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  • 學位論文

探討連接蛋白造成聽障的機制

Investigation of the molecular mechanisms underlying hearing loss mediated by connexin protein

指導教授 : 楊建洲 李娟

摘要


聽障是全世界最常見的感音神經性疾病。許多研究證明連接蛋白(connexin; CX)所組成的間隙連接通道(gap junction; Gj)在聽覺形成的功能上扮演重要角色。本研究論文的具體目標,我們將探討連接蛋白基因[ CX30.2/CX31.3 (GJC3); CX30.3(GJB4)和CX31( GJB3)]變異與聽障之間的關連和其造成聽障的分子機制。 先前實驗室的研究發現在253位台灣非症候群聽障病人中有11位帶有GJB4基因的變異。在此次研究中,我們進一步分析了這11位GJB4變異的聽障患者的外表型與基因型的相關性。結果發現,大多數帶有GJB4基因變異的聽障患者,都是雙側性、穩定性的先天性聽力損失,這些病人皆無過去文獻所報告某些特定GJB4基因變異常造成皮膚異常的的。且這些患者的聽力圖呈現consistency、severe-to-profound 和 flat shapes dominant的型態,這些特徵將可做為臨床上診斷GJB4基因突變的參考依據。根據上述的結果我們認為在台灣造成聽障的成因中,GJB4基因突變可能是其中一個遺傳因子。而且這些研究結果將可提供作為臨床診斷和遺傳諮詢的依據。 在先前的研究中,我們在GJC3(CX30.2/CX31.3)基因中發現一個新的錯意突變點p. W77S和在GJB3(CX31)基因中的一個錯意突變點p.V174M。然而,對於這兩個錯意突變(CX30.2/CX31.3W77S和CX31V174M)是否造成功能的改變和其機制到目前仍然不清楚。在這個研究中,我們的結果顯示,CX30.2/CX31.3W77S的錯意突變蛋白會聚集在HeLa細胞的內質網。此結果不同於正常的CX30.2/CX 31.3蛋白會表現在細胞膜上。同時,我們利用雙向的tet –on蛋白表達系統共同表達WT和p.W77S蛋白得知,這異構的連接蛋白會在細胞質中堆積,從而影響正常CX30.2/CX31.3蛋白在細胞膜上的表現。此外,我們發現, CX30.2/CX31.3 W77S錯意突變蛋白質是經由溶酶體和蛋白體降解,使得轉染的HeLa細胞看不見突變蛋白的表現。基於這些發現,我們認為p.W77S突變對間隙連接的形成和功能有顯性抑制效應(dominant negative effect)。 另外,本研究我們發現正常的CX31蛋白可以正常的表現和被運送到細胞膜,並在兩個鄰近細胞間形成間隙連接。相反的,p.V174M的CX31突變蛋白卻會堆積在細胞質溶酶體中,無法正常送至細胞膜上表現。此外,轉染(dye transfer)的實驗也證明了CX31V174M突變體沒有形成功能性的間隙連接通道,這可能是由於不正確的組裝或是CX31通道性質的改變所造成。另外在上述的研究中我們發現轉殖入CX31V174M突變的HeLa細胞會陸續的死亡,所以我們利用細胞存活檢測方法 (MTT assay) 分析細胞存活率。我們發現細胞如果含p.V174M的CX31突變蛋白,其還原MTT的能力有下降的趨勢,這代表細胞存活率會漸漸降低,亦即這突變可能會造成細胞的死亡。在CX31V174M與CX31WT或CX26WT共同表達的研究中,CX31V174M會損害CX26WT蛋白在細胞內運輸到細胞膜表現,但不影響CX31WT運輸。基於這些發現,我們認為CX31V174M突變可能會影響間隙連接通道的形成與功能,並對於CX26WT的功能有顯性抑制效應(dominant negative effect)。 綜合以上的結果,我們的研究針對台灣地區遺傳性聽障的成因和機制提供了重要的資訊和更深入的瞭解。而此研究結果也針對在連接蛋白(CX)於聽障發展中所扮演的角色提供了一個新的分子解釋。

並列摘要


Hearing loss is the most common sensory disorder worldwide. The crucial role of gap junctions, which are composed of connexin (CX) protein, in auditory functions has been confirmed by numerous studies. In this study we are going to focus on the effect of mutations on the function of specific connexin genes [CX30.2/CX31.3 (GJC3), CX30.3 (GJB4) and CX31(GJB3)]. In a previous study, we have identified one novel missense mutation, p.W77S, in the GJC3 gene encoding connexin30.2/connexin31.3 (CX30.2/CX31.3) and a missense mutation, p.V174M, in the GJB3 gene encoding connexin 31 (Cx31), in the patient with nonsyndromic hearing loss. However, the functional change in the two missense mutations (CX30.2/CX31.3W77S and CX31V174M) remains unknown. In the study, our result indicated that the intracellular distribution of the p.W77S missense mutation proteins is different from that of CX30.2/CX31.3WT, which showed continuous staining along apposed cell membranes. Accumulation of the mutant protein in the endoplasmic reticulum (ER) of the HeLa cell was observed. Furthermore, co-expression of WT and p.W77S mutant proteins by a bi-directional tet-on expression system showed that the heteromeric connexon accumulated in the cytoplasm, thereby impairing the expression of the WT proteins in the cell membranes. In addition, we found that CX30.2/CX31.3W77S missense mutant proteins were degraded by lysosomes and proteosomes in the transfected HeLa cell. Based on these findings, we suggest that p.W77S mutant has a dominant negative effect on the formation and function of the gap junction. Additionally, we found that the p.V174M missense mutation resulted in the accumulation of the mutant protein in the lysosomes rather than in the cytoplasmic membrane in a fluorescent localization assay. This expression pattern is different from that of Cx31WT, which shows the typical punctuate pattern of a gap junction channel between the neighboring expression cells. Moreover, dye transfer experiments have also demonstrated that the CX31V174M mutant did not form functional gap junction channels, probably due to incorrect assemblies or altered properties of the CX31 channels. In addition, we found that CX31V174M-transfection can cause cell death by MTT assay. CX31V174M co-expressed with either CX31WT or CX26WT showed the impairment of the ability of CX26WT proteins to intracellular trafficking and targeting to the plasma membrane, but did not influence the trafficking of CX31WT. Based on these findings, we suggest that the CX31V174M mutant may have an effect on the formation and function of the gap junction and CX31V174M has a trans-dominant negative effect on the function of wild types CX26. Previously, we identified eleven patients with GJB4 gene variants in 253 unrelated Taiwanese patients with nonsyndromic hearing loss. In the study, we investigate the phenotype-genotype correlation in eleven deaf patients with variants of GJB4. Analytical results revealed that most probands had congenital hearing loss that is bilateral, stable and without associated dermatological manifestation or morphological changes of the inner ear. An audiometric profile including observed consistency, severe-to-profound and flat shapes dominant, may enable screening for variant of GJB4. On the basis of the results of investigation, we suggest that the GJB4 may be genetic risk factor for the development of nonsyndromic hearing loss in the Taiwanese, and our data can be applied to direct the clinical evaluation and effectively counsel families of children with GJB4.In conclusion, our study provides information for understanding the importance of genetic factors in nonsyndromic deafness of Taiwanese. These results provide a novel molecular explanation for the role CX plays in the development of hearing loss.

並列關鍵字

Hearing loss nonsyndromic hearing loss GJC3 GJB3 GJB4

參考文獻


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