病毒介導的傳染病導致每年嚴重的發病率和死亡率。哺乳動物先天免疫系統逐漸清楚是由多種內體TLR和細胞質病毒感測器組成並偵測病毒核酸，在早期病毒感染時誘發第一型干擾素的抗病毒防禦。TBK1是參與RLRs, TLR3, TLR4以及DNA sensor活化第一型干擾素的重要蛋白激酶。我們先前發現一個先天免疫調控分子稱為TAPE (TBK1-associated protein in endolysosomes)，在哺乳類細胞中TAPE是一個位在endolysosomal的銜接蛋白並參與在TLR3, TLR4和RLRs產生第一型干擾素的路徑中。由於這些原因，我們對於利用遺傳學及生物化學方法探討TAPE在調節RLR和內體TLR路徑的角色感到興趣。不同於先前的Tape -/-小鼠胚胎纖維母細胞(MEFs) 仍然具有一段TAPE截斷N端區域，我建立了新的TapeKO/KO確定其TAPE基因完整剃除無法偵測到TAPE蛋白來進行體外實驗。從酵素免疫分析法結果得到在初代小鼠胚胎纖維母細胞中TAPE基因缺失時會減低RLR活化，並且在TAPE基因缺失的骨隨分化之樹突細胞中也看到相同的結果。由於TAPE與RIG-I和MAVS會形成一個複合物，並且TAPE的N端區域會與RIG-I 的CARD區域相互作用。因此，我主要探討TAPE和MAVS分別經由哪些區域去相互作用。由共免疫沉澱實驗指出TAPE的C端會與MAVS的C端區域作用。我評估了TAPE對RIG-I泛素化的影響。由生物化學結果顯示，TAPE基因下調減少了RIG-I的K63多聚合泛素化。另外實驗也顯示TAPE與TRIM25有相互作用並進一步調節TRIM25的K63連鎖多聚合泛素化。此外，先前實驗室研究已知在哺乳類細胞中發現TAPE參與在TLR3的路徑中。我的論文進一步利用基因剔除的實驗方法確定TAPE在內體TLR3和TLR7的路徑當中之角色。體外實驗指出在初代小鼠胚胎纖維母細胞、骨隨分化之樹突細胞和巨噬細胞中TAPE基因缺失時會降低TLR3的活化。反之，漿細胞樣樹突細胞（pDC）中的TAPE基因缺失時在TLR7路徑中沒有顯著地影響。綜合上述，我的論文工作證明了在RIG-I的訊號傳導過程中，TAPE會調控TRIM25和RIG-I的泛素化，並且也可能扮演一個連接RIG-I到MAVS的介質。此外，在初代細胞中TAPE確實在TLR3的訊號傳導路徑扮演一個重要的角色。

Virus-mediated infectious diseases cause the severe morbidity and mortality every year. It becomes clear that the mammalian innate immune system consists of several endosomal TLRs and cytosolic viral sensors, which can detect viral nucleic acids to trigger type I IFN-mediated antiviral defenses at the early viral infection. TBK-1 is an important protein kinase linking RIG-I-like receptors (RLRs), TLR3, TLR4 and DNA sensors to the activation of type I IFNs. Our previous work uncovered an innate immune regulator called TAPE (TBK1-associated protein in endolysosomes), which is an endolysosomal adaptor involved in linking the TLR3, TLR4 and RLRs signaling pathways to type I IFN production in mammalian cells. Given these facts, we were interested in further understanding how TAPE regulates the RIG-I-like receptor (RLR) pathway and endosomal TLR pathways by genetic and biochemical approaches. Distinct from a Tape -/- mouse embryonic fibroblast (MEF) line which still encodes a truncated N-terminal region of TAPE, a new TapeKO/KO MEF line, which has no detectable TAPE protein, has been generated for ex vivo experiments. Results from ELISA analyses showed that TAPE deficiency impaired RLR activation in primary MEFs. Similarly, TAPE-deficienct bone marrow derived dendritic cells (BMDCs) were impaired in RLR signaling. Given the facts that TAPE forms a complex with RIG-I and MAVS and the N terminal domain of TAPE interacts with the RIG-I CARD domains, I thus focused on mapping the interaction domains between TAPE and MAVS. Co-immunoprecipitation experiments indicated that the C terminal domain of TAPE interacted with the C terminal region of MAVS. It is known that RIG-I activation and signaling is regulated by ubiquitination and oligomerization of RIG-I. I thus assessed the effect of TAPE on the ubiquitination of RIG-I. Biochemical results revealed that TAPE knockdown reduced K63-linked polyubiquitination of RIG-I. Moreover, TAPE was shown to interact with TRIM25 and regulate the K63-linked polyubiquitination of TRIM25. In addition, previous work from our lab demonstrated that TAPE is involved in the TLR3 pathway in a mammalian cell line. My thesis work further used the genetic knockout approach to assess the role of TAPE in the endosomal TLR3 and TLR7 pathways. Ex vivo studies showed that TAPE deficiency impaired TLR3 signaling in MEFs, BMDCs and bone marrow derived macrophages (BMDMs). In contrast, TAPE deficiency in plasmacytoid dendritic cells (pDCs) showed no obvious defect in the TLR7 pathway. Together, my work suggests that during RIG-I signaling, TAPE functions to regulate the ubiquitination of TRIM25 and RIG-I, and also may bridge RIG-I to MAVS. Also, TAPE is required for in the TLR3 pathways in primary cells.

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