蛋白質亞硫酸化之生化機制與生物調控

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(TPST, EC 2.8.2.20)將3’磷酸腺甘酸5’磷酸硫(PAPS)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。相較於TPST的重要性，其酵素反應與生理調控之機制，卻仍鮮少研究。在此計畫中，希望藉由發展高效率之生化工具與酪胺酸亞硫酸化偵測平台進行系統性與跨物種的方式來探討TPST之酵素反應、生理調控、其演化意義和功能。過去三年計畫中，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TPST，並發展其他關鍵技術，如PAPS原位生合成系統、酚之亞硫酸酸基轉移酶與酪氨酸亞硫酸基轉移酶螢光量測平台，以及利用酵素連結免疫分析法(ELISA)為基礎之快速檢測流程，這些方法有別於傳統量測TPST酵素的方式，將以高靈敏的特性來監測不同物種間之TPSTs活性，並進一步觀測亞硫酸化蛋白質使否與其特定蛋白質之交互作用。除此之外，我們將與同步輻射中心合作解析TPST1與TPST2的結構、TPST與其受質共結晶結構和亞硫酸化蛋白質於其作用蛋白質間之結晶複合結構，透過這些結構資訊來了解TPST之催化機制和亞硫酸化蛋白質與其作用蛋白質間之作用機制，未來將有利於進行電腦輔助虛擬藥物設計與開發。另外，將利用本研究開發出來的量測工具來探討TPST上的單點突變如何導致甲狀腺低下所引發的侏儒症之原因與其詳細生化機制。未來結合蛋白質體技術與蛋白質晶片技術，將可發掘更多新穎的與亞硫酸化蛋白質交互作用之蛋白質及其反應路徑。此計畫的完成將有助於系統性了解蛋白質酪氨酸亞硫酸化之功能。

Tyrosine O-sulfation catalyzed by tyrosylprotein sulfotransferases (TPST, EC 2.8.2.20) is a post-translational modification that transfers 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. It is surprising that little research has been devoted to study enzymatic actions of TPST-mediated pathways. The purpose of this research is to explore the catalytic mechanism of TPST and its role in the regulation of protein sulfation. We will first develop feasible biochemical tools and protein sulfation assay to analyze evolution, enzyme actions, and biological functions of TPSTs from cross-species. During the last three-year project, we have successfully developed a heterologous TPST expression system and purification procedure to produce enzymatically active recombinant TPSTs at homogeneity. An in situ radioactive [S35] 3’-phosphoadenosine-5’- phosphosulfate (PAPS) generating system, a real-time fluorescence assay of TPST and an ELISA-based assay are also developed to efficiently determine the activities of recombinant TPSTs and to identify sulfated proteins/peptides. Recently, we are also able to observed protein-protein interactions regulated through protein sulfation. The structure-function relationship of protein tyrosine sulfation will be studied via X-ray crystal structures (in collaboration with National Synchrotron Radiation Research Center) of sulfated protein-protein complex, TPST and co-crystallize with its substrates, such as HIV co-receptor, CCR5, and leukocyte counter-receptor, PSGL-1. The structural information will be used to clarify the catalytic mechanism of TPST and protein-protein interaction regulated through protein sulfation and to help for the screening of potential inhibitors/drugs candidates against TPST by computational simulation. A disease target, dwarfism-associated hypothyroidism, that is related to a single point mutation of TPST will be further examined. Our preliminary study indicates that point mutation at H269Q significantly affects Drosophila melanogaster TPST, which resulted in weak enzyme activity. In the future, protein sulfation will be examined by 2-D electrophoresis or protein chips and following by mass spectrometry analysis. By utilizing these tools, TPST actions and related signaling pathways can be elucidated. We expect that the completion of this project will help to uncover more about the significances of protein tyrosine sulfation and to understand its biological roles from molecular level to complex organism.