Phosphorylation mimetic motif of cryptochrome proteins blocks IRF3 activation

项目总结 先天免疫系统使用生殖线编码的模式识别受体(PRR)来检测常见的 病原体相关的分子模式。然而，只有几个PRR信号通路，包括RIG-I- MAV、cGAS-STING和TLR3/4-TRIF激活干扰素调节因子3(IRF3)。最近的研究表明 先天免疫适配器(MAV、STING和TRIF)在其各自的C末端被磷酸化 共有基序，pLxIS(p，亲水性残基；x，任何残基；S，磷酸化位点)。这个 先天免疫接头蛋白中pLxIS基序的磷酸化是一种基本和保守的 选择性地招募IRF3以激活I型干扰素产生的机制。然而，东道主是否以及如何 或者病原体利用这个基序来减弱或阻断IRF3信号尚不清楚。我们在这方面的总体目标 应用是确定隐花色素(CRY)蛋白在IRF3信号通路和宿主中的作用 对病毒感染的防御。我们的初步蛋白质组学数据显示，隐色素(CRY)蛋白CRY1 和CRY2，与IRF3相互作用。有趣的是，CRY1和CRY2有两个和一个保守的pLxIE基序， 分别进行了分析。由于谷氨酸(E)是一种磷酸化模拟物，所以这个pLxIE基序可能是一个结构性的 磷酸化pLxIS。我们的初步研究表明，pLxIE中E到丙氨酸(A)的突变消除了 与IRF3结合，表明pLxIE基序也是IRF3的对接位点。此外，两者的不足之处 CRY蛋白也增加了IRF3信号和I型干扰素的产生。然而，CORT蛋白是如何抑制 先天免疫反应还没有得到很好的阐明。根据现有的文献和我们的初步数据，我们 假设CORT蛋白通过pLxIE基序和结合区块是IRF3的诱饵适配子 IRF3和天然免疫适配器之间的相互作用，从而抑制IRF3的磷酸化和激活。 目的1确定CRY1/2在RIG-I介导的IRF3信号通路中的作用。《目标2》将剖析 CRY1/2如何抑制IRF3介导的天然免疫的分子机制。 许多人类免疫疾病的症状和病理表现出昼夜节律，包括 哮喘、类风湿性关节炎和其他免疫系统疾病。我们的研究将弥补 了解昼夜节律蛋白CRY1/2在宿主先天免疫反应中的作用。这部小说 在我们的应用中提出的机制将促进我们对先天之间串扰的理解 免疫力和昼夜节律。

Project summary The innate immune system employs germline-encoded pattern recognition receptors (PRRs) to detect common pathogen-associated molecular patterns. However, only several PRR signaling pathways, including RIG-I- MAVS, cGAS-STING, and TLR3/4-TRIF, activate interferon regulatory factor 3 (IRF3). Recent studies showed that the innate immune adaptors (MAVS, STING, and TRIF) are phosphorylated at their respective C-terminal consensus motif, pLxIS (p, hydrophilic residue; x, any residue; S, phosphorylation site) by TBK1. The phosphorylation of the pLxIS motif in innate immune adaptor proteins is an essential and conserved mechanism that selectively recruits IRF3 to activate type I IFN production. However, whether and how the host or pathogen utilizes this motif to attenuate or block IRF3 signaling is unknown. Our overall objective in this application is to determine the role of the cryptochrome (CRY) proteins in IRF3 signaling pathways and host defense to viral infection. Our preliminary proteomic data showed that cryptochrome (CRY) proteins, CRY1 and CRY2, interacted with IRF3. Interestingly, CRY1 and CRY2 have two and one conserved pLxIE motifs, respectively. As glutamic acid (E) is a phosphorylation mimic, this pLxIE motif might be a constitutively phosphorylated pLxIS. Our pilot studies showed that mutation of E to alanine (A) in the pLxIE abolished the binding to IRF3, suggesting that the pLxIE motif is also a docking site for IRF3. Furthermore, deficiency of both CRY proteins also augmented IRF3 signaling and type I IFN production. However, how CRY proteins inhibit innate immune responses is not well elucidated. Based on the existing literature and our preliminary data, we hypothesize that CRY proteins are decoy adaptors for IRF3 via the pLxIE motif and the binding blocks interaction between IRF3 and innate immune adaptors, thereby inhibiting IRF3 phosphorylation and activation. Aim 1 will determine the role of CRY1/2 in the RIG-I-mediated IRF3 signaling pathway. Aim 2 will dissect the molecular mechanisms of how CRY1/2 suppresses IRF3-mediated innate immunity. Many human immune diseases exhibit circadian rhythmicity in their symptoms and pathology, including asthma, rheumatoid arthritis, and other disorders of the immune system. Our study will bridge the gaps in understanding the role of the circadian proteins CRY1/2 in host innate immune response. This novel mechanism proposed in our application will advance our understanding of the crosstalk between innate immunity and circadian.