Towards a molecular understanding of Myddosome organization and regulation of IRAK kinase activity

先天免疫反应是在toll样受体（TLRs）等模式感知受体识别危险信号和白细胞介素-1受体（IL-1R）等细胞因子受体激活后触发的第一道防线。激活这些受体后的第一步是组装大型多蛋白信号平台，称为髓质体。在这些平台中，il - 1r相关激酶（IRAKs）被共价修饰并被激活以触发信号级联，最终形成形成免疫反应的转录程序。尽管在过去的二十年中成为了密集研究的焦点，但我们对Myddosomes的组织，IRAK家族成员如何被招募到这些复合体以及调节IRAKs活性的机制的理解仍然有限。这是因为到目前为止，大多数结构研究都是孤立地研究了粘粒体的成分。此外，由于该系统具有巨大的时空复杂性，以及冗余和补偿机制，对髓粒体和细胞内先天免疫信号的分析并不能提供清晰的图像。本建议的总体目标是在体外重建这些先天免疫信号的中心枢纽，以了解Myddosome复合物组织的支配原则，并揭示IRAK激活和调节的机制。为了实现这些目标，我们将使用纯化的粘粒体来定义控制超分子复合物组装的规则。这种方法将使我们能够系统地评估支配Myddosome组装的原理，并解决迄今仍未得到解答的重要基本问题：Myddosome的组成是否在受体水平上受到控制？哪个伊拉克人可以在一个给定的Myddosome中共存？纳入高阶复合体如何调节IRAK的活性和功能？我们将通过低温电子显微镜（cryo- EM）来确定这些完全组装的配合物的结构。我们最近解决了IRAK3伪激酶结构域的晶体结构，揭示了它可能与IRAK4的自磷酸化激酶结构域异二聚化的新机制。这些研究提出了一种假设，即这种失活的假激酶如何调节IRAK4的活性，我们将对此进行研究。确定髓粒体结构的核心是捕捉复合体的不同状态。在这里，我们将利用我们最近获得的关于IRAK相互作用的关键见解，并将采用模拟该复合体激活状态的突变体。我们还将使用我们设计的纳米体与IRAK2和IRAK3结合，以稳定复合物。由于Myddosome复合物和IRAK家族成员是先天免疫的核心，这项工作将为免疫反应如何被激活提供重要的基础见解，并揭示它们如何被调节以治疗炎症性疾病，如关节炎、动脉粥样硬化、系统性红斑狼疮和牛皮癣。

Innate immune responses are the first line of defence that are triggered following the recognition of danger signals by pattern sensing receptors such as the Toll-like receptors (TLRs) and activation of cytokine receptors such as the interleukin-1 receptor (IL-1R). A first step following the activation of these receptors is the assembly of large multi-protein signalling platforms called Myddosomes. Within these platforms, the IL-1R-associated kinases (IRAKs) are modified covalently and are activated to trigger a signalling cascade that culminates in a transcriptional program that shapes the immune response. Despite being the focus of intense study over the past two decades, our understanding of the organization of Myddosomes, how the IRAK family members are recruited to these complexes and the mechanisms regulating the activity of the IRAKs is still limited. This is because most structural studies to date have investigated components of Myddosomes in isolation. Further, analysis of Myddosomes and innate immune signalling in cells do not provide a clear picture owing to the enormous temporospatial complexities of the system, as well as redundancy and compensatory mechanisms. The overall goal of this proposal is to reconstitute these central hubs of innate immune signalling in vitro to understand the principles governing the organization of Myddosome complexes and to uncover mechanisms of IRAK activation and regulation. To achieve these goals, we will use purified Myddosomes to define the rules governing the assembly of the supramolecular complex. This approach will allow us to systematically evaluate the principles governing Myddosome assembly and to address important basic questions that are still unanswered to date: Is the composition of Myddosomes controlled at the receptor level? Which of the IRAKs can co-exist in a given Myddosome? How does incorporation into higher order complexes regulate IRAK activity and function? We will determine the structures of these fully assembled complexes by cryo-electron microscopy (cryo- EM). We recently solved the crystal structure of the IRAK3 pseudokinase domain, which revealed a novel mechanism by which it may heterodimerize with the autophosphorylated kinase domain of IRAK4. These studies suggest a hypothesis for how this inactive pseudokinase may modulate the activity of IRAK4, a premise we will investigate. Central to determining the structures of Myddosomes will be to capture distinct states of the complex. Here, we will take advantage of the key insights into IRAK interactions that we recently obtained and will employ mutants that mimic the activated state of the complex. We will also use nanobodies that we have engineered to bind to IRAK2 and IRAK3 to stabilize the complexes. Since the Myddosome complex and IRAK family members are at the heart of innate immunity, this work will provide important fundamental insights into how immune responses are activated and reveal how they could be modulated to treat inflammatory diseases such as arthritis, atherosclerosis, systemic lupus erythematosus and psoriasis.