Poster Presentation AUS-oMicS 2025

Mutations in the vicinity of the IRAK3 guanylate cyclase centre impact its subcellular localisation and ability to modulate inflammatory signalling (122596)

Ilona Turek 1 2 , Trang Nguyen 2 3 4 , Charles Galea 5 , Isaiah Abad 5 , Lubna Freihat 5 , David Manallack 5 , Tony Velkov 6 , Helen Irving 2 3 4
  1. Australian Centre for Disease Preparedness, Commonwealth Scientific and Industrial Research Organisation, East Geelong, Victoria, Australia
  2. Department of Rural Clinical Sciences, La Trobe University, Bendigo, Victoria, Australia
  3. La Trobe Institute for Molecular Science, La Trobe University, Bendigo, Victoria, Australia
  4. Holsworth Initiative for Medical Research, Rural People, La Trobe University, Bendigo, Victoria, Australia
  5. Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria, Australia
  6. Department of Microbiology, Monash University, Clayton, Victoria, Australia

Interleukin-1 receptor-associated kinase 3 (IRAK3) modulates the magnitude of cellular responses to ligands perceived by interleukin-1 receptors (IL-1Rs) and Toll-like receptors (TLRs), leading to decreases in pro-inflammatory cytokines and suppressed inflammation. Although IRAK3 was proposed as a useful diagnostic and prognostic marker in inflammation, and possibly a target for intervention, the exact mechanism of action and the selectivity of IRAK3 is still largely unclear. Prior studies using bioinformatic search tools identified IRAK3 as a potentially novel guanylate cyclase catalysing cyclic guanosine monophosphate (cGMP) synthesis, and IRAK3 was shown to contain a guanylate cyclase (GC) centre within its pseudokinase domain. We demonstrate that wild type IRAK3 is capable of producing cGMP, whereas point mutations in the vicinity of the GC centre reduce cGMP production and influence distribution of the protein in mammalian cells, suggestive of changes in the interactome. Either IRAK3 or membrane permeable cGMP alone suppresses downstream signalling through modulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in the presence of lipopolysaccharide (LPS). IRAK3 mutants with reduced cGMP-generating capacity failed to suppress LPS-induced NFκB activity in the absence of cGMP, suggesting that the cGMP generated by IRAK3 may be involved in its regulatory function. The presence of cGMP may selectively affect downstream signalling pathway(s) by modulating the binding and/or activity of nearby interacting proteins involved in the cascade and thus contribute to the selectivity and functionality of IRAK3 in the inflammatory signalling cascade. Mutant IRAK3 variants with reduced cGMP generating capacity and differential regulation of NFκB activity influence subcellular localisation of IRAK3 in HEK293T cells and fail to rescue IRAK3 function in stimulated IRAK3 knock-out THP-1 monocytes unless the cGMP analog is present. Together, our results shed new light on the mechanism by which IRAK3 and its enzymatic product control the downstream signalling, affecting inflammatory responses.