REPOSITORY > RESULTS

Abstract

Programmed cell death is a highly organized process by which multicellular organisms eliminate
damaged, superfluous and potentially harmful cells. Apoptosis is the most studied form of programmed cell
death. It is characterized by typical morphological and biochemical changes which are a consequence of
activation of cysteine proteases, caspases. Apoptosis is mediated through two major pathways, the extrinsic
or death receptor pathway and the intrinsic or mitochondrial pathway. In the extrinsic pathway, the ligation
of death ligands with their receptors results in formation of the death-inducing signaling complex (DISC),
in which caspases -8 or -10 are activated. Activation of the intrinsic pathway is a consequence of
intracellular stress, resulting in cytochrome c release from the mitochondria into the cytosol, leading to the
formation of the apoptosome complex and subsequent activation of effector caspases. In certain cells,
signals from the extrinsic pathway are amplified by recruitment of the intrinsic pathway. This is achieved
by caspase-8 mediated clevage of the protein Bid leading to mitochondrial permeabilization. Bid can also
be cleaved by calpains, granzyme B, and lysosomal proteases.
Apoptosis can be mediated by mechanisms other than traditional caspase-mediated cleavage cascade.
There is increasing evidence that alternative proteolytic enzymes such as lysosomal proteases (cathepsins)
can initiate or propagate proapoptotic signals. In vivo, lysosomal membrane permeabilization occurs during
aging and in different neurodegenerative diseases. In vitro, lysosomal membrane permeabilization can be
induced by sphingosine, oxidative stress, activation of death receptors, as well as by lysosomotropic agents
such is L-leucyl-L-leucine methyl ester (LeuLeuOMe). The main function of lysosomal proteases is to
recycle proteins within lysosomes, but they can be also active when released into the cytosol. Once in the
cytosol they activate proapoptotic molecules such as Bid or inactivate antiapoptotic Bcl-2 proteins, thereby,
leading to mitochondrial pathway. However, they have been also suggested to trigger caspase-independent
cell death.
The aim of our work was to understand the molecular mechanisms by which lysosomal proteases induce
apoptosis triggered by the lysosomotropic agent LeuLeuOMe and to investigate the role of cathepsins in
this model of apoptosis. The destabilization of lysosomes by LeuLeuOMe in HaCaT cell line caused
morphological and biochemical changes typical for apoptosis, such as phosphatidylserine externalization,
caspase activation, mitochondrial damage, and PARP cleavage. We showed that the addition of
LeuLeuOMe resulted in lysosomal destabilization prior to mitochondrial damage. Apoptosis induced by
LeuLeuOMe resulted in the cleavage of Bid to its truncated form. The latter could be prevented by E-64d, a
broad spectrum inhibitor of papain-like lysosomal cysteine proteases, but not by the pancaspase inhibitor z-
VAD-fmk. Moreover, we also showed that cathepsins degrade the antiapoptotic protein Bcl-xL. This
degradation was prevented by E-64d, but not by z-VAD-fmk. These results suggest that apoptosis triggered
by LeuLeuOMe is caspase-dependent.
It was reported that lysosomal destabilization was also involved in the extrinsic pathway of apoptosis.
The exact mechanisms by which lysosomal destabilization lead to cell death are still not well understood.
Some authors suggested that lysosomal destabilization is an early event in apoptosis, thus preeceding
mitochondrial destabilization. On the other side, we recently showed that in Fas-induced apoptosis,
lysosomes were destabilized after the mitochondria.
In order to address this question, TNF-α induced apoptosis was investigated in wild-type and cathepsin
B and cathepsin L-deficient primary mouse embryonic fibroblasts. In mouse embryonic primary
fibroblasts, we showed that in TNF-α induced apoptosis, destabilization of mitochondria precede
destabilization of lysosomes. Moreover, the reactive oxygen species produced by mitochondria, were
responsible for destabilization of lysosomes. This effect was almost completely prevented by reactive
oxygen species scavengers, butylated hydroxyanisole and TEMPOL, and a potent iron chelator
desferrioxamine. We further confirmed that cathepsins B and/or L contribute to TNF-α signalling
downstream of lysosomal destabilization. In addition, we showed that Bid cleavage was significantly
reduced in cathepsin B and cathepsin L deficient fibroblasts, thus proving that Bid is a critical substrate for
cathepsins in our cellular model.