探討先天性免疫調控分子TAPE與endolysosomes在RIG-I-like receptor訊息傳遞中的角色

Abstract

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.