酪氨酸亞硫酸化蛋白質轉譯後修飾之生物功能

Abstract

第二型膜蛋白的蛋白質酪氨酸亞硫酸基轉移酶(TPST, EC 2.8.2.20)，催化蛋白質酪氨 酸亞硫酸化，進而調控許多生理與病理功能，例如人類免疫缺陷病毒(HIV)入侵、發炎 與凝血反應、不孕等徵候。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。我們將探討人類、果蠅(Drosophila melanogaster)與阿拉伯芥(Arabidopsis thaliana)等模式生物之TPST 酵素反應、生理調控、其演化意義和功能。首先我們建立 一套重組蛋白質表現系統，生產具酵素活性且高純度之TPST 膜蛋白，並發展其他關鍵 技術，如3’磷酸腺甘酸5’磷酸硫(PAPS)原位生合成系統，以及利用酵素連結免疫分析法 (ELISA)為基礎之快速檢測流程，用以高靈敏監測TPST 活性。重組TPST 也將應用於研 究蛋白質交互作用反應。另外我們將解析TPST 與其受質共結晶結構，受質將為HIV 共 受體(CCR5)與白血球配對受體(PSGL-1)，藉此了解TPST 作用機制並進行電腦輔助虛擬 藥物設計。TPST 基因轉殖果蠅與阿拉伯芥也將應用於了解此酵素表現位置與時期、受 調控蛋白質與訊息路徑及其生理功能。結合蛋白質體技術、酵素活性分析與生物顯性檢 測，將可發掘更多新穎TPST 受質。未來，我們將設計核甘酸cross-linker 衍生物，藉由 共價鍵結、西方墨點法與質譜儀分析鑑定TPST 及其蛋白質結合體之身分。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。

Biological Functions of Post-translational Protein Tyrosine O-sulfation Tyrosine O-sulfation, catalyzed by a type II membrane protein, tyrosylprotein sulfotransferase (TPST, EC 2.8.2.20), regulates many important physiological and pathological functions, such as HIV entry, inflammation, coagulation, and sterility. It is surprising that little research has been devoted to study such important enzymatic actions and TPST-mediated pathways. In the proposed research, we will examine TPSTs from Homo sapiens, Drosophila melanogaster, and Arabidopsis thaliana to comprehend their enzymatic actions and relationship in evolution. The purpose of this research is first to develop a heterologous expression system and other biochemical techniques for the characterization of TPSTs. An in situ radioactive [S35]3’-phosphoadenosine-5’-phosphosulfate (PAPS) generating system and an ELISA-based assay are developed to efficiently determine the activities of recombinant TPSTs. The structure-function relationship of protein tyrosine sulfation will be studied via X-ray crystal structures of TPST and co-crystallize with its substrates, such as HIV co-receptor, CCR5, and leukocyte counter-receptor, PSGL-1, respectively. The structural information will be used to clarify the catalytic mechanism of TPST and to further screen potential drugs candidates against TPST by computational simulation. The recombinant TPST will also be applied to pull-down assay to discover its interactive proteins amongst proteome and further analyze via Mass identification. TPST-transgenic Drosophila melanogaster and Arabidopsis thaliana will be generated and used to investigate the locations and developmental stages of TPST gene, TPST-regulatory proteins, and physiological functions of protein tyrosine sulfation. The combination of proteomic approaches, TPST enzyme assay, and phenotype examinations will be used to discover novel TPST substrates and further understand their significances in biological system. In the future, the design of synthetic PAP nucleotide analogues will be undertaken to fit into TPST active site and crosslink with TPST and TPST-interactive proteins. These strings of complex could be spread by 2-D gel electrophoresis and monitored via anti-TPST antibody. By utilizing these tools, TPST actions and related signaling pathways can be elucidated. We expect that the completion of this project will uncover more about the significances of protein tyrosine sulfation and help us to understand its biological roles from molecular level to complex organism.