- 學位論文
利用結構生物學探討XIAP與Caspase-3之間的相互抑制作用
Structural Analysis on the Reciprocal Inhibition Between XIAP and Caspase-3
摘要
X染色體連鎖凋亡抑制蛋白（XIAP）為一多功能蛋白質，屬於IAP蛋白家族，其最大的特點是具有抗細胞凋亡的功能，藉由與caspase-3 結合能夠直接抑制其活性。矛盾的是，caspase-3 能夠於特定位點(Asp242)將XIAP切割成兩個片段，從而減弱 XIAP 對細胞凋亡的抑制。而先前的研究證據顯示，XIAP 能夠與未完全活化的caspase-3 p20/p12結合，使其停留在該型態而無法透過自催化作用活化成p17/p12。本篇研究提出 XIAP 主要藉由與 caspase-3 p20/p12 相互作用來抑制 caspase-3 活性，阻止其完全活化，而非直接抑制有活性的 caspase-3。對於成熟的 caspase-3，XIAP 是被其水解的受質，該水解會導致其功能之喪失。在本篇研究中發現，與成熟的 p17/p12 相比，XIAP 對 caspase-3 p20/12 表現出更高的親和力，這表明 XIAP 與這兩種形式的 caspase-3 之間的結合模式可能是不同的。為了證明這個想法，我純化了 XIAP/caspase-3 複合物（XIAP/caspase-3 p20/p12 和 XIAP/caspase-3 /p17/p12），並嘗試利用冷凍電子顯微鏡解析其結構。利用戊二醛(glutaraldehyde)將蛋白複合體進行交叉鍵接(cross-link)反應，穩定XIAP/caspase-3 p20/p12複合體後，我們得到一低解析度(~12Å)的三維重建。後續研究將繼續專注於解析出高解析度的 XIAP/caspase-3 複合物的結構，以闡明 XIAP 與 caspase-3 之間的相互作用。
並列摘要
X-chromosome-linked inhibitor of apoptosis protein (XIAP) is a multi-functional protein which belongs to the IAP protein family and is most characterized by its antiapoptotic function. XIAP was reported to be a direct inhibitor of active caspase-3, a main executioner caspase in apoptosis. Paradoxically, caspase-3 was shown to be able to cleave XIAP into half at Asp242, attenuating the inhibition of apoptosis exerted by XIAP. Besides, previous studies have indicated that caspase-3 was arrested in intermediate form (p20/p12) due to XIAP binding. Here, I propose that XIAP inhibits caspase-3 activity mainly by interacting with caspase-3 p20/p12, preventing it from full activation instead of directly inhibiting the active caspase-3. For mature caspase-3, XIAP is a substrate to be cleaved and this cleavage event leads to the loss of its function. In my study, XIAP exhibited much higher affinity toward partially processed caspase-3 p20/12 than mature p17/p12, suggesting the binding modes between XIAP and this two forms of caspase-3 might be distinct. To prove this idea, I have purified XIAP/caspase-3 complexes (XIAP/caspase-3 p20/p12 and XIAP/caspase-3 /p17/p12) with high purity and attempted to determine the structures by cryo-EM. After improving the sample stability by chemical crosslinking, we could only obtained a low-resolution EM density map (~12 Å) of XIAP/caspase-3 p20/p12 complex by 3D reconstruction. On the other hand, I have also investigated the autoprocessing of caspase-3 p20/p12 into mature p17/p12 and discovered that this process is salt-dependent in vitro and relevant to its aggregation. The detailed mechanism of the autocatalysis of caspase-3 p20/p12 and how XIAP prohibits this process still require further investigation. The follow-up research will keep focusing on resolving the structure of XIAP/caspase-3 complexes with high resolution to elucidate the interplay between XIAP and caspase-3.