Molecular Determinants of TLR Trafficking

描述(申请人提供)：TLR9是一种内体定位的模式识别受体(PRR)，可检测病原体相关DNA。TLR9从内质网(ER)运输到内溶酶体，在内溶酶体被切割，使其有能力进行信号激活。我们最近证明TLR9是泛素化的，TLR9靶向早期内体是一个泛素依赖的过程，该过程是由ESCRT蛋白肝细胞生长因子调节的酪氨酸激酶底物(HRS)介导的。这种区分在调节信号激活以及降低引发自身免疫性疾病的可能性方面至关重要。然而，使TLR9从内质网转移到内聚体的分子辅助因子大多还没有确定。在这里，我们建议使用高通量遗传学方法鉴定TLR9泛素化所需的E3泛素连接酶，并表征ESCRT途径组件在TLR9运输中的非典范作用。此外，我们建议验证TLR7/9信号的全基因组RNAi筛查确定的15种蛋白质在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功能在表达TLR9细胞亚群中的体内作用。了解TLR9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，这种疾病是通过内体TLR信号过度激活引起的。

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.