Identification and characterization of signal-transducing and regulatory kinases involved in the RIG-I/IRF3-mediated induction phase of the innate antiviral immune-response

参与 RIG-I/IRF3 介导的先天抗病毒免疫反应诱导期的信号转导和调节激酶的鉴定和表征

• 批准号: 236528794
• 负责人: Dr. Marco Binder
• 金额: --
• 依托单位: Abteilung Virus-assoziierte Karzinogenese
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/236528794?language=en
• 关键词: Identification; characterization; signal; transducing; regulatory

Every cell (from bacterial to human) possesses innate mechanisms to perceive and promptly defend against intruding microorganisms. In case of virus infections in higher animals, the interferon system is the single major player. A panel of sensor molecules on and within cells detects invariant components of the pathogen and elicits a signaling cascade that culminates in the production and secretion of various cytokines, most prominently type I (and III) interferons. In both, the infected and surrounding cells, these messengers subsequently trigger an antiviral program, which by means of a multitude of molecular effectors impedes or even prevents viral replication and spread. Along with this antiviral program inflammatory processes are mounted, which, however, must be strictly regulated in order to avoid severe damage of the affected organ.The molecular mechanisms of regulation during induction of the interferon system are incompletely understood thus far. In the here proposed follow-up project, we are going to characterize factors, which significantly affect antiviral signal transduction and its regulation. For this, we will build upon our previous project, for which we had performed a high-throughput RNAi-based screen, in which we silenced the genes of 719 human kinases and assessed their impact on the activation of the essential antiviral transcription factor IRF-3. Those genes that exhibited the most significant effects have undergone robust validation, and basal functional characterization. One particularly impactful gene, the kinase DAPK1, has been investigated in detail and shown to function as a novel negative feedback regulator of the antiviral defense program of the cell.In the now proposed project, we are going to unravel the molecular mechanism of how the antiviral RIG-I/IRF3 signaling pathway activates its negative regulator DAPK1. We will further investigate how phosphorylation of RIG-I by DAPK1 affects the molecular recognition of virus infection. To this end, we have already observed that phosphorylation leads to a completely unexpected response-behavior of RIG-I towards viral RNA. We now plan to exploit this observation to study and better understand the mechanism of self-vs-non-self recognition of RNAs by RIG-I. In a second work package, we will investigate wether the closely related proteins DAPK2 and DAPK3 have a similar regulatory role for the RIG-I pathway as DAPK1. For this purpose, we will compare all three DAPK molecules functionally and also across different cell types.Lastly, within the proposed project, we are also going to deeper characterize further kinase genes that exhibited a strong phenotype in antiviral signaling in the first funding period of the project. For three of these kinases, we have already accumulated substantial amounts of data, which we will now deepen further and extend to full-fledged molecular characterizations.

每一个细胞（从细菌到人类）都具有感知和迅速防御入侵微生物的先天机制。在高等动物病毒感染的情况下，干扰素系统是唯一的主要参与者。细胞上和细胞内的一组传感器分子检测病原体的不变组分，并激发信号级联，其最终产生和分泌各种细胞因子，最突出的是I型（和III型）干扰素。在受感染的细胞和周围的细胞中，这些信使随后触发了一个抗病毒程序，通过大量的分子效应器阻止甚至阻止病毒的复制和传播。沿着该抗病毒程序的是炎症过程，然而，为了避免受影响器官的严重损伤，必须严格调节炎症过程。在这里提出的后续项目中，我们将表征显著影响抗病毒信号转导及其调节的因素。为此，我们将建立在我们以前的项目基础上，我们已经进行了一个高通量的基于RNAi的筛选，在该筛选中，我们沉默了719种人类激酶的基因，并评估了它们对必需的抗病毒转录因子IRF-3激活的影响。那些表现出最显着影响的基因已经过了稳健的验证和基本的功能表征。其中一个特别有影响力的基因，激酶DAPK 1，已被详细研究，并被证明是一种新的负反馈调节剂的抗病毒防御程序的细胞。在现在提出的项目中，我们将解开抗病毒RIG-I/IRF 3信号通路如何激活其负调节剂DAPK 1的分子机制。我们将进一步研究DAPK 1对RIG-I的磷酸化如何影响病毒感染的分子识别。为此，我们已经观察到磷酸化导致RIG-I对病毒RNA的完全出乎意料的反应行为。我们现在计划利用这一观察结果来研究和更好地理解RIG-I对RNA的自我与非自我识别的机制。在第二个工作包中，我们将研究密切相关的蛋白DAPK 2和DAPK 3是否与DAPK 1对RIG-I通路具有类似的调节作用。为此，我们将比较所有三种DAPK分子的功能以及不同细胞类型。最后，在拟议的项目中，我们还将进一步深入表征在项目的第一个资助期内在抗病毒信号传导中表现出强表型的激酶基因。对于其中三种激酶，我们已经积累了大量的数据，我们现在将进一步深化并扩展到全面的分子表征。