Viral invasion triggers a swift and decisive response of the immune system to the site of infection. This defense mechanism is to attenuate the ability of the virus to replicate, cause systemic havoc or even death. The activation of the immune system is thus crucial to fight infection and this response is critically dependent on the production of type I interferon. Post-translational modification of proteins by ubiquitination is essential in the interferon-mediated innate antiviral immune response. During this process, the ubiquitin machinery covalently links either ubiquitin monomers or polymeric chains to a lysine residue on the peptide of interest. For instance, polymeric ubiquitin chains can be formed by sequential linkage of an ubiquitin molecule to lysine 48 (K48) or lysine 63 (K63) of the preceding ubiquitin. K48-linked polyubiquitin signals the degradation of the protein, whereas K63-linked ubiquitin modulates the signal function of the target protein. Chen ZJ et al (2004) have shown that TNF receptor-associated factor (TRAP) protein family members such as TRAF6 are involved in ubiquitination of higher order kinase complexes. These complexes such as the TAB/TAK1 and ?/? kinases, when modified by K63-linked polyubiquitin, activate the NF-?B pathway. Sequence analysis of TRAF3, another TRAF family member, reveals that it contains a RING finger domain. Truncation of this domain hinders the protein's E3 ubiquitin-protein ligase activity and consequently resulted in less interferon production (Saha SK, 2006). Preliminary studies showed that TRAFS's downstream target, TBK1, undergo K63-linked and K-48 linked polyubiquitination. Taken together, these findings suggest that the ubiquitination status of TBK1 may be critical in regulating its activity and determining whether or not IRF3 and IRF7 become phosphorylated by TBK1 to promote interferon gene expression. We thus hypothesize that K63-linked ubiquitination of specific residues on TBK1 is required for activation of this kinase and conversely, K48-linked ubiquitination of TBK1 results in its degradation and down-modulation of the IFN response.
In specific aim 1, we will identify K63-linked and K48-linked ubiquitin acceptor site(s) on TBK1.
In specific aim 2, we will study the functionality of the newly identified sites.
In specific aim 3, we will determine whether ubiquitination is necessary for TBK1 to form signaling complexes during viral infection and whether TBK1 stability can be altered by ubiquitination. The end result of this project will be a better understanding on the role of ubiquitination on TBK1-mediated antiviral response. A greater understanding of TBK1-mediated antiviral defense may suggest potential therapeutic targets in clinical applications.
