title: Functional studies for the TRAF6 mutation associated with hypohidrotic ectodermal dysplasia.
abstract: BACKGROUND
  Hypohidrotic ectodermal dysplasia (HED) is a rare condition characterized by hypotrichosis, hypohidrosis and hypodontia. A de novo heterozygous mutation in the tumour necrosis factor receptor-associated factor 6 gene (TRAF6) was recently identified in a patient with HED, while functional consequences resulting from the mutation remained unknown.
  
  
  OBJECTIVE
  To determine the mechanism by which the TRAF6 mutation results in HED.
  
  
  METHODS
  We performed coimmunoprecipitation (co-IP) studies to determine whether the mutation would affect the interaction of TRAF6 with transforming growth factor β-activated kinase 1 (TAK1), TAK1-binding protein 2 (TAB 2) and ectodysplasin-A receptor-associated death domain protein (EDARADD). We then performed co-IP and glutathione S-transferase-pulldown assays to determine the TRAF6 binding sequences in EDARADD. In addition, we analysed the effect of the mutant TRAF6 protein on the affinity between wild-type TRAF6 and EDARADD, as well as on EDARADD-mediated nuclear factor (NF)-κB activation.
  
  
  RESULTS
  The mutant TRAF6 protein was capable of forming a complex with TAK1 and TAB 2 in a similar way to wild-type TRAF6. However, the mutant TRAF6 protein completely lost the affinity to EDARADD, while the wild-type TRAF6 bound to the N-terminal domain of EDARADD. Furthermore, the mutant TRAF6 inhibited the interaction between the wild-type TRAF6 and EDARADD, and also potentially reduced the EDARADD-mediated NF-κB activity.
  
  
  CONCLUSIONS
  We conclude that the mutant TRAF6 protein shows a dominant negative effect against the wild-type TRAF6 protein, which is predicted to affect the EDARADD-mediated activation of NF-κB during the development of ectoderm-derived organs, and to lead to the HED phenotype.
authors.affiliation: Laboratory of Genetic Skin Diseases Division of Dermatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Fujikawa
authors.forename: M
authors.initials: M
authors.lastname: Farooq
authors.forename: A
authors.initials: A
authors.lastname: Fujimoto
authors.forename: M
authors.initials: M
authors.lastname: Ito
authors.forename: Y
authors.initials: Y
authors.lastname: Shimomura
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D004347:Drug Interactions; D053360:Ectodermal Dysplasia, Hypohidrotic, Autosomal Recessive / Q000235:genetics*; D053339:Edar Receptor / Q000235:genetics / Q000378:metabolism; D053362:Edar-Associated Death Domain Protein / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D009154:Mutation / Q000235:genetics*; D016328:NF-kappa B / Q000378:metabolism; D048029:TNF Receptor-Associated Factor 6 / Q000235:genetics* / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C506008:EDAR protein, human; C506125:EDARADD protein, human; D053339:Edar Receptor; D053362:Edar-Associated Death Domain Protein; D016328:NF-kappa B; C486300:TAB1 protein, human; C410585:TAB2 protein, human; D048029:TNF Receptor-Associated Factor 6