Molecular Determinants of TLR Trafficking

描述（由申请人提供）：TLR 9是一种内体定位的模式识别受体（PRR），可检测病原体相关DNA。TLR 9从内质网（ER）运输到内溶酶体，在那里它被切割并成为信号转导激活的胜任者。我们最近证明TLR 9是泛素化的，并且TLR 9靶向早期内体是由ESCRT蛋白肝细胞生长因子调节的酪氨酸激酶底物（HRS）介导的泛素依赖性过程。这种区室化在介导信号激活以及降低触发自身免疫性疾病的可能性方面至关重要。然而，使TLR 9能够从ER转运到内溶酶体的分子辅因子大多未被表征。在这里，我们建议确定E3泛素连接酶，需要使用高通量遗传学方法的TLR 9泛素化，以及表征非典型作用的ESCRT途径的TLR 9贩运组件。此外，我们建议验证通过全基因组RNAi筛选TLR 7/9信号转导的15种蛋白质在TLR 7/9内体运输中的潜在作用。最后，我们建议在体内研究TLR 9泛素化和ESCRT功能在TLR 9表达细胞亚群中的作用。了解TLR 9的运输系统对于开发治疗自身免疫性疾病的药物至关重要，这些疾病是通过内体TLR信号传导的混杂激活引起的。

DESCRIPTION (provided by applicant): TLR9 is an endosomally localized Pattern Recognition Receptor (PRR) that detects pathogen-associated DNA. TLR9 traffics from in the Endoplasmic Reticulum (ER) to the endolysosome, where it is cleaved and made competent for signaling activation. We have recently demonstrated that TLR9 is ubiquitinated and targeting of TLR9 to the early endosome is an ubiquitin-dependent process that is mediated by the ESCRT protein Hepatocyte Growth Factor Regulated Tyrosine Kinase Substrate (HRS). This compartmentalization is critical in mediating the signaling activation, as well as reducing the likelihood of triggering autoimmune disease. However, the molecular cofactors that enable TLR9 to transit from the ER to the endolysome are mostly uncharacterized. Here, we propose to identify the E3 ubiquitin ligase that is required for TLR9 ubiquitination using a high throughput genetic approach, as well as characterize the non-canonical role of ESCRT pathways components in TLR9 trafficking. In addition, we propose to validate 15 proteins identified by a genome-wide RNAi screen for TLR7/9 signaling for their potential roles in TLR7/9 endosomal trafficking. Finally, we propose to study the in vivo role of TLR9 ubiquitination and ESCRT function in TLR9 expressing cell subsets. Understanding the trafficking system of TLR9 is critical for the development of drugs for autoimmune diseases that results from promiscuous activation through endosomal TLR signaling.