NLRP1 is an inflammasome-forming pattern recognition receptor that triggers release of inflammatory interleukins and pyroptotic cell death in response to pathogens or cellular stress. NLRP1 is highly expressed in epithelial barrier tissues and certain rare NLRP1 variants are responsible for localised and systemic autoinflammatory diseases in patients. These include keratosis of various epithelial tissues, papillomatosis of extremities or mucosal surfaces and multiple self-healing palmoplantar carcinoma. Over 600 non-synonymous, exonic variants of NLRP1 identified from human genomic sequencing have been reported on databases such as ClinVar and Infevers. Many variants are classified as likely benign, however, most have not been thoroughly analysed and are thus listed as variants of uncertain significance (VUS).
To systematically assess the functional impacts of these uncharacterised variants, we measured inflammasome activation in a HEK293T overexpression model both spontaneously and in response to known NLRP1 activators anisomycin and talabostat. This approach allowed direct comparison of variants with the wild-type protein and has identified 18 previous VUS that are highly autoactive at baseline and therefore can be reclassified as likely pathogenic, and additional variants with differential responses to the tested NLRP1 agonists. Through ongoing structural modelling and experimental molecular analyses of these variants, we have uncovered new details of the many ways NLRP1 can be activated, including structural rearrangement of the N-terminus and areas of the protein important for autoproteolytic cleavage and interaction with the natural NLRP1 inhibitor DPP9. These results contribute to our understanding of NLRP1 biology and will aid with stratification of NLRP1-asscoiated autoinflammatory diseases.