利用細胞模式探討CX30.2/CX31.3蛋白的功能

Connexins (CXs) 基因族在許多研究已經被報導和非症候群遺傳性聽障有關。我們實驗室先前利用免疫組織染色法發現，老鼠的耳蝸表現 Cx29 (同義於人類的 CX30.2/CX31.3 ) 與其他 Cxs 蛋白家族成員 (如 Cx26 及 Cx30 ) 所表現的位置相似，且這些位置對於維持耳蝸內的鉀離子再循環相當重要。此外，目前對於 CX30.2/CX31.3 蛋白功能的研究相對其他 CX 蛋白還是很少。本論文將分兩部分來探討：第一部分我們想了解正常 CX30.2/CX31.3 蛋白在細胞膜上形成半通道和 GJ 通道的特性以及其運送路徑。在 GJ 通道特性的研究，我們發現 GJ 半通道在低鈣濃度環境下，可以有意義的釋放 ATP 至細胞外。同時我們利用藥物 18α-glycyirrhetinic acid (18α-GA) 以及 Carbenoxolone (CBX) 抑制半通道的的功能證明 ATP 確實是由半通道所釋放。因此，我們認為 CX30.2/CX31.3 蛋白，主要扮演的功能角色為半通道功能，而不是像一般 CX 蛋白所形成的 GJ 通道功能。在 GJ 通道形成機制方面，我們利用藥物 Brefeldin A 、 Nocodazole 以及 Cytochalasin B 破壞胞器的方式來證明。由研究結果我們認為 CX30.2/CX31.3 蛋白在細胞內的運送過程中，主要依賴高基氏體的修飾，並且藉由肌動蛋白絲快速的運送至細胞膜上。另外我們也發現 CX30.2/CX31.3 和 ZO-1 蛋白在細胞膜上有交互作用。第二部分我們繼續探討 p.W77S 突變蛋白無法持續表現在轉殖細胞內和在細胞內造成影響機制。根據 genomic DNA 的分析發現 p.W77S 的突變蛋白並沒有造成細胞進行細胞凋亡 (apoptosis) 的情況產生，進一步觀察發現含 p.W77S 突變細胞內其 GJC3 mRNA 表現情況和正常型 mRNA 表現情況一致，顯示出轉錄轉譯功能正常。我們加入溶酶體抑制劑 Chloroquine 和蛋白酶體抑制劑 MG132 藥物於表現 p.W77S 融合綠螢光的細胞，證明突變型 p.W77S 蛋白進行了降解 (degradation) 作用。在這研究中，我們知道了 CX30.2/CX31.3 蛋白的主要功能和運送路徑，並且藉由 p.W77S 的研究了解此突變造成聽障的可能機制，更進一步的連結了 CX30.2/CX31.3 和聽障的關係。

關鍵字

CX30.2/CX31.3 ；功能；運送路徑；聽障

並列摘要

Connexins (CXs) are known to be involved in human nonsyndromic genetic deafness. In our previous study, mouse Cx29, orthologs of human CX30.2/CX31.3, was detected in the inner ear of mouse, all of which resemble those of other mouse Cx family (ex. Cx26 and Cx30) by immunohistochemistry analysis. At least, the functional characteristics of CX30.2/CX31.3 are unclear. We divided this thesis into two part. In the first part, we assess the normal CX30.2/CX31.3 protein how trafficking to membrane form hemichannel and then to study the functional properties of hemichannel. A significant amount of ATP was released from the HeLa cells that stably expressed CX30.2/CX31.3, in a medium with low calcium ion concentration. Furthermore, we use non-specific GJ hemichannel inhibitor 18α-glycyirrhetinic acid (18α-GA) and Carbenoxolone (CBX) to confirm that CX30.2/CX31.3 shares functional properties with hemichannels rather than gap junction channels. In the formation pathway of CX30.2/CX31.3 channels, we found that the targeting of CX30.2/CX31.3 to membrane form channels in stably expressing HeLa cells was affected by brefeldin A, cytochalasin B or nocodazole treatment. Base on these results, we suggest that formation pathway of CX30.2/CX31.3 channels may be a Golgi-dependent pathway and the actin filaments are important components in the processes of CX30.2/CX31.3 channels. Furthermore, we detect protein complex from CX30.2/CX31.3 interact with ZO-1 protein. In the second part, we investigate the molecular mechanisms of missense mutation, p.W77S, in CX30.2/CX31.3 using HeLa cells. We extract genomic DNA from p.W77S tranfacted HeLa cells and were then resolved by conventional agarose gel electrophoresis to evaluate the potential apoptotic DNA fragmentation. The results confirmed absence of the characteristic DNA laddering of those cells. Therefore, we suggest that there are not apoptosis in the p.W77S transfected HeLa cells. Furthermore, the quantity of p.W77S mRNA and wild type mRNA are consistency in the q-PCR analysis. Besides, we use lysosome inhibitor chloroquine and proteasome inhibitor MG132 to confirm that missense mutation CX30.2/CX31.3 p.W77S was degraded in HeLa cells. Base on the above results, we suggest that missense mutation CX30.2/CX31.3 p.W77S was degraded in HeLa cells. In summary, we display the functional property and formation pathway of normal CX30.2/CX31.3 in the study. In addition, we reveal the reason of hearing loss caused by mutation p.W77S and provide a novel molecular explanation for the role CX30.2/CX31.3 plays in the development of hearing loss.

並列關鍵字

CX30.2/CX31.3 ； functional properties ； secretary pathway ； hearing loss

參考文獻

Ahmad, S., Chen, S., Sun, J. &Lin, X. (2003). Connexins 26 and 30 are co-assembled to form gap junctions in the cochlea of mice. Biochem Biophys Res Commun 307(2): 362-368.

Ahmad, S. &Evans, W. H. (2002). Post-translational integration and oligomerization of connexin 26 in plasma membranes and evidence of formation of membrane pores: implications for the assembly of gap junctions. Biochem J 365(Pt 3): 693-699.

Ahn, M., Lee, J., Gustafsson, A., Enriquez, A., Lancaster, E., Sul, J.-Y., Haydon, P. G., Paul, D. L., Huang, Y., Abrams, C. K. &Scherer, S. S. (2008). Cx29 and Cx32, two connexins expressed by myelinating glia, do not interact and are functionally distinct. Journal of Neuroscience Research 86(5): 992-1006.

Altevogt, B. M., Kleopa, K. A., Postma, F. R., Scherer, S. S. &Paul, D. L. (2002). Connexin29 is uniquely distributed within myelinating glial cells of the central and peripheral nervous systems. J Neurosci 22(15): 6458-6470.

ANDREW T. CHAYTOR, W. L. M., IAIN R. HUTCHESON* and TUDOR M. GRIFFITHt (2000). Comparison of Glycyrrhetinic Acid Isoforms and Carbenoxolone as Inhibitors of EDHF-Type Relaxations Mediated via Gap Junctions. Overseas Publishers Aasociiition.

國際替代計量

利用細胞模式探討CX30.2/CX31.3蛋白的功能

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