先天性免疫系統是人體對抗外來病原菌的第一道防線，當受到感染的時候，宿主的pattern-recognition receptors (PRRs)會辨識病原菌具有高保留性的結構pathogen-associated molecular patterns (PAMPs)並且活化許多訊息傳遞路徑。當有病毒入侵的時候，宿主細胞質中的重要病毒RNA接受器RIG-I-like receptors (RLRs)，包括RIG-I與MDA5，會偵測病毒RNA之後活化下游訊息傳遞路徑並產生proinflammatory cytokines與type I interferons。目前文獻大部分認為在RIG-I辨識到病毒RNA活化之後會位移至mitochondria，與位在mitochondrial的調控蛋白IPS-1/MAVS交互作用活化下游訊息。然而，並沒有直接的證據證明RIG-I受到刺激活化之後會與mitochondria以及IPS-1位於相同位置。TAPE (TBK1-Associated Protein in Endolysosomes)是我們實驗室發現的一個新的先天免疫調控分子。實驗結果指出，TAPE是一個位於endolysosome的調控蛋白，並且位於IPS-1上游參與RLR的訊息傳遞路徑。因此，我們推測RIG-I在受到病毒刺激之後會先位移到endolysosomes。我論文主要的工作是探討TAPE以及endolysosomes在RLR的訊息傳遞中扮演的角色。我們已發表的文獻以及我的結果顯示，TAPE在細胞受到刺激的前後都會與endolysosomes的標示蛋白位於相同位置，並且TAPE是一個會與磷脂質結合的蛋白，根據以上結果推斷TAPE是一個能結合於endolysosomes的蛋白。另外，我的結果顯示TAPE-N與RIG-I-CARD會有交互作用，並且刺激之後的TAPE會與RIG-I位於相同位置。因此我想進一步確認RIG-I在受到刺激之後是否會位移到endolysosomes。根據我的螢光結果，RIG-I在受到刺激之後會與early endosome的標示蛋白Rab5位於相同位置。另外，knockdown endolysosome標示蛋白Rab5以及Rab7的時候，會抑制RIG-I及MDA5活化IFN-β，而knockdown Rab5以及Lamp1的時候則會抑制RIG-I及MDA5活化NF-κB，進一步用抑制lysosome酸化的藥物作用後也會抑制RIG-I訊息傳遞路徑。不同於目前的想法，RLR的訊息傳遞中mitochondria是關鍵胞器，我的結果指出，endolysosomes也參與在RLR的訊息傳遞路徑中。之後的工作則會更深入探討TAPE是如何調控RLR的訊息傳遞。

The innate immune system is the first line of defense against invading pathogen. Following infection, host pattern-recognition receptors (PRRs) detect highly conserved components of pathogen known as pathogen-associated molecular patterns (PAMPs) and trigger multiple signaling pathways. During virus invasion, host RIG-I-like receptors (RLRs), including RIG-I and MDA5, are key cytosolic sensors for detecting viral RNA to induce the activation of proinflammatory cytokines and type I interferons. Studies suggested that viral RNA activates RIG-I translocation to mitochondria, so that RIG-I can interact with a mitochondrial adaptor IPS-1/MAVS for triggering downstream signaling. However, direct evidences indicating that RIG-I is co-localized with IPS-1 at mitochondria after virus infection is still lacking. TAPE (TBK1-Associated Protein in Endolysosomes), an innate immune regulator, was previously identified in our lab. Our data suggest that TAPE is an endolysosomal adaptor acting upstream of IPS-1 in RLR signaling. Therefore, we are interested to know whether RIG-I translocates to endolysosomes after viral stimulation. The work at my thesis aims to determine the role of TAPE adaptor and endolysosomal mediators in RLR signaling. The previous studies of our lab and my current results revealed that TAPE is co-localized with endolysosomal proteins before and after stimulation. My results also showed that TAPE is able to bind phospholipids. These data support the idea that TAPE is able to target endolysosomes. In addition, my results showed that TAPE-N interacts with RIG-I-CARD and TAPE is co-localized with RIG-I after stimulation. Then, I further confirmed whether RIG-I translocates to endolysosomes after stimulation. According to my fluorescent data, RIG-I is co-localized with early endosomal protein Rab5 after stimulation. Moreover, my results also showed that knockdown of endolysosomal protein Rab5 and Rab7 impaired the IFN-β pathway mediated by RIG-I and MDA5, while knockdown of Rab5 and Lamp1 impaired the NF-κB pathway mediated by RIG-I and MDA5. Furthermore, treatment of cells with lysosomal inhibitors blocked the RIG-I mediated IFN-β activation. Distinct from the current idea which mitochondria are key compartments for RLR signaling, our preliminary data suggest that endolysosomes are also involved in the RLR pathways. Future work will determine how TAPE regulates the RLR signaling pathways.

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