Structure-function analysis reveals the molecular determinants of the impaired biological function of DAX-1 mutants in AHC patients

Abstract

Mutations in the DAX-1 (NR0B1) gene cause the X-linked form of adrenal hypoplasia congenita (AHC), which is constantly found associated with hypogonadotropic hypogonadism (HHG). DAX-1 encodes an atypical orphan member of the nuclear hormone receptor superfamily. DAX-1 acts at multiple levels to repress the expression of genes involved in steroid hormone metabolism through a potent transcriptional repression domain present in its C-terminus, which is similar to the nuclear receptors' ligand binding domain. All DAX-1 mutations causing AHC/HHG alter the protein C-terminal domain, impairing its nuclear localization and, consequently, its transcriptional repression activity. Here we show that DAX-1 AHC mutants have a misfolded conformation, which correlates with their cytoplasmic retention. Extensive structure-function analysis reveals that the chemical nature of amino acid residues at positions interested by AHC mutations and critical determinants in helix 12 affect DAX-1 nuclear localization and transcriptional silencing. Surprisingly, mutations in a conserved putative corepressor binding surface have a negative effect upon DAX-1 transcriptional repression only when they also affect protein expression levels. These data suggest that a folding defect underlies the impaired function of DAX-1 missense mutants found in AHC/HHG patients and that interactions with transcriptional cofactors different from known corepressors mediate DAX-1 silencing properties.

Role of Orphan Nuclear Receptor DAX-1/NR0B1 in Development, Physiology, and Disease

“…Among corepressors, DAX-1 was shown to interact with N-CoR [63] and Alien [64], even if further studies have ruled out a role of N-CoR as a bona fide DAX-1 corepressor [65]. However, site-directed mutagenesis of conserved surface residues in DAX-1, which in other nuclear receptors are involved in corepressor interaction, does not impair DAX-1 transcriptional silencing properties (see Section 6) [66]. These data suggest that cofactors different from known nuclear receptor corepressors mediate DAX-1 transcriptional silencing activity.…”

“…Third, the Dax-1 mutant ΔV271, which corresponds to the human AHC mutant ΔV269, behaves similarly to the wild type. However, the localization of this mutant is shifted to the cytoplasm (see below Section 6) [65,66] and it is difficult to explain how it may regulate promoter expression in the nucleus. Further studies are then needed to assess the role of potential interactions between DAX-1 and RNA transcriptional cofactors in the regulation of gene expression.…”

“…A significant breakthrough to identify the mechanism explaining the impaired transcriptional activity by DAX-1 missense mutants came from the discovery that they all have a defect in nuclear localization, which is of variable severity according to the position of the residue affected by the mutation [65,66]. An inverse relationship exists between the repression activity of the mutant DAX-1 and the degree of its cytoplasmic localization (Figure 2).…”

“…An inverse relationship exists between the repression activity of the mutant DAX-1 and the degree of its cytoplasmic localization (Figure 2). Limited proteolysis experiments showed that DAX-1 AHC missense mutations alter the structure of the protein C-terminal domain probably determining its misfolding and allowing the interaction of the protein with anchoring sites that retain it in the cytoplasm, since the protein nuclear localization signal (NLS) lies in the DAX-1 N-terminal domain and is not affected by the mutations [65,66]. Remarkably, the misfolded DAX-1 Cterminal domain can also induce cytoplasmic retention of a fused heterologous DNA-binding domain (yeast Gal4) possessing its own NLS.…”

Molecular Basis of Adrenal Insufficiency

“…One missense mutation (L466R), which is located in the activation function 2 (AF2) core of a highly conserved region in the C-terminus, was shown to be defective in nuclear localization in spite of having an intact N-terminus and to impair the repression activity of DAX-1 (31). It has been also observed that several mutants from patients with AHC cannot localize into the nucleus, and thus this would partly explain the loss of function (53,54).…”