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Study of the cellular response to type I interferons : role of cysteine protease USP18

Abstract : Type I and type III IFNs form two multigenic families of pathogen-induced cytokines that bind to different receptors but exhibit common bioactivities. In humans, Type I IFN comprises 17 highly related subtypes, broadly referred to as IFN α/β, all binding a ubiquitously expressed receptor complex constituted of two subunits, IFNAR1 and IFNAR2 chains. The type III IFN (3 λs) binds to a receptor complex made of cell type-restricted IFNLR1 and the broadly expressed IL-10R2. Downstream of these receptor complexes is a shared Jak/STAT pathway, involving the Janus kinases Jak1 and Tyk2 and the transcription factors STAT1/2/3. Thus, the Type I and III IFN families induce the same gene subset and exert antiviral activity through independent receptor complexes. Among the human subtypes induced in vivo in response to multiple stimuli, IFN β is especially potent in bioactivities requiring long term stimulation, such as proliferation inhibition. However, the molecular basis of the α2/β differential is unknown. A critical feature of the IFN response concerns its negative regulation and indeed, its perturbation leads to auto-immune manifestations. Signaling feedback controls operate at immediate-early times and include Ser/Thr kinases and ubiquitin ligase(s) targeting the IFNAR1 receptor subunit as well as SOCS-mediated action on receptor/Jaks and STATs. An additional type of negative feedback control becomes effective at late time of IFN stimulation and involves USP18, an IFN-induced isopeptidase that cleaves ubiquitin-like ISG15 from conjugates. In the first part of my thesis work I studied how prolonged exposure (priming) of various cell types to type I or III IFNs interferes with their subsequent ability to respond to IFNs. I found that primed cells retain sensitivity to IFN β but are desensitized to IFNs α subtypes. Differential desensitization is not consequent to down-regulation of surface receptor but is dependent of induction of the isopeptidase USP18. Using 125I-radiolabeled ligands, I found that desensitized cells, ie expressing USP18, are impaired in their ability to bind IFN α2 but not IFN β. These data suggest that USP18, by targeting the assembly of functional IFN α binding sites, is responsible for the differential desensitization state (Francois-Newton et al., 2011). In the second part of my thesis, I analyzed to what extent induced USP18 affects bioactivities requiring long term IFN treatment. For this, I monitored STAT activation and ISG accumulation at the mRNA and protein levels in control cells and in cells silenced for 7 USP18. At late stimulation times (>10 hrs), an α2/β differential ISG accumulation became manifest at both transcript and protein levels. Importantly, this α2/β differential was almost totally abrogated in cells that had been silenced for USP18. I also assessed the long term (72 hrs) response to IFNs of control and USP18-silenced cells in an antiproliferative assay and found that the α2/β differential is remarkably decreased in cells silenced for USP18. Overall, these data show that upon prolonged treatment, the dose-dependent accumulation of USP18 progressively restrains IFN α2-induced signaling (Francois-Newton et al., Biochem J. in revision). In the third part of my work, I investigated whether the isopeptidase activity of USP18 is required for differential desensitization. To address this question two approaches were used. In the first one, I generated clones expressing a catalytically inactive USP18 mutant and analysed their response to IFN α2 and IFN β. I showed that the catalytic activity of USP18 is required for differential desensitization, unless the protein is very abundant. In a second approach the enzymes involved in the ISGylation machinery were silenced and the response to type I IFN was monitored. I found that the ISGylation machinery is essential for USP18 to exert its function and that the E3 enzyme EFP/TRIM25 is implicated in ISGylation of a putative USP18 substrate(s) that may contribute to efficient IFN α driven receptor complex formation. Finally, I showed that endogenous USP18 expression is fine-tuned by free ISG15. Overall, these studies demonstrate the importance of USP18 in making primed cells refractory to IFN α and in establishing differential activities of IFN α2 and IFN β.
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Véronique François-Newton. Study of the cellular response to type I interferons : role of cysteine protease USP18. Development Biology. Université Pierre et Marie Curie - Paris VI, 2012. English. ⟨NNT : 2012PAO66191⟩. ⟨tel-00829540⟩

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