Molecular mechanism of RIG-I and RIPLET in antiviral signaling

摘要 对微生物感染的有效免疫防御依赖于对病原体的有效检测 免疫受体。在确保这些免疫受体正常运作的蛋白质中，有泛素 (UB)和E3连接酶，通过蛋白酶体依赖和非依赖机制工作。在这 Grant，我们探索了RIPLET的分子机制，RIPLET是一种E3连接酶，发挥着蛋白酶体非依赖性的作用 激活抗病毒天然免疫受体RIG-I的功能。这笔赠款建立在我们之前关于钻井平台的工作基础上- 我和我们在RIPLET上的最新发现。 RIG-I是一种保守的细胞质天然免疫受体，识别来自广泛的 病毒。RIG-I包含N端信令域(串联卡或2CARD)和C端RNA 结合结构域。我们实验室和其他实验室的研究已经确定了激活至少涉及三个步骤 RIG-I：(I)RNA结合，(Ii)释放2CARD自抑制，(Iii)2CARD四聚。这个 2CARD四聚体随后激活下游接头MAVS，进而刺激抗病毒信号 小路。特别是，K63连接的多泛素刺激了2CARD四聚的第三步 链(K63-UBN)，它结合并稳定了2CARD四聚体，如我们的晶体结构所示。 尽管对K63-UBN在RIG-I上的作用有了详细的了解，但关于K63-UBN如何 当K63-UBN放置在RIG-I上时。越来越多的证据表明，RIPLET，一种鲜为人知的E3 连接酶在2CARD四聚所需的K63-UBN的结合中起着至关重要的作用。我们发现 RIPLET识别RIG-I的RNA结合域，但只有当它在dsRNA上预先寡聚时才能识别 丝状的形状。我们进一步揭示了RIPLET通过两种不同的结合模式与RIG-I细丝结合： 丝内结合和丝间桥接。后者在较长的dsRNA上的rig-I细丝中占主导地位， 从而导致RIG-I聚集并以依赖于dsRNA长度的方式进一步扩增RIG-I信号。 这些发现表明E3连接酶作为辅助受体的意外作用，它直接参与 受体寡聚和配体识别(Cadena等人，正在修订中，可在BioRxiv中获得)。 我们的这些发现现在从全新的角度提出了关于RIG-I机制的新的、更深层次的问题 RIPLET的观点：RIG-I究竟如何被RIPLET泛化(目标1)，RIG-I是如何被识别的 RIPLET(目标2)，RIG-I的寡聚体状态如何被RIPLET(目标3)改变，以及RIPLET是否可以 用于确定RIG-I的配体(目标4)。我们在这里提出了生物化学、结构生物学 和细胞生物学来回答这些问题，我们相信这是解决下一层 RIG-I信号通路的复杂性。这四个目标将独立实现，但高度 协同效应。这四个目标建立在我们强大的初步数据和已建立的合作网络之上 以及我们实验室在过去几年里开发的生化和功能分析。 好了！

SUMMARY Effective immune defense against microbial infection depends upon efficient detection of pathogens by innate immune receptors. Among the proteins that ensure proper functioning of these immune receptors are ubiquitin (Ub) and E3 ligases that work through both proteasome-dependent and -independent mechanisms. In this grant, we explore the molecular mechanism of RIPLET, an E3 ligase that plays a proteasome-independent function in activating antiviral innate immune receptor, RIG-I. This grant builds upon our previous work on RIG- I and our recent findings on RIPLET. RIG-I is a conserved cytosolic innate immune receptor that recognizes RNAs from a broad range of viruses. RIG-I contains an N-terminal signaling domain (tandem CARD or 2CARD) and C-terminal RNA binding domain. Studies from our lab, and others, have identified at least three steps involved in the activation of RIG-I: (i) RNA binding, (ii) release of 2CARD auto-repression, and (iii) tetramerization of 2CARDs. The 2CARD tetramer then activates the downstream adaptor, MAVS, which in turn stimulates the antiviral signaling pathways. In particular, the third step of 2CARD tetramerization is stimulated by K63-linked polyubiquitin chains (K63-Ubn), which binds and stabilizes the 2CARD tetramer, as demonstrated by our crystal structures.! Despite the detailed understanding of the action of K63-Ubn on RIG-I, much remains debated about how and when K63-Ubn is placed on RIG-I. Accumulating evidence suggests that RIPLET, a poorly understood E3 ligase, plays an essential role in conjugating K63-Ubn required for 2CARD tetramerization. We found that RIPLET recognizes the RNA-binding domain of RIG-I, but only when it is pre-oligomerized on dsRNA in a filamentous form. We further revealed that RIPLET binds RIG-I filaments through two distinct binding modes: intra-filament binding and inter-filament bridging. The latter dominates for RIG-I filaments on longer dsRNAs, leading to RIG-I clustering and further amplification of RIG-I signaling in a dsRNA length-dependent manner. These findings showed the unexpected role of an E3 ligase as a co-receptor that directly participates in receptor oligomerization and ligand discrimination (Cadena et al, under revision, available in BioRxiv). These findings of ours now raise new and deeper questions about the RIG-I mechanism from the fresh perspective of RIPLET: precisely how RIG-I is ubiquitinated by RIPLET (Aim 1), how RIG-I is recognized by RIPLET (Aim 2), how the oligomeric state of RIG-I is altered by RIPLET (Aim 3), and whether RIPLET can be utilized to identify ligands for RIG-I (Aim 4). We here propose a combination of biochemistry, structural biology and cell biology to answer these questions, which we believe are the key to resolving the next layers of complexity in the RIG-I signaling pathway. The four aims will be pursued independently, but are highly synergistic. These four aims build upon our strong preliminary data, an established network of collaboration and biochemical and functional assays that our lab has developed over the last several years. !