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

结构性

• 批准号: 9893784
• 负责人: Hao Wu
• 金额: $ 53.1万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9893784
• 关键词: Adaptor Signaling Protein; Area; Binding; Biological; C-terminal; Cell Line; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complex; Cryoelectron Microscopy; Crystallization; Data; Death Domain; Disease; Drug Kinetics; Fluorescence Microscopy; Goals; IRAK1 gene; IRAK2 gene; IRAK3 gene; IRAK4 gene; Image; Immune response; Immune system; In Vitro; Inflammation; Inflammatory; Interleukin-1; Interleukin-1 Receptors; Interleukin-18; Lipid Bilayers; Lymphoma; Membrane; Methods; Molecular; Molecular Conformation; Mus; N-terminal; Pathway interactions; Pattern; Permeability; Pharmacology; Phosphotransferases; Process; Receptor Activation; Resolution; Signal Transduction; Signaling Protein; Specificity; Structure; System; TIRAP gene; TLR3 gene; Testing; Toll-like receptors; Transcription Factor AP-1; X-Ray Crystallography; activating transcription factor; base; cellular imaging; cytokine; gain of function mutation; human disease; inhibitor/antagonist; innate immune pathways; light microscopy; macrophage; medical schools; mutant; pathogen; receptor; reconstitution; recruit; research clinical testing; response; small molecule inhibitor; two-dimensional

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.

Toll样受体（TLR）和促炎细胞因子IL-1和IL-18的受体共享一个 在它们的细胞内区域中具有共同的TIR结构域，并且属于TLR/IL 1-R超家族。TLRs 识别病原体相关分子模式（PAMPs）以启动保护性免疫 应答这些受体的分子途径是复杂的，并且它们的失调是 与免疫系统内外的许多人类疾病有关。信号 这些受体的转导由受体TIR结构域的接近启动 在PAMP和细胞因子结合后。这导致细胞内含有TIR的细胞的募集。 衔接子如MyD 88、TIRAP/Mal、TRIF和TRAM。MyD 88是关键的 IL-1、IL-18和除TLR 3之外的所有TLR的信号传导应答。除了其C-末端TIR外， MyD 88含有N-末端死亡结构域（DD）。通过DD，MyD 88与 IRAK，包括IRAK 1、IRAK 2、IRAK 4和IRAK-M，其特征在于N末端 DD和C-末端Ser/Thr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IRFs以引发抗病原体应答 和炎症。尽管TLR/IL-1 R信号传导系统的生物学重要性， 结构和机械信息是可用的。在本申请中，我们 我建议继续我们的研究，在这个系统中使用结构的方法，如X射线 晶体学和冷冻电子显微镜，以及药理学和光学显微镜 接近。 在延长期内，我们将重点关注四个相关领域： 1.阐明野生型和疾病中TIR-TIR相互作用的分子机制 突变衔接蛋白。我们将根据这方面的初步数据， 追求这些低聚复合物的高分辨率结构测定。 2.利用脂质双层上的重构系统探究TLR/IL-1 R信号转导 和全内反射荧光显微镜。这些研究将更好地模拟 因为它们提供了基于膜的二维平台 用于信号复合体组装。在我们的初步数据中，我们已经确定了可行性 重建和成像的过程。 3.阐明TLR/IL-1 R信号转导及其与其他先天性 CRISPR修饰的巨噬细胞中的免疫途径，其中内源性信号传导 蛋白质被荧光标记的形式取代。我们已经生成了一个 这些细胞系的数量，并显示它们可以被激活。我们将这些图像 细胞在不同的激活条件下，以获得定量分析的时间 以及对这些受体的空间控制 4.在淋巴瘤的治疗中鉴定IRAK的小分子抑制剂。我们有 先前结晶的IRAK 4，并揭示了在 反式自磷酸化的过程。在我们的初步数据中，我们已经结晶 IRAK 4和IRAK 1与许多小分子抑制剂复合。我们建议 使用结构引导的理性方法来发现特异性、有效性和细胞渗透性 靶向IRAK中不同活化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， 进行临床试验。直接的里程碑是体外低nM Ki和体外低nM Gi 50。 细胞对抗IRAKs。一个好的细胞候选者将在小鼠中进行合作测试 威尔康奈尔医学院的阿里·梅尔尼克博士。