TMPRSS3基因之功能分析

Functional analysis of the TMPRSS3 gene

楊璨鴻(Cen-Hong Yang)

指導教授 ： 李宣佑

中山醫學大學/醫學科技學院/生物醫學科學學系碩士班/碩士(2011年)

https://doi.org/10.6834/CSMU.2011.00147

摘要

有研究指出TMPRSS3 (transmembrane protease, serine 3) 基因與非症候群聽障相關。TMPRSS3是屬於第二型穿膜絲氨酸蛋白(type II transmembrane serine protease，TTSPs)的超級家族之一。這是第一個被提出與非症候群有關的酵素基因。先前我們實驗室針對台灣地區的非症候群聽障患者進行TMPRSS3 基因的突變分析，結果在230位聽障患者中發現有14位聽障學童有突變的發生，所佔的比例為6.09 % (14/230)，然而針對這些突變所造成的致病機轉我們並不清楚。因此本研究將針對先前發現的TMPRSS3基因錯意突變，利用細胞模式來探討TMPRSS3基因突變所造成的功能影響，進一步釐清其在非症候群聽障中所扮演的角色。本論文將分兩部分來探討：第一部分我們利用tet‐on雙向表現系統，探討這些突變蛋白在HeLa細胞內的表現位置，並分析是否造成TMPRSS3蛋白的功能喪失。首先利用細胞模型來觀察正常及突變TMPRSS3蛋白在細胞內的表現位置，在我們的研究當中發現正常與突變TMPRSS3蛋白表現位置並無差異皆表現在內質網中。雖然正常與突變點TMPRSS3蛋白表現位置無差異，但並不代表功能上沒有受到影響，須進一步探討。第二部分我們將對TMPRSS3蛋白進行功能上的分析，探討除了ENaC可能為受質外，是否還有其他TMPRSS3受質存在。首先我們利用西方點墨法來分析H441細胞中是否有TMPRSS3、ENaC、brain-derived neurotrophic factor (BDNF)和neurotrophin 3 (NT-3)蛋白質表現，結果顯示H441細胞中確實有TMPRSS3、ENaC、BDNF和NT-3蛋白質的表現。進一步利用免疫螢光染色觀察上述蛋白於細胞內的表現情形，結果顯示， ENaC、BDNF和NT-3 在H441細胞中和TMPRSS3有共同表現的現象。然而這些只是初步結果，因此需要更一步釐清ENaC、BDNF和NT-3蛋白質和TMPRSS3之間的關係。綜合以上所述，本研究結果對於進一步釐清TMPRSS3基因突變造成非症候群聽障的成因有很大的幫助。

關鍵字

第二型穿膜絲氨酸蛋白 ； 非症候群聽障 ； 雙向表現系統

並列摘要

TMPRSS3 is a member of the Type II Transmembrane Serine Protease (TTSP). This was the first description of a serine protease involved in deafness. Previously, we have found many mutations in the TMPRSS3 genes from screening of 230 children with non-syndromic deafness (14/230; 6.09 %). However, functional alteration in these mutants of TMPRSS3 gene remains unknown. Moreover, the mechanism of ENaC activation by TMPRSS3 remains elusive. In addition, some studies suggested have presence of other substrates for TMPRSS3. Therefore, further research is worthy to conduct. We divided this thesis into two parts. First, we compared the intracellular distribution of mutants TMPRSS3 with that of the wild-type TMPRSS3 in HeLa cells and the effect the mutant protein had on those cells. Localization assay of all missense mutations in TMPRSS3 gene reveals their distributions in the ER of the HeLa cell, which is similar to the WT. Therefore, we suggest that all mutants do not affect the intracellular trafficking. However, the functional significance of all mutations is required further investigation. Secondary, we will study the functional interaction of TMPRSS3 with ENaC and new substrates in the inner ear and study the functional interaction between TMPRSS3 and new substrates. First, we found that four proteins, including TMPRSS3, ENaC, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3), were expressed in H441 cells using western blot analysis. Further, we also found that ENaC, BDNF or NT-3 was expressed in H441 cell and colocalizated with TMPRSS3 using immunofluorescence staining analysis. Base on above results, this research will help us to clarify the reason after the TMPRSS3 mutations cause hearing loss.

並列關鍵字

TMPRSS3 ； ENaC ； BDNF ； NT-3

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