分析和c-Maf有交互作用之蛋白質研究以及c-Maf對IL-10之調控

Abstract

轉錄因子c-Maf是調控IL-4基因之特異性轉錄因子，並且在第二型輔助型T細胞(T helper 2 cells)的分化中扮演一個極為重要的角色。在被刺激之後，c-Maf的表現量會在Th2細胞以及巨噬細胞中大大提升，並引發大量的IL-4以及IL-10之表現，抑制IL-12的表達。因此，我們假設c-Maf之所以在不同的細胞中會有不同功能，可能是透過和不同的蛋白質有交互作用所致。 利用酵母菌雙雜交系統，我們實驗室已經篩選出SUMOylation的E2 conjugating enzyme Ubc9，E3 ligase PIAS1，以及蛋白酪氨酸磷酸脢PTPN22可能和c-Maf有交互作用。於是藉由螢光共振能量轉移的方法，再次確認了c-Maf的確跟這三個蛋白質有交互作用。另外一項假設是既然c-Maf可以和SUMOylation以及去磷酸脢等酵素有交互作用，暗示了c-Maf可以被SUMOylation以及磷酸化，而這些轉錄後修飾也許會對於c-Maf的轉譯能力造成影響。因此，藉由螢光酵素實驗，發現c-Maf的SUMOylation以及磷酸化突變種對於IL-10基因啟動子有較差的轉錄能力。

C-Maf is an IL-4-specific transcription factor that plays a vital role in Th2 cells differentiation. Upon stimulation, c-Maf is highly upregulated in Th2 cells as well as macrophages, and induces significant IL-4 and IL-10 expression but downregulates IL-12 expression. Thus, we hypothesized that c-Maf may have different functions through interacting with cell-specific proteins in different types of cells. Using yeast two-hybrid system, our group demonstrated that c-Maf can interact with Ubc9, the SUMOylation-specific E2 conjugating enzyme, PIAS1, the SUMOylation E3 ligase, and protein tyrosine phosphatase non-receptor type22 (PTPN22). Therefore, by Fluorescence Resonance Energy Transfer (FRET) assay, the interaction between c-Maf, Ubc9, c-Maf, PIAS1, and c-Maf, PTPN22 are confirmed. Since c-Maf can interact with SUMOylation enzymes and de-phosphorylation enzyme, which suggests that c-Maf undergoes SUMOylation and phosphorylation, these post-translational modification may affect the transactivity of c-Maf. Thereafter, we found that both c-Maf SUMOylation and phosphorylation mutants have poorer transactivity on IL-10 promoter by luciferase assay.