Comunicación

CHARACTERIZATION OF NLRC4 MUTATIONS ASSOCIATED TO AUTOINFLAMMATORY SYNDROMES

Autores:

ALEJANDRO ELEAZAR PEÑIN FRANCH1, Ana Tapia Abellán2, DIEGO ANGOSTO BAZARRA1, Juan Ignacio Arostegui Gorospe3, PABLO PELEGRIN VIVANCOS1

Afiliaciones:

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

Comunicación:

Antecedentes:

Nucleotide-binding domain leucine-rich repeat containing a CARD domain 4 (NLRC4) inflammasome forms oligomers upon recognition of intracellular flagellin by NAIPs and activate a type of inflammasome to drive an inflammatory response. Structural studies have suggested that oligomerization is mediated by conformational changes in the NLRC4 protein structure, moving from a close to an open structure. Different NLRC4 mutations have been related with autoinflammatory syndromes as the macrophage activation syndrome. This study aims to address the conformation of mutated NLRC4.

Métodos:

Construction of plasmids to express wild-type and different mutated NLRC4 under the control of the CMV promoter were developed by overlapping PCR, sequenced and transfected in HEK293 cells. In these constructions NLRC4 were fused at the amino-terminal to YFP and at the carboxy-terminal to luciferase. After 24 h, transfected cells were seeded into white 96-well plates with opaque bottom, and after 16 h, 5 µM of the luciferase substrate coelenterazine H was added. Bioluminescence resonance energy transfer (BRET) was measured in a plate reader at 480 and 530 nm every 5 minutes during 30 minutes. Results were expressed in milliBRET units (mBUs) and allow us to interpret the theoretical distance among YFP and luciferase in wild-type and the different NLRC4 mutants, evidencing differential protein conformations.

Resultados:

Wild-type NLRC4 present a BRET signal of 662.7 ± 71.5 mBUs. Only mutated NLRC4 whose amino acids change polarity or charge (S445P, C697S, and D1009G) showed a decreased BRET signal. Also, BRET signal decrease was dependent on the domain affected by the mutation, being lower BRET found when the winged-helix domain (WHD) was mutated, and higher BRET when the leucine-rich repeat (LRR) domain was mutated.

Conclusiones:

Mutations in NLRC4 that present a change in polarity or charge of the amino acid are able to change the conformational state of the protein to an open structure, resulting in a distancing of YFP and luciferase, with a concomitant decrease of the BRET signal. The NLRC4 BRET signal variation also depends on the domain mutated, presenting a lower BRET when the mutation is in the WHD, which is close to the hinge domain of the protein, than mutations in the distal LRR. Mutated NLRC4 with an open state could produce spontaneous activation without the requirement of any further stimuli and could explain the association of this mutation with autoinflammatory syndromes.


Dirección

Campus de Ciencias de la Salud
Carretera Buenavista s/n, 30120 El Palmar
Murcia, España

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