Cathepsin D triggers Bax activation, resulting in selective AIF relocation in T lymphocytes entering the early commitment phase to apoptosis

Authors:
Bidère N, Lorenzo HK, Carmona S, Laforge M, Harper F, Dumont C and Senik A
In:
Source: J Biol Chem
Publication Date: (2003)
Issue: 278(33): 31401-31411
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
Platform:
Nucleofectorâ„¢ I/II/2b
Experiment
Cat D, Bax and AIF were efficiently knocked down by nucleofection of specific siRNA in primary human T cells. Subsequent stimulation of apoptosis with staurosporine showed that Cat D triggers Bax activation and AIF release.
Abstract
Activated human T lymphocytes exposed to apoptotic stimuli targeting mitochondria (i.e. staurosporine), enter an early, caspase-independent phase of commitment to apoptosis characterized by cell shrinkage and peripheral chromatin condensation. We show that during this phase, AIF is selectively released from the intermembrane space of mitochondria, and that Bax undergo conformational change, relocation to mitochondria, and insertion into the outer mitochondrial membrane, in a Bid-independent manner. We analyzed the subcellular distribution of cathepsins (Cat) B, D, and L, in a search for caspase-independent factors responsible for Bax activation and AIF release. All were translocated from lysosomes to the cytosol, in correlation with limited destabilization of the lysosomes and release of lysosomal molecules in a size selective manner. However, only inhibition of Cat D activity by pepstatin A inhibited the early apoptotic events and delayed cell death, even in the presence of bafilomycin A1, an inhibitor of vacuolar type H+-ATPase, which inhibits acidification in lysosomes. Small interfering RNA-mediated gene silencing was used to inactivate Cat D, Bax, and AIF gene expression. This allowed us to define a novel sequence of events in which Cat D triggers Bax activation, Bax induces the selective release of mitochondrial AIF, and the latter is responsible for the early apoptotic phenotype.