從結構方向解析死亡結構域堆疊形成複合體之機制及Apaf-1細胞凋亡複合體活化凋亡蛋白酶九號之機制

Structural Insights into DD-Fold Assembly and Caspase-9 Activation by the Apaf-1 Apoptosome

蘇琮為(Tsung-Wei Su)

指導教授 ： 林世昌

共同指導教授 ： 梁博煌

國立臺灣大學/生命科學院/生化科學研究所/博士(2018年)

https://doi.org/10.6342/NTU201800045

摘要

調控細胞存活與凋亡的訊息傳遞中死亡結構域之堆疊扮演一個重要的角色。本篇論文中發表了第一個細胞凋亡複合體(the Apaf-1 apoptosome)上碗盤狀複合體(the CARD-CARD disk)的蛋白質晶體結構。我們發現有三對由Apaf-1 與凋亡蛋白酶九號(procaspase-9)組成的一比一死亡結構域原聚體(protomer)，進一步於細胞凋亡複合體平台上堆疊出一個全新的螺旋狀排列。小角度X光散射及多角度光散射儀的實驗結果也顯示，於液體狀態三對的一比一死亡結構域原聚體，可以堆疊出與晶體結構相似的碗盤狀複合體。有趣的是在quasi-equivalent 環境中，死亡結構域CARD可以組成二種不同的四級結構。我們同時發現第二種形態的交互作用存在於所有已知的死亡結構域堆疊中， 然而第一種形態的交互作用只存在於螺旋狀排列的死亡結構域堆疊中。這篇研究解析了Apaf-1細胞凋亡複合體活化凋亡蛋白酶九號之機制是如何受死亡結構域CARD堆疊的調控，以及更加了解死亡結構域堆疊形成複合體之機制。

關鍵字

細胞凋亡 ； 凋亡蛋白酶活化 ； 螺旋狀排列 ； 死亡結構域 ； 原聚體 ； 晶體結構 ； 小角度X光散射

並列摘要

Death domain (DD)-fold assemblies play a crucial role in regulating the signaling to cell survival or death. Here we report the crystal structure of the caspase recruitment domain (CARD)-CARD disk of the human apoptosome. The structure surprisingly reveals that three 1:1 Apaf-1: procaspase-9 CARD protomers form a novel helical DD-fold assembly on the heptameric wheel-like platform of the apoptosome. The small-angle X-ray scattering and multi-angle light scattering data also support that three protomers could form an oligomeric complex similar to the crystal structure. Interestingly, the quasi-equivalent environment of CARDs could generate different quaternary CARD assemblies. We also found that the type II interaction is conserved in all DD-fold complexes, whereas the type I interaction is found only in the helical DD-fold assemblies. This study provides crucial insights into the caspase activation mechanism, which is tightly controlled by a sophisticated and highly evolved CARD assembly on the apoptosome, and also enables better understanding of the intricate DD-fold assembly.

並列關鍵字

Apoptosis ； caspase activation ； helical assembly ； CARD ； DED ； DARK ； CED-4 ； protomer ； crystal structure ； SAXS

參考文獻

Adams, P.D., Grosse-Kunstleve, R.W., Hung, L.W., Ioerger, T.R., McCoy, A.J., Moriarty, N.W., Read, R.J., Sacchettini, J.C., Sauter, N.K., and Terwilliger, T.C. (2002). PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954.

Bansal, M., Kumar, S., and Velavan, R. (2000). HELANAL: a program to characterize helix geometry in proteins. J. Biomol. Struct. Dyn. 17, 811–819.

Chao, Y., Shiozaki, E.N., Srinivasula, S.M., Rigotti, D.J., Fairman, R., and Shi, Y. (2005). Engineering a dimeric caspase-9: a re-evaluation of the induced proximity model for caspase activation. PLoS Biol. 3, e183.

Cheng, T.C., Hong, C., Akey, I.V., Yuan, S., and Akey, C.W. (2016). A near atomic structure of the active human apoptosome. Elife 5, e17755.

Czabotar, P.E., Lessene, G., Strasser, A., and Adams, J.M. (2014). Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat. Rev. Mol. Cell Biol. 15, 49–63.