title: Alanine Expansions Associated with Congenital Central Hypoventilation Syndrome Impair PHOX2B Homeodomain-mediated Dimerization and Nuclear Import.
abstract: 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 characterized 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 localization 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. Because 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 a co-immunoprecipitation assay and the mammalian two-hybrid system. Our findings show that PHOX2B forms homodimers and heterodimerizes 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.
authors.affiliation: From the Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, 20129 Milan, Italy and.
authors.forename: Simona
authors.initials: S
authors.lastname: Di Lascio
authors.affiliation: From the Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, 20129 Milan, Italy and.
authors.forename: Debora
authors.initials: D
authors.lastname: Belperio
authors.affiliation: From the Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, 20129 Milan, Italy and the National Research Council (CNR) Neuroscience Institute, 20129 Milan, Italy.
authors.forename: Roberta
authors.identifier: 0000-0001-5396-5429
authors.initials: R
authors.lastname: Benfante
authors.affiliation: From the Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, 20129 Milan, Italy and the National Research Council (CNR) Neuroscience Institute, 20129 Milan, Italy diego.fornasari@unimi.it.
authors.forename: Diego
authors.identifier: 0000-0002-9668-3103
authors.initials: D
authors.lastname: Fornasari
mesh_terms: D021581:Active Transport, Cell Nucleus; D001483:Base Sequence; D002467:Cell Nucleus / Q000201:enzymology*; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007040:Hypoventilation / Q000151:congenital* / Q000201:enzymology / Q000235:genetics; D010455:Peptides / Q000235:genetics; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D020182:Sleep Apnea, Central / Q000201:enzymology* / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D019680:Trinucleotide Repeat Expansion
chemical_list: D018398:Homeodomain Proteins; C109220:NBPhox protein; C491061:PHOX2A protein, human; D010455:Peptides; D014157:Transcription Factors; C019529:polyalanine
keywords: PHOX2A; PHOX2B; congenital central hypoventilation syndrome; dimerization; homeobox; mammalian two-hybrid system; protein import; protein misfolding; transcription factor; trinucleotide repeat disease