Lack of interaction between between NEMO and SHARPIN impairs linear ubiquitination and NF-κB activation, and leads to incontinentia pigmenti

Publication date: Available online 27 February 2017
Source:Journal of Allergy and Clinical Immunology
Author(s): E. Bal, E. Laplantine, Y. Hamel, V. Dubosclard, B. Boisson, A. Pesacatore, C. Picard, S. Hadj-Rabia, G. Royer, J. Steffann, J.P. Bonnefont, V.M. Ursini, P. Vabres, A. Munnich, J.L. Casanova, C. Bodemer, R. Weil, F. Agou, A. Smahi
BackgroundIncontinentia pigmenti (IP; MIM308300) is a severe, male-lethal, X-linked, dominant genodermatosis resulting from loss-of–function mutations in the IKBKG gene encoding NF-κB essential modulator (NEMO, the regulatory subunit of the IKK complex). In 80% of cases of IP, the deletion of exons 4 to 10 leads to the absence of NEMO and total inhibition of NF-κB signaling. Here, we described a new IKBKG mutation responsible for IP resulting in an inactive truncated form of NEMO.ObjectivesTo identify the mechanism(s) by which the truncated NEMO protein inhibits the NF-kB signalling pathway.MethodsWe sequenced the IKBKG gene in IP patients and performed complementation and transactivation assays in NEMO-deficient cells. We also used immunoprecipitation assays, immunoblotting and an in situ proximity ligation assay to characterize the truncated NEMO protein interactions with IKK-α, IKK-β, TRAF6, TRAF2, RIP1 HOIL-1, HOIP and SHARPIN. Lastly, we assessed NEMO linear ubiquitination by immunoblotting and investigated the formation of NEMO-containing structures (using immunostaining and confocal microscopy) after cell stimulation with IL-1β.ResultsWe identified a novel splice mutation in IKBKG (c.518+2T>G, resulting in an in-frame deletion: p.DelQ134_R256). The mutant NEMO lacked part of the CC1 coiled-coil and the HLX2 helical domain. The p.DelQ134_R256 mutation caused inhibition of NF-κB signaling, although the truncated NEMO protein interacted with proteins involved in the activation of NF-κB signaling. The IL-1β-induced formation of NEMO-containing structures was impaired in IP fibroblasts carrying the truncated NEMO form (as also observed in HOIL-1^-/- cells). The truncated NEMO interaction with SHARPIN was impaired in a male fetus with IP, leading to defective linear ubiquitination.ConclusionWe identified a hitherto unreported disease mechanism (defective linear ubiquitination) in IP.Clinical implicationsA splice mutation in IKBKG gene responsible for IP result in an inactive truncated form of NEMO, unable to interact specifically with SHARPIN leading to defect in NEMO linear ubiquitination.

Teaser

We identified for the first time in human the NEMO domain involved in SHARPIN interaction which is dispensable for NF-κB signaling. Beside, HOIL-1, HOIP, and OTULIN deficiency we confirmed that defect of linear ubiquitination is responsible for human diseases.


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