Deregulation of autophagy plays an important role in the pathogenesis of diseases such as cancer, neuronal degenerative or cardiovascular disease. Autophagy is a process to engulf the cytoplasmic contents into autophagosome and deliver them for lysosomal degradation. Its major function is to clear unfolded protein or damage organelles for maintaining proper metabolic homeostasis and normal cell physiological activities. Autophagy and multivesicular bodies, MVBs, cooperate to regulate the turnover of intracellular macromolecule, defective organelles and signaling receptor. Endosomal sorting complex required for transport, ESCRT, is important for the formation of MVBs, which regulates membrane receptor recycling, protein sorting and vesicular trafficking. Tumor Susceptibility Gene 101(TSG101) is a member of ESCRT-I that plays an important role on MVBs formation and maintaining ESCRT function. Previous report indicated that autophagosome accumulation upon deprivation of TSG101, implying possible role of TSG101 during autophagic process. In this study, we observed the increase of TSG101 and autophagic marker proteins, such as LC3-II and ATG upon nutrient starvation. Furthermore, knockdown TSG101 in cervical carcinoma HeLa cell resulted in the elevation of LC3-II, ATG3 and ubiquitinated protein aggregates marker protein p62, which is congruous to other reports. However, in neuroblastoma SH-SY5Y cell, transfection of siRNA led to the decrease of LC-II and ubiquitinated protein level. These results indicated that TSG101 might be critical for autophagy and the maintenance of steady-state level of cellular ubiquitinated proteins. Ectopic upregulatory expression of HA-TSG101 led to the increase of LC3-II in both cell type. The elevation of ATG3 level is also observed in HeLa cell. Therefore, we speculated that TSG101 might be important for the formation of autophagosome, but our data did not exclude the possible role of TSG101 in regulation of the fusion of autophagosome and lysosome, because the increase of ATG3 indicated ectopic HA-TSG101 might facilitate the execution of autophagic flow. In addition, we have established GFP-LC3 expression cell lines. Our imaging data showed the colocalization of TSG101 and GFP-LC3 in both cytoplasm and nucleus that might be an interesting research topic for investigation the role of TSG101 in autophagic pathway.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0217112-160301 |
| Date | 17 February 2012 |
| Creators | Yeh, Chun-Cheng |
| Contributors | Yi-Ren Hong, Jiin-Tsuey Cheng, Chi-Liang Chern |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0217112-160301 |
| Rights | user_define, Copyright information available at source archive |
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