Abstract
<title>Abstract</title> <p>Cathepsins, the principle proteases of the endolysosomal compartment, can escape into the cytosol and nucleus following lysosomal membrane permeabilization (LMP). LMP-associated toxicity of nucleo-cytosolic cathepsins is restricted by endogenous, competitive and reversible inhibitors known as stefins/cystatins. These inhibitors bind at a 1:1 stoichiometry, limiting the buffering capacity during acute LMP, raising the question of whether additional systems co-regulate nucleo-cytosolic cathepsins. Here we show that dipeptidyl-peptidase 9 (DPP9) controls the steady-state levels of cytosolic/nuclear cathepsins, conveying resistance to LMP. N-terminomics on HEK293 DPP9 knockout cells identified cathepsin Z (CTSZ) as a DPP9 substrate. By cleaving mature CTSZ, DPP9 removes the N-terminal Leu-Pro dipeptide and unmasks a latent N-degron, targeting CTSZ for proteasomal degradation. Together, mass-spectrometry analysis, molecular docking simulations, and cell-based assays employing DPP9-depleted cells, revealed DPP9-dependent N-degron exposure across multiple mature cathepsins. In contrast to cystatins, this mechanism is an irreversible, non-saturable response to the toxic accumulation of lysosomal-escaped cathepsins.</p>