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

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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.