Amino acid substitution equivalent to human chorea-acanthocytosis I2771R in yeast Vps13 protein affects its binding to phosphatidylinositol 3-phosphate.
Amino acid substitution equivalent to human chorea-acanthocytosis I2771R in yeast Vps13 protein affects its binding to phosphatidylinositol 3-phosphate.
复制标题
DOI：
10.1093/hmg/ddx054
复制
发表时间：
2017-04-15
影响因子：
3.5
通讯作者：
Zoladek T
中科院分区：
文献类型：
--
作者：
Rzepnikowska W;Flis K;Kaminska J;Grynberg M;Urbanek A;Ayscough KR;Zoladek T
罕见的人类疾病舞蹈病-棘细胞增多症（ChAc）是由hVPS13A基因突变引起的。hVps13A蛋白与肌动蛋白相互作用，调节神经细胞膜中磷脂酰肌醇4-磷酸（PI4P）水平。酵母Vps13参与液泡蛋白运输，与hVps13A一样，参与PI4P代谢。Vps13蛋白在真核生物中是保守的，但其分子功能尚不清楚。在ChAc患者中发现的一种突变导致氨基酸取代I2771R，影响骨骼肌中hVps13A的定位。为了剖析I2771R的发病机制，我们创建并分析了一个携带等效突变的酵母菌株。本研究表明，在酵母中，I2749R替代导致Vps13蛋白内吞作用和空泡运输功能障碍，尽管蛋白水平不受影响，表明功能丧失。我们还发现Vps13和hVps13A一样，在免疫沉淀实验中影响肌动蛋白细胞骨架组织并结合肌动蛋白。Vps13-I2749R结合肌动蛋白，但在肌动蛋白细胞骨架组织中不起作用。此外，我们发现Vps13通过其SHR_BD和APT1结构域结合磷脂，特别是磷脂酰肌醇3-磷酸（PI3P）。取代I2749R削弱了这种能力。最后，当PI3P的细胞水平降低时，Vps13-GFP的定位发生改变，这表明PI3P在内体膜系统内运输。这些结果表明，PI3P调节Vps13的功能，包括蛋白质运输和肌动蛋白细胞骨架组织。突变体hVps13A蛋白中pi3p结合能力的减弱可能是其在ChAc患者细胞中定位错误和功能破坏的原因之一。
The rare human disorder chorea-acanthocytosis (ChAc) is caused by mutations in hVPS13A gene. The hVps13A protein interacts with actin and regulates the level of phosphatidylinositol 4-phosphate (PI4P) in the membranes of neuronal cells. Yeast Vps13 is involved in vacuolar protein transport and, like hVps13A, participates in PI4P metabolism. Vps13 proteins are conserved in eukaryotes, but their molecular function remains unknown. One of the mutations found in ChAc patients causes amino acids substitution I2771R which affects the localization of hVps13A in skeletal muscles. To dissect the mechanism of pathogenesis of I2771R, we created and analyzed a yeast strain carrying the equivalent mutation. Here we show that in yeast, substitution I2749R causes dysfunction of Vps13 protein in endocytosis and vacuolar transport, although the level of the protein is not affected, suggesting loss of function. We also show that Vps13, like hVps13A, influences actin cytoskeleton organization and binds actin in immunoprecipitation experiments. Vps13-I2749R binds actin, but does not function in the actin cytoskeleton organization. Moreover, we show that Vps13 binds phospholipids, especially phosphatidylinositol 3-phosphate (PI3P), via its SHR_BD and APT1 domains. Substitution I2749R attenuates this ability. Finally, the localization of Vps13-GFP is altered when cellular levels of PI3P are decreased indicating its trafficking within the endosomal membrane system. These results suggest that PI3P regulates the functioning of Vps13, both in protein trafficking and actin cytoskeleton organization. Attenuation of PI3P-binding ability in the mutant hVps13A protein may be one of the reasons for its mislocalization and disrupted function in cells of patients suffering from ChAc.