Alanine Expansions Associated with Congenital Central Hypoventilation Syndrome Impair PHOX2B Homeodomain-mediated Dimerization and Nuclear Import.
Alanine Expansions Associated with Congenital Central Hypoventilation Syndrome Impair PHOX2B Homeodomain-mediated Dimerization and Nuclear Import.
复制标题
DOI：
10.1074/jbc.m115.679027
复制
发表时间：
2016-06-17
期刊：
影响因子：
--
通讯作者：
Fornasari D
中科院分区：
文献类型：
--
作者：
Di Lascio S;Belperio D;Benfante R;Fornasari D
人类 PHOX2B 基因（自主神经系统发育的关键调节因子）的杂合突变会导致先天性中枢通气不足综合征 (CCHS)，这是一种以呼吸自主控制失败为特征的神经发育障碍。 PHOX2B C 末端 20 个残基区域的多聚丙氨酸扩展是导致 CCHS 的主要突变。 PHOX2B 中丙氨酸的延伸导致蛋白质的 DNA 结合、转录活性和核定位发生改变，并可能形成细胞质聚集体；此外，各种研究的结果支持这样的观点，即 CCHS 并非纯粹的功能丧失机制所致，还涉及 PHOX2B 突变的显性负面效应和/或毒性功能获得。由于 PHOX2B 在体外与其旁系同源物 PHOX2A 形成同二聚体和异二聚体，因此我们使用免疫共沉淀测定和哺乳动物双杂交系统测试了以下假设：突变蛋白的显着负面影响是由于与野生型蛋白或 PHOX2A 的非功能性相互作用所致。我们的研究结果表明，PHOX2B 与突变蛋白形成同二聚体和异二聚体较弱，排除了聚丙氨酸束直接参与二聚体形成，并表明突变蛋白保留了与 PHOX2A 形成异二聚体的部分能力。此外，在这项研究中，我们研究了最长的聚丙氨酸扩展对同源域介导的核输入的影响，我们的数据清楚地表明扩展的 C 末端干扰了这一过程。这些结果为丙氨酸束扩张对 PHOX2B 折叠和活性的影响提供了新的见解。
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.