Discovery and Characterization of Cellular Factors Modulating Type III Interferon Induction and Signaling

原理：干扰素(IFN)是一种细胞因子，在抵御真菌、细菌和病毒病原体方面发挥着重要作用。它们还在抗肿瘤反应、粘膜免疫和脂质代谢中发挥积极作用。干扰素的表达在细胞中受到严格调控。我的长期目标是了解细胞蛋白在调节细胞内各种IFN的诱导和信号传递中的作用。根据进化的亲缘关系，干扰素分为三类，即I型、II型和III型，其中最新发现的是III型干扰素，包括干扰素lambda 1-4(IFN-L1-4)。尽管是宿主屏障免疫反应的主要组成部分，但对调节III型干扰素诱导和信号转导的细胞蛋白的系统筛选尚未完成。我们假设，几种细胞蛋白和途径可能在调节干扰素系统中发挥关键作用。在这项发现拨款中，我们建议使用全基因组CRISPR基因敲除和激活文库筛选和基于TurboID的邻近标记交互作用组筛选来全面定位积极或消极调控干扰素-L的诱导和信号转导的细胞蛋白质。目的1研究两个PRRs RIG-I和TLR-3被其同源配体双链RNA激活后，调节干扰素-L3诱导的细胞蛋白的特征。通过CRISPR基因标签产生的在基因组环境中表达标记了干扰素-L3基因的霓虹灯绿色荧光蛋白的细胞将被用于基于FACS的全基因组CRISPR敲除和激活文库筛选，以识别候选基因。最相关候选细胞的作用机制和细胞类型特异性将由标准分子生物学技术确定。目的2定位通过III型干扰素信号调节ISG表达的蛋白质。这些候选基因将在干扰素-L2治疗后在霓虹灯绿色标记的OAS-2蛋白报告细胞系中通过基于FACS的全基因组CRISPR敲除和激活文库筛选来识别。在选定的候选者得到验证后，将研究干扰素信号通路中调控的确切步骤和涉及的潜在机制。意义：III型干扰素在屏障组织中抵抗入侵病原体的宿主免疫中发挥核心作用。全面了解调控干扰素-L途径的细胞蛋白将使我们能够了解这些组织中宿主细胞与病原体之间的复杂相互作用，并揭示病原体与这一途径相互作用和破坏的新途径。在干扰素-L途径中发现新的可药物靶点也将增强我们在感染和自身免疫性疾病中调节它的能力。

Rationale: Interferons (IFNs) are cytokines that play an essential role in protection against fungal, bacterial and viral pathogens. They also play an active role in antitumor response, mucosal immunity and lipid metabolism. Expression of interferons are tightly regulated in cells. My long-term objective is to understand the role of cellular proteins in regulation of induction and signaling of various IFNs in cells. Based on evolutionary relatedness, interferons are grouped into three classes namely type I, type II and type III. Among these, the recently identified group is type III IFNs which include IFN lambda 1-4 (IFN-L1-4). Despite being a major component of host barrier immune response, a systematic screening for cellular proteins modulating type III IFN induction and signaling has not been done yet. We hypothesise that several cellular proteins and pathways may play a critical role in regulating the interferon system. In this discovery grant we propose to comprehensively map the cellular proteins that positively or negatively regulate the induction and signaling of IFN-L using genome-wide CRISPR knockout and activation library screens and TurboID-based proximity labeling interactome screen. Aim 1 will characterize cellular proteins regulating the induction of IFN-L3 following activation of two PRRs, RIG-I and TLR-3 by their cognate ligand, double stranded RNA. Cells expressing neon green fluorescent protein tagged IFN-L3 gene in genomic context, generated by CRISPR-gene tagging will be used in a FACS-based genome wide CRISPR knock-out and activation library screen to identify gene candidates. The mechanism of action and cell type specificity of the most relevant candidates will be determined by standard molecular biology techniques. Aim 2 will map the proteins modulating ISG expression by type III IFN signaling. The gene candidates will be identified by a FACS-based genome-wide CRISPR knock-out and activation library screen in neon green tagged OAS-2 protein reporter cell line following IFN-L2 treatment. Following verification of selected candidates, the exact steps in the IFN signaling pathway regulated and the potential mechanism involved will be studied. Significance: Type III IFNs play a central role in host immunity against invading pathogens in barrier tissues. A comprehensive understanding of the cellular proteins modulating the IFN-L pathway will enable us to understand the complex interaction between host cells and pathogens in these tissues, and to uncover novel ways in which pathogens interact with and undermine this pathway. Identifying novel druggable targets in IFN-L pathway will also augment our ability to regulate it during infections and autoimmune diseases.