Browsing by Author "Ulke-Lemée, Annegret"
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- ItemOpen AccessApplication 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 AnthonyThe 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.
- ItemOpen AccessBinding of smoothelin-like 1 to tropomyosin and calmodulin is mutually exclusive and regulated by phosphorylation(2017-03-21) Ulke-Lemée, Annegret; Sun, David H; Ishida, Hiroaki; Vogel, Hans J; MacDonald, Justin AAbstract Background The smoothelin-like 1 protein (SMTNL1) can associate with tropomyosin (Tpm) and calmodulin (CaM), two proteins essential to the smooth muscle contractile process. SMTNL1 is phosphorylated at Ser301 by protein kinase A during calcium desensitization in smooth muscle, yet the effect of SMTNL1 phosphorylation on Tpm- and CaM-binding has yet to be investigated. Results Using pull down studies with Tpm-Sepharose and CaM-Sepharose, we examined the interplay between Tpm binding, CaM binding, phosphorylation of SMTNL1 and calcium concentration. Phosphorylation greatly enhanced the ability of SMTNL1 to associate with Tpm in vitro; surface plasmon resonance yielded a 10-fold enhancement in K D value with phosphorylation. The effect on CaM binding is more complex and varies with the availability of calcium. Conclusions Combining both CaM and Tpm with SMTNL1 shows that the binding to both is mutually exclusive.