| Summary: | 博士 === 高雄醫學大學 === 醫學研究所 === 91 === A chicken anemia virus-derived protein VP-3 (Apoptin) was reported to cause apoptosis in human transformed cell lines but not in normal cells. Apoptosis induced by Apoptin is p53-independent and Bcl-2-insensitive, making Apoptin a potential agent for treatment of cancers, including those lacking p53 or overexpressing Bcl-2. However, the molecular mechanism of Apoptin-induced apoptosis has not yet been elucidated. In this study, we had identified four Apoptin-associated proteins, named APAP1, APAP2, APAP3 and APAP4, by yeast two-hybrid screening. Sequence analysis of APAP1 revealed that APAP1 is identical to DEDAF( death effector domain associated factor) and RYBP( Ring 1 and YY1 binding protein). DEDAF is a protein with the ability to interact with the death effector domains (DEDs) of caspase-8 and caspase-10. Sequence analysis of APAP4 revealed that APAP4 is identical to Hippi, a protein interactor of huntingtin interacting protein 1 (HIP-1). Interestingly, both HIP-1 and Hippi contain a domain with homology to DED. The binding of APAP1 and APAP4 to Apoptin in human cells was demonstrated by GST pull down assay and co-immunoprecipitation assays. Overexpression of APAP1 induced apoptosis in both cancerous HeLa cells and normal HEL cells, however less apoptotic toxicity was observed when HeLa and HEL cells were overexpressed with APAP4. Both APAP1 and APAP4 were bound to the identical region of Apoptin which was localized in the N terminus of a.a. 1-59. The immunofluorescence assay was performed to study the cellular localization of APAP1 and APAP4 in HeLa and HEL cells. APAP1 was localized mainly in the nuclear region of both cell lines, the colocalization of APAP1 and Apoptin was seen in the nucleus region of tumorous HeLa cells. However, only little amount of Apoptin was seen colocalized in the nucleus region of HEL cells. The colocalization of Apoptin and APAP4 was not observed. However, Apoptin and APAP4 were seen localized perfectly in HEL cells. The protein expression level of APAP1 and APAP4 was also studied. Higher APAP1 expression level was observed in various tumor cell lines compared to the normal cell lines. In contrast, the expression level of APAP4 was lower in the tumor cell lines compared to the normal cells. In addition, by using APAP4 as a bait for the further screening of APAP4-associated proteins, an N-myc interactor ( Nmi ) protein was identified. Interestingly, Nmi was also a binding partner of APAP3/IFP35, an interferon induced protein. In this study, by the cloning and characterization of the Apoptin associated proteins APAP1 and APAP4, we had proposed the possible roles of APAP1 and APAP4 in the apoptotic pathway induced by Apoptin in human tumor cells, and hopefully the suggested model will be beneficial for the clinical application of Apoptin.
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