효모세포 MAPK 신호 전달계에서의 티로신 질산화 단백질의 동정과 분석

Abstract

학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2015. 2. 박상현.Identification and characterization of a novel posttranslational modification is crucial for understanding of accurate signaling regulation mechanism. As many signaling pathways are conserved in eukaryotes from yeast to human, yeast could be a good model system for studying the mechanism of posttranslational modifications in cellular signaling pathways. Protein tyrosine nitration is a selective posttranslational modification that is involved in many diseases caused by oxidative stress. Recently, it is suggested that many signaling proteins are supposed to be nitrated on specific tyrosine residues and these tyrosine nitration regulate protein activity or localization and signaling flux. However, due to low abundance or tyrosine nitration and relevance of specific disease, protein tyrosine nitration in yeast was not studied well. In this study, we profiled and investigated the role of protein tyrosine nitration in yeast S. cerevisiae. Introducing enrichment methods using chemical and immunoprecipitation into LC-MS/MS, we successfully identified tyrosine nitrated proteins in yeast in vivo. This is the first nitroproteome study in yeast during signal transduction. 23 proteins were identified as nitrated in enrichment methods, and the overall level of nitration was increased after pheromone stimulation. Sequence and structural analysis showed that most tyrosine residues, surrounded by acidic residues and located in solvent accessible site, could be easily nitrated. These results imply that the environment of tyrosine residue is important to allow of tyrosine nitration, thus protein tyrosine nitration is a selective and reversible modification regulated during mating signal transduction. In addition, we showed that tyrosine nitration of Ste7 was increased during mating signaling and predicted Tyr 381 as a putative nitration residue. Substitution of Tyr 381 to Phe reduced mating signaling flux by inhibition of Ste7 activity, and this mechanism seems to be conserved in other MAPK signaling pathway in yeast such as Hog signaling pathway. Taken together, we suggest that tyrosine nitration is a novel modification which is regulated during MAPK signaling pathway in yeast. Also, proteins identified in this study could provide a clue for searching nitration targets in other signaling pathway or other species.ABSTRACT i COPYRIGHT INFORMATION iii TABLE OF CONTENTS iv LIST OF FIGURES ix LIST OF TABLES xi ABBREVIATION xii Chapter 1. Introduction 1 Chapter 2. Identification and analysis of protein tyrosine nitration in yeast model system 5 SUMMARY 6 INTRODUCTION 7 MATERIALS AND METHODS 10 Yeast strains and plasmids construction 10 Preparation of S. cerevisiae cellular extracts 10 Tryptic digestion of total cell lysate 11 Enrichment of nitroproteins using fluorinated carbon tags 11 LC-MS/MS analysis 12 Identification of nitrated peptides 12 Immunoprecipitation and western blot 13 Comparison of protein expression and nitration levels 14 Peptide sequences and protein structures analysis 14 Inference of nitroprotein functions from a functional network 15 Construction of network model 15 RESULTS 17 Identification of nitrated proteins from S. cerevisiae in vivo 17 Relative quantification of nitrated peptide in S.cerevisiae 19 Relationship between protein tyrosine nitration and the amount of protein in yeast 19 Selectivity of protein tyrosine nitration 20 Inference of functions and network analysis of nitroproteins in mating signal transduction pathway 23 DISCUSSION 36 Chapter 3. Protein tyrosine nitration in yeast mating signaling pathway 39 SUMMARY 40 INTRODUCTION 41 MATERIALS AND METHODS 44 Yeast strains and plasmids construction 44 Preparation of S. cerevisiae cellular extracts 44 Immunoprecipitation and western blot 45 Tryptic digestion of total cell lysate 45 LC-MS/MS analysis 46 Identification of nitrated peptides 46 Mating assay 47 RESULTS 48 Identification of nitrated proteins from Ste5 mating complex 48 Sequence and structural analysis of nitrated proteins from mating complex 49 Functional study of nitrated proteins from mating complex 51 DISCUSSION 62 Chapter 4. Catalytic activity of Ste7 MAPKK is regulated by tyrosine nitration at conserved Tyr 381 residue. 64 SUMMARY 65 INTRODUCTION 66 MATERIALS AND METHODS 68 Yeast strains and plasmids construction 68 Preparation of S. cerevisiae cellular extracts 68 Immunoprecipitation and western blot 69 Mating assay 69 RESULTS 71 Ste7 MAPKK is a candidate protein tyrosine nitration target. 71 Tyrosine nitration on Y381 ismay be important for proper mating signal transduction. 72 Mutation on Ste7 Y381 does not affect interactions with kinases or scaffold protein. 73 Y381F affects phosphorylation states of Ste7 74 Y381F decreases Ste7 catalytic activity itself 75 Ste7 Y381F act as dominant negative to inhibit endogenous Ste7 75 Hog pathway is downregulated by mutation on conserved nitration residue Y541 of Pbs2 MAPKK 76 DISCUSSION 101 Chapter 5. Conclusion 105 REFERENCES 107 ABSTRACT IN KOREAN 118Docto