Oral Presentation 15th Lorne Infection and Immunity 2025

Mechanisms of NLRP3 activation and inhibition elucidated by functional analysis of disease-associated variants (#33)

Shouya Feng 1 2 3 4 , Matthew Wierzbowski 2 , Katja Hrovat-Schaale 2 , Yaoyuan Zhang 2 , Andreas Dumortier 2 , Thomas Reygaerts 2 , Annemarie Steiner 2 , Dominic De Nardo 5 , Dhanya Lakshmi Narayanan 1 , Raju P Khubchandani 6 , Florian Milhavet 7 , Guilaine Boursier 7 , Seth L. Masters 1 2 3 4
  1. Hudson Institute of Medical Research, Clayton, VICTORIA, Australia
  2. Inflammation, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia
  3. Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia
  4. Department of Molecular and Translational Science, Monash University, Melbourne, Victoria, Australia
  5. Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia
  6. Department of Pediatric Rheumatology, Jaslok Hospital and Research Centre, Mumbai, India
  7. Department of Medical Genetics, Rare and Autoinflammatory diseases unit, University of Montpellier, Montpellier, France

The NLRP3 inflammasome is a multiprotein complex that mediates caspase-1 activation and the release of proinflammatory cytokines including interleukin (IL)-1β and IL-18. Gain-of-function mutations in the gene encoding NLRP3 (also called cryopyrin) lead to constitutive inflammasome activation and excessive IL-1βproduction in cryopyrin-associated periodic syndromes (CAPS). Here we present functional screening and automated analysis of 529 NLRP3 variants from the international INFEVERS registry and the ClinVar database. This resource captures the effect of NLRP3 variants on ASC speck formation spontaneously, at low temperature, after inflammasome stimulation, and with the specific inhibitor MCC950. Most importantly, our analysis facilitated the updated classification of NLRP3 variants in INFEVERS. Structural analysis suggested multiple mechanisms by which CAPS mutations activate NLRP3, including enhanced ATP binding, stabilizing the active NLRP3 conformation, destabilizing the inactive NLRP3 complex, and promoting oligomerization of the pyrin domain. Furthermore, we identified pathogenic variants that can hypersensitize the activation of NLRP3 in response to nigericin and cold temperature exposure. We also found that most CAPS-related NLRP3 variants can be inhibited by the small molecule MCC950. However, NLRP3 variants with changes to proline affecting helices near the inhibitor binding site are resistant to MCC950, as are variants in the pyrin domain which likely trigger activation with the ASC pyrin domain directly. Our findings could help stratify the CAPS population for NLRP3 inhibitor clinical trials and our automated methodologies can be implemented for molecules with a different mechanism of activation, and in labs worldwide that are interested in adding new functionally validated NLRP3 variants to the resource. Overall, our study provides improved diagnosis for patients with CAPS, mechanistic insight into the activation of NLRP3, and stratification of patients for the future application of targeted therapeutics.