囊泡型H+-ATPase的B1亚基在其囊泡靶向转运至细胞顶膜的分子机制研究

项目名称： 囊泡型H+-ATPase的B1亚基在其囊泡靶向转运至细胞顶膜的分子机制研究

项目编号： No.30871005

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 杨琼琼

作者单位： 中山大学

项目金额： 30万元

中文摘要： 囊泡型H+-ATPase（V-ATPase）是调节真核细胞中细胞器及细胞内外pH的关键质子泵。其囊泡转运障碍会导致肾小管酸中毒、神经性耳聋、骨质硬化症和肿瘤等许多严重疾病，但其调控机制尚不清楚。本课题就是在我们已经建立的稳定表达B1亚基突变融合蛋白的IMCD细胞模型(研究V-ATPase靶向转运到细胞顶膜的理想模型)，在B1亚基突变体的基础上通过基因突变和切除技术等分子生物学技术、合成B1和B2亚基C末端小片段肽进行蛋白质竞争抑制实验等分别证实了以下结果：（1）B1亚基,而不是B2亚基，是介导V-ATPase靶向转运到IMCD细胞顶膜的关键亚基；（2）调控细胞酸化后B1亚基靶向细胞顶膜的结构可能位于C末端20个氨基酸系列，特别是DTAL结构域；（3）DTAL结构域T位点磷酸化在介导B1亚基靶向细胞顶膜起着关键的调控作用。这一研究的结果为阐明V-ATPase胞内靶向转运的分子机制及其V-ATPase囊泡转运障碍相关疾病的机制提供一定的理论依据，并为将来研发防治新药提供一种新的治疗靶位。

中文关键词： V-ATPase B1亚基；基因突变；囊泡靶向转运；肾脏内髓集合管细胞

英文摘要： Vacuolar H+-ATPase(V-ATPase) is a key proton pump regulating intracellular and extracellualr pH of organelles and eukaryotes.The defects in V-ATPase trafficking can lead to a lot of diseases such as renal tubular acidosis, sensorneural deafness, osteopetrosis, and cancer, but the mechanism is still unknown. Based on our prevously established the stable B1 mutant transfected IMCD cell lines ( an ideal cell model for studying the vesicle trafficking and targeting to the apical membrane of the V-ATPase), and using the molecular biological techniques by traucation and mutation of B1 mutant or competitive inhibition test by B1 and B2 C-terminal synthetic peptide, our study was investigated that: (1) B1 subunit was a key subunit regulating the V-ATPase trafficking and targeting to apical membrane,but B2 was not; (2) C-terminal 20 aa in B1 subunit may contain a motif, including the DTAL that encodes information for apical V-ATPase trafficking; (3) phosphorylation of T point in DTAL motif may regulate the B1 for apical V-ATPase trafficking. These results would help to understant the molecular mechanisms of V-ATPase trafficking and pathogenesis of its associated diseases, and may be a new therapy target of V-ATPase related diseases.

英文关键词： V-ATPase B1 subunit; mutation; vesicle trafficking; IMCD cells