The Mechanisms in activation and secretion of procathepsin L in accordance with transformation

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This project was started by the point of view that a large amount of procathepsin L is excreted from v-Ha-ras transformed NIH3T3 cells in which mRNA for cathepsin L increases whereas a large amount of procathepsin L is excreted from its revertant cells in which mRNA for cathepsin L although does not increase, suggesting that v-ras activated the signal transduction pathways to not only the activation of cathepsin L gene but also the change in trafficing of procathepsin i to lysosomeg for excretion. Although I tried to isolate the NIH3T3 cells stably expressing rab7, its dominant-active forms and dominant negative forms, I failed to isolate the cell lines expressing rab7, its dominant active, and dominant-negative forms. Next, I try to isolate the cells expressing of rab7, its dominant-active and dominant-negative forms under the control of doxycyclin induction systems.By using peptidyl antibodies that recognized 31kDa, 30kDa, and the mature forms of cathepsin L, respectively, it was revealed that the processing of cathepsin L in vivo was the same as that in vitro.When the cells were treated with Bafilomycin A1, the intralysosomal pH increased resulting degradation of lysosomal proteinases. After this treatment, the cells were further culture in the presence of proteinase inhibitors showing the processing of legumain, cathepsins B, D, and L were carried out by papain type cysteine proteinase except for cathepsin B.This result suggested the same processing proteinase might participate in the processing of legumain, cathepsins B, D, and L.