane domain required for insertion in mitochondrial outermembranes, implying that L. major mitochondrion may express proteins able to interact with Bax. The recruitment of cysteine proteinases and mitochondria to the cell death machinery may be of very ancient evolutionary origin. Alternately, host/parasite interactions may have exerted selective pressures on the cell death phenotype of kinetoplastid parasites, resulting in the more recent emergence of an apoptotic machinery through a process of convergent evolution.
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Abbreviations
- BAF:
-
Boc-Asp(OMe)-CH2F
- E64:
-
trans-Epoxysuccinyl-L-Leucylamido-(4-Guanido)Butane
- AIF:
-
Apoptosis Inducing Factor
- CAD:
-
Caspase Activated Dnase
- ΔΨm:
-
mitochondrial transmembrane potential
- ICAD:
-
Inhibitor of Caspase Activated Dnase
- PARP:
-
Poly(ADP-ribose) polymerase
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Acknowledgements
We are very grateful to B Antonsson (Serono Pharmaceutical research Institute, Serono International, SA, Geneva, Switzerland) and JC Martinou (Departement de Biologie Cellulaire, Universite de Geneva, Geneva, Switzerland) for providing the recombinant full length and ΔTM Bax and for helpful discussions; to R Kluck (La Jolla Institute for Allergy and Immunology, San Diego, CA, USA) for providing an anti-yeast cytochrome c antibody; to. YJ-F Garin (Hopital Saint Louis, France) for providing L. major; and to G Kroemer and S Susin (Villejuif, France) for providing an anti-mammalian AIF antibody. EMI-U 9922 is supported by Institut National de la Sante et de la Recherche Medicale (INSERM) and Universite Paris 7, and by grants from Agence Nationale de Recherches sur le Sida (ANRS), Ensemble contre le Sida (ECS), Fondation pour la Recherche Medicale (FRM), Etablissement Francais des Greffes (to JC Ameisen). D Arnoult was supported by doctoral fellowships from Delegation Generale de l'Armement (DGA) and FRM and K Akarid by a post-doctoral fellowship from ECS.
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Arnoult, D., Akarid, K., Grodet, A. et al. On the evolution of programmed cell death: apoptosis of the unicellular eukaryote Leishmania major involves cysteine proteinase activation and mitochondrion permeabilization. Cell Death Differ 9, 65–81 (2002). https://doi.org/10.1038/sj.cdd.4400951
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DOI: https://doi.org/10.1038/sj.cdd.4400951
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