輔助蛋白質在RIG-I樣受體活化中的分子機制研究

Abstract

先天性免疫透過模式識別受體（PRRs）來偵測病毒核酸，以啟動抗病毒訊號傳遞。在RIG-I樣受體（RLR）訊息傳遞中，多種輔助蛋白可以共同協調抗病毒免疫反應。在本研究中，我們著重於14-3-3和TRIM25這兩種輔助蛋白，它們分別能夠增強黑色素瘤分化相關蛋白5（MDA5）和視黃酸誘導基因I（RIG-I）的活化。14-3-3蛋白家族是重要的伴護蛋白(Chaperone protein)，它們廣泛表達於真核細胞中，並用來調控不同的訊息傳遞路徑。先前的研究曾指出，在RNA病毒感染期間，14-3-3η能夠正向調控MDA5介導的第一型干擾素（IFN）的生成。為了防止訊息傳遞路徑過度活化，凋亡蛋白酶-3(Caspase-3)相關的細胞凋亡(apoptosis)路徑會被MDA5活化所觸發，進而切割MAVS和IRF3等信號分子。在本研究中，我們發現當MDA5活化時，14-3-3η會被凋亡蛋白酶-3切割而產生sub-14-3-3η，我們也觀察到冠狀病毒(coronavirus)和腸病毒(enterovirus)感染時也會導致sub-14-3-3η在細胞中積累，此外，抑制凋亡蛋白酶-3就能夠抑制sub-14-3-3η的生成，證實了14-3-3η是由凋亡蛋白酶-3所切割的。另外我們也發現缺少了第9個α螺旋(the 9th α-helix)的sub-14-3-3η與MDA5 之間有很強的交互作用，它們的結合改變了MDA5從細胞質移動到粒線體的狀況，並顯著降低了MDA5介導的第一型干擾素的產生，這些都證明了sub-14-3-3η可作為MDA5相關訊息傳遞路徑的負調節因子。此外，在冠狀病毒感染的細胞當中，sub-14-3-3η可以去抑制第一型干擾素的生成，使得病毒複製數量增加。在本論文的第二章中，我們的研究顯示了14-3-3η被凋亡蛋白酶-3切割可調控MDA5的活化與去活化，形成一種負向回饋機制以調節MDA5介導的第一型干擾素的生成。 另外，先前的研究顯示，TRIM蛋白家族參與了先天性免疫的調控，其中TRIM25被報導參與了RIG-I介導的抗病毒先天性免疫反應。TRIM25包含一個RING區域、兩個B-box結構、一個coiled-coil結構以及一個SPRY區域，其中RING區域具有泛素連接酶(ubiquitin ligase)的活性，可以協助RIG-I進行K63鏈結泛素化(K63-linked ubiquitination)，這顯示了TRIM25在RIG-I的活化中扮演了重要角色。蛋白質體學的研究顯示，RIG-I和TRIM25在未感染的細胞中可以被乙醯化(acetylation)，而且先前研究也指出，RIG-I的去乙醯化能夠促進其形成多聚體(oligomer)，進而在急性病毒感染期間誘導第一型干擾素的表達。在本論文的第三章中，我們發現在細胞中表現模擬乙醯化的TRIM25能夠抑制第一型干擾素的生成，因此接下來我們評估了TRIM25的乙醯化在RIG-I介導的抗病毒先天性免疫中的作用。我們的實驗數據顯示乙醯化的TRIM25能使其形成二聚體(dimer)的比例下降，但其他具體影響的詳細機制尚需進一步探討。另一方面，我們發現去乙醯酶(deacetylase) HDAC10和內源性TRIM25有交互作用，顯示了HDAC10可能在病毒感染期間調控了TRIM25的去乙醯化。在第三章中，我們的研究顯示，TRIM25的乙醯化調控了RIG-I介導的抗病毒訊息傳導路徑，而進一步的分子機制探討能夠揭示TRIM25的乙醯化在抗病毒先天性免疫中的其他調控作用，有助於發現病毒感染時的潛在治療靶點。

Detection of viral nucleic acids through pattern recognition receptors (PRRs) triggers antiviral innate immunity. The RIG-I-like receptor (RLR) signaling pathway relies on various accessory proteins to orchestrate the antiviral immune response. In this study, we focused on two accessory proteins, 14-3-3 and tripartite motif-containing protein 25 (TRIM25), which enhance the activations of melanoma differentiated-associated protein 5 (MDA5) and retinoic acid inducible gene-I (RIG-I), respectively. The 14-3-3 family is a group of important chaperone proteins which are ubiquitously expressed in eukaryotic cells to modulate different signaling pathways. Previous studies have reported that during RNA virus infection, 14-3-3η positively regulates MDA5-dependent type I interferon (IFN) induction. To prevent overactivation, MDA5 activation triggers Caspase-3-dependent apoptosis to cleave certain signaling molecules such as MAVS and IRF3. In Chapter 2, we reported that 14-3-3η is cleaved by apoptotic Caspase-3 to form sub-14-3-3 upon MDA5 activation. We observed that coronavirus and/or enterovirus infections caused the accumulation of sub-14-3-3η. Also, inhibition of Caspase-3 suppressed the sub-14-3-3η production, confirming that 14-3-3η was cleaved by Caspase-3. We found that sub-14-3-3η lacking the 9th α-helix has a stronger interaction with MDA5, altering MDA5 distribution and dramatically impairing MDA5-dependent IFNβ induction, indicating that sub-14-3-3η is a negative regulator of MDA5-dependent signaling. In addition, in coronavirus infected cells, sub-14-3-3η could inhibit IFNβ induction and result in higher viral replication. In Chapter 2, our study revealed temporal control of MDA5 activation/deactivation by Caspase-3-dependent 14-3-3η cleavage, which provides a negative feedback mechanism of MDA5-mediated type I IFN induction. Previous reports showed that members of the tripartite motif (TRIM) protein family are involved in the regulation of innate immunity. Among the TRIM family, TRIM25 has been reported to be involved in RIG-I-mediated antiviral innate immunity. TRIM25 contains a RING-finger domain, two B-box domains, a coiled-coil domain, and a SPRY domain. The RING domain of TRIM25 has an E3 ubiquitin ligase activity, which is essential for K63-linked ubiquitination of RIG-I. It is indicated that TRIM25 plays an important role in RIG-I activation. Previous proteomics reports have shown that RIG-I and TRIM25 were acetylated in the uninfected cells. In addition, according to a previous study, deacetylation of RIG-I promotes its oligomerization to induce type I IFN expression during acute virus infection. In Chapter 3, we found that expression of acetyl-mimetic mutants of TRIM25 suppressed IFNβ induction. We assessed the role of TRIM25 acetylation in RIG-I-dependent antiviral innate immunity and found that acetyl-TRIM25 regulates its dimerization. However, the detailed mechanism requires further studies. On the other hand, specific deacetylases, HDAC10, could interact with endogenous TRIM25, suggesting that HDAC10 may mediate TRIM25 deacetylation during viral infection. In Chapter 3, the role of TRIM25 acetylation in RIG-I-dependent antiviral signaling was described and discussed. Further investigations in molecular mechanisms may reveal additional regulatory roles of TRIM25 acetylation in antiviral immunity and uncover potential therapeutic targets for viral infections.