A Non-Canonical IRAK1 Signaling Pathway Triggered by Ionizing Radiation

项目摘要 白介素1受体(IL-1R)相关激酶1是Toll样受体的核心转导因子 (TLR)和IL-1R介导的苍蝇对人的先天免疫信号。作为对病原体感染的反应， 结扎的TLR/IL-R受体通过形成MyDDosome复合体几乎瞬间激活IRAK1 (IL-1R/TLR-MyD88-IRAK4-IRAK1)。一旦被IRAK4及其自身磷酸化，完全 激活的IRAK1从平台上解离，参与核因子-kB和其他信号级联，最终达到 在急性炎症反应中。直到最近，脊椎动物IRAK1还没有被牵连到过程中 而不是微生物反应。 在一个无偏见的斑马鱼筛选中，我们最近发现IRAK1对细胞生存是必不可少的，以响应 电离辐射(GIR)(Liu等人，NAT Cell Biol 2019；参考文献1)。这一功能在人类细胞中是保守的， 在肿瘤模型中促进细胞对放射治疗(R-RT)的抵抗。与其采取行动刺激核因子-kB， IRAK1通过对抗PIDDosome复合体(PIDD-Raidd-)介导的细胞凋亡而促进细胞存活 Caspase-2)。与经典的IRAK1/4免疫信号进一步对比，我们的初步数据表明 IRAK1对GIR的反应：(I)完全需要其激酶活性；(Ii)不需要IL-1R/TLR-IRAK1/4 接头MyD88；最引人注目的是，(Iii)在受辐射的细胞的细胞核中起始，而不是在细胞表面。 然而，GIR诱导的IRAK1激活确实在刺激的几分钟内发生，并且绝对需要IRAK4， 提示存在一种新的寡聚平台，负责协调GIR诱导的IRAK1 取代MyDDosome的激活。 虽然证据令人信服地指出了脊椎动物中一种新的IRAK1应激反应途径，但 GIR诱导IRAK1激活的细胞和分子基础尚不清楚。在目标1中，我们将监控 GIR后活跃的和天然的IRAK1的定位作为时间的函数；识别细胞信号(S) 有效地在受辐射的细胞中激活IRAK1，伴随着DNA断裂、微核、细胞因子和危险- 相关的分子模式(DAMP)作为主要候选；并探索环境压力是否 (例如，紫外线照射)可以触发该途径。在目标2中，我们将剖析irak1的分子机制。 响应GIR的激活，首先通过关注IRAK4和IRAK1本身的作用；其次通过识别 DD蛋白替代MyD88作为GIR诱导的IRAK4/IRAK1激活平台的支架 采用无偏见、更大规模的蛋白质组学方法无偏见地鉴定上游 在受辐射的细胞核中协调IRAK1激活的传感器、传感器和调节器。超越照亮 作为脊椎动物中一种新的应激反应途径，我们的建议探索了与肿瘤R-RT有关的途径。 因此，我们工作的另一个直接影响可能是发现新的药物靶点以克服 大多数接受放疗作为其治疗一部分的癌症患者的R-RT。

Project Summary Interleukin-1 Receptor (IL-1R)-Associated Kinase 1, IRAK1, is a core transducer of Toll-like receptor (TLR) and IL-1R-mediated innate immune signaling from flies to humans. In response to pathogen infection, ligated TLR/IL-R receptors almost instantaneously activate IRAK1 via formation of the MyDDosome complex (IL-1R/TLR—MyD88-IRAK4-IRAK1) at the cell surface. Once phosphorylated by IRAK4 and itself, fully activated IRAK1 dissociates from the platform and engages NF-kB and other signaling cascades, culminating in the acute inflammatory response. Until recently, vertebrate IRAK1 had not been implicated in processes other than the microbial response. In an unbiased zebrafish screen, we recently identified IRAK1 as essential for cell survival in response to ionizing radiation (gIR) (Liu et al., Nat Cell Biol 2019; ref. 1). This function is conserved in human cells and drives cellular resistance to radiotherapy (R-RT) in tumor models. Rather than acting to stimulate NF-kB, IRAK1 drives cell survival by countering apoptosis mediated by the PIDDosome complex (PIDD-RAIDD- caspase-2). In further contrast with canonical IRAK1/4 immune signaling, our preliminary data indicate that the IRAK1 response to gIR: (i) fully requires its kinase activity; (ii) does not require the IL-1R/TLR—IRAK1/4 adaptor MyD88; and most strikingly, (iii) initiates in the nucleus of irradiated cells and not at the cell surface. gIR-induced IRAK1 activation does however occur within minutes of stimulus and absolutely requires IRAK4, suggesting the existence of a novel oligomeric platform responsible for orchestrating gIR-induced IRAK1 activation in place of the MyDDosome. While the evidence convincingly points to a novel IRAK1 stress response pathway in vertebrates, the cellular and molecular bases of gIR-induced IRAK1 activation remain to be defined. In Aim 1, we will monitor the localization of both active and native IRAK1 as a function of time after gIR; identify the cellular signal(s) that effectively trigger IRAK1 activation in irradiated cells, with DNA breaks, micronucleation, cytokines and danger- associated molecular patterns (DAMPs) as primary candidates; and explore whether environmental stresses (e.g., UV irradiation) can trigger the pathway. In Aim 2, we will dissect the molecular mechanism of IRAK1 activation in response to gIR, first by focusing on the roles of IRAK4 and IRAK1 itself; second by identifying the DD protein substituting for MyD88 as scaffold for the gIR-induced IRAK4/IRAK1 activation platform; and finally taking unbiased, larger scale proteomic approaches toward the unbiased identification of the upstream sensors, transducers and regulators that orchestrate IRAK1 activation in irradiated nuclei. Beyond illuminating a novel stress response pathway in vertebrates, our proposal explores a pathway implicated in tumor R-RT. Thus, an additional immediate impact of our work might be the discovery of novel drug targets for overcoming R-RT in the majority of cancer patients that receive RT as part of their treatment.