Cathepsin S Cleaves BAX as a Novel and Therapeutically Important Regulatory Mechanism for Apoptosis

Abstract

Certain lysosomal cathepsin proteins have come into focus as being good candidates for therapeutic targeting, based on them being over-expressed in a variety of cancers and based on their regulation of the apoptotic pathway. Here, we report novel findings that highlight the ability of cathepsin S expression to be up-regulated under Paclitaxel-stimulatory conditions in kidney cell lines and it being able to cleave the apoptotic p21 BAX protein in intact cells and in vitro. Consistent with this, we demonstrate that this effect can be abrogated in vitro and in mammalian cells under conditions that utilize dominant-inhibitory cathepsin S expression, cathepsin S expression-knockdown and through the activity of a novel peptide inhibitor, CS-PEP1. Moreover, we report a unique role for cathepsin S in that it can cleave a polyubiquitinated-BAX protein intermediate and is a step that may contribute to down-regulating post-translationally-modified levels of BAX protein. Finally, CS-PEP1 may possess promising activity as a potential anti-cancer therapeutic against chemotherapeutic-resistant Renal Clear Cell Carcinoma kidney cancer cells and for combined uses with therapeutics such as Paclitaxel.

Intrinsically Connected: Therapeutically Targeting the Cathepsin Proteases and the Bcl-2 Family of Protein Substrates as Co-regulators of Apoptosis

“…Collectively, such findings highlight a novel approach for inhibitor design targeted at BAX stabilization as a foresight, for this regulatory axis of the intrinsic apoptotic pathway. As proof of principle, CS-PEP1 enhanced apoptotic cell numbers at low doses, in good agreement with its intended purpose [ 79 ]. Such findings do suggest that pro-apoptotic proteins (such as BAX), along with their upstream regulator proteases, can be simultaneously targeted by a single therapeutic that can serve a dual purpose.…”

“…While this could have been due to a number of reasons, from recent studies, our findings were supportive of BAX being directly cleaved to p18BAX in vitro by cathepsin S (at pH 5 or 7) and to complete digestion within intact mammalian cells. Moreover, the inhibition of cathepsin S had the effect of stabilizing p21BAX [ 79 ]. Based on the functional role of BAX and BAK up-regulating MOMP and apoptosis, many structural studies have revealed the potential to disrupt the interaction between the BAK BH3-domain with the hydrophobic groove of its cognate anti-apoptotic proteins, through designing BH3-mimetics and thus increasing the availability of monomeric BAK protein for MOMP and apoptosis induction [ 44 ].…”

“…Being mindful of such effects, in our recent study, we demonstrated BAX to be a novel and cathepsin S-targeted substrate. We also described the design of a novel cathepsin S-directed peptide inhibitor (CS-PEP1), based on a peptide sequence from the fungal papain protease inhibitor protein pit2, and which was identical to a sequence found within α-5 helix of the hydrophobic groove of anti-apoptotic Bcl-xL [ 79 ]. Mechanistically, CS-PEP1 was observed to act through the stabilization of BAX (through cathepsin S inhibition), or through possibly interfering with BAX-Bcl-xL protein binding.…”

“…Such a pH-sensitive cleavage reaction appeared fortuitously to favor the inhibition of Bcl-xL cleavage (by CS-PEP1) at a relatively higher pH, in relation to the lower pH that can be prevalent during tumor development and apoptosis. Favorably, cathepsin S-mediated cleavage of BAX (and the inhibition of this reaction by CS-PEP1) was observed to be effective at both pH 5 and 7 [ 79 ]. Collectively, such findings offer an alternative approach in targeting this inhibitory axis of the intrinsic pathway for apoptosis with greater flexibility, through designing a more selective therapeutic that is based upon combining the principles of BH3-mimetics with the classical approach of cathepsin S-directed allosteric inhibition [ 79 ].…”

“…Favorably, cathepsin S-mediated cleavage of BAX (and the inhibition of this reaction by CS-PEP1) was observed to be effective at both pH 5 and 7 [ 79 ]. Collectively, such findings offer an alternative approach in targeting this inhibitory axis of the intrinsic pathway for apoptosis with greater flexibility, through designing a more selective therapeutic that is based upon combining the principles of BH3-mimetics with the classical approach of cathepsin S-directed allosteric inhibition [ 79 ]. Importantly, the inhibition of cathepsin-mediated BAX (or possibly BAK) and Bcl-xL cleavage, may also be modulated further in a pH-dependent manner by such a therapeutic, thus permitting the favorable cleavage of an anti-apoptotic protein better than its pro-apoptotic counterpart.…”