Interplays between ADAR1 and MDA5 in the pathogenesis of Aicardi-Goutière Syndrome

 描述（由申请人提供）：MDA 5是一种保守的先天性免疫受体，可识别病毒双链RNA（dsRNA）并诱导I型干扰素免疫应答。虽然MDA 5的适当功能对于针对病毒感染的有效免疫防御是重要的，但我们最近的研究表明，其通过功能获得性（GOF）突变的失调活性可导致严重的炎性疾病Aicardi-Goutière综合征（AGS）（Rice et al，Nat Genetics，2014）。该提案的目标是进一步阐明这些突变导致慢性炎症和AGS的分子机制。特别是，我们专注于定义MDA 5和dsRNA修饰酶ADAR 1之间的潜在关系，ADAR 1的功能丧失（LOF）突变也被证明会导致AGS。我们假设MDA 5和ADAR 1代表了免疫耐受关系的两个平衡臂。也就是说，细胞dsRNA，如反向Alu元件，通常通过ADAR 1将腺苷（A）修饰为肌苷（I）而被阻止刺激MDA 5，而ADAR 1中的LOF突变或MDA 5中的GOF突变可能允许这些内源性dsRNA刺激MDA 5。 为了验证这些假设，并进一步扩大我们对AGS发病机制的理解，我们在这里提出了一个涉及生物化学，细胞生物学和计算建模相结合的多学科研究项目。我们将首先使用我们在先前关于MDA 5的机制研究中开发的一系列生化测定来确定A-to-I修饰对MDA 5：dsRNA相互作用的影响（Aim 1A）。为了获得更详细的结构见解，我们将根据我们过去确定的MDA 5：dsRNA复合物的晶体结构（Aim 1B），通过计算建模来补充这种生化分析。我们接下来将使用源自AGS患者的细胞鉴定野生型和GOF突变体MDA 5的内源性刺激性dsRNA的来源（目的2A-B）。这将与波士顿儿童医院的Luigi Notarangelo博士密切合作进行，他是遗传性免疫疾病领域的专家，已经从患者中产生并鉴定了诱导多能干细胞系。 该项目建立在我们以前对MDA 5的结构和生化机制的工作以及支持我们假设的初步数据的基础上。我们相信，这项研究将为AGS和其他相关炎症性疾病的发病机制提供新的见解，并帮助我们定义病原体传感机制和细胞RNA修饰之间复杂相互作用的新范式。

 DESCRIPTION (provided by applicant): MDA5 is a conserved innate immune receptor that recognizes viral double-stranded RNA (dsRNA) and induces type I interferon immune response. While an appropriate function of MDA5 is important for effective immune defense against viral infection, our recent study showed that its dysregulated activity via gain-of-function (GOF) mutations can lead to a severe inflammatory disease, Aicardi-Goutière Syndrome (AGS) (Rice et al, Nat Genetics, 2014). The goal of this proposal is to further elucidate the molecular mechanisms by which these mutations cause chronic inflammation and AGS. In particular, we focus on defining the potential relationship between MDA5 and a dsRNA modifying enzyme, ADAR1, of which loss-of-function (LOF) mutation has been also shown to cause AGS. We hypothesize that MDA5 and ADAR1 represent two balancing arms of the immune-tolerance relationship. That is, cellular dsRNAs, such as inverted Alu elements, are normally prevented from stimulating MDA5 through modification of adenosine (A) to inosine (I) by ADAR1, whereas LOF mutations in ADAR1 or GOF mutations in MDA5 may allow stimulation of MDA5 by these endogenous dsRNAs. To test these hypotheses and further expand our understanding of the AGS pathogenesis, we here propose a multi-disciplinary research project involving a combination of biochemistry, cell biology and computational modeling. We will first determine the impact of A-to-I modifications on the MDA5:dsRNA interaction using a series of biochemical assays that we have developed in our previous mechanistic studies on MDA5 (Aim 1A). To obtain more detailed structural insights, we will complement this biochemical analysis with computational modeling based on the crystal structure of the MDA5:dsRNA complex that we have determined in the past (Aim 1B). We will next identify the source of the endogenous stimulatory dsRNA for both wild-type and GOF mutant MDA5 using cells derived from AGS patients (Aims 2A-B). This will be performed in close collaboration with Dr. Luigi Notarangelo at Boston Children's Hospital, an expert in the field of inherited immune disorders and has generated and characterized induced pluripotent stem cell lines from patients. This project builds upon our previous work on the structural and biochemical mechanisms of MDA5 and the preliminary data that support our hypothesis. We believe that the proposed research would provide novel insights into the pathogenesis of AGS and other related inflammatory diseases, and also help us define a new paradigm of the complex interplays between the pathogen sensing mechanism and cellular RNA modification.