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E3 ligase SIAH and its interaction with PHC2 , and EEF1D protein research

Author: WuHuiLing
Tutor: HuoKeKe
School: Fudan University
Course: Genetics
Keywords: SIAH PHC2 Degradation Ubiquitination EEF1D Suppression
CLC: Q55
Type: PhD thesis
Year: 2009
Downloads: 65
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Abstract

This paper is about the human E3 ubiquitin ligase SIAH-1b (hereinafter referred to as SI AH) and its interacting protein research. By yeast two-hybrid technology SIAH as bait protein in human fetal liver library screening to the two new interactions of protein: PHC2 EEF1D. Around the the two protein with SIAH relationship, we have carried out in two parts: The first part is about SIAH the degradation PHC2 the second part of the study; E3 ligase activity on EEF1D regulation SIAH. E3 ligase SIAH and Drosophila SIAN (seven-in-absentia) homologous identify substrate proteins so degraded by the ubiquitin - proteasome system. SIAH in cell cycle regulation and physiological processes play a role in tumorigenesis and neurodegeneration. In the first part, we found the SIAH the combined PHC2 and through ubiquitination - proteasome pathway degradation. PHC2 (polyhomeotic homolog 2) is one of the PCG gene group. The PcG gene cluster encoding a class of proteins, the transcriptional expression of poly complex binds to chromatin and stably suppress some developmental gene required (such as Hox gene cluster), is formed. Our experimental results show that: the SIAH able and PHC2 occur in vivo and in vitro interaction, and can be co-localized in the nucleus of HeLa cells; the SIAH the Cys-rich domain, PHC2 the PxVxAxP motif play an important role in the combination of the two in the; In mammalian cells, 293T, SIAH promotes PHC2 ubiquitination and degradation by the ubiquitin - proteasome pathway. To verify both the E3 enzyme - substrate relationships, we constructed a the two mutant SiahR The Siahm, the former with PHC2 combination but lost E3 ligase activity, which retains E3 adapter capacity greatly decreased enzyme activity, but combined with the PHC2 . The results showed that the two mutants ubiquitination degradation PHC2 ability inferior to wild type, confirming the PHC2 is the SIAH the ubiquitination degradable substrate. PcG genes the mutations cause the animal developmental defects, tumor formation PHC2 as class Ⅱ (PcG) composite material important Yi Yuan, in the embryo to form the brain's early development also has an important role, therefore research SIAH of PHC2 the degradation regulation of exploration PHC2 important in the early development of the regulatory mechanism. In the second part, we found EEF1D interaction with SIAH and inhibit its E3 ligase activity. EEF1D (eukaryotic translation elongation factor 1 delta, and also called eEF1Bδ or eEF1δ) to extend a subunit of the complex factors eEFIB eukaryotic translation. eEFIB complex is eEFIA conversion factor (exchange factor, GEF), activates the conversion of nucleoside eEF1A exits the ribosome inactive eEF1A-GDP into rejoin ribosome activity eEF1A-GTP, thus completing eEFIA vitality circulation. Previous studies show that the EEF1D have played a role in the virus infection, cell cycle, tumorigenesis. We found SIAH with EEF1D, occurred in vivo interaction; co-localization of the experimental results show that both can be co-localization in the cytoplasm of HeLa cells; series of deletion experiments found that of SIAH the Cys-rich domain in the combination of the two in the play an important role. In 293T cells, EEF1D protein level does not change with the increase of the SIAH changes, the results suggest that it is not SIAH substrate. However, SIAH protein levels will with the EEF1D expression amount increases protein stability test results showed that this increase is due to EEF1D SIAH inhibit the degradation, resulting SIAH protein levels increase. The addition, EEF1D can inhibit the SIAH self ubiquitination, also inhibited the degradation of the SIAH pairs PHC2. Based on the above results, we found EEF1D inhibit of SIAH the E3 ligase activity, leading to its own levels of degradation and the degradation of the decline in the ability of certain substrates, which provide new clues for research SIAH regulatory mechanism.

E3 ligase SIAH and its interaction with PHC2 , and EEF1D protein research

Abstract

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