Alanine Expansions Associated With Congenital Central Hypoventilation Syndrome Impair PHOX2B Homeodomain-Mediated Dimerisation And Nuclear Import

Heterozygous mutations of the human PHOX2B gene, a key regulator of autonomic nervous system development, lead to Congenital Central Hypoventilation Syndrome (CCHS), a neurodevelopmental disorder characterised by a failure in the autonomic control of breathing. Polyalanine expansions in the 20-residues region of the C-terminus of PHOX2B are the major mutations responsible for CCHS. Elongation of the alanine stretch in PHOX2B leads to a protein with altered DNA-binding, transcriptional activity and nuclear localisation, and the possible formation of cytoplasmic aggregates; furthermore, the findings of various studies support the idea that CCHS is not due to a pure loss of function mechanism, but also involves a dominant-negative effect and/or toxic gain of function for PHOX2B mutations. As PHOX2B forms homodimers and heterodimers with its paralogue PHOX2A in vitro, we tested the hypothesis that the dominant-negative effects of the mutated proteins are due to non-functional interactions with the wild-type protein or PHOX2A using co-immunoprecipitation assay and the mammalian two-hybrid system. Our findings show that PHOX2B forms homodimers, heterodimerise weakly with mutated proteins, exclude the direct involvement of the polyalanine tract in dimer formation, and indicate that mutated proteins retain partial ability to form heterodimers with PHOX2A. Moreover, in this study we investigated the effects of the longest polyalanine expansions on the homeodomain-mediated nuclear import and our data clearly show that the expanded C-terminus interferes with this process. These results provide novel insights into the effects of the alanine tract expansion on PHOX2B folding and activity.