感染性海綿狀腦病是一種藉由變性普恩蛋白所引起的致命家族性神經退行性疾病。正常普恩蛋白可藉由結構的錯誤折疊而轉變成不可溶聚集型澱粉樣纖維。此外，正常普恩蛋白在高溫和低酸鹼值的環境下較容易使其局部結構展開，並誘發錯誤折疊，進而造成可能的聚集行為。本文利用分子動力學模擬，研究人類普恩蛋白(胺基酸序列90-231)於不同酸鹼值和溫度下的結構轉變特性，並從中發現可能最容易造成結構聚集的致病機制。結果顯示，在450 K下其結構特性相較於常溫下300 K時更為緊密，而600 K則呈現較為鬆散的構形。此外，溫度達450 K時，初始結構中的第二個阿法螺旋結構(Helix B；HB)的熱穩定性最差，並可將 HB視為結構聚集中的一個晶種成核點。另一方面，當結構由450 K回溫到室溫300 K時，結果顯示結構的β-sheet含量增加，尤其在低pH值時，造成β-sheet的誘發又更為明顯。這種高含量的β-sheet結構可能是經由錯誤折疊，而形成PrPSc聚集前的中間態產物。這種由高溫加速結構展開並利用酸鹼值誘發錯誤折疊以及回溫後的複折路徑，將使我們更深入瞭解普恩蛋白結構轉變的致病機制。

Transmissible spongiform encephalopathies (TSE) is the fatal family neurodegenerative diseases cased by prion protein. Normal cellular prion protein (PrPC) undergoes structural conversion from misfolding and denaturing of the prion protein, resulting in insoluble aggregated amyloid fibrils. PrPC unfolded and misfolded occure more readily at high temperature and low pH value that finally have the possible aggregation. To gain insight into the structural transition of pathogenic mechanism, the structural characteristics of huPrP 90-231 have examined using molecular dynamics simulations. The results shown the PrPC structure became more compact at 450 K than 300 K, whereas the 600 K cases exhibit extended states. Furthermore, the results indicated that HB local structure is instable than HA and HC at 450 K, i.e. HB like a seed of nucleation for structural aggregates. On the other hand, while temperature from 450 K drops to 300 K, the β-sheet structures are obviously increased, especially at acidic pH condition. These β-sheet-rich misfolded structures may be the intermediates before amyloid fibrils. Eventually, the strategy in the present study, i.e. raise temperature, equilibrium and refolding, will shed light on the relationship between structural transition and pathogenic mechanism.

1. 呂建榮, 陳., 台灣大學醫學院附設醫院神經部 高雄長庚紀念醫院神經科*, Prion 疾病的觀念演變. Formosan J Med, 2004. 8: p. 6.
2. Prusiner, S.B., Shattuck lecture - Neurodegenerative diseases and prions. New England Journal of Medicine, 2001. 344(20): p. 1516-1526.
3. Prusiner, S.B. and S.J. Dearmond, PRION DISEASES AND NEURODEGENERATION. Annual Review of Neuroscience, 1994. 17: p. 311-339.
4. Weissmann, C., Molecular biology of transmissible spongiform encephalopathies. Febs Letters, 1996. 389(1): p. 3-11.
5. Aguzzi, A., C. Sigurdson, and M. Heikenwaelder, Molecular mechanisms of prion pathogenesis. Annual Review of Pathology-Mechanisms of Disease, 2008. 3: p. 11-40.
6. Cobb, N.J. and W.K. Surewicz, Prion Diseases and Their Biochemical Mechanisms. Biochemistry, 2009. 48(12): p. 2574-2585.
7. Riek, R., et al., NMR structure of the mouse prion protein domain PrP(121-231). Nature, 1996. 382(6587): p. 180-182.
8. Riek, R., et al., NMR characterization of the full-length recombinant murine prion protein, mPrP(23-231). Febs Letters, 1997. 413(2): p. 282-288.
9. Donne, D.G., et al., Structure of the recombinant full-length hamster prion protein PrP(29-231): The N terminus is highly flexible. Proceedings of the National Academy of Sciences of the United States of America, 1997. 94(25): p. 13452-13457.
10. Garcia, F.L., et al., NMR structure of the bovine prion protein. Proceedings of the National Academy of Sciences of the United States of America, 2000. 97(15): p. 8334-8339.
11. Zahn, R., et al., NMR solution structure of the human prion protein. Proceedings of the National Academy of Sciences of the United States of America, 2000. 97(1): p. 145-150.
12. Calzolai, L. and R. Zahn, Influence of pH on NMR structure and stability of the human prion protein globular domain. Journal of Biological Chemistry, 2003. 278(37): p. 35592-35596.
13. Haire, L.F., et al., The crystal structure of the globular domain of sheep prion protein. Journal of Molecular Biology, 2004. 336(5): p. 1175-1183.
14. Knaus, K.J., et al., Crystal structure of the human prion protein reveals a mechanism for oligomerization. Nature Structural Biology, 2001. 8: p. 770-774.
15. Abalos, G.C., et al., Identifying Key Components of the PrPC-PrPSc Replicative Interface. Journal of Biological Chemistry, 2008. 283(49): p. 34021-34028.
16. Brown, D.R., J. Herms, and H.A. Kretzschmar, MOUSE CORTICAL-CELLS LACKING CELLULAR PRP SURVIVE IN CULTURE WITH A NEUROTOXIC PRP FRAGMENT. Neuroreport, 1994. 5(16): p. 2057-2060.
17. Forloni, G., et al., NEUROTOXICITY OF A PRION PROTEIN-FRAGMENT. Nature, 1993. 362(6420): p. 543-546.
18. Supattapone, S., et al., Prion protein of 106 residues creates an artificial transmission barrier for prion replication in transgenic mice. Cell, 1999. 96(6): p. 869-878.
19. Hornshaw, M.P., J.R. McDermott, and J.M. Candy, COPPER-BINDING TO THE N-TERMINAL TANDEM REPEAT REGIONS OF MAMMALIAN AND AVIAN PRION PROTEIN. Biochemical and Biophysical Research Communications, 1995. 207(2): p. 621-629.
20. Stockel, J., et al., Prion protein selectively binds copper(II) ions. Biochemistry, 1998. 37(20): p. 7185-7193.
21. Pauly, P.C. and D.A. Harris, Copper stimulates endocytosis of the prion protein. Journal of Biological Chemistry, 1998. 273(50): p. 33107-33110.
22. Linden, R., et al., Physiology of the prion protein. Physiological Reviews, 2008. 88(2): p. 673-728.
23. Le Pichon, C.E. and S. Firestein, Expression and localization of the prion protein PrPc in the olfactory system of the mouse. Journal of Comparative Neurology, 2008. 508(3): p. 487-499.
24. Millhauser, G.L., Copper and the prion protein: Methods, structures, function, and disease. Annual Review of Physical Chemistry, 2007. 58: p. 299-320.
25. Singh, A., et al., Prion Protein Modulates Cellular Iron Uptake: A Novel Function with Implications for Prion Disease Pathogenesis. Plos One, 2009. 4(2).
26. Viles, J.H., M. Klewpatinond, and R.C. Nadal, Copper and the structural biology of the prion protein. Biochemical Society Transactions, 2008. 36: p. 1288-1292.
27. Kupfer, L., W. Hinrichs, and M.H. Groschup, Prion Protein Misfolding. Current Molecular Medicine, 2009. 9(7): p. 826-835.
28. van der Kamp, M.W. and V. Daggett, Influence of pH on the Human Prion Protein: Insights into the Early Steps of Misfolding. Biophysical Journal, 2010. 99(7): p. 2289-2298.
29. Abid, K. and C. Soto, The intriguing prion disorders. Cellular and Molecular Life Sciences, 2006. 63(19-20): p. 2342-2351.
30. Prusiner, S.B., Prions. Proceedings of the National Academy of Sciences of the United States of America, 1998. 95(23): p. 13363-13383.
31. Safar, J., et al., THERMAL-STABILITY AND CONFORMATIONAL TRANSITIONS OF SCRAPIE AMYLOID (PRION) PROTEIN CORRELATE WITH INFECTIVITY. Protein Science, 1993. 2(12): p. 2206-2216.
32. Pan, K.M., et al., CONVERSION OF ALPHA-HELICES INTO BETA-SHEETS FEATURES IN THE FORMATION OF THE SCRAPIE PRION PROTEINS. Proceedings of the National Academy of Sciences of the United States of America, 1993. 90(23): p. 10962-10966.
33. Castilla, J., et al., In vitro generation of infectious scrapie prions. Cell, 2005. 121(2): p. 195-206.
34. Aguzzi, A. and A.M. Calella, Prions: Protein Aggregation and Infectious Diseases. Physiological Reviews, 2009. 89(4): p. 1105-1152.
35. Weissmann, C., The state of the prion. Nature Reviews Microbiology, 2004. 2(11): p. 861-871.
36. Soto, C., Diagnosing prion diseases: Needs, challenges and hopes. Nature Reviews Microbiology, 2004. 2(10): p. 809-819.
37. Wayne M. Becker, L.J.K., Jeff Hardin, Gregory Paul Bertoni, The World of the Cell. Seventh Edition ed. 2009, ISBN-13: 978-0-8053-9393-4.
38. Cashman, N.R. and B. Caughey, Prion diseases - Close to effective therapy? Nature Reviews Drug Discovery, 2004. 3(10): p. 874-884.
39. Riek, R., et al., Prion protein NMR structure and familial human spongiform encephalopathies. Proceedings of the National Academy of Sciences of the United States of America, 1998. 95(20): p. 11667-11672.
40. Harris, D.A., Biosynthesis and cellular processing of the prion protein, in Advances in Protein Chemistry, Vol 57. 2001, Academic Press Inc: San Diego. p. 203-228.
41. Taraboulos, A., et al., CHOLESTEROL DEPLETION AND MODIFICATION OF COOH-TERMINAL TARGETING SEQUENCE OF THE PRION PROTEIN INHIBIT FORMATION OF THE SCRAPIE ISOFORM. Journal of Cell Biology, 1995. 129(1): p. 121-132.
42. Gorodinsky, A. and D.A. Harris, GLYCOLIPID-ANCHORED PROTEINS IN NEUROBLASTOMA-CELLS FORM DETERGENT-RESISTANT COMPLEXES WITHOUT CAVEOLIN. Journal of Cell Biology, 1995. 129(3): p. 619-627.
43. Sunyach, C., et al., The mechanism of internalization of glycosylphosphatidylinositol-anchored prion protein. Embo Journal, 2003. 22(14): p. 3591-3601.
44. Lee, R.J., S. Wang, and P.S. Low, Measurement of endosome pH following folate receptor-mediated endocytosis. Biochimica Et Biophysica Acta-Molecular Cell Research, 1996. 1312(3): p. 237-242.
45. Hornemann, S. and R. Glockshuber, A scrapie-like unfolding intermediate of the prion protein domain PrP(121-231) induced by acidic pH. Proceedings of the National Academy of Sciences of the United States of America, 1998. 95(11): p. 6010-6014.
46. Kelly, J.W., The environmental dependency of protein folding best explains prion and amyloid diseases. Proceedings of the National Academy of Sciences of the United States of America, 1998. 95(3): p. 930-932.
47. DeMarco, M.L. and V. Daggett, Local environmental effects on the structure of the prion protein. Comptes Rendus Biologies, 2005. 328(10-11): p. 847-862.
48. Swietnicki, W., et al., pH-dependent stability and conformation of the recombinant human prion protein PrP(90-231). Journal of Biological Chemistry, 1997. 272(44): p. 27517-27520.
49. Cobb, N.J., A.C. Apetri, and W.K. Surewicz, Prion Protein Amyloid Formation under Native-like Conditions Involves Refolding of the C-terminal alpha-Helical Domain. Journal of Biological Chemistry, 2008. 283(50): p. 34704-34711.
50. Alonso, D.O.V., C. An, and V. Daggett, Simulations of biomolecules: characterization of the early steps in the pH-induced conformational conversion of the hamster, bovine and human forms of the prion protein. Philosophical Transactions of the Royal Society of London Series a-Mathematical Physical and Engineering Sciences, 2002. 360(1795): p. 1165-1178.
51. Alonso, D.O.V., et al., Mapping the early steps in the pH-induced conformational conversion of the prion protein. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(6): p. 2985-2989.
52. Colacino, S., et al., The determinants of stability in the human prion protein: Insights into folding and misfolding from the analysis of the change in the stabilization energy distribution in different conditions. Proteins-Structure Function and Bioinformatics, 2006. 62(3): p. 698-707.
53. DeMarco, M.L. and V. Daggett, Molecular mechanism for low pH triggered misfolding of the human prion protein. Biochemistry, 2007. 46(11): p. 3045-3054.
54. Gu, W., et al., Molecular dynamics simulation of the unfolding of the human prion protein domain under low pH and high temperature conditions. Biophysical Chemistry, 2003. 104(1): p. 79-94.
55. Langella, E., R. Improta, and V. Barone, Checking the pH-induced conformational transition of prion protein by molecular dynamics simulations: Effect of protonation of histidine residues. Biophysical Journal, 2004. 87(6): p. 3623-3632.
56. Langella, E., et al., Assessing the acid-base and conformational properties of histidine residues in human prion protein (125-228) by means of pK(a) calculations and molecular dynamics simulations. Proteins-Structure Function and Bioinformatics, 2006. 64(1): p. 167-177.
57. El-Bastawissy, E., M.H. Knaggs, and I.H. Gilbert, Molecular dynamics simulations of wild-type and point mutation human prion protein at normal and elevated temperature. Journal of Molecular Graphics & Modelling, 2001. 20(2): p. 145-154.
58. Shamsir, M.S. and A.R. Dalby, One gene, two diseases and three conformations: Molecular dynamics simulations of mutants of human prion protein at room temperature and elevated temperatures. Proteins-Structure Function and Bioinformatics, 2005. 59(2): p. 275-290.
59. Barducci, A., et al., Misfolding pathways of the prion protein probed by molecular dynamics Simulations. Biophysical Journal, 2005. 88(2): p. 1334-1343.
60. Zhong, L.H., Exposure of Hydrophobic Core in Human Prion Protein Pathogenic Mutant H187R. Journal of Biomolecular Structure & Dynamics, 2010. 28(3): p. 355-361.
61. van der Kamp, M.W. and V. Daggett, Pathogenic Mutations in the Hydrophobic Core of the Human Prion Protein Can Promote Structural Instability and Misfolding. Journal of Molecular Biology, 2010. In Press, Corrected Proof.
62. Chen, W., M.W. van der Kamp, and V. Daggett, Diverse Effects on the Native β-Sheet of the Human Prion Protein Due to Disease-Associated Mutations. Biochemistry, 2010. 49(45): p. 9874-9881.
63. Bamdad, K. and H. Naderi-Manesh, Contribution of a putative salt bridge and backbone dynamics in the structural instability of human prion protein upon R208H mutation. Biochemical and Biophysical Research Communications, 2007. 364(4): p. 719-724.
64. Yong, Y., et al., Molecular dynamics study on the conformational transition of prion induced by the point mutation: F198S. Thin Solid Films, 2006. 499(1-2): p. 224-228.
65. Santini, S. and P. Derreumaux, Helix H1 of the prion protein is rather stable against environmental perturbations: molecular dynamics of mutation and deletion variants of PrP(90-231). Cellular and Molecular Life Sciences, 2004. 61(7-8): p. 951-960.
66. Sekijima, M., et al., Molecular dynamics simulation of dimeric and monomeric forms of human prion protein: Insight into dynamics and properties. Biophysical Journal, 2003. 85(2): p. 1176-1185.
67. Okimoto, N., et al., Computational studies on prion proteins: Effect of Ala(117) -> Val mutation. Biophysical Journal, 2002. 82(5): p. 2746-2757.
68. Levy, Y. and O.M. Becker, Conformational polymorphism of wild-type and mutant prion proteins: Energy landscape analysis. Proteins-Structure Function and Genetics, 2002. 47(4): p. 458-468.
69. Gsponer, J., P. Ferrara, and A. Caflisch, Flexibility of the murine prion protein and its Asp178Asn mutant investigated by molecular dynamics simulations. Journal of Molecular Graphics & Modelling, 2001. 20(2): p. 169-182.
70. van der Kamp, M.W. and V. Daggett, The consequences of pathogenic mutations to the human prion protein. Protein Engineering Design & Selection, 2009. 22(8): p. 461-468.
71. Daggett, V., alpha-sheet: The toxic conformer in amyloid diseases? Accounts of Chemical Research, 2006. 39(9): p. 594-602.
72. 岳俊杰, 梁., 馮華, 蛋白質結構預測實驗指南. 2010.
73. 孫慶姝, Homology Modeling (Knowledge-Based Structure Modeling ) 蛋白質三級結構預測之同源模擬. 中央研究院生物醫學科學研究所, 2002.
74. Bordoli, L., et al., Protein structure homology modeling using SWISS-MODEL workspace. Nature Protocols, 2009. 4(1): p. 1-13.
75. Guex, N. and M.C. Peitsch, SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis, 1997. 18(15): p. 2714-2723.
76. 李軍, 張., 溫珍昌, 生物軟件選擇與使用指南. 2008.
77. James, T.L., et al., Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform. Proceedings of the National Academy of Sciences of the United States of America, 1997. 94(19): p. 10086-10091.
78. Lindahl, E., B. Hess, and D. van der Spoel, GROMACS 3.0: a package for molecular simulation and trajectory analysis. Journal of Molecular Modeling, 2001. 7(8): p. 306-317.
79. Scott, W.R.P., et al., The GROMOS biomolecular simulation program package. Journal of Physical Chemistry A, 1999. 103(19): p. 3596-3607.
80. H.J.C.Berendsen, J.P.M.P., W.F.van Gunsteren and J. Hermans*, INTERACTION MODELS FOR WATER IN RELATION TO PROTEIN HYDRATION. Intermolecular Forces, 1981. pp. 331-342.
81. Ryckaert, J.P., G. Ciccotti, and H.J.C. Berendsen, NUMERICAL-INTEGRATION OF CARTESIAN EQUATIONS OF MOTION OF A SYSTEM WITH CONSTRAINTS - MOLECULAR-DYNAMICS OF N-ALKANES. Journal of Computational Physics, 1977. 23(3): p. 327-341.
82. Hess, B., et al., LINCS: A linear constraint solver for molecular simulations. Journal of Computational Chemistry, 1997. 18(12): p. 1463-1472.
83. Darden, T., D. York, and L. Pedersen, PARTICLE MESH EWALD - AN N.LOG(N) METHOD FOR EWALD SUMS IN LARGE SYSTEMS. Journal of Chemical Physics, 1993. 98(12): p. 10089-10092.
84. Wolfgang Kabsch, C.S., Dictionary of protein secondary structure: Pattern recognition of hydrogen-bonded and geometrical features. 1983. 22(12): p. 2577-2637.
85. Humphrey, W., A. Dalke, and K. Schulten, VMD: Visual molecular dynamics. Journal of Molecular Graphics, 1996. 14(1): p. 33-&.
86. Watanabe, Y., et al., Identification of pH-sensitive regions in the mouse prion by the cysteine-scanning spin-labeling ESR technique. Biochemical and Biophysical Research Communications, 2006. 350(3): p. 549-556.
87. Zhang, J.P., Comparison studies of the structural stability of rabbit prion protein with human and mouse prion proteins. Journal of Theoretical Biology, 2011. 269(1): p. 88-95.
88. Shamsir, M.S. and A.R. Dalby, beta-Sheet containment by flanking prolines: Molecular dynamic simulations of the inhibition of beta-sheet elongation by proline residues in human prion protein. Biophysical Journal, 2007. 92(6): p. 2080-2089.
89. Blinov, N., et al., Structural Domains and Main-Chain Flexibility in Prion Proteins. Biochemistry, 2009. 48(7): p. 1488-1497.
90. Torrent, J., et al., Modulation of prion protein structure by pressure and temperature. Biochimica Et Biophysica Acta-Proteins and Proteomics, 2006. 1764(3): p. 546-551.
91. Rezaei, H., Prion Protein Oligomerization. Current Alzheimer Research, 2008. 5(6): p. 572-578.
92. Villa, V., et al., Characterization of the proapoptotic intracellular mechanisms induced by a toxic conformer of the recombinant human prion protein fragment 90-231, in Signal Transduction Pathways, Pt a - Apoptotic and Extracellular Signaling, M. Diederich, Editor. 2006, Blackwell Publishing: Oxford. p. 276-291.
93. Palladino, P., et al., Peptide Fragment Approach to Prion Misfolding: The Alpha-2 Domain. International Journal of Peptide Research and Therapeutics, 2009. 15(3): p. 165-176.
94. Adrover, M., et al., Prion Fibrillization Is Mediated by a Native Structural Element That Comprises Helices H2 and H3. Journal of Biological Chemistry, 2010. 285(27): p. 21004-21012.
95. Huang, Z.W., S.B. Prusiner, and F.E. Cohen, Scrapie prions: A three-dimensional model of an infectious fragment. Folding & Design, 1996. 1(1): p. 13-19.
96. Fontaine, S.N. and D.R. Brown, Mechanisms of Prion Protein Aggregation. Protein and Peptide Letters, 2009. 16(1): p. 14-26.
97. Huang, L.Q., et al., Macromolecular crowding converts the human recombinant PrPC to the soluble neurotoxic beta-oligomers. Faseb Journal, 2010. 24(9): p. 3536-3543.