SUMO化修飾減弱c-Maf相關之介白素四與介白素二十ㄧ之表現

Abstract

轉錄因子的功能是藉由控制其生成、活性與降解來嚴格調控的，而SUMO化修飾是在轉錄後層級調控蛋白質的活性。c-Maf蛋白是屬於大Maf家族的鹼性白氨酸拉鍊蛋白，在第二或第十七型輔助T細胞上分別為介白素四與介白素二十一的專一性轉錄因子。我們藉由酵母雙雜交系統來尋找c-Maf的交互作用蛋白。我們發現兩個SUMO化修飾的關鍵酵素：Ubc9與PIAS1，可以與c-Maf蛋白交互作用。在T細胞中，PIAS1可以與c-Maf蛋白交互作用；這兩個SUMO化修飾的關鍵酵素也與c-Maf蛋白共同位於細胞核中。我們也確認c-Maf蛋白在活體外與活體內都可被SUMO修飾。c-Maf蛋白N端第33個離胺酸是SUMO蛋白的修飾位。SUMO化修飾會降低c-Maf蛋白對介白素四的轉錄活性，而無法被SUMO化修飾的c-Maf蛋白卻有較強的轉錄活性。同時，我們也發現c-Maf是介白素二十一的專一性轉錄因子。SUMO化修飾也會影響c-Maf蛋白相關之介白素二十一的產生。SUMO化修飾不會影響c-Maf蛋白的穩定性，但無法SUMO化修飾的c-Maf蛋白匯集到啟動子的能力卻較強。我們的結論是c-Maf蛋白的SUMO化修飾對其在輔助T細胞中的活性十分重要。

The function of transcription factor is tightly regulated by controlling their synthesis, activity and degradation. SUMOylation modulates target protein activity on post-translational level. c-Maf, the cellular homologue of v-Maf, is a basic-leucine zipper protein and belongs to the large Maf family. In helper T cells, c-Maf is the specific transcription factor of the IL-4 and IL-21 genes in type 2 T helper (Th2) and type 17 T helper (Th17) cells, respectively. In our study, we performed the yeast two-hybrid assay to identify the c-Maf interacting proteins. We found that c-Maf can interact with Ubc9 and PIAS1, the key enzymes of SUMOylation system. In T cells, c-Maf interacts with PIAS1 in primary T cells and also co-localizes with these two SUMO ligases in the nucleus. We also demonstrated that c-Maf can be SUMOylated in vitro and also in vivo. We identified the c-Maf SUMO acceptor site(s) by mutated the putative conjugating lysine residues. We demonstrated that N-terminal lysine-33 within the activation domain is the SUMO acceptor site for c-Maf. SUMO modification attenuates Wt-c-Maf transcriptional activity. Conversely, c-Maf SUMO deficient mutant is more potent to drive IL-4 production in Th2 cells. Furthermore, we showed that c-Maf, but not other transcription factor, transactivates IL-21 gene expression. SUMOylation also affects c-Maf dependent IL-21 production. In addition, SUMO deficient c-Maf does not alter the localization and the protein stability, but further enhances its recruitment to the Il4-promoter. We conclude that post-translational lysine-33 SUMOylation is critical for c-Maf activity in helper T cells.