臺灣博碩士論文加值系統

中文摘要
實驗室先前利用蛋白質體學的方法，找出一個與PAK-2有交互作用的蛋白質Myo18A。我們想進一步研究Myo18A在細胞凋亡的過程中所扮演的角色。分析Myo18A的胺基酸序列，發現在Myo18A C端末端的位置含有多個caspase-3的偏好切位DXXD序列。利用UV照射或是 staurosporine誘發細胞凋亡，發現利用C端末端的抗體進行西方墨點法分析時，會偵測到Myo18A的含量下降的情形。另外，也利用重組caspase-3 蛋白進行體外分解實驗證明Myo18A可以被caspase-3切割。為了明確證明Myo18A的確可以在細胞中被切割成兩個片段，我將Myo18A-EGFP的融合蛋白表現於細胞中，接著誘發細胞凋亡，將偵測到的裂解片段分子量減去GFP的分子量，可確定Myo18A在細胞凋亡後被caspase-3切成了222 kDa及8 kDa兩個片段，並據以推算出兩個可能的切位(DSDVD1973及DRVD1981)，再利用定點突變的方法進一步證明 DRVD1981 序列為caspase-3的切割位點。接下來利用NF-Myo18A(1-1981)及CF-Myo18A(1982-2054)轉染到細胞中，分析Myo18A切割後產生的片段，對細胞形態及細胞凋亡的過程的影響，發現了NF-Myo18A可能幫助細胞凋亡過程的進行，而CF-Myo18A則有在細胞核表現的情形，可能與基因調控相關。

英文摘要
Previously, our lab has employed a proteomic approach to identify Myo18A as a novel PAK2-binding partner. To further investigate the biological functions of Myo18A, I performed sequence analysis of Myo18A, which revealed that several consensus caspase-3 cleavage sites are present in the Myo18A’s C-terminal part. We first monitored the fate of Myo18A protein in apoptosis. I used UV irradiation or staurosporine (STS) to induce apoptosis in human A431 cells, and found that Myo18A could be cleaved in response to the two apoptotic stimuli. To determine whether Myo18A is cleaved by caspase3, I performed an in vitro protease assay using recombinant caspase-3 to process Myo18A-GFP fusion protein. I further confirmed the presence of two fragments during apoptosis using a C-terminally tagged Myo18A-GFP construct. As the consensus caspase-3 cleavage sequence is DXXD, and Myo18A was cleaved into two fragments (222 kDa and 8 kDa), we deduced the putative cleavage site and generated two point mutants. Among the two mutants, the D1981N was resistant to cleavage during apoptosis. Taken together, these results demonstrated that the DRVD1981 sequence in Myo18A underwent caspase3-mediated cleavage during apoptosis. Finally, I generated NF-Myo18A(1-1981) and CF-Myo18A(1982-2054) truncation mutants that mimic the cleavage products of Myo18A. I found that NF-Myo18A could induce apoptosis when transfected into A431 cells, and that CF-Myo18A could be localized to the nucleus.

目 錄
指導教授推薦書
口試委員會審定書
授權書
誌謝 iv
中文摘要 v
英文摘要 vi
目錄 vii
圖表目錄 viii
第一章 緒論 1
第二章 材料與方法 5
第三章 結果 12
第四章 討論 19
附圖 23
參考文獻 38

參考文獻
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