繆希椿臺灣大學：免疫學研究所謝婉云Hsieh, Wan-YunWan-YunHsieh2007-11-292018-07-092007-11-292018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/63331c-Maf是主要表現在第二型輔助型T細胞中的轉錄因子，能有效的促進間白素- 4 ( IL-4 )的表現，並大量驅使原生型輔助型T細胞朝第二型輔助型細胞的方向分化。此外，在吞噬細胞(Macrophage)中，c-Maf則受間白素-10( IL-10 )活化並抑制間白素-12 ( IL-12 ) 的p35及 p40 次單元( subunit )表現。但c-Maf在不同細胞中扮演不同功能的調節機制仍舊不明。 為了解c-Maf在細胞中的調解機制，我們利用酵母菌雙雜交系統( yeast-two-hybride system )在輔助型T細胞中篩選出可能和c-Maf有交互作用的蛋白質，並詳加研究其相關的分子調節及生理影響。 c-Maf胺基酸端功能區為活化功能區，而羧基端區則負責和另一個c-Maf分子形成雙分子並結合至目標DNA。過去的研究指c-Maf的胺基酸端功能區中含有一段最小活化區( mini-transactivation domain；MTD) 能和TATA結合蛋白(TATA-binding protein；TBP)結合，在酵母菌雙雜交系統中直接活化下游報告基因(reporter gene)。因此，我們選用不含最小活化區的胺基端功能區作為餌( bait )篩選出和這段區域有交互作用的可能蛋白質。 我們共篩選約300個蛋白質，在分類定序後確定為UBC-9及 PIAS1。我們進一步以免疫共同沈澱法(co-immunoprecipitation)確定c-Maf與細胞蛋白質Ubc9及PIAS1之間的結合的確存在。 之前的研究指出，Ubc9及PIAS1的主要功能是參與細胞內small ubiquitin-like modifiers (SUMO)轉譯後修飾作用的蛋白質。SUMO轉譯後修飾作用具有許多不同的功能。爲了解SUMO轉譯後修飾作用是否調節c-Maf功能，我們利用生物冷光素(luciferase)為報告基因偵測c-Maf在接受SUMO的修飾後,是否影響其活化IL-4基因的能力。本實驗結果也證明,經SUMO修飾後的c-Maf調控IL-4基因表現的能力些微下降。After engagement of the TCR by the appropriate peptide-MHC complex, Helper T (Th) cells differentiate into Th1 or Th2 cells, which specialize in producing distinct cytokines to mediate different types of immune responses. c-Maf, a basic region/leucine zipper transcription factor, is induced during normal T cell differentiation along a Th2 but not Th1 lineage. In Th2 cells, c-Maf directs the production of IL-4 but not other Th2 cytokines; in macrophage, c-Maf selectively inhibits transcriptional activation of IL-12 p40 and p35 genes while potently activating IL-10 and IL-4 expression. These phenomena suggest that c-Maf displays different roles in different cell types, perhaps through interacting with distinct cell-specific proteins. For finding out the possible interacting partners, yeast-two-hybrid system was performed by using a 138bp c-Maf as the bait, and the activated Th library as the prey. Finally, PIAS1 and UBC9 were identified. PIAS1 and UBC9 both participate in the process of sumoylation as the E2-conjugating enzyme and E3-ligase, respectively. Sumoylation is a post-translational modification that modifies many proteins. In these studies, I demonstrated the in vivo interaction between PIAS1, UBC9, and c-Maf by co-immunoprecipitation. The functional analysis of sumoylated c-Maf was also performed by luciferase assay. The data show that sumoylation of c-Maf reduced the transactivation activity on IL-4 promoter.致謝 i 中文摘要 i Abstract iii Introduction 1 Part I. An overview of type II helper T cells 1 Part II. An overview of c-Maf in immune system 3 Part III. Rationale to study the cytokine regulation by c-Maf 6 Part IV. An overview of sumoylation 8 Material and method 10 Part I. Experimental procedures for identifying the interacting proteins of c-Maf by yeast-two-hybrid system 10 1.1 Construction of c-maf (bait) 10 1.2 Yeast transformation 11 1.3 Library transformation 12 1.4 Determination of replating efficiency 14 1.5 Survival test and lift assay 14 1.6 Isolation of plasmid DNA from positive yeast clones 15 1.7 Preparation of electrocompetent KC8 cells 17 1.8 Electroporation of KC8 18 1.9 PCR analysis of positive plasmids 18 1.10 AluI analysis of positive plasmids 19 Part II. Experimental procedures for studying the effect of UBC9 and PIAS1 on expression of IL-4 20 2.1 Maxi-preparation of plasmids 20 2.2 Cell culture 20 2.3 Co-Immunoprecipitation assay- Generation of proteins 21 2.4 Co-Immunoprecipitation assay- Immunoprecipitation 22 2.5 Co-Immunoprecipitation assay- Western blot 23 2.6 Transfection of M12, EL4, and Jurkat 23 2.7 Luciferase assay 24 Part III. Media and buffers 26 3.1 Media – LB, SOB, SOC, YPAD 26 3.2 Media – Dropped out media for yeast 27 3.3 Media – M9-Trp 28 3.4 Buffers – Lift assay 29 3.5 Buffers – Isolation of plasmids from yeast 30 3.6 Buffers – Transfection by calcium-phosphate 30 3.7 Buffers – Co-Immunoprecipitation, Protein electrophoresis and western blot 31 3.8 PMA and Ionomycin solution 32 Result 33 Part I. Yeast-two-hybrid system 33 Part II. Demonstration of the in vivo interaction between c-Maf, PIAS1, and UBC9 by Co-immunoprecipitation 35 Part III. Functional analysis of sumoylated c-Maf by Luciferase assay 36 Discussion 38 Part I. Identification of c-Maf interacting proteins by yeast-two-hybrid system 38 Part II. Demonstration of in vivo interaction between c-Maf/PIAS1 and c-Maf/UBC9 39 Part III. Functional analysis of sumoylated c-Maf by luciferase assay 39 Reference 44 Figure 1. Experimental design for yeast-two-hybrid screening. 51 Figure 2. Survival test for different length of c-maf constructions. 52 Figure 3. Lift assay for different length of c-maf constructions. 53 Figure 4. The results of library screening. 54 Figure 5. The classification of candidates by PCR analysis. 55 Figure 6. The classification of PCR products with AluI digestion 56 Figure 7. Binding of c-Maf and PIAS1 in vivo. 57 Figure 8. Binding of c-Maf and UBC9 in vivo. 58 Figure 9. The effect of sumolyation on c-Maf induced IL-4 production in M12 cells 59 Figure 10. The effect of sumoylated c-Maf on IL-4 induction in M12 cells 60 Figure 11. The effect of sumoylated c-Maf on IL-4 induction in different cell lines 61 Figure 12. The effect of mutated c-Maf on IL-4 induction in different cell lines 63 Table 1. Autoactivation assay for different length of c-Maf 64 Table 2. The data of sequencing analysis. 651446634 bytesapplication/pdfen-US細胞激素c-Mafotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/63331/1/ntu-94-R92449010-1.pdf