The events that follow the disruption of ΔΨm result in protease and nuclease activation, responsible for dismantling the respective cells. The main executors of apoptosis in mammalian cells are a group of cysteine proteases, the caspases . However, in the past few years, accumulating evidence in the literature supports the existence of pathways of caspase-independent apoptosis with central players proteases being cathepsins, calpains, granzymes A and B and the proteases of the proteasome . Trypanosomatids do not have caspase genes, and therefore they undergo a caspase-independent apoptosis.
An example of a non-caspase executor of apoptosis involves the proteasomal proteases in L. amazonensis amastigotes (Table 1) treated with *NO donors . Other putative executors of apoptosis are metacaspases (MCAs) (Table 1), i.e. cysteine proteases with similar folds as caspases . The genome of T. brucei possesses five metacaspases (Tb MCA1-5) , whereas two genes are present in T. cruzi (Tc MCA3, Tc MCA5) , two in L. donovani (Ld MC1 and Ld MC2)  and one in L. major (Lmj MCA) [130, 131]. These proteases have arginine/lysine proteolytic activity, and are unable to cleave caspase-specific substrates [129, 131, 132]. Heterologous expression of Tb MCA4 in yeast caused loss of respiration competence and clonal cell death , whereas the L. major metacaspase could replace the endogenous yeast metacaspase YCA1 in apoptosis . The role of metacaspases as executors of apoptosis in trypanosomatids still remains controversial. For example, it has been suggested that the two T. cruzi MCAs might be involved in human serum-induced apoptosis , and that over-expression of L. donovani MCAs renders the parasites more sensitive to hydrogen-peroxide . While Tb MCAs (2, 3 and 5) and the L. major metacaspase could play a functional role in key steps of the cell-cycle and division [130, 133], their function in trypanosomatid apopotosis awaits confirmation.
Although caspases are not present in the trypanosomatid genomes, many investigators have reported the presence of caspase-like activity assessed by the cleavage of caspase-specific substrates and the inhibitory effect of caspase-specific inhibitory peptides [15, 26, 44, 46, 49–51, 68, 92, 114, 134, 135]. This activity was described in Leishmania parasites treated with different drugs , hydrogen peroxide , inhibitors of protein kinase C [49, 100], and in T. cruzi epimastigotes treated with human serum , as well as stationary phase or nutrient deprived parasites . Therefore it is evident that proteases with little homology, but with overlapping activity to metazoan caspases, may be involved in the execution of apoptosis in trypanosomatids. Indeed, Zangger et al. showed that cleavage of the caspase-specific substrate, a DEVD peptide in a 10 day axenic culture, was inhibited by E-64, an inhibitor of cathepsin-like cysteine proteases  that does not inhibit caspases . Moreover the DEVDase activity was not present in a double mutant of the cathepsin L-like cysteine CPA/CPB proteases , indicating that this activity is likely due to one of the two cysteine proteases . In addition, the intracellular binding of the cell permeate pancaspase inhibitor Z-VAD-FMK, upon heat shock induced apoptosis, was attributed to the binding to the cathepsin B-like cysteine proteinase c (CPC) . CPC was not only shown to bind z-VAD but also its knocking out appeared to make parasites survive better when exposed hydrogen peroxide , therefore providing strong evidence that at least part of the execution of apoptosis in Leishmania spp. may function via the involvement of CPC . Finally cruzipain, the major cysteine protease of T. cruzi, was able to act on caspase substrates at low rates . Overall these data suggest that the caspase substrate activity in trypanosomatids may be stimulated by the lysosomal cathepsin-like proteases (Table 1).
Using protease inhibitors, several investigators have demonstrated that proteases stimulate nucleases to degrade DNA. This was shown with the cysteine protease inhibitor E-64 in staurosporine treated L. donovani promastigotes  and with caspase inhibitors in Leishmania and Trypanosoma upon different triggers of cell death [15, 44, 46, 51, 135]. However, there are many examples of apoptosis, where DNA fragmentation was shown to be insensitive to caspase inhibitors or to caspase-like activity [22, 28, 32, 47, 54, 66, 69, 139, 140], suggesting that DNA degradation may be under the control of multiple proteases.
Although DNA fragmentation is commonly observed in trypanosomatids undergoing apoptosis, effectors of this pathway have only recently been described (Table 1). From the genome data it is known that trypanosomatids do not contain homologues of caspase-activated DNAase (CAD), one of the best characterised nucleases in mammalian apoptosis. In addition to CAD, mammalian cells possess a mitochondrial endonuclease G (EndoG) (Table 1) that translocates to the nucleus during caspase-independent apoptosis [141, 142]. EndoG is encoded in the trypanosomatid genome [140, 143, 144], as a mitochondrial enzyme [140, 144] that upon oxidative [114, 143] and/or drug induced apoptosis [32, 140, 144], translocates to the nucleus (Figure 1; [143, 144]). This enzyme, in the nucleus, forms separate complexes with Flap endonuclease-1 and TatD-like nuclease to generate the degradosome in L. donovani promastigotes . Over-expression of this endonuclease strongly promoted apoptotic cell death under oxidant or differentiation-induced stress in Leishmania, while conversely down-regulation of EndoG conferred resistance to oxidative induced cell death in T. brucei , indicating that it is an essential effector of apoptosis in trypanosomatids.
During activation of apoptosis, ions and pH may play an important role in the execution process, affecting both nuclease and protease activity. This was demonstrated in camptothecin-treated L. donovani promastigotes, where treatment of the drug was followed by a significant decrease in intracellular pH and the impairment of the Na+-K+ ATPase pump by oxidative stress . The reduction of the K+ concentration and the pH change propagated the protease activity (DEVDase) of untreated cytosolic L. donovani extracts . Moreover, a nuclease present within the nuclei of untreated extracts of L. donovani that became activated in the presence of Mg2+ and/or Ca2+ ions was strongly repressed at physiological concentrations of K+ . The authors suggested that K+ efflux from the cells during apoptosis is an important regulator of the nuclease activity . Different ion requirements were observed for the nuclease activity from stationary phase L. major parasites that was inhibited by Zn2+ ions, and was not dependent on Ca+2 or Mg2+ ions, although the addition of Mg2+ ions improved this activity . These differences in ion concentrations suggest that more than one nuclease is present in Leishmania spp. that is induced by different apoptosis triggers. In addition the L. infantum EndoG required Mg2+, Mn2+ or Co2+ ions for optimal activity, whereas moderate K+ concentrations (150 mM) or higher Na+ concentrations (300 mM) inhibited the enzyme . Therefore, imbalances of intracellular ion concentrations and pH values during apoptosis in trypanosomatids may trigger the caspase-independent activation of proteases and nucleases to execute cell death.