Endolysosomal proteolysis and its regulation.
The endolysosomal system is a multifunctional system and is involved in catabolism, antigen presentation and regulation of hormones. The descriptions of, and functions assigned to organelles within the system are often applied using different criteria. Further, the properties of the hydrolases within the system, and the organelles that house them are usually studied in isolation from one another. Considering that the endolysosomal system may be extremely dynamic, an understanding of the system as an integrated whole is a necessary first step. Thus, a review of the endolysosomal system was undertaken. It was determined that the enzymes within the endolysosomal system are probably subject to 'organelle-dependent' regulation, i.e. the organelles create the appropriate luminal conditions for these enzymes to work. It is also likely that the effectors of these luminal conditions are regulated in a manner that is related to GTPase networks that control much of the cell's functions. The organisation of the endolysosomal system may be hierarchical, with proteases being downstream effectors of a system that is regulated at the whole body level. The main endoprotease class within the endolysosomal system are cysteine proteases. A literature review suggested that these enzymes may not be redox regulated within the endolysosomal system. However, the lysosomal endoprotease cathepsin B has been implicated in many pathologies where it is operating in pH and redox conditions different from the endolysosomal system. To study this, cathepsin B was isolated from bovine livers using a novel procedure that exploits the ability of tertiary butanol to temporarily inhibit protease interactions in tissue homogenates. This would prevent artefactual, as well as protease-inhibitor interactions. This novel procedure resulted in increased yields of cathepsin B. Cathepsin D, an aspartic protease, was isolated using established methods. In order to test the hypothesis that cathepsin B may be redox regulated in vivo, cathepsin B activity and stability were measured in cysteine and/or cystine-containing redox buffers. Cathepsin B activity in cysteine-containing buffers was similar at pH 6.0 and pH 7.0, over all thiol concentrations tested. In contrast, the stability of the enzyme was greater at pH 6.0 than at pH 7.0. This suggests that the enzyme's operational pH in vivo may be < pH 7.0. The activity of the enzyme was depressed in glutathione-containing buffers. When assessed in cysteine:cystine redox buffers (pH 6.0 - 7.0) cathepsin B was active over a broad redox potential range, suggesting that cathepsin B activity may not be redox regulated.