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J. Biol. Chem., Vol. 281, Issue 10, 6334-6348, March 10, 2006

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A Point Mutation in NEMO Associated with Anhidrotic Ectodermal Dysplasia with Immunodeficiency Pathology Results in Destabilization of the Oligomer and Reduces Lipopolysaccharide- and Tumor Necrosis Factor-mediated NF-B Activation^*

Emilie Vinolo, Hélène Sebban, Alain Chaffotte^¶, Alain Israël^||, Gilles Courtois, Michel Véron, and Fabrice Agou¹

From the Unité de Regulation Enzymatique des Activités Cellulaires, the ^¶Unité de Repliement et Modélisation des Protéines, CNRS URA 2185 and the ^||Unité de Signalisation Moléculaire et Activation Cellulaire, CNRS URA 2582, Institut Pasteur, 25/28 Rue du Dr. Roux, 75724 Paris Cedex 15, France and INSERM U697, Hôpital Saint-Louis, 1 Av. Claude Vellefaux, 75475 Paris Cedex 10, France

The NEMO (NF-B essential modulator) protein plays a crucial role in the canonical NF-B pathway as the regulatory component of the IKK (IB kinase) complex. The human disease anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) has been recently linked to mutations in NEMO. We investigated the effect of an alanine to glycine substitution found in the NEMO polypeptide of an EDA-ID patient. This pathogenic mutation is located within the minimal oligomerization domain of the protein, which is required for the IKK activation in response to diverse stimuli. The mutation does not dramatically change the native-like state of the trimer, but temperature-induced unfolding studied by circular dichroism showed that it leads to an important loss in the oligomer stability. Furthermore, fluorescence studies showed that the tyrosine located in the adjacent zinc finger domain, which is possibly required for NEMO ubiquitination, exhibits an alteration in its spectral properties. This is probably due to a conformational change of this domain, providing evidence for a close interaction between the oligomerization domain and the zinc finger. In addition, functional complementation assays using NEMO-deficient pre-B and T lymphocytes showed that the pathogenic mutation reduced TNF- and LPS-induced NF-B activation by altering the assembly of the IKK complex. Altogether, our findings provide understanding as to how a single point mutation in NEMO leads to the observed EDA-ID phenotype in relation to the NEMO-dependent mechanism of IKK activation.

Received for publication, September 14, 2005 , and in revised form, December 26, 2005.

^* This work was supported by Association pour la Recherche sur le Cancer Grant 5795 and grants from the Cancéropôle Ile-de-France, the Ligue Nationale Contre le Cancer (équipe labelisée; to A. I.), and the Fondation pour la Recherche Médicale (to G. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 33-1-44-38-95-69; Fax: 33-1-45-68-83-99; E-mail: fagou{at}pasteur.fr.

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