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-κB Activation

Emilie Vinolo,Michel Véron,Fabrice Agou,Gilles Courtois,Alain Israël,Hélène Sebban,Alain Chaffotte

doi:10.1074/jbc.m510118200

Abstract

The NEMO (NF-kappaB essential modulator) protein plays a crucial role in the canonical NF-kappaB pathway as the regulatory component of the IKK (IkappaB 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-alpha and LPS-induced NF-kappaB 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.

Highlights

The NF-␬B family of eucaryotic transcription factors plays a crucial role in immune and inflammatory responses, development, and control of apoptosis [1]
The fragment called C-terminal domain (C-ter), which is responsible for the regulatory role of the protein through connecting the IKK complex to different upstream activators [26], contains the minimal oligomerization domain of NEMO [27]
The missense C 3 G mutation at nucleotide 863 identified in a patient with ectodermal dysplasia with immunodeficiency (EDA-ID) leads to a Ala 3 Gly replacement at position 288 within the CC2 motif of human NEMO [5], which corresponds to residue 281 in murine NEMO (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

Reagents—n-Dodecylmaltoside (DDM) and n-octyl glucoside (OG) were from Roche Applied Science. Flame Atomic Absorption Spectroscopy—Zinc titration was performed as previously described [39] with samples of the C-ter proteins extensively dialyzed against a buffer containing 25 mM Tris-HCl, pH 8.0, 150 mM KCl, 1 mM EDTA, 0.2 mM DDM, and 1 mM DTE. The beads were collected, washed twice in 20 mM sodium phosphate buffer, pH 7.0, containing 150 mM NaCl, 20 mM imidazole, 0.1 mM DTE, 1 mM DDM and 5% glycerol, and incubated for 1 h at room temperature with S100 extracts from wild-type 70Z/3 or NEMOϪ/Ϫ 1.3E2 pre-B cells (300 ␮g of proteins) in the same buffer supplemented with an EDTA-free protease inhibitor mixture (Complete; Roche Applied Science). Bound proteins were eluted with 40 ␮l of a 20 mM sodium phosphate buffer, pH 7.0, containing 150 mM NaCl, 300 mM imidazole, 0.1 mM DTE, 1 mM DDM, and 5% glycerol and analyzed by Western blotting using an anti-IKK␣ antibody (Imgenex)

RESULTS

Deduced molecular mass

DISCUSSION