Browsing by Author "Sandall, Christina F."

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    Application of immobilized ATP to the study of NLRP inflammasomes
    (2019-01-11) Liao, Kuo Chieh; Sandall, Christina F.; Carlson, David A.; Ulke-Lemée, Annegret; Platnich, Jaye; Hughes, Philip Floyd; Muruve, Daniel A.; Haystead, Timothy Arthur James; MacDonald, Justin Anthony
    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.
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    Effects of phosphorylation on the NLRP3 inflammasome
    (2019-03-05) Sandall, Christina F.; MacDonald, Justin Anthony
    The pyrin domain containing Nod-like receptors (NLRPs) are a family of pattern recognition receptors known to regulate an array of immune signaling pathways. Emergent studies demonstrate the potential for regulatory control of inflammasome assembly by phosphorylation, notably NLRP3. Over a dozen phosphorylation sites have been identified for NLRP3 with many more suggested by phosphoproteomic studies of the NLRP family. Well-characterized NLRP3 phosphorylation events include Ser198 by c-Jun terminal kinase (JNK), Ser295 by protein kinase D (PKD) and/or protein kinase A (PKA), and Tyr861 by an unknown kinase but is dephosphorylated by protein tyrosine phosphatase non-receptor 22 (PTPN22). Since the PKA- and PKD-dependent phosphorylation of NLRP3 at Ser295 is best characterized, we provide detailed review of this aspect of NLRP3 regulation. Phosphorylation of Ser295 can attenuate ATPase activity as compared to its dephosphorylated counterpart, and this event is likely unique to NLRP3. In silico modeling of NLRP3 is useful in predicting how Ser295 phosphorylation might impact upon the structural topology of the ATP-binding domain to influence catalytic activity. It is important to gain as complete understanding as possible of the complex phosphorylation-mediated mechanisms of regulation for NLRP3 in part because of its involvement in many pathological processes.
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    Structural Dissection and Catalytic Properties of the NLRP (Nucleotide-Binding Domain and Leucine-Rich Repeat-Containing Gene Family, Pyrin Domain Containing) Family of Inflammatory Proteins
    (2022-09-19) Sandall, Christina F.; MacDonald, Justin; Muruve, Daniel; Lees-Miller, Susan
    Inflammasomes are high molecular weight hetero-oligomeric protein complexes nucleated by innate immune cytosolic pattern recognition receptors (PRRs) including the NOD-like receptors (NLRs). A subset of NLR proteins include those with N-terminal Pyrin domains (NLRPs) which play critical roles in the detection and response to both endogenous and exogenous danger signals. The NLRP3 inflammasome is the most highly studied and contributes to a multitude of inflammatory and autoimmune conditions. Thus, this work provides a more detailed understanding of NLRP3 activation mechanisms that will be essential for the rationalised development of future pharmacological interventions. First, the impact of orientation and linkage on NLRP3 capture with immobilised ATP demonstrates ATP binds this protein with an exposed phosphate tail and buried adenine ring. Decreased competitive recovery with free ATP indicated NLRP3 underwent a conformational change upon ATP binding. Additionally, P-linked ATP Sepharose provides a strategy for capture of the entire NLRP family and enrichment of NLRP3 containing samples for mass spectrometry analyses. Next, the ATP hydrolysis kinetics of NLRP proteins and hyperactive NLRP3 disease mutant R262W were evaluated with GFP nano-trap beads and a bioluminescent ATPase assay. NLRP proteins displayed distinct ATPase kinetic profiles, suggesting variable ATP sensitivity and kinetics of assembly for some NLRP proteins. Classical Michaelis-Menten kinetics were observed for NLRP1, 3 and 12, and positive Hill cooperativity was revealed for NLRP3R262W as well as NLRP6 and 7. Furthermore, NLRP3 inhibitors targeting ATPase activity demonstrated promising results in blocking inflammasome activation. Next, the impacts of NLRP3 phosphorylation at Ser295 on protein structure, ATP binding & inflammasome activation were evaluated in silico. These results suggest that modifications to the NACHT domain are conveyed globally and result in variable nucleotide hydrolysis and inflammasome activation. Finally, unique mechanisms of ATP and ADP binding and the detailed structural impacts were illuminated by molecular dynamics simulations with NLRP3. NLRP3-ADP simulations indicate high protein stability and few global rearrangements, while NLRP3-ATP binding was thermodynamically favourable, induced protein flexibility, and resulted in global structural rearrangements that were initiated from the NACHT domain. To conclude, a detailed mechanism of ATP induced structural changes provided a basis for rationale design of NLRP3 inhibitors, and a region of NLRP3 in a cleft between the HD2 and LRR domains was proposed for pharmacological targeting.