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

摘要 Toll样受体(TLR)介导的炎症启动生理性免疫反应；然而， 在炎症性疾病中观察到的不适当的TLR活性导致免疫病理，如全身性 红斑狼疮(SLE)。在TLR途径中起作用的各种蛋白质，如Src激酶Lyn和 转录因子IRF5与系统性红斑狼疮有遗传关系。相关的小鼠模型证实了它们的关键作用。林恩- 缺陷小鼠会发展成一种自发性狼疮样疾病，这种疾病可以通过IRF5或UP基因的缺失来挽救 流作用TLR接头蛋白MyD88。因此，LYN是一种重要的负性调节蛋白，而IRF5 促进狼疮炎症。尽管如此，TLR/MyD88、Lyn和IRF5之间的分子机制是 不完全理解。在初步研究中，我们发现MyD88通过一系列序列控制Lyn的活性 明确的蛋白质相互作用，在TLR刺激过程中抑制Lyn，从而允许IRF5激活。 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Lyn活性和 抑制IRF5的激活。这一机制与MyD88之间的酪氨酸磷酸化事件相耦合， LYN和IRF5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 观察我们假设，这一机制对于维持TLR驱动的 生理性和病理性炎症。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)这一途径在体内的相关性，特别是与狼疮样 炎症性疾病和可能的治疗意义以及(Ii)IRF5调节的机制，在 特别是与未知的IRF5 E3连接酶有关。 在这个项目中，我们将研究三个新的具有控制点突变的敲入(KI)小鼠 提到了蛋白质的相互作用。如果我们的假设是正确的，这些点突变中的两个(作用于 Lyn)将由于抑制IRF5而在小鼠模型中预防TLR驱动的狼疮样疾病，而一种突变 (作用于LYN下游)有望介导IRF5功能获得表型，从而驱动狼疮 病理学。我们将使用基于定量质谱学的生化方法来鉴定 提到，到目前为止还没有定义的E3连接酶。 总而言之，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 级联作为关键的TLR调节机制。鉴于TLRs和IRFs在许多生理过程中的中心作用 和病理情况，我们期望在这个项目中获得的结果的意义超出 IRF5和狼疮生物学。

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.