Application of immobilized ATP to the study of NLRP inflammasomes

Abstract

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 fluorescencelinked 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. . The NLRP Inflammasome. The inflammasome is a protein complex and caspase-activating platform that drives inflammation and plays a crucial role in the innate immune response [1][2][3][4].Following stimulation by pathogen or danger/damage associated molecular patterns (PAMPs or DAMPs), inflammasome assembly induces the recruitment and autocatalytic cleavage of procaspases-1, 8 and 11, which are cysteine proteases and the key effectors of the inflammasome [5][6][7]. Activated caspases in turn regulate key processes involved in inflammation and host defense that includes cytokine maturation, metabolism, reactive oxygen species (ROS) generation, apoptosis and pyroptosis. For example, pro-IL-1, an endogenous pyrogen that induces fever as well as other immune responses, is cleaved by caspase-1 into mature IL-1 and is released into the extracellular environment to fulfill its physiological functions [8].Each inflammasome has scaffold proteins that determine inflammasome specificity and mediate its assembly. The nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing-3 (NLRP3) protein is the most extensively studied inflammasome scaffold protein. This protein belongs to the 14-member NLRP subfamily of the immune sensor Nod-like receptor (NLR) family [9]. NLRP3 possesses an N-terminal pyrin domain (PYD), a central NAIP, CIITA, HET-E and TP1 (NACHT) domain, and a C-terminal leucine rich repeat (LRR) domain [10]. The PYD of NLRP3 has been demonstrated to interact with pro-caspase-1 ...

ATP-Binding and Hydrolysis in Inflammasome Activation

“…These structural models revealed a similar spatial arrangement of nucleotide-binding domain (NBD), helical domain 1 (HD1) and winged helix domain (WHD) for the entire NLRP family. Biochemical studies of the NLRP family members with P-linked ATP-Sepharose also support effective ATP-binding potential [ 80 ].…”

“…This result contrasted with the abolition of activity observed when a similar silencing mutation was introduced into the Walker A motif of NLRP3. While the ATP-binding properties of NLRP1 to immobilized ATP-Sepharose are similar to other human NLRP family members [ 80 ], the NACHT domain of murine NLRP1b may have different requirements for ATP than that of human NLRP1. Ultimately, future clarification will be required since the studies to date have employed both murine and human homologues as well as different model systems for the assessment of inflammasome signaling (i.e., cell-based and recombinant proteins).…”

“…While structural modeling supports the ability of NLRP2 to bind ATP [ 47 ], NLRP2 was unique amongst the NLRPs in displaying low elution efficiency from immobilized ATP-Sepharose resin. This could indicate lower comparative efficacy in ATP binding [ 80 ]. Of note, evidence suggests that the NLRP2 inflammasome also contains CARD8, a caspase recruitment domain-containing protein.…”

“…Although the role of the NACHT domain in regulating the assembly of NLRP9 inflammasome has not been examined, a few reports suggest a functional importance for ATP. NLRP9 could be recovered from immobilized ATP-Sepharose resin [ 80 ], which suggests effective ATP-binding. Moreover, 3D modeling of the NACHT domain suggests stable coordination of ADP and ATP in the binding pocket of NLRP9, although comparatively fewer H-bonds were identified with molecular dynamic simulations using a manually-docked complex [ 47 ].…”

“…A recent study of NLRP6 provided some evidence for ATP-binding properties; LPS-induced NLRP6 dimers were assembled into higher molecular oligomers only in the presence of ATP [ 139 ]. Additionally, both NLRP6 and NLRP8 could be captured with immobilized γ-linked ATP-Sepharose and eluted in a concentration-dependent manner with ATP [ 80 ].…”