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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 分子生物學研究所 > 依資料類型分類 > 碩博士論文 >  1. TCP theta 類泛素後修飾研究2. PAX3 配對結構域突變蛋白的轉錄調控機制

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/93132

標題: 1. TCP theta 類泛素後修飾研究2. PAX3 配對結構域突變蛋白的轉錄調控機制
1. The study on TCP theta NEDDylation 2. The studies on transcription activity of PAX3 PD mutants
作者: 陽森
Contributors: 陳良築;姚雅莉
Liang-Jwu Chen;Ya-Li Yao
Wen-Ming Yang
關鍵字: NEDDylation
日期: 2011
Issue Date: 2012-08-31 12:48:40 (UTC+8)
Publisher: 分子生物學研究所
摘要: PART 1:
Post-translational modifications (PTM) play a major role in regulating various cellular processes like cell cycle regulation, protein trafficking and signal transduction. NEDD8 is one such post-translational modifier involved in cell cycle regulation and cell proliferation. NEDD8 modifies its target proteins by conjugation to their lysine amino acid. This process is called NEDDylation. Until now the knowledge of NEDD8 target proteins and how NEDD8 conjugation regulates them is very little. We aimed to identify more NEDD8 conjugated proteins and identify the role of NEDDylation on them. NEDD8 stable complex was purified and stably associated proteins were identified by proteomic analysis. Of the many proteins found in the complex, TCP theta, a subunit of TCP-1 chaperonin drew our attention since it is critical for regulating the folding of many ingredients needed for normal cell cycle regulation and cell proliferation. We show that NEDD8 can conjugate to TCP theta. And we further found that TCP theta was NEDDylated at K459. To confirm that TCP theta is NEDDylated at K459, a lysine to arginine mutant was generated by site-directed mutagenesis. Surprisingly, the TCP theta (K459R) mutant was still conjugated by NEDD8. This suggests that TCP theta might have other lysines that can be conjugated by NEDD8. Hence, we pursued to identify NEDDylation sites in TCP theta K459R. TCP theta (K459R) overexpressed with NEDD8 was affinity purified and the NEDDylation site of TCP theta (K459R) was identified as K509 by LC-MS/MS. Over-expression of NEDD8 with TCP theta did not affect TCP theta's poly-glutamine folding function. This suggests that NEDDylation may not affect TCP theta's chaperonin ability . Our study has found TCP theta as a novel target of NEDD8 and TCP theta is conjugated by NEDD8 at K459 and K509. The function of NEDDylation on TCP theta still remains unknown.

PAX3 is a transcription factor essential for normal development of mammals. PAX3's PD and HD domains are DNA binding domains important for normal functioning of PAX3. Mutation in these domains results in Warrdenburg syndrome. Little is known about the role of PD domain mutants in transcription regulation. Hence, we sought to understand the mechanism of transcription regulation by PAX3 PD mutants. Transcription regulation assays showed that all PAX3 PD mutants repressed Gal4 promoter more strongly than wild type PAX3, but derepressed MITF and TRP1, two genes controlling the melanocyte lineage. We proposed that PAX3 PD mutants lost their ability to bind native promoters, and their interaction with co-factors and histone modifiers kept them from accessing the native promoters. In order to prove this model, PAX3 PD mutants' interaction with co-factors and recruitment of PAX3 PD mutants to PAX3 native promoters was determined. Indeed, all PAX3 PD mutants interacted with co-factors like KAP1 and HDAC10. These interactions suggest that the cofactors may quench all of the PD mutants from the promoter region, thereby derepressing the native promoters. Interestingly, PAX3 PD mutant were recruited to MITF promoter. To conclude, our study shows that PAX3 PD mutants derepress MITF and TRP-1 promoters. Also they can interact with HDAC10 and KAP1, which are previously known to help PAX3 derepress MITF and TRP-1. Interestingly PAX3 PD mutants were still recruited to MITF promoter. Our study has made a preliminary analysis of mechanism of PAX3 PD mutants' transcription and shown that they derepress MITF and TRP-1. More studies are needed to understand the PAX3 PD mutants' mechanism of transcription regulation.
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