UC San Diego

The recognition of foreign pathogens by innate pattern recognition receptors (PRRs) serves as the first line of defense against harmful microorganisms. One family of PRRs, known as Toll-like receptors (TLRs), are expressed on plasma and endocytic membranes in order to detect extracellular or endocytosed bacteria-, fungus-, or virus- derived ligands. The sensing of pathogen-encoded patterns by TLRs activates these receptors, which transmit downstream signals resulting in the activation of NF- [kappa]B transcription factors and the production of pro- inflammatory cytokines, as well as the activation of IRF (interferon regulatory factor) transcription factors and the induction of type I interferons. The production of both mediators is critical for the immediate innate response to pathogen infection and for further signaling to engage long-term adaptive immune responses. Altogether, these pathways are exquisitely regulated to ensure clearance of invading microbes while limiting harmful autoimmune responses. Here, we report the characterization of novel regulatory co-factors that are required for TLR- directed innate immune signaling responses. These factors were identified using two screening approaches. First, the set of cellular components associated with TLR7- and TLR9- dependent responses were uncovered using an integrative systems-based genome-wide RNAi screen. This loss-of- function screen characterized 190 genes necessary for TLR7 /9 signaling, and further mapped 90 of these factors to a relative position on the TLR signaling cascade. HRS (hepatocyte growth factor-regulated tyrosine kinase substrate) was also found to be essential for the ubiquitin-dependent targeting of TLR9 to endolysosomes. Second, a biased gain-of-function screening approach was used to identify genes that increased NF-[kappa]B signaling. This method classified the gamma subunit of protein phosphatase 1 (PP1-[gamma]) as a specific regulator of MyD88-dependent pro-inflammatory responses, and a more extensive mechanistic study demonstrated that PP1-[gamma] enhances the E3 ubiquitin ligase activity of TRAF6 towards itself and IKK--[gamma]. Collectively, this analysis has identified proteins that are critical regulators of TLR-dependent innate responses and has provided significant insight into the molecular events underlying this complex signaling pathway