The caspase-independent algorithm of programmed cell death in Leishmania induced by baicalein: the role of LdEndoG, LdFEN-1 and LdTatD as a DNA ‘degradesome’

Abstract

In the post-genomic perspective, the quest of programmed cell death (PCD) mechanisms in kinetoplastid parasites lies in the identification and characterization of cell death executer proteins. Here, we show that baicalein (BLN), a potent topoisomerase IB inhibitor, generates an oxidative stress in the parasites leading to altered physiological and morphological parameters, which are characteristic of PCD. For the first time we elucidate that, caspase-independent activation of a novel effector molecule, endonuclease G (LdEndoG), mediates BLN-induced cell death. Functional characterization of LdEndoG identifies Flap endonuclease-1 (LdFEN-1) and LdTatD-like nuclease as other effector molecules. BLN treatment translocates LdEndoG from mitochondria to nucleus, where it forms separate complexes with LdFEN-1 and LdTatD to constitute a DNA 'degradesome' unique to these parasites. Conditional antisense knockdown of LdEndoG provides protection against PCD. This knowledge paves the path toward a better understanding of the PCD pathway in simpler systems, which could be exploited in antileishmanial chemotherapy.

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

“…From our results on the basis of subcellular fractionation and Western blot, the nuclear localization of Endo G is detected in TM-treated apoptotic Leishmania cells. Several groups established that Endo G acts as an apoptotic DNase when released from the mitochondria to the nucleus (8,40,44). Preincubation with Z-VAD-FMK (caspase inhibitor) or antipain (Leishmania metacaspase inhibitor) cannot inhibit DNA fragmentation and PS externalization in TM-treated apoptotic Leishmania cells, although caspase or metacaspase-like activity is ϳ1.5-fold higher after 16 h of treatment with TM.…”

“…In Leishmania, both Cyt c and Endo G have been reported to play important roles in apoptosis (6). The mitochondrial release of Endo G and its involve- ment in DNA fragmentation have also been shown in kinetoplastid parasites during oxidative stress-induced apoptosis (8,44). Subcellular fractionation and Western blot analysis were performed to check the possibility of TM-induced release of cytochrome c and Endo G from mitochondria.…”

Staurosporine-Induced Cell Death in <em>Trypanosoma brucei</em> and the Role of Endonuclease G during Apoptosis

“…Its increased formation in the mitochondrion leads probably to the rupture of the mitochondrial membrane and thus release of EndoG into the cytosol. Here, EndoG, together with other nucleases seems to form a DNA-degradation complex [16] [21], which fragmentizes the parasite's DNA in the last step of trypanosomal apoptosis. As trypanosomes belong to one of the most ancient diverging branches of the eukaryotic phytogenic tree and are amongst the first eukaryotes with a mitochondrion, our results offer an insight in apoptosis development and how complex these simple organisms can react on different stimuli.…”

“…Since data from another study indicate that the mls of TbEndoG might not been cleaved in T. brucei [36], we favour the idea that TbEndoG is not translocated to the matrix, but to the intermembrane space of the mitochondrion. During permeabilization of the outer mitochondrial membrane in the course of apoptosis, TbEndoG may be released into the cytosol, and translocates most likely as part of a DNA-degradation complex together with other nucleases into the nucleus to cleave DNA [16] [21].…”

“…Interestingly, endonuclease G (EndoG), another nuclease active during apoptosis in higher eukaryotes [14] [15], is, however, also expressed in kinetoplastids [16] [17]. This nuclease is encoded in nuclear DNA and is translocated into the mitochondrion, where it may be involved in replication and repair functions [18].…”