The Responses Of Quantitative Proteomics In The HeLa Organelles To Cathepsin D Knockdown

Cathepsin D is peptidase belonging to the family of aspartic peptidases. Several in vitro and in vivo studies found many evidences that Cathepsin D is closely related to cancer development and progression. However, the mechanism which Cathepsin D take to function in tumorigenesis and tumor progression remains unclear. In our study, Based on the cell sencescence model induced in T-RExTM HeLa cells by the knockdown of Cathepsin D, We carried out a preliminary study on its underlying molecular mechanism.We first investigated the effect of Cathepsin D knockdown on cell cycle progression. The cell cycle analysis showed that G2/M phase progression was significantly inhibited by Cathepsin D knockdown, about 88.1±2.3% cell population arrested in G2/M phase. Next, we conducted quantitative proteomics analysis using stable isotope labeling with amino acids in cell culture (SILAC) to determine the cytoplasmic, nuclear and lysosomal proteomes regulated by Cathepsin D knockdown in T-RExTM eLa cells. We first enriched the organelles using the methods differential centrifugation and density gradient centrifugatioa Then we used SDS-PAGE to fractionate the proteins into 10 or 15 fractions according to its molecular sieve effect. Every gel slice was subjected to in-gel digestion with trypsin. At last we applied the high-precision mass spectrometer LTQ OrbitrapVelos to detect the isolated organelle proteins and utilized the software MaxQuant to do peptide search and quantificatioa Finally We found that the knockdown of Cathepsin D can induce significantly changes in these three organelle proteomes.266 proteins and 161 proteins were found to be significantly upregulated or downregulated in cytoplasmic proteome.71 proteins and 84 proteins were significantly upregulated or downregulated in nuclear proteome.42 proteins and 107 proteins were significantly upregulated or downregulated in lysosomal proteome. Compared with cytoplasmic and nuclear proteome data, the number of downregulated proteins in lysosome increases significantly. Among them, seven proteins belong to the component of lysosome. And five of them which participate in the molecular transport process were all downregulated, suggesting that the knockdown of Cathepsin D may affect the tranport process of proteins from cytoplasm to lysosome. Ingenuity Pathway Analysis (IPA) further revealed that among the proteins which were sensitive to Cathepsin D knockdown in nucleus, about 17% percentage proteins were cell cycle regulatory protein. These included proteins like HMGA1, PARP1, TOP2A, NPM1, TPR, which had been reported to be associated with the G2/M cell cycle process. While in the cytoplasm part, we found that all ribosomal proteins related to the protein synthesis were upregulated, indicating that the knockdown of Cathepsin D may cause increase in the number of ribosome assembly, then causing an increase in protein synthesis. To sum up, Our quantitative organelle proteomics study provided molecular basis for the in-depth study of the mechanism how Cathepsin D knockdown induces cell senescence and G2/M cell cycle arrest.