NOD1與NOD2是位在細胞質中用於偵測細菌肽聚醣(peptidoglycan)的蛋白，藉由驅動NF-κB來活化發炎反應。NOD2是第一個被發現與克羅恩病(Crohn’s disease)相關的基因。NOD2在自噬作用(autophagy)中因可以標的入侵的胞內菌而發揮關鍵作用，特別是NOD2顯示與自噬蛋白ATG16L1有相互作用。NOD2缺少transmembrane domains，然而一些報導顯示NOD2會被招募到胞內體(endosome)並在那裡偵測細菌入侵。TAPE (TBK1-Associated Protein in Endolysosomes)是與TLR3、TLR4與RIG-I路徑調節有關的先天免疫蛋白，我們實驗室先前的研究發現TAPE缺陷的巨噬細胞在活的Salmonella感染後所活化的炎性細胞因子(inflammatory cytokine) 較正常細胞低，並且也缺乏抑制Salmonella感染時菌數的生長，但不影響E. coli感染時細菌的清除。此外生化分析結果顯示在Salmonella感染時TAPE缺陷的巨噬細胞活化的自噬蛋白LC3也較正常細胞少，這些數據都說明TAPE在防禦Salmonella感染的自噬作用中發揮作用。另外在HEK293細胞給予Salmonella感染或NOD2 ligand MDP刺激後，TAPE也顯現可以提升NOD2-mediated 的NF-κB活化。在本篇論文中我主要的工作重點是探討TAPE在細菌感染過程中調節NOD2和NOD1路徑以及自噬作用的功能和機制研究。我的研究結果顯示TAPE在NOD1 ligand iE-DAP刺激後可以提升NOD1-mediated 的NF-κB活化，以及TAPE也可以加乘NOD1/2下游kinase RIPK2 對NF-κB的活化。此外在HEK293T細胞中降低TAPE表達量會減少NOD1和RIPK2活化的NF-κB表達量，但較不影響更下游分子TRAF6活化的NF-κB表達量。另外TAPE在巨噬細胞中缺陷會降低NOD2 ligand刺激後所激活的IL-6表達量。同時TAPE還顯現與RIPK2有相互作用。這些數據都支持TAPE參與NOD1/2路徑並扮演重要角色。我另外的研究結果還顯現TAPE的缺乏在Salmonella感染期間會減少巨噬細胞中自噬蛋白(autophagic proteins )TBK1的磷酸化。這樣的結果支持TAPE是通過激活自噬作用來增強對胞內菌感染的保護。此外TAPE顯示與幾種自噬蛋白如ATG16和p62有相互作用。這些數據表明TAPE參與自噬作用。之後可以更深入研究TAPE是如何與這些分子有相互作用，以釐清TAPE是如何調控NOD路徑以及自噬作用的活化。

NOD1 and NOD2 are cytosolic sensors for detecting bacterial peptidoglycan to trigger NF-κB activation leading to inflammation. NOD2 plays a key role in the regulation of autophagy clearance of bacterial infection via recruiting the autophagy protein ATG16L1. Notably, NOD2 is shown to translocate to endosomes to detect invading bacteria. TAPE (TBK1-Associated Protein in Endolysosomes) is an innate immune regulator implicated in the TLR3, TLR4 and RIG-I pathways. The previous studies of our lab results showed that TAPE-deficient macrophages were impaired in inflammatory cytokine production and bacterial clearance upon live Salmonella infection. Moreover, biochemical analyses showed that TAPE-deficient macrophages were impaired in cleavage of an autophagy marker LC3. These data suggest that TAPE play a role in autophagic defense Salmonella. In addition, TAPE was also showed positively regulated NOD2-mediated NF-κB activation upon Salmonella infection or NOD2 ligand MDP stimulation. Here my results showed that TAPE positively regulated NOD1 or NOD1/2 downstream kinase RIPK2-mediated NF-κB activation. Furthermore, TAPE knockdown significantly lowered NOD1 or RIPK2 but not TRAF6 induced NF-κB activation. In addition, TAPE deficiency impairs NOD2 signaling to inflammatory cytokines in macrophages. Moreover, TAPE showed interaction with RIPK2. These data support that TAPE is essential and involved in NOD1/2 pathway. Besides, my results also showed that TAPE deficiency reduces phosphorylation of TBK1 during the Salmonella infection in macrophages. Furthermore, TAPE showed interaction with several autophagic proteins such as ATG16 and p62. It supports that TAPE enhances protection against intracellular bacterial infections by activating autophagy and suggest that TAPE is involved in autophagy.

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