繆希椿臺灣大學：免疫學研究所顏偉峰Yen, Wei-FengWei-FengYen2007-11-292018-07-092007-11-292018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/63319轉錄因子c-Maf 在不同種類的細胞中扮演不同的角色。例如c-Maf 在type 2 T Helper cell (TH2) 為IL-4基因之特異性轉錄因子。而在macrophages細胞中，c-Maf 則為IL-10基因的 activator。此外c-Maf 還能down-regulate IL-12 p40 and p35基因的轉錄作用。於是我們假設c-Maf 之所以在不同的細胞中會有不同功能可能是透過不同的交互作用的蛋白質所調控。而我們利用酵母菌雙雜交系統篩選CD8+T細胞c-Maf的交互作用蛋白質。最後我得到15個可能與c-Maf有交互作用的蛋白質，而其中之ㄧ為PTPN22。在這次研究中，我們發現primary resting or activated CD4+ T helper cells 會表現PTPN22。此外在in vitro 的實驗中可以發現PTPN22與c-Maf 的蛋白質交互作用。我們也發現PTPN22可以抑制c-Maf在IL-4 promoter 上的轉錄能力。同時在HEK 293T 細胞中我們可以發現c-Maf的蛋白酪胺酸有磷酸化的情形。而異位表現DsRed2-c-Maf 與EYFP-PTPN22時在細胞核中可以觀察到co-localization的現象。然而關於在in vivo 環境中c-Maf磷酸化的意義則需要更進一步去研究探討。The transcription factor c-Maf plays different roles in different types of cells. For instance, c-Maf is the IL-4–specific transcription factor in TH2 cells. Besides, c-Maf is a potent activator of the IL-10 gene expression whereas c-Maf could suppress the IL-12 p40 and p35 gene transcription in macrophages. We hypothesize that c-Maf could have distinct functions through interacting with cell-specific protein in different types of cells. We screened the c-Maf interacting protein in CD8+ T cell library by yeast two-hybrid system. Fifteen candidate genes were obtained. One of the candidate genes encoded a protein-tyrosine phosphatase, PTPN22. Here, we demonstrated that PTPN22 can express in primary resting or activated CD4+ T helper cells and c-Maf can interact with PTPN22 in vitro. Furthermore, we demonstrated that the PTPN22 had the ability to diminish the transactivity of c-Maf on IL-4 promoter. We also found that the tyrosine residues of c-Maf could be phosphorylated in HEK 293T cell line. Moreover, we found that ectopic-expressed DsRed2-c-Maf and EYFP-PTPN22 could co-localize in the nucleus. However, the functions of phosphorylated c-Maf in vivo need to be further elucidate.Acknowledgement i Abstract ii CHAPTER I Introduction 1 1.1.1 T helper cell development and differentiation 1 1.2 An overview of c-Maf transcription factor 2 1.2.1 The discovery of c-Maf 2 1.2.2 Protein structure of c-Maf 3 1.2.3 Regulation of c-Maf 4 1.3 An overview of PTPN22 5 1.3.1 Introduction of PTPN22 5 1.3.2 The discovery of PTPN22 5 1.3.3 Biological functions of PTPN22 6 1.4 Rationale to study the relationship between c-Maf and PTPN22 7 CHAPTER II Material and Methods 9 2.1 Experimental procedures 9 2.1.1Constructions 9 2.1.2 Tyrosine phosphorylation sites prediction 14 2.1.3 Yeast two-hybrid system 14 2.1.4 Cell culture 15 2.1.5 HEK 293T cells transfection 15 2.1.6 Luciferase assay 16 2.1.7 Purification of GST-PTPN22(201aa) fusion protein for the production of PTPN22 polyclonal antibodies 16 2.1.8 In vitro CD4+ T Helper cells differentiation ..17 2.1.9 Co-immunoprecipitation assay ..17 2.1.10 Nuclear and cytoplasmic extracts assay 20 2.1.11 Immunoprecipitation of phosphorylated c-Maf 20 2.1.12 Confocal microscopy 21 2.2 Experimental materials 22 2.2.1 Enzymes 22 2.2.2 Kits 22 2.2.3 Instruments and softwares 23 2.2.4 Chemicals, reagents and materials 23 2.2.5 Media, solutions and buffers 25 CHAPTER III Results 31 3.1 Identify potential c-Maf interacting proteins from CD8+ T cell cDNA library by yeast two-hybrid system 31 3.2 PTPN22 expresses in primary TH1 and TH2 31 3.3 PTPN22 could be the interacting partner of c-Maf 33 3.4 PTPN22 could reduce c-Maf transactivity in IL-4-luciferase reporter system 33 3.5 c-Maf could be tyrosine phosphorylated 34 3.6 The transactivity of c-Maf Y298F mutant was impaired 35 3.7 Cellular localizations of c-Maf and PTPN22 were analysed by confocal microscopy and nuclear and cytoplasmic extracts assay 36 3.8 PTPN22 and c-Maf could co-localize in the nucleus 36 CHAPTER IV Discussions 38 References 42 Figures and Tables 46en-US細胞激素輔助型T細胞c-Mafother