LAURA HURTADO NAVARRO¹, DIEGO ANGOSTO BAZARRA¹, Ana Tapia Abellán², Juan Ignacio Arostegui Gorospe³, PABLO PELEGRIN VIVANCOS¹

(1) CIRUGÍA DIGESTIVA, ENDOCRINA Y TRASPLANTE DE ÓRGANOS ABDOMINALES, IMIB-Arrixaca, España
(2) Eberhard Karls Univesität Tübingen, Alemania
(3) Hospital Clinic de Barcelona, España (Cataluña)

Nucleotide-binding leucine-rich repeat receptor with a pyrin domain-containing protein 3 (NLRP3) is an innate immune sensor of the nucleotide-binding leucine-rich repeat receptor (NLR) family. Activation of NLRP3 oligomerizes and form inflammasomes that uncontrolled, due to gain-of-function mutations on NLRP3, is one of the major causes of numerous autoimmune diseases like Cryopirin-associated periodic syndrome (CAPS). Some CAPS patients present mutations in NLRP3 that only appear in mosaicism and others in the germ line. Recently our group (Tapia-Abellán et al. 2019) demonstrated that MCC950, a potent and selective inhibitor of NLRP3, could block the oligomerization and activation of mutant NLRP3 by changing its structure. However, as the inhibitor effect of MCC950 depends on the specific mutation, studying every case in particular could raise a possibility to develop personalized therapy for individuals with monogenic autoinflammatory syndromes carrying specific alterations. Tapia-Abellán A, Angosto-Bazarra D, Martínez-Banaclocha H, de Torre-Minguela C, Cerón-Carrasco JP, Pérez-Sánchez H, Arostegui JI, Pelegrin P. MCC950 closes the active conformation of NLRP3 to an inactive state. Nat Chem Biol. (2019); 15(6): 560-564. doi: 10.1038/s41589-019-0278-6.

Plasmid constructs that contain unique auto-active mutations of NLRP3 and wild-type NLRP3 tagged at N-terminus with YFP and at C-terminus with Luciferase were produced by overlapping PCR, sequenced and transiently transfected in HEK293T. The expression of the different NLRP3 were monitored by reading YFP fluorescence. Bioluminescence resonance energy transfer (BRET) was used to study the conformation of the wild-type and mutant NLRP3, expressing the results in milliBRET units (mBUs).

NLRP3 mutations within the central NACTH domain, which is associated to the activation of NLRP3, implicate an open/active conformation of the inflammasome since the BRET signal decrease with regards to the wild-type control. Mutations found only in mosaicisms presented lower BRET signal. In all cases studied, MCC950 is associated to changes in the conformation to a close/inactive structure, but with different potency depending the type of NLRP3 mutation.

Overall, NLRP3 mutations that implicate changes in the polarity or charge of the amino acids result in an open state of the inflammasome. This open/active state was more evident in NLRP3 mutations associated to mosaicisms and could explain the relationship between the mutations studied and the autoinflammatory syndromes associated to them.