2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38805Recently, some studies have shown that mutations in TMPRSS3 were responsible for non-syndromic deafness (DFNB8 and DFNB10). TMPRSS3 is a member of the Type II Transmembrane Serine Protease (TTSP). This was the first description of a serine protease involved in deafness. However, in Taiwan, the data of TMPRSS3 gene associated with non-syndromic deafness is still insufficient. 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. In the proposed three-year project, we will focused on study the functional consequence of TMPRSS3 mutations, and functional interaction of TMPRSS3 with ENaC, and potential substrates of TMPRSS3 in the inner ear, using approaches of molecular biological, cell biological, animal model and electrophysiology. In the first year, we will investigate their functional properties and interaction amongst these mutant proteins using sGASP (secretory Genetic Assay for Site specific Proteolysis) and Xenopus oocyte expression system. In addition, we will also investigate intron mutation of TMPRSS3 gene using in vitro splice analysis. Based on previous study and results from the first year, we will study functional interaction of TMPRSS3 with ENaC in the second year. Finally, we will search new substrates for TMPRSS3 in the inner ear and study functional interaction between TMPRSS3 and new substrates. Further more, we will study functional interaction of TMPRSS3 and TMPRSS1 or TMPRSS5 We anticipate that the study will be comprehensively understanding of the functional role of TMPRSS3 gene in the inner ear. We believe that the etiology and mechanism of pathogenesis in hereditary deafness will be further appreciated upon completion of this research. These data will insight into strategies for their prevention and treatment.TMPRSS3 (transmembrane protease, serine 3) 已經發現牽涉在非症候群聽障中的一個新基因，當TMPRSS3基因發生突變後已知會影響到耳蝸內Na+的再吸收因而造成聽障(DFNB8和DFNB10)。TMPRSS3 (transmembrane protease, serine 3)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。台灣地區對於TMPRSS3基因在非症候聽障患者中的相關研究相當闕乏，在之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。而且TMPRSS3在耳蝸內活化epithelial sodium channe (ENaC)促使Na+再吸收的機制也很模糊，可能是直接作用或者間接作用。再者有些研究推測in vivo的狀態下TMPRSS3在耳蝸內的受質可能不只有ENaC一種。因此本計畫將針對下列四點：(1) 探討TMPRSS3基因突變後造成非症候群聽障的功能影響；(2) 瞭解TMPRSS3與ENaC在Na+的再吸收作用機制中彼此的相互關係；(3) TMPRSS3基因在耳蝸內除了ENaC外是否還有其他受質；(4) TMPRSS3與TMPRSS1或TMPRSS5之間在聽力的產生上是否會相互影響。 本計劃擬利用分子生物學、細胞生物學、動物模式和電生理等研究法分三年來加以探討。第一年：擬利用兩個分析系統--- sGASP(secretory Genetic Assay for Site specific Proteolysis)系統和爪蟾卵母細胞(Xenopus oocyte)表現系統來探討TMPRSS3基因missence突變後造成功能上的影響。另外我們也將利用in vitro splicing analysis來探討intron突變所造成的影響；第二年：進一步的利用human lung carcinoma cell (H441) 來探討TMPRSS3基因missence突變造成功能上的影響，另外我們也將利用瞭解正常和突變的TMPRSS3蛋白在細胞內的表現位置，同時利用siRNA技術、基因轉殖技術、whole-cell electrophysiology、雷射細胞擷取技術、TnT-coupled reticulocyte lysate system和共同免疫沈澱法探討TMPRSS3與ENaC之交互作用機制；第三年：探討neurotrophin家族的成員是否當TMPRSS3的受質之一，另外也想探討TMPRSS3與TMPRSS1或TMPRSS5之間的相互關係。相信如這計畫如獲得支持，在這計畫完成後將可進一步的瞭解TMPRSS3在內耳的功能角色和我們所發現的TMPRSS3突變點造成聽障的致病機轉，這些資料的建立將可提供預防或治療的一個策略。基因非症候群聽障功能研究鈉離子通道TMPRSS3nonsyndromic deafnessfunctional studyepithelial sodium channelProject