Toll-Like Receptors are activated by substances produced by microbial pathogens, such as lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria. This induces the formation of the Myddosome, a multi-protein complex, which recruits and activates at least three E3 ubiquitin ligases, termed TRAF6, Pellinos and LUBAC. These E3 ligases then generate hybrid ubiquitin chains containing both Lys63- and Met-1-ubiquitin linkages. One of their functions is to recruit and activate the “master” TAK1 and IKK kinases that drive production of the inflammatory mediators needed to combat microbial infections. Another function is to recruit negative regulators of the pathway, such as ABIN1 and A20, which restrict its activation and so prevent the uncontrolled production of inflammatory mediators that is a major cause of inflammatory and autoimmune diseases. TRAF6 has other essential functions in the pathway that are independent of its E3 ligase activity (reviewed Cohen and Strickson, 2017, Cell Death Diff. 24, 1153-1159).
Recently, the atypical mammalian protein kinase ALPK1 was reported to be the receptor for the bacterial metabolite ADP-heptose, which is required for the synthesis of LPS. ADP-heptose activates ALPK1 enabling it to phosphorylate TIFA (TRAF-interacting protein with Forkhead-Associated domain). TIFA then induces the dimerization of TRAF6 and activation of the TAK1 and IKK kinases. This is likely to be a novel pathway for combatting gram negative bacteria that have evaded the membrane-associated TLR systems and have entered the cytosol of infected mammalian cells. The aim of the project is to build on these novel and exciting findings to understand the role of ubiquitin chains in this pathway. By analogy with the TLR signalling network, does the ADP-hexose-ALPK1-TIFA pathway induce the formation of hybrid ubiquitin chains and does this involve Pellinos and LUBAC or other E3 ligases, as well as TRAF6? Which proteins undergo ubiquitylation in response to ADP-Hexose and which protein deubiquitylases control the hydrolysis of these ubiquitin chains and how are they regulated? As in the TLR pathway, does TRAF6 have both E3 ligase-dependent and independent roles? Further work will depend on the outcome these experiments and on the ingenuity of the student that tackles this problem. This is a multidisciplinary project that connects with a number of branches of the life sciences.