探討ADP核糖基化因子於類鐸受體9之免疫訊息的功能

Involvement of ADP-ribosylation Factors in Toll-Like Receptor 9-mediated Immune Signaling Pathway

吳京穎(Jing-Yiing Wu)

指導教授 ： 郭呈欽 ； 詹鴻霖

國立清華大學/生命科學院/生物資訊與結構生物研究所/博士(2013年)

摘要

摘要
類鐸受體9經由辨識致病性CpG去氧核醣核酸(CpG DNA)/CpG寡聚核苷酸(CpG ODN)會產生促發炎因子。在這過程中，短片段CpG寡聚核苷酸(CpG ODN)進入核內體以及類鐸受體9從內質網運輸至核內體則是引起初級免疫反應的兩個重要步驟。而ADP核糖基化因子(ARF)是屬於與鳥嘌呤核苷酸結合的Ras蛋白，主要參與細胞中的胞飲作用和囊泡運輸。在本研究中，我們發現抑制ARF3或ARF6會降低CpG ODN所調控的反應，而持續活化ARF3或ARF6則會提高此反應。進一步的結果指出在ARF6活化的細胞中會增加CpG ODN被攝入細胞，但於ARF6缺失的細胞中則會減少CpG ODN被攝入細胞的結果。細胞預先施與CpG ODN則會誘發ARF6的活性進而增加CpG ODN被攝入細胞；此外並證明Class III PI3K為下游ARF6調控CpG ODN進入細胞所需。這些結果推得出一條以class III PI3K-ARF6為軸的路徑，此路徑經由調控CpG ODN進入細胞的過程，從而調節類鐸受體9的訊息。相對於ARF6參與CpG ODN/類鐸受體9所調控的反應，ARF3的活化則不影響CpG ODN進入細胞，但是參與類鐸受體9運輸至核內體的過程，也因此影響了裂解型式的功能性類鐸受體9形成。而於免疫沉澱的實驗中則指出具活性的ARF3會與類鐸受體9以及受體的運送蛋白UNC93B1相互作用，所以證明ARF3是經由調控類鐸受體9從內質網運輸至核內體的過程，進而調控類鐸受體9的訊息。總結來說，class I ARF3和class III ARF6在類鐸受體9的訊息路徑中各自扮演不同的角色，ARF6參與CpG ODN進入細胞內的過程，而ARF3則調控類鐸受體9的運輸從而活化CpG ODN/類鐸受體9的免疫反應。

關鍵字

類鐸受體9 ； ADP核糖基化因子 ； CpG寡聚核苷酸進入細胞 ； 類鐸受體9運輸

並列摘要

Abstract
Toll-like receptor 9 (TLR9) is responsible for proinflammatory cytokine production via recognizing pathogenic unmethylated CpG DNA. In the process, CpG oligodeoxynucleotides (CpG ODN) uptake into endosomes and TLR9 trafficking from endoplasmic reticulum (ER) to endosomes are two critical steps in eliciting innate immune responses. ADP-ribosylation factors (ARFs) are members of the Ras superfamily of guanine nucleotide-binding proteins, which are involved in mediating a wide variety of cellular events including endocytosis and vesicle trafficking. Here, we found that inhibition of ARF3 or ARF6 by dominant mutants and siRNA impaired CpG ODN-mediated responses, whereas constitutive active ARF3 or ARF6 mutant enhanced these responses. Additional studies showed that CpG ODN uptake was increased in ARF6-activated cells but impaired in ARF6-defective cells. Cells pre-treated with CpG ODN had increased CpG ODN uptake due to CpG ODN-induced ARF6 activity. Further studies with ARF6-defective and ARF6-activated cells demonstrated that class III phosphatidylinositol 3-kinases (PI3K) were required for downstream ARF6 regulation of CpG ODN uptake. These results propose that a novel class III PI3K-ARF6 axis pathway mediates TLR9 signaling by regulating the cellular uptake of CpG ODN. In contrast to the involvement of ARF6 in CpG ODN/TLR9-mediated responses, ARF3 activity is essential for TLR9 trafficking into endolysosomes without affecting CpG ODN uptake, thereby regulating the formation of functional cleaved TLR9. In addition, experiment with immnuoprecipitation indicated that activation of ARF3 interacted with the TLR9 and the carrier protein, UNC93B1. Thus, the results demonstrate that ARF3 mediates CpG ODN-driven responses by regulating TLR9 trafficking from ER into endolysosomes. Collectively, class I ARF3 and class III ARF6 play distinct roles in CpG ODN/TLR9-mediated responses. ARF6 directly participates in CpG ODN internalization, while ARF3 drives TLR9 trafficking to activate CpG ODN/TLR9-induced immune responses.

並列關鍵字

Toll-like receptor 9 ； ADP-ribosylation factor ； CpG ODN uptake ； TLR9 trafficking

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