Introduction: Polyalanine expansions in the 20-residues region of the human PHOX2B gene, a key regulator of autonomic nervous system development, lead to Congenital Central Hypoventilation Syndrome, a neurodevelopment disorder characterised by a failure in the autonomic control of breathing. Elongation of the alanine stretch leads to a protein with altered DNA-binding, transcriptional activity, nuclear localisation, and cytoplasmic aggregates formation. Previous studies support the idea that PHOX2B mutant proteins display both loss and new toxic gain of function, resulting in a dominant negative effect. As PHOX2B forms homodimers and heterodimers with its paralogue PHOX2A in vitro, we tested the hypothesis that the dominant-negative effect of the mutated proteins are due to non-functional interactions with the wild-type protein or PHOX2A. Moreover we investigated the effects of the longest polyalanine expansions on the homeodomain mediated nuclear import. Material and methods: To study hetero- and homo-dimerisation we used co-immunoprecipitation assay, and mammalian two-hybrid system. Deletion analysis and immunocytochemistry were used to study the consequences of alanine expansion on the sub-cellular localisation. Results and conclusions: 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, with a possible role in the pathogenic process. Moreover our data show that the expanded C-terminus interferes with nuclear import process. These results provide novel insights into the effects of the alanine tract expansion on PHOX2B folding and activity.
Alanine expansions associated with Congenital Central Hypoventilation Syndrome (CCHS) impair PHOX2B homeodomainmediated dimerisation and nuclear import
2016
Abstract
Introduction: Polyalanine expansions in the 20-residues region of the human PHOX2B gene, a key regulator of autonomic nervous system development, lead to Congenital Central Hypoventilation Syndrome, a neurodevelopment disorder characterised by a failure in the autonomic control of breathing. Elongation of the alanine stretch leads to a protein with altered DNA-binding, transcriptional activity, nuclear localisation, and cytoplasmic aggregates formation. Previous studies support the idea that PHOX2B mutant proteins display both loss and new toxic gain of function, resulting in a dominant negative effect. As PHOX2B forms homodimers and heterodimers with its paralogue PHOX2A in vitro, we tested the hypothesis that the dominant-negative effect of the mutated proteins are due to non-functional interactions with the wild-type protein or PHOX2A. Moreover we investigated the effects of the longest polyalanine expansions on the homeodomain mediated nuclear import. Material and methods: To study hetero- and homo-dimerisation we used co-immunoprecipitation assay, and mammalian two-hybrid system. Deletion analysis and immunocytochemistry were used to study the consequences of alanine expansion on the sub-cellular localisation. Results and conclusions: 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, with a possible role in the pathogenic process. Moreover our data show that the expanded C-terminus interferes with nuclear import process. These results provide novel insights into the effects of the alanine tract expansion on PHOX2B folding and activity.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
