Molecular Determinants of TLR Trafficking

描述（由申请人提供）：TLR9是一种检测病原体相关DNA的内体细胞定位模式识别受体（PRR）。TLR9从内质网（ER）运输到内溶酶体，在那里它被切割并使其具有信号激活能力。我们最近证明了TLR9是泛素化的，TLR9靶向早期内体是一个泛素依赖的过程，由ESCRT蛋白肝细胞生长因子调节酪氨酸激酶底物（HRS）介导。这种区隔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TLR9从内质网转运到内溶体的分子辅助因子大多未被表征。在这里，我们建议使用高通量遗传方法鉴定TLR9泛素化所需的E3泛素连接酶，并表征ESCRT通路组分在TLR9运输中的非规范作用。此外，我们建议验证通过全基因组RNAi筛选TLR7/9信号的15种蛋白在TLR7/9内体运输中的潜在作用。最后，我们建议研究TLR9泛素化和ESCRT功能在TLR9表达细胞亚群中的体内作用。了解TLR9的转运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.