由生化分子層次到模式動物實驗之系統性探討酪氨酸亞硫酸化蛋白質體研究

Abstract

酪氨酸亞硫酸化為一種蛋白質轉譯後修飾，主要利用酪氨酸亞硫酸基轉移酶將3’磷酸-5’磷酸亞硫酸腺苷的亞硫酸基轉移至特定胜肽的酪氨酸而調節許多重要的生理與病理反應，如：發炎反應、趨化細胞激素引發的免疫反應、以及病毒入侵等。在此論文中，希望藉由發展高效率之生化工具與蛋白質體平台進行系統性探討酪氨酸亞硫酸化的酵素機制與生理功能。首先我們利用原核表現系統成功純化出具有酵素活性且高純度之酪氨酸亞硫酸基轉移酶，並進而發展出一套原位3’磷酸-5’磷酸亞硫酸腺苷生產平台以供應予酪氨酸亞硫酸基轉移酶進行催化反應。結果顯現，利用此套平台可比一般傳統的方法高出40倍以上的催化效率。另外，先前文獻指出，在酪氨酸亞硫酸基轉移酶上的單點突變（在果蠅中此胺基酸在酵素的位置為H269Q）將會導致甲狀腺低下所引發的侏儒症。此研究發現，將His-269取代成其他不同特性的胺基酸會造成酵素活性大幅減弱，其詳細機制將探論之。最後，我們製備酪氨酸亞硫酸基轉移酶缺陷的果蠅，並結合此研究所開發的生化分析工具、蛋白質體技術、與果蠅抗壓能力測試，發現酪氨酸亞硫酸基轉移酶缺陷的果蠅對於抗氧化壓力與飢餓有明顯較差的耐受性，也另外發現其他新穎的酪氨酸亞硫酸化蛋白質與其可能參與的生理功能。在此論文中，除了提供重要的生化分析方法，更是第一個提出酪氨酸亞硫酸化蛋白質體研究。藉由這些工具與平台的搭配整合，不僅可以發現更多酪氨酸亞硫酸化的蛋白質與功能，更可便於分子層次與生理反應雙向研究的交互配合。

Tyrosine O-sulfation, catalyzed by tyrosylprotein sulfotransferases (TPST, EC 2.8.2.20), is a post-translational modification, which transfers a moiety of sulfuryl group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to specific tyrosine residues within polypeptides. Protein tyrosine sulfation regulates many important physiological and pathological functions including inflammation, chemokine signaling, and virus entry. In this dissertation, I developed feasible biochemical tools and a sulfoproteomics platform to comprehend TPST actions and biological functions. Firstly, a heterologous TPST expression system and purification procedure was developed to produce the enzymatically active recombinant TPSTs at near homogeneity. An in situ PAPS generating system was further established to efficiently determine the activities of recombinant TPSTs. Our data indicated that the combination of in situ PAPS generating system with TPST-catalyzed reaction significantly improved the apparent productive rate of tyrosine-sulfated proteins over 40-fold magnitude. A single point mutation, H269Q of Drosophila melanogaster TPST which resulted in dwarfism-associated hypothyroidism, would contribute to weak enzyme activity. Detailed mechanism regarding structure-function relationship was discussed. Furthermore, TPST-knockdowned D. melanogaster was generated and investigated using the biochemical tools developed in this research and conventional proteomics technology. By the identified proteins in proteomics process and the stress paradigm of paraquat treatment and dietary restriction, the TPST-deficient flies were confirmed to be labile under both stresses of oxidation and starvation. This research provides feasible biochemical tools and pioneering sulfoproteomics approach to uncover the significance of protein tyrosine sulfation from molecular level to a complex organism.