Molecular mechanism of RIG-I and RIPLET in antiviral signaling

总结 针对微生物感染的有效免疫防御取决于先天免疫系统对病原体的有效检测。 免疫受体确保这些免疫受体正常运作的蛋白质包括泛素 (Ub)和通过蛋白酶体依赖性和非依赖性机制起作用的E3连接酶。在这 授予，我们探索RIPLET的分子机制，E3连接酶发挥蛋白酶体非依赖性 激活抗病毒先天免疫受体RIG-I的功能。这项赠款建立在我们以前的工作对钻机- 我和我们最近对RIPLET的调查结果。 RIG-I是一种保守的胞质先天免疫受体，其识别来自广泛的免疫细胞的RNA。 病毒。RIG-I含有N-末端信号传导结构域（串联CARD或2CARD）和C-末端RNA 结合域我们实验室的研究和其他人的研究已经确定了至少三个激活步骤 （i）RNA结合，（ii）2CARD自阻遏的释放，和（iii）2CARD的四聚化。的 然后，2CARD四聚体激活下游接头MAVS，MAVS反过来刺激抗病毒信号传导。 途径。特别地，2CARD四聚化的第三步由K63连接的多聚泛素刺激 链（K63-Ubn），其结合并稳定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是如何作用的， 以及当K63-Ubn被放置在RIG-I上时。越来越多的证据表明，RIPLET，一个鲜为人知的E3 连接酶在2CARD四聚化所需的K63-Ubn缀合中起重要作用。我们发现 RIPLET识别RIG-I的RNA结合结构域，但只有当它在dsRNA上以寡聚化的方式预先寡聚化时，才能识别RIG-I的RNA结合结构域。 丝状形式。我们进一步揭示了RIPLET通过两种不同的结合模式结合RIG-I细丝： 丝内结合和丝间桥接。后者在较长dsRNA上的RIG-I细丝中占主导地位， 导致RIG-I成簇并以dsRNA长度依赖性方式进一步扩增RIG-I信号传导。 这些发现显示了E3连接酶作为直接参与细胞凋亡的共受体的意想不到的作用。 受体寡聚化和配体辨别（Cadena等人，修订中，可在BioRxiv中获得）。 我们的这些发现现在从新鲜的角度对RIG-I机制提出了新的和更深层次的问题 RIPLET的观点：RIG-I是如何被RIPLET泛素化的（目的1），RIG-I是如何被RIPLET识别的， RIPLET（目的2），RIG-I的寡聚状态如何被RIPLET改变（目的3），以及RIPLET是否可以被 用于鉴定RIG-I的配体（目的4）。我们在此提出结合生物化学，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问题的关键。 RIG-I信号通路的复杂性。这四个目标将独立地追求，但高度 协同作用。这四个目标建立在我们强大的初步数据基础上， 以及我们实验室在过去几年中开发的生化和功能分析。 !

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. !