Mechanisms and regulation of lysosomal proteolysis.

We have determined the sequence of cDNAs encoding the precursors of rat cathepsins H and L. In order to understand the mechanism and regulation of expression and processing of these genes we have also determined the genomic restriction maps and the genomic structures in the extension sequences of the 5'-regions. We have found that the intracellular locations of cathepsins B, H and L in various cells and organs are different. Lysosomes are shown to have different amounts of cathepsins B, H and L and this heterogeneity may reflect their different functions in autophagy and heterophagy. The intracellular role of cystatins, endogenous inhibitors of cathepsins, in suppressing intralysosomal proteolysis has been clarified. Electron microscopic studies of islet cells of the pancreas using double gold particle immunostaining for insulin and cystatin beta, indicate that cystatin beta is located in the insulin secretory granules. We propose the following hypothesis for the mode of action of cystatin beta: cystatin beta is secreted from its primary location in the secretory granules and is then taken up by target cells which direct it into lysosomes for the regulation of intralysosomal cathepsin activities. The inhibitory activity of cystatin beta is controlled by formation of a mixed-disulfate with glutathione and cysteine at position 3: the free form is active and the "glutathionated" form is inactive. The changes in glutathione balance (oxidized/reduced) in cells may regulate the inhibitory activities of cystatin beta. Further, the regulation of cathepsins under various nutritional and hormonal conditions, as well as the abnormal expressions of cathepsins in various pathological conditions, are discussed.