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Protease‐protease interactions as a microenvironment‐dependent regulatory mechanism

Proteases are central to many physiological functions, including protein turnover, immunesystem response, and tissue remodeling. One family of proteases that have been of particular interest in recent years, are the cysteine cathepsins, which were originally characterized for their role in lysosomal protein turnover. Recent findings have shown that unchecked cathepsin activity contributes to the progression of diseases ranging from cancer to osteoporosis; however, all of the clinical trials for drugs targeting cathepsins, to date, have failed. Therapeutically targeting cathepsins is nontrivial due to the existence of a proteolytic network, defined by protease‐protease interactions that regulate concentrations of active enzymes through protease activation/degradation. Perturbation of the proteolytic network through the inhibition of cathepsins may lead to unexpected downstream off‐target effects. Further complexity arises when considering that the favorability of the interactions of the proteolytic network is affected by microenvironmental cues, such as pH. As a result, the direction and significance of connections within the proteolytic network are dependent on where the cathepsin is localized inside/outside the cell. Of particular interest to this work, are recently characterized cathepsin:cathepsin interactions that lead to cathepsin degradation, coined as “cannibalism”, which was first identified for the degradation of cathepsin K (catK) by cathepsin S (catS). We have utilized a hybrid computational and experimental approach to elucidate the pH‐dependence of catS:catK interactions and to quantify the effects of pH on catS on catK cannibalism. We performed computational protein docking, pKa predictions, and continuum electrostatics calculations to identify potential interactions that contribute to catS on catK cannibalism, as well as, to elucidate pH‐dependent effects. Our predictions indicate that interactions predicted for catS on catK cannibalism increase in favorability when increasing pH from 4 to 7.4. In vitro experiments using recombinant enzymes show that catK is degraded faster at pH 7.4 than at pH 4, suggesting that the strong predicted catS:catK interactions contribute to cannibalism. CatS, interestingly degrades catK slowest at pH 6, indicating that there is a preference for catK to be active at pH 6 despite cathepsins typically preferring more acidic environments, such as the lysosome. Our results illustrate a role for cathepsin cannibalism as a microenvironment‐dependent regulatory mechanism of cathepsin activity, which should be further investigated in the context of the proteolytic network and the development of cathepsin targeted therapeutics. Finally, the proposed hybrid approach lays the groundwork for investigating the role of microenvironmental pH in regulating other interactions in the proteolytic network inside and outside the cathepsin family of proteases.