Abstract
Procathepsin D is the inactive zymogen of the lysosomal aspartic proteinase, cathepsin D. The conversion of procathepsin D to an active form of the enzyme involves the removal of the amino terminal propeptide of the protein from the active site cleft of the enzyme and subsequent proteolytic cleavage. Due to the rapid conversion of procathepsin D in vivo to the mature catalytically active forms of the enzyme, it has been difficult to elucidate the precise activation mechanism. This thesis details two complementary approaches used to provide information on the requirements of propeptide processing, and the role this processing event has on the maturation of cathepsin D. Proteinase inhibitor studies were performed on tissue culture cells to block the processing of procathepsin D. Cysteine proteinases were shown to play a role in the processing and delivery of procathepsin D and other lysosomal hydrolases. The second approach used site-directed mutagenesis to study the processing and maturation of procathepsin D to the lysosome. Mutations were made to prevent cleavage at two processing sites in the propeptide and the cDNAs encoding the mutant proteins were expressed in mammalian cells to analyze the effects of the mutations on processing and maturation. The results of these studies indicated that propeptide processing is not necessary for further maturation of cathepsin D.