title: Lack of interaction between NEMO and SHARPIN impairs linear ubiquitination and NF-κB activation and leads to incontinentia pigmenti.
abstract: BACKGROUND
  Incontinentia pigmenti (IP; MIM308300) is a severe, male-lethal, X-linked, dominant genodermatosis resulting from loss-of-function mutations in the IKBKG gene encoding nuclear factor κB (NF-κB) essential modulator (NEMO; the regulatory subunit of the IκB kinase [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 describe a new IKBKG mutation responsible for IP resulting in an inactive truncated form of NEMO.
  
  
  OBJECTIVE
  We sought to identify the mechanism or mechanisms by which the truncated NEMO protein inhibits the NF-κB signaling pathway.
  
  
  METHODS
  We sequenced the IKBKG gene in patients with IP 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, TNF receptor-associated factor 6, TNF receptor-associated factor 2, receptor-interacting protein 1, Hemo-oxidized iron regulatory protein 2 ligase 1 (HOIL-1), HOIL-1-interacting protein, and SHANK-associated RH domain-interacting protein. Lastly, we assessed NEMO linear ubiquitination using immunoblotting and investigated the formation of NEMO-containing structures (using immunostaining and confocal microscopy) after cell stimulation with IL-1β.
  
  
  RESULTS
  We 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 HLX2 helical domain. The p.DelQ134_R256 mutation caused inhibition of NF-κB signaling, although the truncated NEMO protein interacted with proteins involved in activation of NF-κB signaling. The IL-1β-induced formation of NEMO-containing structures was impaired in fibroblasts from patients with IP carrying the truncated NEMO form (as also observed in HOIL-1-/-cells). The truncated NEMO interaction with SHANK-associated RH domain-interacting protein was impaired in a male fetus with IP, leading to defective linear ubiquitination.
  
  
  CONCLUSIONS
  We identified a hitherto unreported disease mechanism (defective linear ubiquitination) in patients with IP.
authors.affiliation: INSERM U1163 Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France.
authors.forename: Elodie
authors.initials: E
authors.lastname: Bal
authors.affiliation: Laboratory of Signaling and Pathogenesis, CNRS UMR 3691, Pasteur Institute, Paris, France.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Laplantine
authors.affiliation: INSERM U1163 Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France.
authors.forename: Yamina
authors.initials: Y
authors.lastname: Hamel
authors.affiliation: Departments of Cell Biology and Infection and of Structural Biology and Chemistry, URA 2185, Pasteur Institute, Paris, France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Dubosclard
authors.affiliation: Rockefeller Branch, St Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller University, New York, NY; Necker Branch, Laboratory of Human Genetics of Infectious Diseases, UMR 1163, Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Boisson
authors.affiliation: Institute of Genetics and Biophysics "Adriano Buzzati-Traverso" (CNR), Naples, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Pescatore
authors.affiliation: Rockefeller Branch, St Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller University, New York, NY; Necker Branch, Laboratory of Human Genetics of Infectious Diseases, UMR 1163, Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France; Immunodeficiency Study Center, Necker Children's Hospital, Paris, France.
authors.forename: Capucine
authors.initials: C
authors.lastname: Picard
authors.affiliation: INSERM U1163 Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France; Department of Dermatology, Referral Center for Genodermatoses (MAGEC), Imagine Institute, Necker-Enfants Malades Hospital (AP-HP), Paris, France.
authors.forename: Smaïl
authors.initials: S
authors.lastname: Hadj-Rabia
authors.affiliation: INSERM U1163 Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France.
authors.forename: Ghislaine
authors.initials: G
authors.lastname: Royer
authors.affiliation: INSERM U1163 Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France.
authors.forename: Julie
authors.initials: J
authors.lastname: Steffann
authors.affiliation: INSERM U1163 Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France.
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Bonnefont
authors.affiliation: Institute of Genetics and Biophysics "Adriano Buzzati-Traverso" (CNR), Naples, Italy.
authors.forename: Valeria M
authors.initials: VM
authors.lastname: Ursini
authors.affiliation: Department of Dermatology, Dijon CHU, Medicine Faculty and Bourgogne University, EA427 Genetic of Development Abonomalies, Bocage Hospital, Dijon, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Vabres
authors.affiliation: INSERM U1163 Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France.
authors.forename: Arnold
authors.initials: A
authors.lastname: Munnich
authors.affiliation: Rockefeller Branch, St Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller University, New York, NY; Necker Branch, Laboratory of Human Genetics of Infectious Diseases, UMR 1163, Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France; Pediatric Hematology, Immunology & Rheumatology Unit, Necker Children's Hospital, Paris, France.
authors.forename: Jean-Laurent
authors.initials: JL
authors.lastname: Casanova
authors.affiliation: INSERM U1163 Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France; Department of Dermatology, Referral Center for Genodermatoses (MAGEC), Imagine Institute, Necker-Enfants Malades Hospital (AP-HP), Paris, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Bodemer
authors.affiliation: Laboratory of Signaling and Pathogenesis, CNRS UMR 3691, Pasteur Institute, Paris, France.
authors.forename: Robert
authors.initials: R
authors.lastname: Weil
authors.affiliation: Departments of Cell Biology and Infection and of Structural Biology and Chemistry, URA 2185, Pasteur Institute, Paris, France.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Agou
authors.affiliation: INSERM U1163 Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France. Electronic address: asma.smahi@inserm.fr.
authors.forename: Asma
authors.initials: A
authors.lastname: Smahi
mesh_terms: D003001:Cloning, Molecular; D005260:Female; D005347:Fibroblasts / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism*; D007184:Incontinentia Pigmenti / Q000235:genetics / Q000378:metabolism*; D008297:Male; D009154:Mutation / Q000235:genetics; D016328:NF-kappa B / Q000378:metabolism; D010375:Pedigree; D011485:Protein Binding; D015398:Signal Transduction; D012867:Skin / Q000473:pathology*; D015533:Transcriptional Activation; D054875:Ubiquitination; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: C491731:IKBKG protein, human; D016328:NF-kappa B; C575183:SHARPIN protein, human; D014452:Ubiquitins; D051550:I-kappa B Kinase
keywords: IKBKG; Incontinentia pigmenti; NF-κB essential modulator; SHANK-associated RH domain–interacting protein; linear ubiquitin assembly complex; linear ubiquitination; nuclear factor κB