ATP-binding to the CATERPILLER protein Monarch-1, is required for its inhibitory function (44.19)

Abstract The newly discovered CATERPILLER (NOD, NLR) family is defined by a nucleotide-binding domain (NBD) that consists of the Walker A and B motifs and ten additional motifs. It is conserved from plants to humans, and is important in immune regulation and several genetic disorders. Although the NBD is a defining feature of this family, there is a paucity of data to indicate that these proteins bind nucleotides, or the binding of nucleotides modulate their functions. Monarch-1/Pypaf7 is a negative regulator of IRAK-1 and NIK. In this report, we successfully purified recombinant Monarch-1 proteins to homogeneity. Purified Monarch-1 binds ATP but not CTP, GTP and UTP and exhibits ATP hydrolysis activity. Intact Walker A/B sequences are required for nucleotide binding. Overexpression of Monarch-1 nucleotide-binding defective mutant in THP-1 cells resulted in a dramatic increase of IL-6, chemokine CCL1, CXCL6 and CXCL13 to the extent that is comparable to the THP-1 Monarch-1 shRNA knock down cells in response to TLR2 agonist. This indicates that ATP binding is required for the inhibitory function of Monarch-1 and the nucleotide binding-defective mutant displays a dominant negative effect. Intact nucleotide-binding domain is also required for IRAK-1 dephosphorylation and p100 processing suggesting a nucleotide binding-dependent mechanism of Monarch-1 function in both canonical and non-canonical NF-κB pathway.