Sumoylation 對於c-Maf調控細胞激素之研究

Abstract

原生型輔助性T細胞可被朝向第一型輔助型T細胞或第二型輔助型T細胞的方向分化。c-Maf 是間白素-4（IL-4）專一的轉錄因子，主要表現在第二型輔助型T細胞中。c-Maf藉由促進間白素-4（IL-4）的產生，能造成更多未分化的輔助性T細胞分化為第二型輔助型T細胞。然而，在吞噬細胞（Macrophage）中，c-Maf可直接造成間白素-10（IL-10）活化並間接抑制間白素-12（IL-12）的p35及p40次單元表現。此外，c-Maf也被報導參與在癌化以及眼球晶體的發育過程中。這些現象暗示了也許有不同的因子與c-Maf交互作用進而調控其功能。 之前，我們實驗室利用酵母菌雙雜交系統（yeast two-hybrid 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。共篩選約300個，分類定序後確定為UBC-9 及PIAS1。這兩個蛋白質的主要功能是參與細胞內small ubiquitin-like modifiers （SUMO）轉譯後修飾作用。因此我們假設，c-Maf可被SUMO修飾，進而影響它活化IL-4的能力。 我們發現，c-Maf在HEK293T和DO11.10細胞中都可被SUMO修飾。此外，在HEK293T和DO11.10細胞，利用生物冷光素（Luciferase）為報告基因的偵測系統中，我們證明SUMO修飾對於c-Maf 調控IL-4基因表現上扮演負向調控的角色。此外，部分被SUMO修飾後的c-Maf可出現在細胞質。而在電泳遷移率改變分析（EMSA）的實驗中可看出，不能夠被SUMO修飾的c-Maf似乎有較高的與IL-4 promoter結合的能力。以上兩個現象，也許是造成被SUMO修飾後的c-Maf ，在調控IL-4基因表現的能力下降的原因。

Naïve CD4+ T helper (Th) cells can be polarized towards either the Th1 or the Th2 subset. Interleukin-4 (IL-4) is crucial for the differentiation toward Th2 effector cells that promote humoral immunity and provide protection against intestinal helminthes. Many transcription factors are involved in the regulation of the gene encoding IL-4. Notably, c-Maf, a basic-leucine zipper protein belongs to the MAF family, has been proved to be the IL-4 gene-specific transactivator. On the other hand, c-Maf also acts as a potent activator of IL-10 gene and an inhibitor of IL-12 p40 and p35 gene expression in monocytes and macrophages. Besides, c-Maf was reported to be involved in lens development and oncogenesis. These phenomena implied that some cell-type specific factors would interact with c-Maf and thus to regulate its function. Our lab has identified two c-Maf interacting factors, UBC9, and PIAS1, by the yeast two-hybrid system using c-Maf as the bait. The two proteins are involved in the sumoylation. Therefore, we assume that the sumoylation might play some important roles in the function of c-Maf and further regulate IL-4 production in Th2 cells. To study this issue, we want to investigate whether the sumoylation would occur in vivo and dissect how the post translational modifications affect c-Maf function. In this study, we found that c-Maf could undergo sumoylation in HEK293T and DO11.10 T cells. The functional analysis of sumoylated c-Maf was also performed by luciferase assay in HEK293T and DO11.10 cells indicating that sumoylation acts as a negative regulator to c-Maf in IL-4 transactivation. Sumoylated c-Maf could be revealed in cytosol. In addition, enhanced DNA binding ability could be observed when c-Maf without sumoylation. The two phenomena might contribute to reduced IL-4 gene transactivation by sumoyalted c-Maf.