陰道滴蟲Myb3蛋白質降解機制之分析

Analysis of Myb3 protein degradation in Trichomonas vaginalis

張怡婷(Yi-Ting Chang)

指導教授 ： 翁秀貞 ； 戴榮湘

國立臺灣大學/醫學院/微生物學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.03400

摘要

陰道滴蟲Myb3是活化受鐵調控ap65-1基因的轉錄因子，將陰道滴蟲細胞轉換到新鮮培養液後，發現Myb3會被快速的運送出細胞核，且有加速降解的現象。
根據Myb3的胺基酸序列比對分析，發現Myb3蛋白質C端中有PEST序列存在。將Myb3之PEST序列刪除，導致Myb3穩定性大幅提升，顯示Myb3可能經由PEST序列所參與的泛素-蛋白酶體路徑降解。故利用鋅離子螯合劑TPEN及蛋白酶體活性抑制劑MG-132進行蛋白質降解分析，結果Myb3降解被抑制，顯示此一現象與鋅離子主導之泛素化-蛋白酶體路徑相關。此外，藉由免疫沉澱分析及液相層析串聯質譜技術(Liquid chromatography-tandem mass spectrometry)，發現S87及S203為Myb3主要的磷酸化位置，而後者位於PEST序列中；另外，也發現了和Myb3鍵結的泛素分子。
以定點突變分析發現S203的磷酸化促進Myb3降解，而S87的磷酸化則為穩定Myb3，這些觀察顯示S87與S203的磷酸化反向調控Myb3降解。以上結果顯示，當陰道滴蟲細胞轉換到一個營養充裕的環境，會經由一個嚴密的機制來調控Myb3的命運。未來將會進一步探討上述與Myb3蛋白質降解相關的因子，研究Myb3於陰道滴蟲基因調控上所扮演的角色。

關鍵字

陰道滴蟲 ； Myb3轉錄因子 ； 蛋白酶體 ； 泛素 ； 蛋白質降解分析 ； 免疫沉澱法分析 ； 西方轉漬分析

並列摘要

A Myb3 transcription factor was previously demonstrated to up regulate iron-inducible ap65-1 gene transcription of the protozoan parasite Trichomonas vaginalis . In the present study, Myb3 was found to undergo nuclear export followed by a zinc-dependent ubiquitination and proteasomal degradation right after passages of the parasite to fresh medium. The protein stability increased with the deletion of a PEST sequence from the carboxyl terminus of Myb3. In addition, the zinc chelator TPEN and proteasomal inhibitor MG-132 stabilized the expression of Myb3. The data presented here demonstrate that Myb3 is likewise degraded by the ubiquitin-proteasome system. Using the immunopreciptation coupled with liquid chromatography-tandem mass spectrometry, two phosphorylation sites S87 and S203 in Myb3, the later which lies within the PEST sequence, and the ubiquitin molecule conjugated with Myb3 was identified. Site directed mutagenesis of serine into alanine or the phosphomimetic aspartic acid revealed that the phosphroylation of S87 or S203 may respectively slowdown or accelerate ubiquitination and degradation of Myb3. Together, these observations suggest that the fate of Myb3 must be tightly regulated when aging cells are replete with ample nutrients.

並列關鍵字

Trichomonas vaginalis ； Myb3 ； proteasome ； ubiquitin ； immunoprecipitation ； Western blot assay

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