652V Poster Online - Virtual Posters
Wednesday April 06, 4:00 PM - 7:00 PM

Authors:
Alison Julio ¹; Paloma Dias e Vasconcellos ¹; Priscila Gomes ²; Maira Cardoso ¹; Pedro Pascutti ²; Ethan Bier ^3,4; Paulo Bisch ²; Helena Araujo ^1,5

Affiliations:
1) Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, RJ. Brazil; 2) Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, RJ. Brazil; 3) Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, USA; 4) Tata Institute for Genetics and Society, University of California, San Diego, La Jolla, CA, USA; 5) Instituto Nacional de Entomologia Molecular (INCT-EM), Universidade Federal do Rio de Janeiro, RJ. Brazil

Keywords:
h. NF-kB; a. axis specification

Toll pathway activation during embryogenesis and during the innate immune response leads to N-terminal phosphorylation and subsequent degradation of inhibitor Cactus/IκB proteins through the proteasome. This event releases Dorsal/NFκB transcription factors that translocate to the nucleus and regulate gene expression. Cactus is also a target of proteolysis by Calpain A, producing a Cactus E10 fragment and regulating the levels of free Cactus that may interact with the Toll pathway. However, structural and biophysical requirements involved in the interaction of Calpain A and Cactus remains unknown. Here, we report the structural requirements for the action of Calpain A in the control of NFκB in Drosophila. We have modeled the Calpain A:Cactus, Cactus E10 and Dorsal:Cactus complexes, to define the previously unknown domains involved for complex establishment and maintenance. Molecular dynamic simulations suggested that the N-terminal region of Cactus undergoes wide conformational rearrangements upon interaction with Calpain A. Our results indicate that Cactus acts as a modulator in the cleavage process, by approximating the cleavage site to the catalytic site of Calpain A supporting a fit induced model. In addition, our simulations indicate that the Calpain A:Cactus complex is sustained by salt-bridges formed along the Cactus alpha helix anti-parallel ankyrin repeats and Calpain A EF hands 2, 3, 5 and CBSW domains. Subsequent mutational analysis confirms our structural predictions. Functional analysis using Cactus alleles mutated at the cleavage site and catalytically-dead Calpain A mutants demonstrates that the Cactus cleavage event by Calpain A is fundamental in modulating NFκB responses triggered by Dorsal nuclear translocation. Interestingly, our results revealed an unexpected function for Calpain A independent of catalysis, suggesting that Calpain A may also modulate Toll signal transduction by sequestering Cactus. These results indicate that competition between Calpain A:Cactus and Dorsal:Cactus complexes is an important aspect in Toll responses. New CRISPR Calpain A alleles reinforce the action of Calpain A to modify the Dorsal gradient, affecting target genes during early embryogenesis, and to modulate immune responses mediated by Toll. Taken together, these results provide a mechanistic view for the structural requirements involved in Calpain A modulating NFκB signaling.