本文選題：PCDH15 + PIST��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：在聽覺傳導(dǎo)過程中,聲波通過機電轉(zhuǎn)導(dǎo)(MET)的過程轉(zhuǎn)換為電信號,這個過程發(fā)生在耳蝸毛細胞的聽纖毛。受到機械信號刺激后,MET裝置的核心元件-頂連接(Tip-Link)張力增加,MET通道開放,這導(dǎo)致細胞受體電位的產(chǎn)生。Tip-Link破壞的話,聽覺轉(zhuǎn)導(dǎo)就不能發(fā)生,會導(dǎo)致耳聾。作為毛細胞Tip-Link的組成成分之一的原鈣粘蛋白15即PCDH15,在聽覺轉(zhuǎn)導(dǎo)中起著十分重要的作用。PCDH15有三種不同的剪接本,在聽纖毛發(fā)育期間的機械-電轉(zhuǎn)導(dǎo)過程中冗余地發(fā)揮作用。PCDH15基因的突變能夠引起聽力損失。但是到目前為止,有關(guān)PCDH15細胞內(nèi)運輸?shù)恼{(diào)控機制的研究仍然沒有文獻報道。在本論文中,我們對PCDH15細胞內(nèi)運輸?shù)恼{(diào)控機制進行了初步探討。首先我們用酵母雙雜交來篩選與PCDH15相互結(jié)合的蛋白,我們發(fā)現(xiàn)PIST(一個與高爾基體相聯(lián)系,具有PDZ結(jié)構(gòu)域的蛋白)能夠與PCDH15-CD3相互作用。在酵母雙雜交的基礎(chǔ)上,本論文用共免疫沉淀和亞細胞共定位的實驗方法驗證了PCDH15-CD3與PIST的相互結(jié)合。我們發(fā)現(xiàn)這種相互作用是通過PCDH15-CD3 C末端的PDZ結(jié)構(gòu)域結(jié)合界面(PBI)以及PIST的PDZ結(jié)構(gòu)域介導(dǎo)的。通過二者的相互作用,PIST把PCDH15-CD3滯留在反面高爾基網(wǎng)(TGN),從而減少了 PCDH15-CD3在細胞膜上的表達。我們之前的工作發(fā)現(xiàn)PIST能夠調(diào)控Tip-Link的另外一個核心元件-鈣粘蛋白23(CDH23)的細胞膜表達。綜合起來,我們的發(fā)現(xiàn)表明PIST能夠調(diào)控與Tip-Link相關(guān)的鈣粘蛋白PCDH15-CD3和CDH23的細胞內(nèi)運輸和細胞膜靶向定位。有研究表明PCDH15可以與含有PDZ結(jié)構(gòu)域的Usher蛋白Harmonin、PDZD7、Whirlin結(jié)合,但不同PCDH15亞型與這些蛋白之間的作用模式未見報道。我們通過免疫共沉淀實驗發(fā)現(xiàn)Whirlin及PDZD7與PCDH15-CD3相互作用,但不與PCDH15-CD1或PCDH15-CD2結(jié)合。此外,免疫共沉淀實驗沒有檢測到PCDH15的任何一個亞型能夠與Harmonin共沉淀。免疫熒光實驗也得到了與免疫共沉淀一致的結(jié)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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q25

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相關(guān)期刊論文 前1條

1 梁玲芝;鄭斌嬌;鄭靜;方芳;伍越;管敏鑫;;纖毛束功能的分子機制:遺傳性耳聾小鼠模型研究[J];生理學(xué)報;2012年04期

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本文編號：1843245