Inclusion body myopathy associated with Paget’s disease of bone and frontotemporal dementia, also known as IBMPFD, is a hereditary, autosomal dominant and ultimately lethal multi-system degenerative disorder due to single missense mutations in VCP (Valosin-containing Protein). TER94/VCP is an evolutionary highly conserved AAA-ATPase (ATPases Associated with diverse cellular Activities), which is involved in a great variety of cellular mechanisms such as protein degradation, ERAD (Endoplasmic Reticulum (ER)-associated Protein Degradation), membrane fusion, cell cycle control, transcriptional control, and DNA repair. The mechanisms of how mutations of VCP lead to IBMPFD remain mysterious. Here we utilize a powerful technique, two-dimensional difference gel electrophoresis (2D-DIGE), that when combined with mass spectrometry, can be applied to study IBMPFD disorder at the protein level. With this set up, we are able to employ comparative proteomics to analyze IBMPFD disease using Drosophila melanogaster as our disease model organism. Head proteome of transgenic D. melanogaster expressing TER94/VCP-WT is respectively compared with the head proteome of transgenic D. melanogaster expressing TER94/VCP mutants that correspond to human IBMPFD disease alleles (TER94R152H, TER94R188Q, TER94A229E). Of all the proteins identified, a significant fraction of proteins altered in TER94A229E and TER94R188Q mutants belong to the same functional categories, i.e. eye formation, apoptosis and metabolism. Transferrin, a blood plasma protein for iron ion delivery, is observed to be significantly up-regulated in mutant flies expressing TER94A229E. Knock-down experiment proves transferrin to be a potential key player in IBMPFD disease. The molecular analysis of IBMPFD disease greatly benefit from a proteomics approach that combines the advantages of high throughput analysis and the focus on protein levels.

IBMPFD 是一種顯性遺傳，因為VCP的點突變所導致的多重器官退化性疾病。TER94/VCP是一種在演化上具有高度保留的AAA-ATPase。它參與了許多細胞機制，例如：蛋白質降解、內質網相關蛋白質的降解(ERAD) 、膜融合、調控細胞週期、轉錄調控以及DNA修補。然而VCP的突變是如何導致IBMPFD的機轉目前仍是神秘未知的。因此我們利用了2D-DIGE及質譜儀的技術去比較IBMPFD這個疾病在蛋白質體表現上的差異。本研究我們利用了果蠅當作這個疾病的模式生物去比較分析IBMPFD這個疾病的蛋白質表現。將正常表現 ter94基因的果蠅與其他三種ter94突變的果蠅 (TER94R152H, TER94R188Q, TER94A229E) 比較，結果顯示他們共同有眼睛形成蛋白、apoptosis 和代謝相關蛋白表現量的差異。其中，運鐵蛋白 (Transferrin) 在突變果蠅TER94A229E中明顯具有高度表現。因此我們利用了 Knock-down 果蠅運鐵蛋白的實驗中發現它在IBMPFD這個疾病中扮演一個重要的角色。IBMPFD這個疾病經過2D-DIGE及質譜儀的技術分析後對於它蛋白質的表現以及修飾得到非常大的瞭解及助益。

1. V. E. Kimonis, E. Fulchiero, J. Vesa, G. Watts, Biochim Biophys Acta 1782, 744 (Dec, 2008).
2. V. E. Kimonis et al., Am J Med Genet A 146A, 745 (Mar 15, 2008).
3. G. D. Watts et al., Nat Genet 36, 377 (Apr, 2004).
4. C. C. Weihl, A. Pestronk, V. E. Kimonis, Neuromuscul Disord 19, 308 (May, 2009).
5. M. J. Kovach et al., Mol Genet Metab 74, 458 (Dec, 2001).
6. C. U. Hubbers et al., Brain 130, 381 (Feb, 2007).
7. M. H. Helfrich, L. J. Hocking, Arch Biochem Biophys 473, 172 (May 15, 2008).
8. S. H. Ralston, Bone 43, 819 (Nov, 2008).
9. J. B. Guinto, G. P. Ritson, J. P. Taylor, M. S. Forman, Acta Neuropathol 114, 55 (Jul, 2007).
10. R. M. Liscic, Arh Hig Rada Toksikol 60, 117 (Mar, 2009).
11. I. T. Pleasure, M. M. Black, et al Nature 365(6445), 459 (1993).
12. M. Egerton et al., EMBO J 11, 3533 (Oct, 1992).
13. S. Jentsch, S. Rumpf, Trends Biochem Sci 32, 6 (Jan, 2007).
14. C. Mori-Konya et al., Genes Cells 14, 483 (Apr, 2009).
15. T. Huyton et al., J Struct Biol 144, 337 (Dec, 2003).
16. Q. Wang, C. Song, X. Yang, C. C. Li, J Biol Chem 278, 32784 (Aug 29, 2003).
17. Q. Wang, C. Song, C. C. Li, J Struct Biol 146, 44 (Apr-May, 2004).
18. F. Beuron et al., EMBO J 25, 1967 (May 3, 2006).
19. R. J. Braun, H. Zischka, Biochim Biophys Acta 1783, 1418 (Jul, 2008).
20. V. E. Pye et al., Proc Natl Acad Sci U S A 104, 467 (Jan 9, 2007).
21. N. Vij, J Cell Mol Med 12, 2511 (Dec, 2008).
22. I. Dreveny et al., EMBO J 23, 1030 (Mar 10, 2004).
23. C. C. Weihl, S. Dalal, A. Pestronk, P. I. Hanson, Hum Mol Genet 15, 189 (Jan 15, 2006).
24. F. Madeo, E. Frohlich, K. U. Frohlich, J Cell Biol 139, 729 (Nov 3, 1997).
25. F. Madeo, J. Schlauer, K. U. Frohlich, Gene 204, 145 (Dec 19, 1997).
26. B. DeLaBarre, A. T. Brunger, Nat Struct Biol 10, 856 (Oct, 2003).
27. K. U. Frohlich et al., J Cell Biol 114, 443 (Aug, 1991).
28. V. Pamnani et al., FEBS Lett 404, 263 (Mar 10, 1997).
29. K. J. Venken, H. J. Bellen, Development 134, 3571 (Oct, 2007).
30. K. J. Venken, H. J. Bellen, Nat Rev Genet 6, 167 (Mar, 2005).
31. M. D. Adams et al., Science 287, 2185 (Mar 24, 2000).
32. M. Mutsuddi, J. R. Nambu, Curr Biol 8, R809 (Nov 5, 1998).
33. A. M. Celotto, M. J. Palladino, Mol Interv 5, 292 (Oct, 2005).
34. A. H. Brand, N. Perrimon, Development 118, 401 (Jun, 1993).
35. J. A. Fischer, E. Giniger, T. Maniatis, M. Ptashne, Nature 332, 853 (Apr 28, 1988).
36. J. B. Duffy, Genesis 34, 1 (Sep-Oct, 2002).
37. R. Aebersold, M. Mann, Nature 422, 198 (Mar 13, 2003).
38. J. F. Timms, R. Cramer, Proteomics 8, 4886 (Dec, 2008).
39. H. L. Chan et al., FEBS Lett 580, 3229 (May 29, 2006).
40. R. Marouga, S. David, E. Hawkins, Anal Bioanal Chem 382, 669 (Jun, 2005).
41. J. Minden, Biotechniques 43, 739 (Dec, 2007).
42. R. Westermeier, B. Scheibe, Methods Mol Biol 424, 73 (2008).
43. A. Gorg, W. Weiss, M. J. Dunn, Proteomics 4, 3665 (Dec, 2004).
44. J. D. Dunn, G. E. Reid, M. L. Bruening, Mass Spectrom Rev 29, 29 (Jan-Feb, 2010).
45. T. H. Kang et al., Proteomics 7, 2624 (Aug, 2007).
46. C. L. Maarouf et al., Curr Alzheimer Res 6, 399 (Aug, 2009).
47. L. C. Lee et al., Clin Chim Acta 400, 56 (Feb, 2009).
48. P. G. Woodman, J Cell Sci 116, 4283 (Nov 1, 2003).
49. V. E. Pye et al., J Struct Biol 156, 12 (Oct, 2006).
50. M. Hirabayashi et al., Cell Death Differ 8, 977 (Oct, 2001).
51. Y. Mizuno, S. Hori, A. Kakizuka, K. Okamoto, Neurosci Lett 343, 77 (Jun 5, 2003).
52. C. Ericsson, Methods Mol Biol 112, 35 (1999).
53. H. N. Shah, C. J. Keys, O. Schmid, S. E. Gharbia, Clin Infect Dis 35, S58 (Sep 1, 2002).
54. H. L. Chan et al., Proteomics 5, 2908 (Jul, 2005).
55. S. Buckingham, Nature 425, 213 (Sep 11, 2003).
56. S. P. Gygi, Y. Rochon, B. R. Franza, R. Aebersold, Mol Cell Biol 19, 1720 (Mar, 1999).
57. J.-F. H. a. M. E. W. Jenny Renauta, Physiologia Plantarum 126, 97 (2006).
58. A. Q. Li, A. Popova-Butler, D. H. Dean, D. L. Denlinger, J Insect Physiol 53, 385 (Apr, 2007).
59. J. R. Lamb, V. Fu, E. Wirtz, J. D. Bangs, J Biol Chem 276, 21512 (Jun 15, 2001).
60. S. Imamura, N. Ojima, M. Yamashita, FEBS Lett 549, 14 (Aug 14, 2003).
61. J. M. Muller, K. Deinhardt, I. Rosewell, G. Warren, D. T. Shima, Biochem Biophys Res Commun 354, 459 (Mar 9, 2007).
62. F. Madeo et al., J Cell Biol 145, 757 (May 17, 1999).
63. A. Markkanen et al., Bioelectromagnetics 25, 127 (Feb, 2004).
64. T. Shirogane et al., Immunity 11, 709 (Dec, 1999).
65. M. Ueffing, THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, 25757 (2006).
66. C. W. Gourlay, K. R. Ayscough, Biochem Soc Trans 33, 1260 (Dec, 2005).
67. C. W. Gourlay, K. R. Ayscough, FEMS Yeast Res 5, 1193 (Dec, 2005).
68. C. W. Gourlay, K. R. Ayscough, Nat Rev Mol Cell Biol 6, 583 (Jul, 2005).
69. C. W. Gourlay, K. R. Ayscough, J Cell Sci 118, 2119 (May 15, 2005).
70. C. W. Gourlay, L. N. Carpp, P. Timpson, S. J. Winder, K. R. Ayscough, J Cell Biol 164, 803 (Mar 15, 2004).
71. M. Breitenbach, P. Laun, M. Gimona, Trends Cell Biol 15, 637 (Dec, 2005).
72. T. Eisenberg, S. Buttner, G. Kroemer, F. Madeo, Apoptosis 12, 1011 (May, 2007).
73. J. R. Bamburg, O. P. Wiggan, Trends Cell Biol 12, 598 (Dec, 2002).
74. B. W. Bernstein, J. R. Bamburg, J Neurosci 23, 1 (Jan 1, 2003).
75. S. J. Atkinson, M. A. Hosford, B. A. Molitoris, J Biol Chem 279, 5194 (Feb 13, 2004).
76. D. Halawani et al., Mol Cell Biol 29, 4484 (Aug, 2009).
77. X. Li et al., Mol Cell Biol 29, 6106 (Nov, 2009).
78. I. Dreveny et al., Biochem Soc Trans 32, 715 (Nov, 2004).
79. D. Halawani, M. Latterich, Mol Cell 22, 713 (Jun 23, 2006).
80. A. T. Brunger, B. DeLaBarre, FEBS Lett 555, 126 (Nov 27, 2003).
81. K. Uchiyama, H. Kondo, J Biochem 137, 115 (Feb, 2005).
82. M. S. Forman et al., J Neuropathol Exp Neurol 65, 571 (Jun, 2006).
83. L. Guyant-Marechal et al., Neurology 67, 644 (Aug 22, 2006).
84. D. Haubenberger et al., Neurology 65, 1304 (Oct 25, 2005).
85. S. Krause et al., Clin Neuropathol 26, 232 (Sep-Oct, 2007).
86. T. Kobayashi, K. Tanaka, K. Inoue, A. Kakizuka, J Biol Chem 277, 47358 (Dec 6, 2002).
87. Y. Ye, J Struct Biol 156, 29 (Oct, 2006).