c-Maf 及其交互作用蛋白調節細胞激素表現之探討

Abstract

c-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.