PCDH15跨膜运输机制的初步探讨

发布时间：2018-05-04 14:05

本文选题：PCDH15 + PIST　；参考：《山东大学》2017年硕士论文

【摘要】：在听觉传导过程中,声波通过机电转导(MET)的过程转换为电信号,这个过程发生在耳蜗毛细胞的听纤毛。受到机械信号刺激后,MET装置的核心元件-顶连接(Tip-Link)张力增加,MET通道开放,这导致细胞受体电位的产生。Tip-Link破坏的话,听觉转导就不能发生,会导致耳聋。作为毛细胞Tip-Link的组成成分之一的原钙粘蛋白15即PCDH15,在听觉转导中起着十分重要的作用。PCDH15有三种不同的剪接本,在听纤毛发育期间的机械-电转导过程中冗余地发挥作用。PCDH15基因的突变能够引起听力损失。但是到目前为止,有关PCDH15细胞内运输的调控机制的研究仍然没有文献报道。在本论文中,我们对PCDH15细胞内运输的调控机制进行了初步探讨。首先我们用酵母双杂交来筛选与PCDH15相互结合的蛋白,我们发现PIST(一个与高尔基体相联系,具有PDZ结构域的蛋白)能够与PCDH15-CD3相互作用。在酵母双杂交的基础上,本论文用共免疫沉淀和亚细胞共定位的实验方法验证了PCDH15-CD3与PIST的相互结合。我们发现这种相互作用是通过PCDH15-CD3 C末端的PDZ结构域结合界面(PBI)以及PIST的PDZ结构域介导的。通过二者的相互作用,PIST把PCDH15-CD3滞留在反面高尔基网(TGN),从而减少了 PCDH15-CD3在细胞膜上的表达。我们之前的工作发现PIST能够调控Tip-Link的另外一个核心元件-钙粘蛋白23(CDH23)的细胞膜表达。综合起来,我们的发现表明PIST能够调控与Tip-Link相关的钙粘蛋白PCDH15-CD3和CDH23的细胞内运输和细胞膜靶向定位。有研究表明PCDH15可以与含有PDZ结构域的Usher蛋白Harmonin、PDZD7、Whirlin结合,但不同PCDH15亚型与这些蛋白之间的作用模式未见报道。我们通过免疫共沉淀实验发现Whirlin及PDZD7与PCDH15-CD3相互作用,但不与PCDH15-CD1或PCDH15-CD2结合。此外,免疫共沉淀实验没有检测到PCDH15的任何一个亚型能够与Harmonin共沉淀。免疫荧光实验也得到了与免疫共沉淀一致的结果。
[Abstract]:During auditory conduction, sound waves are converted into electrical signals by electromechanical transduction, which occurs in the auditory cilia of the cochlear hair cells. After being stimulated by mechanical signal, the tension of Tip-Link, the core component of met device, increases the opening of the mett channel, which leads to the cell receptor potential production. Tip-Link damage, the auditory transduction will not occur, which will lead to deafness. As one of the components of Tip-Link in hair cells, procalcitonin 15 (PCDH15) plays a very important role in auditory transduction. PCDH15 has three different splices. Mutations in the PCDH15 gene may cause hearing loss during mechanical-electric transduction during auditory cilium development. Up to now, however, there is still no literature about the regulation of intracellular transport in PCDH15. In this thesis, we studied the regulatory mechanism of intracellular transport in PCDH15 cells. First, we used yeast two-hybrid to screen proteins that bind to PCDH15. We found that PIST (a protein associated with Golgi body with PDZ domain) can interact with PCDH15-CD3. On the basis of yeast two-hybrid, co-immunoprecipitation and subcellular co-localization were used to verify the interaction between PCDH15-CD3 and PIST. We find that this interaction is mediated by the PDZ domain binding interface at the C-terminal of PCDH15-CD3 and the PDZ domain of PIST. Through the interaction of the two, PIST stranded PCDH15-CD3 in the reverse Golgi net, thus reducing the expression of PCDH15-CD3 on the cell membrane. Our previous work has shown that PIST regulates the cell membrane expression of cadherin 23 CDH23, another core component of Tip-Link. Taken together, our findings suggest that PIST can regulate intracellular transport and cell membrane targeting of cadherin PCDH15-CD3 and CDH23 associated with Tip-Link. Some studies have shown that PCDH15 can bind to Usher protein Harmonin PDZD7 Whirlin containing PDZ domain, but the interaction patterns between different PCDH15 subtypes and these proteins have not been reported. We found that Whirlin and PDZD7 interact with PCDH15-CD3 by immunoprecipitation, but not with PCDH15-CD1 or PCDH15-CD2. In addition, no single subtype of PCDH15 was detected to co-precipitate with Harmonin in immunoprecipitation assay. The results of immunofluorescence assay were consistent with that of co-immunoprecipitation.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q25

【参考文献】