FIG 5.

Mitochondria-endolysosome contact in hypoxia leads to truncation of VDAC1 at asparagine 214 by the endolysosomal asparagine endopeptidase. (A) Ratios of the abundance of human VDAC1 (hVDAC1) peptides from the normoxic sample to the abundance from the hypoxic sample. Residue numbers are indicated together with the peptide amino acid sequence. The y axis displays the decimal logarithm of the relative abundance of a given peptide in normoxia versus hypoxia. (B) Structure of VDAC1 showing the major cleavage site of VDAC1 C terminal to asparagine 214 (site C) and a minor cleavage site at glycine 213 (site C′). The consensus sequence between human VDAC1, VDAC2, and VDAC3 at the cleavage site is shown by boldface circles. (C) HeLa cells transfected with two different AEP-specific siRNAs (siAEP#1 and siAEP#2) incubated in hypoxia (Hx) and analyzed by immunoblotting (Ctl, control; siCtl, scrambled control siRNA). (D) HeLa cells were transfected with either a control siRNA or asparagine endopeptidase-specific siRNA (siAEP; 100 nM), incubated in normoxia (Nx) or hypoxia, and challenged with STS (1 mM) for 4 h. Apoptosis was evaluated from the level of caspase 3/7. Data from two independent experiments performed in quadruplicate are given as means ± SDs. (E) rAEP was activated by low pH and incubated with recombinant VDAC1 (rVDAC1) reconstituted into either large unilamellar vesicles (LUVs) or N,N-dimethyldodecylamine N-oxide (LDAO) micelles for 180 min at room temperature. The samples were analyzed by SDS-PAGE. Mk, molecular mass marker; rAEP inact., inactive rAEP; rAEP act., pH-activated rAEP. (F) Autoactivated rAEP was added to mitochondria isolated from normoxic HeLa cells, and the mixture was incubated for 30 min at 37°C. The samples were analyzed by immunoblotting for VDAC.