臺灣博碩士論文加值系統

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。

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.

Verification letter from oral examination committee … ………i
Chinese abstract……… …………………………...…………..ii
Abstract…..…………..…………………………………….….iii
Table of contents ………………………………………………iv
List of Figures…………….....…………………………………vi
List of Tables….…………..………………………………….viii
1. Introduction 1
2. Results 5
2.1 The CARD-CARD Disk Contains Three Protomers 5
2.2 The Protomer Exhibits Concentration-Dependent Monomer-Trimer Equilibrium 7
2.3 The CARD-CARD Disk Employs a Novel Helical Assembly that Limits Its Size 8
2.4 A Novel Helical Assembling Mechanism via Composite Self-Assembling Sites 10
2.5 A New Look at the Assembling Mechanisms of DD-Fold Proteins 12
2.6 The Type I, II, and III Interactions among the DD-Fold Complexes 15
3. Discussion 20
3.1 The Crystal Structure Fits the Cryo-EM Map 21
3.2 Comparison with the Recently Published Cryo-EM Structure and Its Implications 22
3.3 The Caspase Activation Mechanism 27
4. Experimental Procedures 31
4.1 Protein Expression, Purification, and Crystallization 31
4.2 Structure Determination and Model Building 32
4.3 EM Docking 32
4.4 Multi-Angle Light Scattering 33
4.5 Small-Angle X-Ray Scattering 34
5. References 68
6. Appendix 76

Acehan, D., Jiang, X., Morgan, D.G., Heuser, J.E., Wang, X., and Akey, C.W. (2002). Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation. Mol. Cell 9, 423–432.
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.
Boatright, K.M., Renatus, M., Scott, F.L., Sperandio, S., Shin, H., Pedersen, I.M., Ricci, J.E., Edris, W.A., Sutherlin, D.P., Green, D.R., et al. (2003). A unified model for apical caspase activation. Mol. Cell 11, 529–541.
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.
D’Amelio, M., Tino, E., and Cecconi, F. (2008). The apoptosome: emerging insights and new potential targets for drug design. Pharm. Res. 25, 740–751.
D’Amelio, M., Cavallucci, V., and Cecconi, F. (2010). Neuronal caspase-3 signaling: not only cell death. Cell Death Differ. 17, 1104–1114.
Hopfner, K.P. (2014). RIG-I holds the CARDs in a game of self versus nonself. Mol. Cell 55, 505–507.
Hu, Q., Wu, D., Chen, W., Yan, Z., Yan, C., He, T., Liang, Q., and Shi, Y. (2014). Molecular determinants of caspase-9 activation by the Apaf-1 apoptosome. Proc. Natl. Acad. Sci. USA 111, 16254–16261.
Hu, Z., Zhou, Q., Zhang, C., Fan, S., Cheng, W., Zhao, Y., Shao, F., Wang, H.W., Sui, S.F., and Chai, J. (2015). Structural and biochemical basis for induced self-propagation of NLRC4. Science 350, 399–404.
Kao, W.P., Yang, C.Y., Su, T.W., Wang, Y.T., Lo, Y.C., and Lin, S.C. (2015). The versatile roles of CARDs in regulating apoptosis, inflammation, and NFkappaB signaling. Apoptosis 20, 174–195.
Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S., and Wang, X. (1997). Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479–489.
Lin, S.C., Lo, Y.C., and Wu, H. (2010). Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling. Nature 465, 885–890.
Lo, Y.C., Lin, S.C., Yang, C.Y., and Tung, J.Y. (2015). Tandem DEDs and CARDs suggest novel mechanisms of signaling complex assembly. Apoptosis 20, 124–135.
Malladi, S., Challa-Malladi, M., Fearnhead, H.O., and Bratton, S.B. (2009). The Apaf-1*procaspase-9 apoptosome complex functions as a proteolytic-based molecular timer. EMBO J. 28, 1916–1925.
Otwinowski, Z., and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326.
Pang, Y., Bai, X.C., Yan, C., Hao, Q., Chen, Z., Wang, J.W., Scheres, S.H., and Shi, Y. (2015). Structure of the apoptosome: mechanistic insights into activation of an initiator caspase from Drosophila. Genes Dev. 29, 277–287.
Park, H.H., Lo, Y.C., Lin, S.C., Wang, L., Yang, J.K., and Wu, H. (2007a). The death domain superfamily in intracellular signaling of apoptosis and inflammation. Annu. Rev. Immunol. 25, 561–586.
Park, H.H., Logette, E., Raunser, S., Cuenin, S., Walz, T., Tschopp, J., and Wu, H. (2007b). Death domain assembly mechanism revealed by crystal structure of the oligomeric PIDDosome core complex. Cell 128, 533–546.
Peisley, A., Wu, B., Xu, H., Chen, Z.J., and Hur, S. (2014). Structural basis for ubiquitin-mediated antiviral signal activation by RIG-I. Nature 509, 110–114.
Petoukhov, M.V., Franke, D., Shkumatov, A.V., Tria, G., Kikhney, A.G., Gajda, M., Gorba, C., Mertens, H.D.T., Konarev, P.V., and Svergun, D.I. (2012). New developments in the ATSAS program package for small-angle scattering data analysis. J. Appl. Crystallogr. 45, 342–350.
Qi, S., Pang, Y., Hu, Q., Liu, Q., Li, H., Zhou, Y., He, T., Liang, Q., Liu, Y., Yuan, X., et al. (2010). Crystal structure of the Caenorhabditis elegans apoptosome reveals an octameric assembly of CED-4. Cell 141, 446–457.
Qin, H., Srinivasula, S.M., Wu, G., Fernandes-Alnemri, T., Alnemri, E.S., and Shi, Y. (1999). Structural basis of procaspase-9 recruitment by the apoptotic protease-activating factor 1. Nature 399, 549–557.
Renatus, M., Stennicke, H.R., Scott, F.L., Liddington, R.C., and Salvesen, G.S. (2001). Dimer formation drives the activation of the cell death protease caspase 9. Proc. Natl. Acad. Sci. USA 98, 14250–14255.
Salvesen, G.S., and Dixit, V.M. (1999). Caspase activation: the induced-proximity model. Proc. Natl. Acad. Sci. USA 96, 10964–10967.
Shi, Y. (2004). Caspase activation: revisiting the induced proximity model. Cell 117, 855–858.
Stefani, I.C., Wright, D., Polizzi, K.M., and Kontoravdi, C. (2012). The role of ER stress-induced apoptosis in neurodegeneration. Curr. Alzheimer Res. 9, 373–387.
Wu, B., Peisley, A., Tetrault, D., Li, Z., Egelman, E.H., Magor, K.E., Walz, T., Penczek, P.A., and Hur, S. (2014). Molecular imprinting as a signal-activation mechanism of the viral RNA sensor RIG-I. Mol. Cell 55, 511–523.
Yuan, S., and Akey, C.W. (2013). Apoptosome structure, assembly, and procaspase activation. Structure 21, 501–515.
Yuan, S., Yu, X., Topf, M., Ludtke, S.J., Wang, X., and Akey, C.W. (2010). Structure of an apoptosome-procaspase-9 CARD complex. Structure 18, 571–583.
Yuan, S., Yu, X., Asara, J.M., Heuser, J.E., Ludtke, S.J., and Akey, C.W. (2011a). The holo-apoptosome: activation of procaspase-9 and interactions with caspase-3. Structure 19, 1084–1096.
Yuan, S., Yu, X., Topf, M., Dorstyn, L., Kumar, S., Ludtke, S.J., and Akey, C.W. (2011b). Structure of the Drosophila apoptosome at 6.9 A resolution. Structure 19, 128–140.
Yuan, S., Topf, M., Reubold, T.F., Eschenburg, S., and Akey, C.W. (2013). Changes in Apaf-1 conformation that drive apoptosome assembly. Biochemistry 52, 2319–2327.
Zhang, L., Chen, S., Ruan, J., Wu, J., Tong, A.B., Yin, Q., Li, Y., David, L., Lu, A., Wang, W.L., et al. (2015). Cryo-EM structure of the activated NAIP2-NLRC4 inflammasome reveals nucleated polymerization. Science 350, 404–409.
Zhou, M., Li, Y., Hu, Q., Bai, X.C., Huang, W., Yan, C., Scheres, S.H., and Shi, Y. (2015). Atomic structure of the apoptosome: mechanism of cytochrome c- and dATP-mediated activation of Apaf-1. Genes Dev. 29, 2349–2361.