Role of SUMO in interferon pathways : Identification of SUMOylated proteins in response to interferon – SUMO and IFN

Each of the SUMO paralog (SUMO1, SUMO or SUMO3) will be expressed in different cell lines. The effects of SUMO on IFN signaling will be observed by immunofluorescence studies, Western blot and electrophoretic mobility gel shift assays and those on transcription by quantitative RT-PCR. The identification of proteins conjugated to SUMO in response to IFN will be done by a new proteomics method, recently validated by us.

Human cells were chosen to stably express each of the SUMO paralog: HeLa cells derived from a patient suffering a cervix cancer and U373MG cells derived from a glioblastoma. The same experiments will be performed on human epithelial HEK293 cells because these cells expressing each of mutated SUMO paralog were used to identify the proteins conjugated to SUMO in response to arsenic (Galisson et al., Mol. Cell Proteomics. 2011:M110.004796). The selection of the clones expressing each SUMO paralog wild type and the characterization of the cells are under investigation.
We have show that the three parental cell lines are sensitive to type I, type II and type III IFN by analyzing the signal transduction, the transcription and the anti-proliferative effect. In addition these cells are sensitive to infection by DNA and RNA viruses. We have tested the susceptibility of HeLa and U373MG cells to infection by two rhabdoviruses, vesicular stomatitis virus (VSV) and rabies virus. Both cells are sensitive to VSV but only U373MG cells are sensitive to rabies virus.

The aim of our project is the identification of proteins conjugated to SUMO in response to IFN and the demonstration that this post-translational modification inhibits IFN signaling leading to a lower IFN-induced biological responses such as inhibition of cell growth, apoptosis or antiviral defense. Our results will be very informative to understand the mechanisms of IFN action and of resistance of some patients to this treatment. The identification of proteins conjugated to SUMO in response to IFN will shed a light on new pathways to explore.

In progress

Interferons (IFNs) are a family of cytokines that exhibit diverse biological activities. Identified and named for their antiviral properties, IFNs have also immunomodulatory, antiproliferative and apoptotic activities. IFNs are successfully used in therapy to treat viral infections, cancer or multiple sclerosis. However, the interferon response is limited and some patients are resistant to treatment. Thus, progress remains to be done to better understand the mechanism of action of IFNs. IFNs consist in multiple type I species (IFN alpha , IFN beta, IFN omega, and IFN kappa), one type II (IFN gamma) and type III species (IFN lambda). They act on cells by binding to their respective receptors to activate the JAK/STAT pathways to trigger the transcription of more than three hundred interferon-inducible genes (ISG), the products of which are believed to mediate the biological effects of IFNs.
It has been established that many of the ISGs or key regulators of IFN signalling are modified by ubiquitin or ubiquitin-like modifiers such as SUMO or ISG15. Little is known about the role of SUMO in the IFN signalling, cell regulation and antiviral response. The SUMOylation status of proteins in response to IFN has not been yet investigated. Indeed, it is unknown whether IFN treatment for short or long periods could alter the SUMOylation of proteins that regulate the IFN response. Therefore, we propose to study the effect of SUMO on the regulation of IFNs pathways in order to better understand the mechanism of action of IFNs.
So far, only eight proteins known to be induced by IFN (Stat1, IRF1, IRF2, IRF3, IRF7, PML, Sp100, p53) have been described to be conjugated to SUMO in transfected cells. Two of them are implicated in IFN synthesis (IRF3 and IRF7), three in IFN signalling (Stat1, IRF1 and IRF2) and three are permanent (PML and Sp100) or transient (p53) proteins associated with PML NBs. SUMO is generally associated with transcriptional gene repression and can have important consequences on the biological activity of a protein by modifying its subcellular localization or its ability to interact with DNA. Also, it has been shown recently that SUMO can act as a signal for the recruitment of E3 ubiquitin ligase, which leads to the ubiquitylation and degradation of the modified protein. Indeed, the Ring finger protein 4 (RNF4) ubiquitinates polySUMO chains and targets SUMO-modified PML for ubiquitin-mediated proteolysis. This suggests that SUMOylation of key regulators of JAK/STAT or PML pathways could have dramatic effects on IFN signalling or ISG products.

Therefore, we will study the impact of SUMO and RNF4 on 1- rhabdovirus (VSV and rabies virus)-induced IFN production, 2- IFN signaling and transcription of ISGs, 3- IFN-induced biological activities and also on the antiviral activity of PML, which play a major role in antiviral defence. Indeed, knockout PML mice are more sensitive to infection with VSV. We have also shown that PML expression confers resistance to VSV and rabies virus. Also, it has been shown recently that RNF4 can regulate the fate of PML. Therefore, we will determine the effect of RNF4 knock down on the replication of these viruses either in cells stably expressing PML or in IFN-treated cells. In addition, we will use a proteomic approach recently validated to identify proteins SUMO-modified in response to IFN. We will measure the changes in SUMOylation in cells treated with IFN for a short or a long period in order to determine if IFN induces a redistribution of SUMO between substrates. This study should shed a new light on the role of SUMOylation on the production and action of IFN and on the innate immunity response.