- 學位論文
表達純化全長 XIAP來檢測 XIAP:CASP7 的可逆抑制劑
Expression and purification of the full-length XIAP for confirming a reversible XIAP:CASP7 inhibitor
摘要
X-linked inhibitor of apoptosis (XIAP) 抑制caspase-3 (CASP3)、7 (CASP7) 和 9，進而阻止細胞凋亡。已在化療抗性癌細胞中發現 XIAP 有過表達的現象。因此，已經開發了幾種小分子的 XIAP 拮抗劑和 SMAC 的模擬物來抑制 XIAP 與這些caspase之間的蛋白質相互作用以殺死腫瘤細胞。XIAP 利用第二個重複 (BIR2) 結構域和 BIR1 和 BIR2 之間的linker來結合並抑制內在凋亡途徑中的 CASP3/7。由這些化合物對缺乏 XIAP:CASP3 和XIAP:CASP7 之間的選擇性，其中一些化合物可能對細胞有毒性。我們之前報導了一種策略，通過選擇性結合CASP7 來破壞 XIAP:CASP7 複合物而不是 XIAP:CASP3，因此該化合物殺死了由化學療法引起的 CASP3 表現量低的 (CASP3/DR) 癌細胞，這些癌細胞中會累積很多 XIAP:CASP7。Shih-Hsun Chen 博士通過電腦模擬鑑定出可逆抑制劑與 CASP7 結合，釋放 XIAP 的linker-BIR2 結構域，並活化 CASP7 以殺死 CASP3/DR 癌細胞（Chen et al.，未發表）。因此，本篇論文旨在表達和純化全長 XIAP，以確認該化合物可以破壞全長 XIAP 和 CASP7 之間的相互作用。我通過使用 CASP7 活性測定、螢光猝滅試驗和 BIAcore 實驗證明了該化合物可以破壞全長 XIAP 與 CASP7 的相互作用。
並列摘要
X-linked inhibitor of apoptosis (XIAP) inhibits caspases 3 (CASP3), 7 (CASP7), and 9, thereby preventing cell apoptosis. XIAP overexpression has been found in chemoresistant cancer cells. Therefore, several small-molecule XIAP antagonists and SMAC mimetics have been developed to inhibit the protein-protein interaction between XIAP and these caspases to kill tumor cells. XIAP utilizes the second repeat (BIR2) domain and the linker between BIR1 and BIR2 to bind and inhibit CASP3/7 in the intrinsic apoptotic pathway. Due to a lack of selectivity between CASP3 and 7 by targeting XIAP, some of these compounds might be toxic to cells. We previously reported a strategy by selectively targeting CASP7 to disrupt XIAP:CASP7 complex but not XIAP:CASP3, so that the compound killed CASP3-downregulated (CASP3/DR) cancer cells resulted from chemotherapy, which have accumulated XIAP:CASP7. A reversible XIAP:CASP7 inhibitor identified by Dr. Shih-Hsun Chen through a multiple-mode virtual screening strategy was found to bind CASP7, release the linker-BIR2 domain of XIAP, and activate CASP7 for killing CASP3/DR cancers cells (Chen et al., unpublished). I therefore aimed to express and purify the full-length XIAP for confirming that the compound can disrupt the interaction between the full-length XIAP and CASP7. I demonstrated that the compound can disrupt the interaction of the full-length XIAP with CASP7 by using CASP7 activity assay, fluorescence quenching, and BIAcore experiments.