Mutations in the Vicinity of the IRAK3 Guanylate Cyclase Center Impact Its Subcellular Localization and Ability to Modulate Inflammatory Signaling in Immortalized Cell Lines

Ilona Turek, Trang H. Nguyen, Charles Galea, Isaiah Abad, Lubna Freihat, David T. Manallack, Tony Velkov, Helen Irving

Research output: Contribution to journalArticleResearchpeer-review

2 Citations (Scopus)

Abstract

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. The molecular mechanism of IRAK3’s action remains unknown. IRAK3 functions as a guanylate cyclase, and its cGMP product suppresses lipopolysaccharide (LPS)-induced nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) activity. To understand the implications of this phenomenon, we expanded the structure–function analyses of IRAK3 through site-directed mutagenesis of amino acids known or predicted to impact different activities of IRAK3. We verified the capacity of the mutated IRAK3 variants to generate cGMP in vitro and revealed residues in and in the vicinity of its GC catalytic center that impact the LPS-induced NFκB activity in immortalized cell lines in the absence or presence of an exogenous membrane-permeable cGMP analog. Mutant IRAK3 variants with reduced cGMP generating capacity and differential regulation of NFκB activity influence subcellular localization of IRAK3 in HEK293T cells and fail to rescue IRAK3 function in IRAK3 knock-out THP-1 monocytes stimulated with LPS 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 signaling, affecting inflammatory responses in immortalized cell lines.

Original languageEnglish
Article number8572
Number of pages19
JournalInternational Journal of Molecular Sciences
Volume24
Issue number10
DOIs
Publication statusPublished - May 2023

Keywords

  • cyclic guanosine monophosphate (cGMP)
  • guanylate cyclase
  • human Toll-like receptor 4 (hTLR4)
  • inflammatory cytokines
  • interleukin-1 receptor-associated kinase 3 (IRAK3 or IRAK-M)
  • lipopolysaccharide
  • nuclear factor kappa-light-chain-enhancer of activated B (NFκB)

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