| Activation of the innate arm of the immune system following pathogen infections rely on the activation of latent transcription factors involved in the induction of a subset of genes responsible for pathogen's clearance. One of these transcription factors, Interferon Regulatory Factor 3 (IRF3), is an essential mediator of the innate immune response. Sustained activation of this transcription factor is also proposed to playa major role in acute and chronic inflammatory diseases. IRF3 is constitutively express in a variety of tissues where it is maintained in a latent conformation in the cytoplasm. Upon viral infection, IRF-3 is phosphorylated within its C-terminus extremity by the IKK-related kinases TANK-binding protein (TBK1) and IKKi inducing a conformationnal change that allows its homodimerization. In this conformation, IRF3 translocates into the nucleus via an atypical nuclear localization sequence (NLS) where its associates with coactivators CREB-binding protein (CBP)/p300 (histone acetyltransferases), inducing its sequestration into this compartment and allowing DNA binding activity. Following its activation, use pharmacological inhibitors has suggested that IRF-3 is targeted to the proteasome machinery. However, the molecular mechanisms involved in this process were unknown.;For several years, phosphorylation was viewed as one of the most important posttranslational modifications leading to the activation of signaling cascades. There is now clear evidence that polyubiquitination and acetylation work in parallel with phosphorylation to orchestrate the overall physiological response. Understanting how these posttranslational modifications affect the activity of IRF3 might pave the way towards new therapeutic avenues.;Keywords: PRR, viral infection, interferon, IRF3, TBK1/IKKi, ubiquitination, SCF complex, acetylation, CBP/p300, NLS.;Thus, the principal objective of this thesis was to characterize the signaling pathways involved in regulating the transcriptional activity of IRF3. We show for the first time that IRF3 is polyubiquitinated following its phosphorylation in its C-terminal end by TBK1 and IKKi. This process, which is mediated in part by a SCF complex E3 ligase, is important for recognition of fRF3 by the proteasome machinery. Lysine residues (K) are the major receptor for covalent attachment of ubiquitin but also for acetyl and methyl motieties. Interestingly, K77 of IRF3 is conserved among all the IRF members and was proposed to be pan of an atypical NLS. Thus, we next addressed the role of K77 in IRF3 activity. Replacing lysine 77 with an arginine residue or mutating the atypical NLS by replacing the two basic residues Lys-Arg to neutral residues Asn-Gly, creating IRF3 KR77/78NG completely abrogated the DNA binding activity of IRF3. In addition, the capacity of IRF3 K77R or IRF3 KR77/78NG to become phosphorylated, to homodimerize and, unexpectedly, to associate with CBP/p300 co-activators and to accumulate into the nucleus upon virus infection was not affected. Furthermore, we show that K77 is subjected to in vivo acetylation by CBP/p300 in a TBK1/IKKi dependent manner. Thus, our data questioned the proposed role of the atypical NLS sequence in the nuclear accumulation of IRF-3 and suggest that acetylation of IRF-3 on K77 by CBP/p300 represent one of last modifications required for its transcriptional activity. |
