Application of immobilized ATP to the study of NLRP inflammasomes

• Published in: Archives of biochemistry and biophysics 2019-07, Vol.670, p.104-115
• Main Authors: Liao, Kuo-Chieh, Sandall, Christina F., Carlson, David A., Ulke-Lemée, Annegret, Platnich, Jaye M., Hughes, Philip F., Muruve, Daniel A., Haystead, Timothy A.J., MacDonald, Justin A.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Drug discovery; FLECS; Fluorescence-linked enzyme chemoproteomic strategy; HEK293 Cells; Humans; Immobilized ATP Sepharose; Inflammasome; Inflammasomes - metabolism; Leucine rich repeat and pyrin domain containing-3; NLR Proteins - metabolism; NLRP; Nucleotide-binding oligomerization domain; Phosphorylation; Protein Processing, Post-Translational; Selected reaction monitoring mass spectrometry; SRM-MS; Ubiquitination
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2018.12.031

The NLRP proteins are a subfamily of the NOD-like receptor (NLR) innate immune sensors that possess an ATP-binding NACHT domain. As the most well studied member, NLRP3 can initiate the assembly process of a multiprotein complex, termed the inflammasome, upon detection of a wide range of microbial products and endogenous danger signals and results in the activation of pro-caspase-1, a cysteine protease that regulates multiple host defense pathways including cytokine maturation. Dysregulated NLRP3 activation contributes to inflammation and the pathogenesis of several chronic diseases, and the ATP-binding properties of NLRPs are thought to be critical for inflammasome activation. In light of this, we examined the utility of immobilized ATP matrices in the study of NLRP inflammasomes. Using NLRP3 as the prototypical member of the family, P-linked ATP Sepharose was determined to be a highly-effective capture agent. In subsequent examinations, P-linked ATP Sepharose was used as an enrichment tool to enable the effective profiling of NLRP3-biomarker signatures with selected reaction monitoring-mass spectrometry (SRM-MS). Finally, ATP Sepharose was used in combination with a fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen to identify potential competitive inhibitors of NLRP3. The identification of a novel benzo[d]imidazol-2-one inhibitor that specifically targets the ATP-binding and hydrolysis properties of the NLRP3 protein implies that ATP Sepharose and FLECS could be applied other NLRPs as well.