Characterization of Metacaspases with Trypsin-Like Activity and Their Putative Role in Programmed Cell Death in the Protozoan Parasite Leishmania

Abstract

In this report, we have characterized two metacaspases of Leishmania donovani, L. donovani metacaspase-1 (LdMC1) and LdMC2. These two proteins show 98% homology with each other, and both contain a characteristic C-terminal proline-rich domain. Both genes are transcribed in promastigotes and axenic amastigotes of L. donovani; however, LdMC1 shows increased mRNA levels in axenic amastigotes. An anti-LdMC antibody was obtained and showed reactivity with a single ϳ42-kDa protein band in both promastigote and axenic amastigote parasite whole-cell lysates by Western blotting. Pulse-chase experiments suggest that LdMCs are not synthesized as proenzymes, and immunofluorescence studies show that LdMCs are associated with the acidocalcisome compartments of L. donovani. Enzymatic assays of immunoprecipitated LdMCs show that native LdMCs efficiently cleave trypsin substrates and are unable to cleave caspase-specific substrates. Consistently, LdMC activity is insensitive to caspase inhibitors and is efficiently inhibited by trypsin inhibitors, such as leupeptin, antipain, and N ␣ -tosyl-L-lysine-chloromethyl ketone (TLCK). In addition, our results show that LdMC activity was induced in parasites treated with hydrogen peroxide, a known trigger of programmed cell death (PCD) in Leishmania and that parasites overexpressing metacaspases are more sensitive to hydrogen peroxide-induced PCD. These findings suggest that Leishmania metacaspases are not responsible for the caspase-like activities reported in this organism and suggest a possible role for LdMCs as effector molecules in Leishmania PCD.Metacaspases constitute a new family of caspase-related proteins recently described by Uren et al. (39). They have been identified by bioinformatic analysis and are found in plants, fungi, and parasitic protozoa but are absent in mammals (39). Metacaspases belong to the CD clan of cysteine peptidases with six other families, including caspases and paracaspases (29). Metacaspases are structurally related to caspases and show conservation of cysteine and histidine amino acid residues involved in the Cys-His catalytic dyad of the active domains of caspase (39). Caspases have been shown to play a central role in programmed cell death of mammalian cells, also called apoptosis (reviewed in reference 14). To date, no caspase gene has been identified in plants, yeasts, or protozoan parasites. However, since these organisms possess one or more metacaspases, it is conceivable that like caspases in mammalian cells, these caspase-related proteases could be involved in the PCD pathways in these organisms. In that regard, the Saccharomyces cerevisiae metacaspase YCA1 and the Norway spruce metacaspase mcII-Pa have been shown recently to be directly implicated in PCD in these organisms (4, 28). To date, however, the possible role of metacaspases in PCD of protozoan parasites remains to be demonstrated.Little is known about the enzymatic properties of metacaspases. In contrast, the related caspase enzymes are wellcharacterized cysteine-dependent aspartate-s...

Inhibition of active nuclear transport is an intrinsic trigger of programmed cell death in trypanosomatids

“…Indeed, the L. major metacaspase has been able to replace the yeast metacaspase in PCD, 14 and the overexpression of L. donovani metacaspase 1 increased the sensitivity of amastigotes to PCD induced by H 2 O 2 . 17 Nevertheless, in this study, (a) metacaspase 1 has a trypsin-like and not a caspase-like activity and (b) the authors did not demonstrate that metacaspases were actually necessary for the initiation and execution of apoptosis. Furthermore, the L. major metacaspase seems to have an essential function in cell-cycle progression rather than in PCD.…”

“…Although metacaspase genes have been identified in the genome of all trypanosomatids, their involvement in apoptosis is still debated. [14][15][16][17] Moreover, no homologue for members of the Bcl-2 protein family has been identified in T. brucei. Finally, two genes whose transcription is upregulated late during the identification of concanavalinA-induced PCD in the procyclic form of T. brucei rhodesiense, a homologue of prohibitin and a protein called TRACK, homologous to the RACK family (receptor for activated kinase C).…”

Endoplasmic Reticulum Stress-induced Apoptosis in Leishmania through Ca2+-dependent and Caspase-independent Mechanism

“…Caspase inhibitor Z-VAD-FMK was added in control reactions at 50 M for 30 min prior to the addition of ER stress inducer. Metacaspase-like protease activity was measured fluorimetrically using Boc-GRR-AMC substrate as described by Lee et al with minor modifications (29,30). Briefly, promastigote cultures were pelleted by centrifugation and resuspended in fresh culture medium containing 20 g/ml TM, and the sample was incubated for 16 h at 26°C.…”

“…Recently, a group of workers has reported about metacaspase genes in Leishmania; these are efficiently able to cleave trypsin substrates and are unable to cleave caspase-specific substrates. Consistently, that activity is insensitive to caspase inhibitors and is efficiently inhibited by trypsin inhibitors, such as antipain (29). To check whether control cells show any difference in metacaspase-like activity in the presence of TM, we used the Leishmania metacaspase-specific fluorogenic substrate, Boc-GRR-AMC.…”

Genes de cisteíno proteases (Catepsina L-like) de Trypanosoma rangeli: polimorfismo, relações filogenéticas e alvos para diagnóstico e genotipagem.

“…Genes de metacaspases têm sido encontrados nos genomas de diversos tripanossomatídeos patogênicos, como T.brucei (MCA1- (SZALLIES et al, 2002;HELMS et al, 2006;KOSEC et al, 2006 a,b;AMBIT et al, 2008;LEE et al, 2007).…”