碩士 / 中興大學 / 生物醫學研究所 / 95 / Apoptosis-inducing factor (AIF) is a mitochondrion-localized flavoprotein with NADH oxidoreductase activity, which is encoded by the nuclear gene. Following induction of apoptosis, AIF is released from mitochondria and translocated to cytosol, and then to the nucleus. The mechanism of AIF translocation from mitochondria to nucleus is still unclear. It has been suggested that translocation is due to the presence of nuclear localization signal (NLS). However, proteins without NLS, such as Smad, PTEN and ERK, could also be translocated into nucleus. There could be a protein that transports protein into nucleus.
Therefore, we used yeast two-hybrid system to find the nuclear transporter and we identified a protein, hHR23A, which could bind to AIF both in cytosol and in nucleus. hHR23A is a mammalian protein that is homologous to yeast Rad23 protein. Although hHR23B has been shown to be correlated with DNA repair, the function of hHR23A is yet to be determined. We propose that hHR23A could be a protein transporter to bring AIF into nucleus following cell stress.
We then raised antibodies to AIF and hHR23A respectively, and by immunoprecipitation we used these antibodies to pull down AIF and hHR23A proteins, of which the protein identities were further determined by MALDI-TOF. We then constructed shRNA(small-hairpin RNA) of hHR23A to study whether down-regulation of hHR23A could interfere nuclear translocation of AIF during apoptosis. Moreover, we constructed deletion mutants of AIF and hHR23A to map out the interaction domains on AIF and hHR23A. Our preliminary data showed that hHR23A bound to AIF directly. Knockdown of hHR23A expression could reduce nuclear translocation of AIF after treating with apoptosis inducer, staurosporine. Furthermore, cell survival rate of hHR23A knockdown cell increased markedly when cells were treated with cisplatin. Addition of DNP, an inhibitor of ATP synthesis, could also diminish nuclear translocation of AIF and hHR23A, and increase cisplatin resistance. Our results suggest that hHR23A is involved in a novel nuclear transportation, which facilitates nuclear translocation of AIF during apoptosis.
| Identifer | oai:union.ndltd.org:TW/095NCHU5114007 |
| Creators | Chih-Yang Huang, 黃智洋 |
| Contributors | 周寬基 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 76 |
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