探討14-3-3蛋白質家族對黑色素瘤分化相關蛋白質-5 (MDA5)活化之調控

Abstract

宿主在受到RNA病毒感染時，宿主細胞內之RIG-I相似受器家族如RIG-I或者MDA5具有辨識細胞質內病毒遺傳物質RNA，並進一步活化下游訊息傳遞路徑，以產生第一型干擾素抵抗病毒感染與複製的功能。RIG-I相似受器家族通常被認為具有不同的雙股RNA辨認親和性，RIG-I被認為會優先辨認長度小於200個鹼基對的雙股RNA，而MDA5則通常被認為會辨認長度大於2000個鹼基對的雙股RNA。根據先前的研究指出，當病毒感染細胞時，RIG-I必須藉由蛋白質14-3-3ε的幫助使RIG-I能夠由細胞質移動到粒線體結合內質網膜(Mitochondria-Associated Membrane; MAM)的位置，以進一步和下游訊息傳遞蛋白質MAVS作用。RIG-I及MDA5會和下游蛋白質MAVS作用進而活化下游第一型干擾素訊息傳遞路徑。目前對於RIG-I活化路徑的研究較為完整及深入，但是對於MDA5活化路徑的探討卻是相對較少。在此研究當中，我們發現14-3-3η對於MDA5活化下游第一型干擾素傳遞路徑具有非常重要的功能。藉由免疫共同沉澱法發現有不同的14-3-3蛋白質亞型可以與MDA5的N端結構域CARDs結合，然而對於MDA5之C端結構域(C-terminal domain)卻沒有結合能力。此外，於細胞中異位表現(ectopic expression)14-3-3η可增強MDA5傳遞路徑所誘發的第一型干擾素啟動子活性(promoter activity)，且此一增強作用是與異位表現的14-3-3η蛋白質表現量呈正相關。而在Huh7肝癌細胞中抑制(knock-down)內源性的14-3-3η蛋白質表現量，對於異位表現全長MDA5或MDA5 N端所引發的第一型干擾素啟動子活性上升的現象會產生抑制作用。為了近一步探討14-3-3η蛋白質對於MDA5傳遞路徑的影響，我們將MDA5 N端序列中預測的14-3-3蛋白質結合基序(binding motif)進行突變，並且發現如果將MDA5第88個胺基酸由絲胺酸(serine)改為天門冬胺酸(aspartic acid)，除了會使MDA5無法正常活化外，也會使14-3-3蛋白質無法和MDA5結合。最後藉由細胞離析方法(cell fractionation)得知內源性MDA5會因為異位表現的N-MDA5而活化細胞第一型干擾素訊息傳遞路徑，並從細胞質移動到粒線體相關區域，而之前發現可以與MDA5結合的14-3-3 蛋白質也同樣可以分布於粒線體相關區域。總結來說，在此研究當中我們發現了14-3-3η蛋白質在MDA5傳遞路徑當中扮演重要的角色。

During RNA virus infection, RIG-I like receptors (RLRs), such as Retinoic acid-inducible gene I (RIG-I) and Melanoma differentiation-associated protein 5 (MDA5), recognize cytoplasmic viral RNA and activate downstream adaptor proteins to produce type I interferon (IFN). It is known that RIG-I preferentially binds to short dsRNA (<200 bp) whereas MDA5 binds to dsRNA longer than 2000 base pairs. It has been reported that upon viral infection, RIG-I interacts with the chaperon protein 14-3-3ε, which facilitates translocation of RIG-I to mitochondrial associated membrane (MAM) for interaction with mitochondrial antiviral-signaling protein (MAVS), which is the common adaptor protein for both RIG-I-dependent and MDA5-dependent signaling to induce type I IFN. However, the molecular mechanisms of MDA5 activation remain less understood. Here we show that 14-3-3η is crucial for MDA5-dependent type I IFN induction. We found that several 14-3-3 isoforms may be co-immunoprecipitated with MDA5 through the CARDs (N-MDA5) but not the C-terminus of MDA5 (C-MDA5). Furthermore, ectopic expression of 14-3-3η, but not other isoforms, could enhance MDA5-dependent activation of IFNβ promoter in a dose-dependent manner. Knock-down of 14-3-3η in Huh7 cells also led to the attenuation of the IFNβ promoter activities which were facilitated by ectopic expression of MDA5 or N-MDA5. To further investigate the essential role of 14-3-3 proteins in MDA5-dependent signaling pathway, we co-immunoprecipitated 14-3-3 proteins with different N-MDA5 mutants and found that a defective mutant form of N-MDA5, N-MDA5-S88D could not interact with 14-3-3 proteins. Besides, in our study we also found that endogenous MDA5 could be translocated from cytosol to mitochondrial membrane fraction in response to the activation of type I IFN signaling pathway due to ectopically expressing N-MDA5. In conclusion, we define 14-3-3η as a novel molecule which is involved in the MDA5-dependent IFNβ signaling pathway.