Structural & Functional Studies of TLR/IL-1R Signaling

Toll样受体和促炎细胞因子IL-1和IL-18受体共有一个 TIR结构域位于其胞内区，属于TLR/IL1-R超家族。TLRS 识别病原体相关分子模式(PAMP)以启动保护性免疫 回应。这些受体的分子通路是复杂的，它们的失调是 与免疫系统内外的许多人类疾病有关。讯号 这些受体的转导是通过受体TIR结构域的近似来启动的 当PAMPs和细胞因子结合时。这导致含有TIR的细胞内的募集 适配器，如MyD88、TIRAP/MAL、TRIF和TRAM。MyD88对以下方面至关重要 IL-1、IL-18和除TLR3外的所有TLRs的信号反应。除了它的C端子轮胎 MyD88含有一个N-末端死亡结构域(DD)。MyD88通过DD进行交互 具有IRAK，包括IRAK1、IRAK2、IRAK4和IRAK-M，其特征在于N终端 DD和C-末端的丝氨酸/苏氨酸激酶或激酶样结构域。最终，随之而来的 途径激活转录因子NF-κB、AP-1和IRF诱导抗病原体反应 和炎症。尽管TLR/IL-1R信号系统在生物学上具有重要意义，但有限 结构和机械信息是可用的。在这份功绩延期申请书中，我们 建议使用X射线等结构方法继续我们在该系统中的研究 结晶学和低温电子显微镜，以及药理学和光学显微镜 接近了。 在延展期间，我们会集中处理四个相关范畴： 1.阐明TIR-TIR在野生型和病害中相互作用的分子机制 突变的接头蛋白。我们将根据我们在这方面的初步数据和 追求这些齐聚物的高分辨率结构测定。 2.利用脂双层重组系统研究TLR/IL-1R信号转导 和全内反射荧光显微镜。这些研究将更好地模仿 细胞信号，因为它们提供了基于膜的二维平台 适用于复杂的信号装配。在我们的初步数据中，我们已经确定了可行性 重建和成像。 3.阐明TLR/IL-1R信号转导途径及其与其他先天因子的功能相互作用 CRISPR修饰的巨噬细胞中内源性信号转导的免疫通路 蛋白质被荧光标记的版本所取代。我们已经生成了一个 这些细胞系的数量，并表明它们可以被激活。我们将想象这些 细胞在不同的激活条件下获得对时间的定量分析 以及对这些受体的空间控制。 4.寻找治疗淋巴瘤中IRAKs的小分子抑制剂。我们有 先前晶化了IRAK4，并揭示了在 反式自磷酸化的过程。在我们的初步数据中，我们已经明确了 IRAK4和IRAK1与一些小分子抑制剂形成复合体。我们建议 使用结构指导的理性方法来发现特定的、有效的和细胞渗透性的 针对IRAK中不同激活阶段和结合口袋的抑制剂。我们的目标是 开发具有足够效力、特异性和药代动力学的化合物 启用临床测试。直接的里程碑是体外低NM KI和低NM GI50 细胞对抗IRAK。一个好的候选细胞随后将在小鼠身上进行合作测试 威尔·康奈尔医学院的阿里·梅尔尼克博士。

Toll-like receptors (TLRs) and receptors for pro-inflammatory cytokines IL-1 and IL-18 share a common TIR domain in their intracellular region and belong to the TLR/IL1-R superfamily. TLRs recognize pathogen-associated molecular patterns (PAMPs) to initiate protective immune responses. The molecular pathways for these receptors are complex and their dysregulation is associated with many human diseases both within and beyond the immune system. Signal transduction of these receptors is initiated by the approximation of the receptor TIR domains upon binding of PAMPs and cytokines. This leads to the recruitment of intracellular TIRcontaining adaptors such as MyD88, TIRAP/Mal, TRIF and TRAM. MyD88 is critical for signaling responses of IL-1, IL-18, and all TLRs except TLR3. In addition to its C-terminal TIR domain, MyD88 contains an N-terminal death domain (DD). Through the DD, MyD88 interacts with IRAKs, including IRAK1, IRAK2, IRAK4 and IRAK-M, which are characterized by an Nterminal DD and a C-terminal Ser/Thr kinase or kinase-like domain. Eventually, the ensuing pathway activates transcription factors NF-κB, AP-1, and IRFs to elicit anti-pathogen responses and inflammation. Despite the biological importance of the TLR/IL-1R signaling system, limited structural and mechanistic information is available. In this application for a MERIT extension, we propose to continue our studies in this system using structural methods such as X-ray crystallography and cryo-electron microscopy, as well as pharmacological and light microscopy approaches. In the extension period, we will focus on four related areas: 1. Elucidating the molecular mechanism of TIR-TIR interactions in wild-type and disease mutant adaptor proteins. We will build from our preliminary data in this regard and pursue high-resolution structure determination of these oligomeric complexes. 2. Interrogating TLR/IL-1R signal transduction using reconstituted systems on lipid bilayers and total internal reflection fluorescence microscopy. These studies will better mimic cellular signaling because they provide the membrane-based, two-dimensional platform for signal complex assembly. In our preliminary data, we have established the feasibility of reconstitution and imaging. 3. Elucidating TLR/IL-1R signal transduction and the functional interaction with other innate immune pathways in CRISPR-modified macrophages in which an endogenous signaling protein is replaced by the fluorescently tagged version. We have already generated a number of these cell lines and shown that they can be activated. We will image these cells under different activating conditions to obtain quantitative analysis on the temporal and spatial control of these receptors. 4. Identifying small molecule inhibitors for IRAKs in the treatment of lymphomas. We have previously crystallized IRAK4 and revealed an inactive conformation of the kinase during the process of trans-autophosphorylation. In our preliminary data, we have crystallized IRAK4 and IRAK1 in complex with a number of small molecule inhibitors. We propose to use a structure-guided rational approach to discover specific, potent and cell permeable inhibitors that target different activation stages and binding pockets in IRAKs. Our goal is to develop compounds with sufficient potency, specificity, and pharmacokinetics to enable clinical testing. The immediate milestone is low nM Ki in vitro and low nM Gi50 in cells against IRAKs. A good cellular candidate will then be tested in mice in collaboration with Dr. Ari Melnick at Weill Cornell Medical College.