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

本项目的出发点是，从v-Ha-ras转化的NIH3T3细胞中分泌大量的原athepsin L，其中组织蛋白酶L的mRNA增加，而从其逆转的细胞中分泌大量的原athepsin L，其中组织蛋白酶L的mRNA虽然没有增加。提示v-ras激活的信号转导通路不仅激活了组织蛋白酶L基因，还改变了组织蛋白酶i原转运到溶酶体排泄的过程。虽然我试图分离稳定表达rab7、其显性活性形式和显性阴性形式的NIH3T3细胞，但我未能分离表达rab7、其显性活性形式和显性阴性形式的细胞系。接下来，我试图在强力霉素诱导系统的控制下分离表达rab7的细胞，以及其显性-活性和显性-阴性形式。利用肽基抗体分别识别组织蛋白酶L的31kDa、30kDa和成熟形式，揭示了组织蛋白酶L在体内的加工过程与体外相同。当细胞用巴菲霉素A1处理时，溶酶体内pH升高，导致溶酶体蛋白酶降解。处理后，细胞在蛋白酶抑制剂的作用下进一步培养，结果显示，除了蛋白酶B，蛋白酶B、蛋白酶D和蛋白酶L的加工都是由木瓜蛋白酶型半胱氨酸蛋白酶进行的。这表明同样的加工蛋白酶可能参与了对蛋白酶B、蛋白酶D和蛋白酶L的加工。

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.