Phosphorylation mimetic motif of cryptochrome proteins blocks IRF3 activation

项目摘要 先天性免疫系统采用种系编码的模式识别受体（PRRs）来检测常见的免疫缺陷。 病原体相关的分子模式。然而，只有几种PRR信号通路，包括RIG-I- MAVS、cGAS-STING和TLR 3/4-TRIF激活干扰素调节因子3（IRF 3）。近年来的研究表明 先天免疫衔接子（MAVS、STING和TRIF）在它们各自的C末端被磷酸化， 共有基序，TBK 1的pLxIS（p，亲水残基; x，任何残基; S，磷酸化位点）。的 在先天性免疫衔接蛋白中，pLxIS基序的磷酸化是一种必需的和保守的 选择性募集IRF 3以激活I型IFN产生的机制。然而，无论是主机， 或病原体利用该基序减弱或阻断IRF 3信号传导是未知的。我们在这方面的总体目标 应用是确定隐花色素（CRY）蛋白在IRF 3信号通路和宿主中的作用。 防御病毒感染。我们的初步蛋白质组学数据表明，隐花色素（CRY）蛋白，CRY 1 与IRF 3相互作用。有趣的是，pLxIE 1和pLxIE 2具有两个和一个保守的pLxIE基序， 分别由于谷氨酸（E）是一种磷酸化模拟物，因此该pLxIE基序可能是一种组成性的磷酸化模拟物。 磷酸化pLxIS。我们的初步研究表明，在pLxIE中E突变为丙氨酸（A）， 结合IRF 3，表明pLxIE基序也是IRF 3的对接位点。此外，两者的不足 CRY蛋白还增强IRF 3信号传导和I型IFN产生。然而，CRY蛋白如何抑制 先天性免疫反应还没有很好地阐明。根据现有文献和我们的初步数据，我们 假设CRY蛋白是IRF 3通过pLxIE基序和结合块的诱饵衔接子 IRF 3和先天免疫衔接子之间的相互作用，从而抑制IRF 3磷酸化和活化。 目的1将确定RIG-I介导的IRF 3信号通路中RIG 1/2的作用。目标2将解剖 研究表明，IL-1/2抑制IRF 3介导的先天免疫的分子机制。 许多人类免疫疾病在其症状和病理学中表现出昼夜节律性，包括 哮喘、类风湿性关节炎和其他免疫系统疾病。我们的研究将弥合 了解昼夜节律蛋白G1/2在宿主先天免疫应答中的作用。这本小说 在我们的申请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.