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Justyna A. Korczeniewska
Interdisciplinary Biomedical Sciences Program
B.A. 2005, Rutgers University, Newark, NJ

Thesis Advisor: Betsy J. Barnes, Ph.D.
Associate Professor
Department of Biochemistry and Molecular Biology

Wednesday, June 27, 2012
1:00 P.M., Cancer Center, G level Conference Room


Interferon Regulatory Factors (IRFs) are a family of transcription factors that play significant roles in innate immunity and tumor suppression. IRF5 is one such family member known to exert crucial functions in the regulation of host immunity against extracellular pathogens, DNA damage-induced apoptosis, death receptor signaling and macrophage polarization.
Like many transcription factors, IRF5 must become "activated" in order to translocate from the cytoplasm to the nucleus where it can then regulate transcription of target genes. Upon exposure of cells to certain extracellular stressors including viral infection, DNA damage, death receptor signaling and Toll-like receptor (TLR) signaling, IRF5 undergoes post-translational modification which results in homodimerization or heterodimerization with other proteins resulting in its translocation to the nucleus and regulation of target gene expression. While much is known of these downstream activation processes, little is still known of IRF5 interacting partners or what IRF5 does in the cytoplasm of unstimulated cells. The goal of this project was to identify new interacting partners of IRF5 in both unstimulated and stimulated cells and characterize the functional consequence of these interactions. Using a variety of proteomics and molecular biology techniques, we were successful in the identification of new IRF5 interacting partners.
Here we report identification of the constitutive photomorphogenesis 9 (COP9) signalosome as a new interacting partner of IRF5 in unstimulated cells. The COP9 signalosome (CSN) is a highly conserved protein complex consisting of eight subunits that is best known for its role in regulating protein stability through the ubiquitin-proteosome pathway. Indeed, we found that interaction of the CSN with IRF5 controls its protein stability. The constitutive CSN/IRF5 interaction was identified by immunoprecipitation of Flag-tagged IRF5 in 293T cells by Mass Spectrometry analysis and confirmed by endogenous immunoprecipitation. All 8 subunits were found to interact with IRF5. Fine-mapping of the interaction led to the identification of both amino- and carboxyl-terminal regions of IRF5 that are necessary and required for interaction with IRF5. Amino-terminal residues 41-50 and carboxyl-terminal residues 455-466 were identified as the critical interacting sites. The subsequent generation of internal deletion mutants at these regions allowed for testing of the functional consequence of the CSN/IRF5 interaction. Utilizing the cycloheximide chase technique to analyze IRF5 protein stability, full-length IRF5 and the ∆455-466 internal deletion mutant were transiently transfected to 293T cells and protein levels detected by immunoblot or flow cytometry analysis. Data clearly showed that loss of the CSN/IRF5 interaction significantly enhanced the rate of degradation of IRF5 proteins.
Using a coupled transcription/translation (TnT) assay, we identified the COP9 subunit 3 (CSN3) as one of the direct interacting partners of IRF5. Next, we sought to confirm data from the internal deletion mutant by knocking down CSN1, CSN3 or both CSN1-CSN3 in an endogenous cellular system and measure IRF5 protein stability. CSN3 was targeted given its direct interaction with IRF5, while CSN1 and CSN3 were targeted together since it had been shown previously that knockdown of these two subunits led to an ~40% reduction in the entire CSN complex. Enhanced degradation of IRF5 proteins was observed with siRNAs targeting CSN3, yet degradation was further enhanced by knockdown of CSN1 and CSN3 together.
In an effort to determine the mechanism by which the CSN prevents IRF5 degradation in unstimulated cells, we tested IRF5 protein stability in the presence of the ubiquitin E1 inhibitor UBEI-41 or the proteosome inhibitor MG132. We found that either inhibitor prevented IRF5 degradation supporting that its stability is regulated by the ubiquitin-proteosome system. Furthermore, we examined the effect of IRF5 activation on the CSN/IRF5 interaction and found that activation of IRF5 by the death receptor ligand TRAIL resulted in enhanced degradation via loss of the CSN/IRF5 interaction. Thus, this study defines the CSN as a new interacting partner of IRF5 that controls its stability. Given that IRF5 has been proposed to play a pathogenic role in diseases such as systemic lupus erythematosus (SLE) and cancer, it may be relevant to look at alterations in the CSN/IRF5 complex in these two disease states since IRF5 expression is significantly upregulated in immune cells of SLE patients and downregulated in many tumor cell types. A clearer understanding of exactly how the CSN complex regulates IRF5 stability as well as its involvement in IRF5 activation may lead to new avenues for targeting this transcription

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