Function and regulation of the ISG15 specific isopeptidase USP18

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Protein modification by ubiquitin (Ub) and ubiquitin like proteins (UBLs) such as SUMO, NEDD8, FAT10 or Hub1 affect pleiotropic biological functions and are counteracted by Ub/UBL deconjugating proteins. Interferon stimulated gene 15 (ISG15) is an UBL, which can be conjugated to target substrates (ISGylation) in a process similar to ubiquitin and represents a major type I interferon (IFN) effector system. ISGylation is directed against viral and bacterial pathogens but is also involved in other processes including DNA repair and tumorigenesis. ISGylation is reversed by the ISG15-specific isopeptidase USP18. Using Knock-In mice, we recently showed that selective inactivation of the protease function of USP18 suppressed viral load suggesting that USP18 protease inhibition could constitute a promising antiviral strategy. So far, only a few proteins interacting with USP18 were identified and little is known about how other proteins influence USP18 or how USP18 regulates the function of binding partners. Using a yeast two hybrid screen, we have now identified several USP18 interacting proteins which hint to a crosstalk of USP18 with the NFk-B signaling pathway and epigenetic regulation. Within the scope of this proposal, we will analyze the functional relationship of these interaction partners and USP18 using biochemical and cell based approaches. Irrespective of its protease activity, USP18 represents a major negative regulator of the IFN response. Consequently, recent work by us and others showed that loss of USP18 results in spontaneous, aberrant activation of microglia in mice and causes fatal interferonopathies in human embryos. Despite the clinical relevance, the nature of the initial trigger for the uncontrolled inflammatory response still remains to be defined. We will address this question by classical genetic rescue approaches using double k.o mice for USP18 in combination with depletion of key signaling molecules of danger/pathogen signal triggered pathways such as MAVS, MYD88, STING etc. Until now, analysis of the function/phenotype relationship was hampered by a great genetic background dependent variability in the phenotype of USP18-/- mice. The conditional USP18fl/fl mice generated in our lab will now allow us to investigate the consequences of spatiotemporal controlled USP18 depletion on the well characterized C57/BL6 strain. Furthermore, embryonic lethality of USP18-/- mice in the C57/BL6 strain, which constitutes a good animal model for the consequences of USP18 deficiency in humans, awaits mechanistic exploration which will be addressed in this study. Collectively we expect that our results will enhance the understanding of USP18 function, its regulation and of its influence on interacting proteins. Results should also help to gain insights into the mechanisms responsible for the death of USP18 deficient human embryos in particular and for detrimental inflammatory responses (e.g. those caused by congenital infections) in general.