E3 Connection Enzyme Siah And The Phc2 And Eef1d Protein The Interaction Studies

The study is focused on the human E3 ubiquitin ligase SIAH-1b (SIAH) and its novel associated proteins. We carried out yeast two-hybrid screening in fetal liver library with SIAH as the bait and found two proteins. PHC2 and EEF1D, as the preys. In the first part, we identified the degradation of PHC2 by SIAH. In the second part, we uncovered the regulation of the E3 ligase activity of SIAH by EEF1D.SIAH, a homologue to Drosophila SINA (seven-in-absentia), is considered as an E3 ubiquitin ligase with abilities to recognize and degrade specific substrate proteins via ubiquitin-proteasome pathway. SIAH play an important role in cell cycle regulation, tumor generation and several eurodegenerative diseases.In the first part I, we identified the interaction between SIAH and PHC2. and the degradation of PHC2 by SIAH via the ubiquitin-proteasome pathway. PHC2 (polyhomeotic homolog 2) is one of the Polycomb Group (PcG) genes which encode proteins that form large multimeric and chromatin-associated complexes implicated in the stable repression of developmentally essential genes (e.g. Hox genes). In our study, SIAH was showed to interact directly with PHC2 both in vitro and in vivo. Subcellular localization assay displayed colocalization of both proteins in the nuclear of HeLa cells. The Cys-rich domain of SIAH and the PxVxAxP motif of PHC2 were demonstrated to be essential for their interaction. SIAH facilitated the ubiquitination and degradation of PHC2 via ubiquitin-proteasome pathway in 293T cells. Two SIAH mutants named SiahR which was defected in E3 ligase activity and Siahm which was defected in the ability of association with PHC2 were constructed for further research. The activities of both mutants to ubiquitinate and degrade PHC2 were severely impaired, which implied PHC2 is the substrate of SIAH. Mutations in the PcG genes result in developmental defects and have been implicated in human cancer. PHC2, as a functional component of classⅡPcG complexes, plays a role in embryogenesis and developing brain. The novel discovery of the degradation of PHC2 by SIAH might help to investigate the road of PHC2 in the early development.In partⅡ, our results indicated that EEF1D could associate with SIAH and inhibit its E3 ligase activity. EEF1D (eukaryotic translation elongation factor 1 delta, also named eEF1Bδor eEF1δ) was considered as a subunit of the eEF1B complex which acts as an exchange factor (GEF) and recycles the inactive eEF1A-GDP released from the ribosome to the active GTP-bound state by stimulating nucleotide exchange on eEF1A. EEF1D is related with cell cycle regulation, tumorigenesis and virus infection. The interaction of SIAH and EEFID was confirmed in vitro and in vivo. Both of the proteins were colocalized in the cytoplasm of HeLa cells. The Cys-rich domain of SIAH was essential for the interaction with EEFID. The protein level of EFF1D didn't change in the cells overexpressing SIAH, which implied that EFF1D was not the substrate of SIAH. In contrast, the amount of SIAH increased significantly in the cells overexpressing EEF1D. The protein stability assay showed that the increasing expression of SIAH was due to the inhibition of degradation by EEFID. Furthermore, the auto-ubiquitination of SIAH and the degradation of PHC2 by SIAH were both inhibited by EEFID. Our data suggests that EFF1D inhibits the E3 ligase activity of SIAH, prevents the degradation of SIAH itself and some substrate of SIAH, which provides new clue on the regulation of SIAH.