Biological Functions of Post-Translational Protein Tyrosine O-Sulfation
|摘要:||第二型膜蛋白的蛋白質酪氨酸亞硫酸基轉移酶(TPST, EC 220.127.116.11)，催化蛋白質酪氨
(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
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
Biological Functions of Post-translational Protein Tyrosine O-sulfation Tyrosine O-sulfation, catalyzed by a type II membrane protein, tyrosylprotein sulfotransferase (TPST, EC 18.104.22.168), 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.