A phospho-tyrosine-based signaling module controlling TLR-mediated inflammatory disease.

一种基于磷酸酪氨酸的信号传导模块，控制 TLR 介导的炎症性疾病。

• 批准号: 10661819
• 负责人: HANS HAECKER
• 金额: $ 73.5万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-07 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10661819
• 关键词: Adaptor Signaling Protein; Address; Amino Acids; B-Cell Antigen Receptor; Binding; Biochemical; Biological Assay; Biology; C-terminal; CBL gene; CRISPR/Cas technology; Cell Line; Cells; Coupled; Disease; Equilibrium; Event; Family; Gene Deletion; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Polymorphism; Goals; Heterozygote; Human; IRF1 gene; Immune; Immune response; Immunologic Receptors; In Vitro; Inflammation; Inflammatory; Interferon Receptor; Interferons; Knock-in; Knock-in Mouse; LYN gene; Link; Lupus; Mass Spectrum Analysis; Mediating; Modeling; Modification; Molecular; Molecular Mechanisms of Action; Mus; Mutant Strains Mice; Mutation; MyD88 protein; Pathologic; Pathology; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Phosphotyrosine; Physiological; Point Mutation; Polyubiquitination; Process; Proteins; Regulation; Resistance; Role; Signal Transduction; Site; Surface; Systemic Lupus Erythematosus; T-Lymphocyte; Therapeutic; Toll-Like Receptor Pathway; Toll-like receptors; Tyrosine; Tyrosine Phosphorylation; Ubiquitination; Work; chronic inflammatory disease; gain of function; genetic regulatory protein; in vitro Assay; in vivo; lupus-like; mouse model; novel; prevent; protein-tyrosine kinase c-src; receptor; recruit; src-Family Kinases; therapeutic target; transcription factor; ubiquitin-protein ligase

ABSTRACT Toll-like receptor (TLR)-mediated inflammation initiates physiological immune responses; however, inappropriate TLR activity leads to immune pathology as observed in inflammatory diseases, such as systemic lupus erythematosus (SLE). Various proteins acting in the TLR pathway, such as the Src kinase LYN and the transcription factor IRF5, are genetically linked to SLE. Related mouse models confirm their critical roles. LYN- deficient mice develop a spontaneous lupus-like disease that is rescued by genetic deletion of IRF5 or the up- stream acting TLR adaptor protein MyD88. As such, LYN is an important negative regulatory protein, while IRF5 promotes lupus inflammation. Still, the molecular mechanisms acting between TLR/ MyD88, LYN and IRF5 are incompletely understood. In preliminary studies, we found that MyD88 controls LYN activity via a sequence of defined protein interactions, which inhibit LYN during TLR stimulation and thus allow for IRF5 activation. Accordingly, point mutations of key interacting surfaces in cell lines results in uncontrolled LYN activity and inhibition of IRF5 activation. This mechanism is coupled with tyrosine phosphorylation events between MyD88, LYN and IRF5 and a hitherto undefined E3 ubiquitin ligase that controls pathway activity. Based on these observations we hypothesize that this mechanism is essential to maintain the balance between TLR-driven physiological and pathological inflammation. While these observations are based on extensive biochemical analyses, they raise important questions as to (i) the in vivo relevance of this pathway, in particular for lupus-like inflammatory disease and possible therapeutic implications and (ii) the mechanism of IRF5 regulation, in particular related to the unknown IRF5 E3 ligase. In this project, we will investigate three novel knock-in (KI) mice with defined point mutations controlling mentioned protein interactions. If our hypothesis is correct, two of these point mutations (acting upstream of LYN) will prevent TLR-driven lupus-like disease in mouse models due to inhibition of IRF5, while one mutation (acting downstream of LYN) is expected to mediate an IRF5 gain-of-function phenotype and thus drive lupus pathology. We will use a biochemical approach based on quantitative mass spectrometry to identify the mentioned, hitherto undefined E3 ligase. Collectively, we expect that this work will establish the described protein interaction and phosphorylation cascade as key TLR-regulatory mechanism. Given the central function of TLRs and IRFs in many physiological and pathological conditions, we expect that the significance of results obtained in this project reaches beyond IRF5 and lupus biology.

摘要 Toll样受体（TLR）介导的炎症启动生理免疫应答;然而， 不适当的TLR活性导致免疫病理学，如在炎性疾病中观察到的，例如全身性炎症， 红斑狼疮（SLE）。在TLR途径中起作用的各种蛋白质，如Src激酶林恩和 转录因子IRF 5在遗传上与SLE相关。相关的小鼠模型证实了它们的关键作用。林恩- 缺陷小鼠发展为自发性狼疮样疾病，其通过IRF 5的基因缺失或up-regulated基因缺失来挽救。 流式作用TLR衔接蛋白MyD 88。因此，林恩是一种重要的负调控蛋白，而IRF 5 促进狼疮炎症。尽管如此，TLR/MyD 88、林恩和IRF 5之间的分子机制仍然是 不完全理解。在初步研究中，我们发现MyD 88通过一个序列控制林恩活性。 确定的蛋白质相互作用，其在TLR刺激期间抑制林恩并因此允许IRF 5活化。 因此，细胞系中关键相互作用表面的点突变导致不受控制的林恩活性， 抑制IRF 5活化。这种机制与MyD 88， 林恩和IRF 5以及迄今未定义的控制途径活性的E3泛素连接酶。基于这些 我们假设这种机制对于维持TLR驱动的 生理和病理炎症。虽然这些观察是基于广泛的生物化学 分析，他们提出了重要的问题，如（i）在体内相关的这一途径，特别是狼疮样 炎症性疾病和可能的治疗意义和（ii）IRF 5调节的机制， 特别是与未知的IRF 5 E3连接酶有关。 在这个项目中，我们将研究三个新的基因敲入（KI）小鼠与确定的点突变控制， 提到蛋白质相互作用。如果我们的假设是正确的，这些点突变中的两个（作用于 林恩）将由于IRF 5的抑制而预防小鼠模型中TLR驱动的狼疮样疾病，而一种突变 （作用于林恩的下游）预期介导IRF 5功能获得性表型，从而驱动狼疮 病理我们将使用基于定量质谱的生化方法来鉴定 E3连接酶是迄今为止未定义的。 总的来说，我们期望这项工作将建立所描述的蛋白质相互作用和磷酸化 级联反应是TLR调控的关键机制。鉴于TLR和IRF在许多生理学过程中的中心功能， 和病理条件下，我们期望在这个项目中获得的结果的意义超越 IRF 5和狼疮生物学。