Background: Bcl2L12是Bcl2家族的一個成員，曾被報導帶有未知功能的BH3相似區段。過去研究指出Bcl2L12在惡性腦瘤中透過多重分子機制，參與後粒線體細胞凋亡的調控，其中包括調控p53的轉錄活性、透過-crystallin (CRYAB)調降caspase-3的活性或是直接抑制caspase-7。然而，Bcl2L12的BH3相似區段是否有參與細胞凋亡的調控仍不清楚。
Specific aims:因此，在本研究中，我們基本上調查Bcl2L12是否具有一功能性的BH3相似區段且透過它與一些Bcl2家族的存活因子，像是Bcl-xL及Bcl2，交互作用參與抗細胞凋亡的作用。除此，我們也測試此一BH3相似區段，可否作為藥物標的，藉以增加惡性腦瘤對於temozolomide (TMZ)的感受性。
Methods:為了達到這些目標，我們首先以電腦程式模擬比較Bcl2L12以及其他Bcl2家族成員的蛋白質次級以及3D結構，且特別鎖定在BH3相似區段。再者，以點突變以及酵母雙雜合試驗來搜尋會影響Bcl2L12-Bcl-xL以及Bcl-xL-BH3交互作用重要的胺基酸位點。第三，為了驗證是否破壞Bcl2L12 BH3相似區段(192-240片段)以及Bcl2家族蛋白的BH3或是忌水區域間的交互作用，會導致細胞凋亡現象的再活化，我們將綠色螢光蛋白融合Bcl2L12野生型蛋白和Bcl2L12 BH3突變型蛋白L213A以及L217A在不同的腦瘤細胞中過表現，並以不同的藥物加以處理(TMZ和staurosporine)。最後，以BH3模擬試藥，ABT-737，來驗證Bcl2L12的BH3相似區段是否與Bcl2L12在腦瘤中的抗細胞凋亡特性以及TMZ誘發的細胞自噬作用有關。
Results: 我們的結果顯示Bcl2L12的BH3區段與其他Bcl2家族蛋白的BH3有高度的相似性，即帶有“LXXXAE(D)”胺基酸組成的特殊區段。另外，我們的結果也顯示h1 (L213), h2 (L217) 以及h4 (I224)胺基酸，在酵母雙雜合系統，扮演影響Bcl2L12與Bcl-xL以及Bcl2交互作用的重要腳色。在腦瘤細胞表現Bcl2L12 h1和h2 BH3突變型蛋白並處理staurosporine (STS) or temozolomide (TMZ)，會導致原本Bcl2L12抑制細胞凋亡蛋白(切分型caspase-3, -9, cytochrome c以及PARP)表現的特性喪失，也會調降細胞自噬作用相關蛋白(Beclin-1和LC3-II)的表現。
Conclusion:總結，本研究中我們建立一個模式來說明Bcl2L12的L213以及L217是其BH3相似區段中重要的氨基酸位點，透過與Bcl-xL和Bcl2的交互作用，可能參與其抗細胞凋亡的腳色且或許也跟TMZ誘發的細胞自噬作用有關。在此，我們首先提出Bcl2L12帶有BH3相似區段且可作為一個標的加以操控，來增加惡性腦瘤對於TMZ敏感性。

Background: Bcl2L12 is a new member of Bcl2 family, which is reported to harbor BH3-like domain with an unidentified function. Previous study showed that Bcl2L12 involved in regulation of post-mitochondrial apoptotic events through multiple molecular mechanisms including controls the transcriptional activity of p53, down regulates caspase-3 activity through -crystallin (CRYAB), and directly antagonist to caspase-7 in glioblastoma (GBM). However, whether BH3-like domain of Bcl2L12 involved in the apoptosis regulation in GBM is still unknown.
Specific aims: Therefore, in this study, we basically investigate whether Bcl2L12 harbors a functional BH3-like domain involved in its anti-apoptotic role in GBM through interacting with pro-survival proteins of Bcl2 family such as Bcl-xL and Bcl2. Besides, we tested whether this BH3-like domain can be as promising target for sensitizing drug response of GBM to temozolomide (TMZ).
Methods: To approach these aims, we firstly computational simulated to compare the similarity of secondary and 3D-structure of Bcl2L12 with others Bcl2 family proteins, especially focused on BH3-like domain. Second, site-directed mutagenesis and yeast two hybrid assay were performed to pinpoint the residues may critical for Bcl2L12-Bcl-xL, Bcl-xL-BH3 interaction. Third, to determine whether disrupt the interaction between Bcl2L12 BH3-like domain (192-240) and BH3 and/or hydrophobic groove of Bcl2 family members led to a reactivation of apoptotic events, GFP-tagged Bcl2L12 wt and Bcl2L12 BH3-mutant L213A and L217A were overexpressed in glioma cell lines under different treatments (TMZ and staurosporine). Lately, the BH3 mimetic agent, ABT-737, was implied to confirm BH3-like domain of Bcl2L12 do responsible for its anti-apoptotic role and TMZ-induced autophagy in glioma.
Results: Our results showed that Bcl2L12 share a highly similarity with Bcl2 family members at BH3 domain, which is a consensus core domain of “LXXXAE(D)” through bioinformatical analysis. Moreover, our data showed that the h1 (L213), h2 (L217) and h4 (I224) residues are essential for the Bcl2L12 interaction with Bcl-xL and Bcl2 in yeast two hybrid system. Ectopically overexpressed Bcl2L12 wt, h1 and h2 mutant in GBM cell lines resulted in lost repression of apoptotic markers (cleaved caspase-3, -9, cytochrome c and PARP) as well as downregulates autophagy markers (Beclin-1 and LC3-II) under either staurosporine (STS) or temozolomide (TMZ) treatment in U87MG cells.
Conclusion: Altogether, we established a model to demonstrate that Bcl2L12 L213 and L217 as critical residues within BH3-like domain that confers an anti-apoptotic role and may involve in TMZ-induced autophagy through interplaying with Bcl-xL and Bcl2 in glioma cells. We, here, reported for the first time that Bcl2L12 harbors BH3-like domain and as a target can be modulated to sensitize drug response of GBM to TMZ.

Table of contents
論文審定書…………………………………………………………….… i

誌謝…………………………………………………………………….… ii

中文摘要………………………………………………………….……….iii

English abstract………………………………………..…………….…….v

Introduction…………………………………………………...……………1

Specific Aims….……………………………………………...…..………12

Materials and Methods……………………………………...…...…....…..13

Results..………………………………………………...……………..…..18

Discussion…………………………………………….…………….….....23

Conclusion………………………………………….…………………..…25

References..……………………………………..……………….………..26

Figures and Tables………………………………..……………….………30

Appendix………………...……………………………………………......39

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