Regulation of the Innate Immune System through SUMOylation of Transcriptional Activators of IFN-B SERGIO J. DAVILA, David Jesse Sanchez, Genhong Cheng Department of Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA ABSTRACT RESULTS Small ubiquitin-like protein modifier (SUMO) has been shown to interact with more than 120 mammalian proteins, in both repressive and inductive manners. SUMO may play a pivotal role in the innate immune system by acting as a repressor of transcriptional activators of the IFN-B antiviral gene after viral infection. IFN-B is a cytokine that commences a cascade causing severe cellular responses such as the induction of apoptosis and cell cycle arrest in an effort to eliminate viral spread. We have shown that SUMO may be involved in repressing excess IFN-B transcription because prolonged, elevated levels of IFN-B would be detrimental to cells and ultimately the entire organism. The IFN-B transcription factor, IRF3, and theoretically other proteins upstream of this pathway, can be employed as cellular tools in understanding when and how much SUMOylation takes place in the cell post viral infection. We will use a wildtype and a mutant IRF3 gene lacking the yKXE motif targeted for SUMOylation to understand the relationship and kinetics of IRF-3, SUMO, and IFN-B levels post viral infection. Furthermore, we will also examine a number of E3 SUMO ligases belonging to the PIAS family in an effort to understand which ligases are responsible for endogenous SUMOylation during and after viral infection. Ultimately, our goal is to elucidate the kinetics and mechanism by which the IFN-B immune response is turned off after viral infection. Setting Up an In vitro SUMOylation System Figure 2:This system is based on overexpressing SUMO-1 and the protein of interest. IP of FLAG tagged proteins and subsequent Western blotting for the Myc tag of SUMO. The IRF3 is FLAG tagged and provides a substrate for SUMOylation. IRF-3 ISRE NFKB AP-1 Figure 10 IFN alpha/beta MAP kinase Activation Inflammatory Cytokine genes pCDNA3 FLAG- Androgen Receptor pCR3.1 FLAG- IRF-3 pCR3.1 FLAG- IRF-3 (K152R) pCDNA3 (Empty) pCR3.1 (Empty) Luciferae (RLUs Normalized to Renilla) Figure 5: Schematic Representation of Promoter Regions on the IFN B gene. ISRE myc-SUMO - + - + - + - + - + Cardif SUMOylated AR Figure 6 IB: anti- myc IP: anti- FLAG Luciferase (RLUs Normalized to Renilla) SUMOylated IRF3 Figures 6-10: These graphs represent the activity of certain regions of the IFN B promoter through the amount of luciferase activity. The blockers, PIAS proteins, all seem to downregulate the activity of the activators, IRF3 and Cardif. The PIAS proteins seem to be involved in the SUMOylation process of the IFN B signaling pathway. IFN B KXE (Hydrophobic Lysine Amino Acids) SUMOlation Consensus DISCUSSION INTRODUCTION IRF3 gene SUMOylation seems to be directly involved in the discontinuation of IFN-b after its activation. Prolonged levels of IFN are detrimental for the cells and ultimately the organism. Thus it is not surprising that the innate immune system has many regulatory mechanisms-like SUMOylation, ubiquitination, and phosphorylation, among others- which work to defend the organism against viral infection and excessive cytokine production. Our results show that PIAS gamma, 1, and 3 are involved in the SUMOylation of IRF3, Cardif and NFkB. PIAS and other E3 ligases are likely contributors to the downregulation of the innate immune response, IFN B, for reasons currently not well understood. We have yet to fully characterize the role of each PIAS ligase by titrimetric experimentation amongst other research avenues. IRF3 is a key player in experiments involving this signaling pathway because its SUMOylation site has already been discovered, Lys 152. We also know that IRF3 acts directly on the IFN-b promoter by nuclear translocation. These two properties make IRF3 a critical protein in the innate anti-pathogen response. Figure 7 Figure 3. Identification and K--->R mutation of 152 site of IRF3. Viral DNA and RNA TRAF3 TANK Luciferase (RLUs Normalized to Renilla) IRF-3 NFKB IKKi TBK1 Cardif IRF3 KB SUMO IFN-beta Luciferase (RLUs Normalized to Renilla) IFN- Pattern Recognition Receptors ? Transcription IFNB ? IRF3 Figure 8 ? FUTURE DIRECTIONS PIAS 1 SUMO Cotransfected: Luciferase (RLUs Normalized to Renilla) PIAS 3 Continue searching for other E3 ligases that may also be involved in the SUMOylation of IFN B transcriptional and signal activators. Apply our results and deductions from these PIAS overexpressions to carry out new experiments that will help us understand their function endogenously and after a viral infection. Vector IRF3 Activator Transfected: ISRE PIAS Gamma Interferon Beta Activation through IRF3 Signaling Pathway Figure 1 Recognition of viral and bacterial components by host pattern recognition receptors (PRR) triggers signaling pathways that induce the transcription of IFN-b. A number of many different PRRs then activate the signaling protein CARDIF, which is directly involved in the activation of TRAF3. The downstream adapter protein TRAF3 activates the associated adapter TANK. The kinases TBK1 and/or IKKi are turned on by activated TANK(1). These two kinases then trigger nuclear translocation of IRF-3 through phosphorylation. IRF3 induces the transcription of IFN-b by binding to the Interferon Stimulated Response Element (ISRE) site on the promoter, thereby activating the first antiviral response from the host. This signaling pathway is regulated by Small Ubiquitin like Modifiers (SUMOs) that covalently bond to IRF3 and possibly other proteins in this pathway. Past literature has shown that SUMOylation is able to impact the substrate’s function by modifying the proteins structure. The PIAS (Protein Inhibitor of Activated STAT) family of protein ligases are able to influence the activity of many transcription factors by SUMOylation(2). PIAS 1,3 and gamma belong to the family of E3 SUMO protein ligases, which may play a fundamental role in the regulation of the IFN-b activation pathway via SUMOylation of one or more components in the figure above(3). ISRE Luciferase (RLUs Normalized to Renilla) ISRE ACKNOWLEDGEMENTS I would like to give thanks to Dr. David Jesse Sanchez for being a dedicated and supportive mentor since day one, Dr. Genhong Cheng for giving me the opportunity to grow as a researcher, and the rest of the Cheng lab for their advice and camaraderie. I would also like to thank Dr. Dwayne Simmons, MARC, and everybody who’s helped me along the way. Figure 9 SUMO Cotransfected: Vector IRF3 ActivatorTransfected: Luciferase (RLUs Normalized to Renilla) REFERENCES Summary of SUMO Modulation of IRF3/7 Gene Induction Figure 4 SUMO has a profound effect on IRF-3 induction of IFN B and ISRE. AP-1 1. Akira S. and Takeuchi (2007). Recognition of viruses by innate immunity. Immunological Reviews, p. 214-224. 2. Kotaja N., et. al. (2002). PIAS Proteins Modulate Transcription Factors by Functioning as SUMO-1 Ligases. Molecular and Cellular Biology, p. 5222-5234. 3. Bischof O, et. al. (2006). The E3 SUMO Ligase PIASy Is a Regulator of Cellular Senescence and Apoptosis. Molecular Cell, p. 783-794. Objectives Establish kinetics and mechanism of SUMOylation in the IRF3 signaling pathway of IFN-b pre- and post viral infection. Identify which ligases are causing SUMOylation in this pathway. UCLA MINOR in BIOMEDICAL RESEARCH