研究生: |
林欣儀 Lin, Hsin-Yi |
---|---|
論文名稱: |
結合化學修飾與質譜分析方法鑑定受特定去乙醯酶作用之乙醯化受質蛋白體 Mass Spectrometry Method for Analysis of Deacetylase-specific Acetylproteome |
指導教授: |
陳玉如
Chen, Yu-Ju |
學位類別: |
碩士 Master |
系所名稱: |
化學系 Department of Chemistry |
論文出版年: | 2017 |
畢業學年度: | 105 |
語文別: | 英文 |
論文頁數: | 78 |
中文關鍵詞: | 化學修飾 、質譜 、去乙醯酶 、乙醯化受質蛋白體 |
英文關鍵詞: | Lysine acetylation, HDAC1, Acetylproteome, Deacetylase-specific acerylproteome |
DOI URL: | https://doi.org/10.6345/NTNU202202244 |
論文種類: | 學術論文 |
相關次數: | 點閱:107 下載:0 |
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離胺酸上的乙醯化(acetylation)是一種可逆的蛋白質後轉譯修飾,藉由乙醯基酶(lysine acetylase)加上乙醯基和去乙醯酶(lysine deacetylases)移除乙醯基所調控。蛋白質上的乙醯化在基因的轉譯上扮演著很重要的角色,包括去氧核醣核酸和蛋白質間的相互作用,蛋白質的穩定性,蛋白質的位置和轉譯的活性等。因此,了解細胞中不同去乙醯酶所特別調控的受質蛋白及修飾位點尤其重要。
我們開發了一個利用化學修飾的方法,分別選用SIRT1和HDAC1兩種去乙醯酶作為此方法開發的模型,此方法結合化學標定以保護離胺酸之一級胺反應、去乙醯基反應,並將去乙醯的位點接上生物素,進而利用生物素和抗生物素蛋白的高親和作用力進行專一性純化,再利用質譜分析以鑑定去乙醯酶之受質蛋白及乙醯化的特定位點。為了驗證新方法的每一實驗步驟,利用兩段合成的胜肽,H3K4(SIRT1受質)和H3K27(HDAC1受質)進行每一步驟的確認,並優化每步驟的條件。再者,為了驗證此方法在複雜樣品的可行性,分別將H3K4和H3K27胜肽混進BSA蛋白質中進行驗證和鑑定,成功地在質譜中鑑定到H3K4和H3K27這兩條胜肽及其修飾位點,結果顯示純化H3K27的專一性為63.8%。我們將此方法應用於癌細胞(Hela cell),希望能分析被特定去乙醯酶作用受體。為了增加鑑定到受質位點的機會,我們改良第一步驟化學標定以保護離胺酸的副產物,利用磺基-N-羥基琥珀酰亞胺-乙酸酯(sulfo-NHS-acetate)作為保護離胺酸的試劑,經過了條件優化後,成功地鑑定到H3K4和H3K27兩段胜肽並鑑定到812個被HDAC1去乙醯酶作用受質蛋白,其中包含1870條受質胜肽,其中40條受質胜肽為前人研究已經報導的受質蛋白。目前,我們尚未成功地鑑定到被SIRT1去乙醯酶作用受質胜肽,希望未來能找出原因並且解決問題。
Lysine acetylation is a reversible posttranslational modification (PTM) of proteins regulated by lysine acetylase (KATs) and lysine deacetylases (KDACs). Protein acetylation plays a key role in regulation of gene expression involves in many biological process, such as protein interaction, activity, and localization. The knowledge for substrate specificity of KDACs is essential for understanding the role of an individual KDAC in a particular cellular process.
In this study, we attempted to develop a chemical modification method, combining lysine blocking, deacetylation, and tagging by biotylation, combined with mass spectrometry analysis for delineating deacetylase-specific acerylproteome. Two synthetic standard peptides H3K4 (SIRT1 substrate) and H3K27 (HDAC1 substrate) was used to verify our workflow. First, the N-terminal amino acids and unmodified lysines (ε-nitrogen of lysine) will be chemically derivatized by adding propionic anhydride. Substrate sites were deacetylated by specific deacetylase, SIRT1 and HDAC1. Here, the reaction time and temperature for deacetylation reaction were optimized. Deacetylated lysine was biotinylated by sulfo-NHS-S-S-Biotin for enrichment of labeled peptides by strepavidin beads. To release the peptides from beads, reduction and alkylation reaction were performed by adding DTT and IAM. The identification of the modified sites was achieved by tandem mass spectrometry. In the preliminary result, the feasibility of this method was demonstrated by identification of H3K4 and H3K27, which were spiked into BSA and the specificity of enrichment efficiency is 63.8%. For reducing side-products which produced from propionylation, we substituted propionic anhydride to sulfo-NHS-acetate for lysine blocking and successfully verified new workflow by spiking H3K4 and H3K27 for identification. We identified 812 substrate proteins including 1870 HDAC1 substrate peptides and 40 substrate-peptides from known substrate proteins in literature. However, we did not identify any SIRT1 substrate proteins yet, in the future, we will further modified the identified substrate and their acetylation sites.
REFERENCE
1. Pickart, C. M., Mechanisms underlying ubiquitination. Annu Rev Biochem 2001, 70, 503-33.
2. Peng, J.; Schwartz, D.; Elias, J. E.; Thoreen, C. C.; Cheng, D.; Marsischky, G.; Roelofs, J.; Finley, D.; Gygi, S. P., A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 2003, 21 (8), 921-6.
3. Martin, C.; Zhang, Y., The diverse functions of histone lysine methylation. Nat Rev Mol Cell Biol 2005, 6 (11), 838-49.
4. Lachner, M.; Jenuwein, T., The many faces of histone lysine methylation. Curr Opin Cell Biol 2002, 14 (3), 286-98.
5. Hu, L. I.; Lima, B. P.; Wolfe, A. J., Bacterial protein acetylation: the dawning of a new age. Mol Microbiol 2010, 77 (1), 15-21.
6. Wang, Q.; Zhang, Y.; Yang, C.; Xiong, H.; Lin, Y.; Yao, J.; Li, H.; Xie, L.; Zhao, W.; Yao, Y.; Ning, Z. B.; Zeng, R.; Xiong, Y.; Guan, K. L.; Zhao, S.; Zhao, G. P., Acetylation of metabolic enzymes coordinates carbon source utilization and metabolic flux. Science 2010, 327 (5968), 1004-7.
7. Phillips, D. M., The presence of acetyl groups of histones. Biochem J 1963, 87, 258-63.
8. Allfrey, V. G.; Faulkner, R.; Mirsky, A. E., Acetylation and Methylation of Histones and Their Possible Role in the Regulation of Rna Synthesis. Proc Natl Acad Sci U S A 1964, 51, 786-94.
9. Vidali, G.; Gershey, E. L.; Allfrey, V. G., Chemical studies of histone acetylation. The distribution of epsilon-N-acetyllysine in calf thymus histones. J Biol Chem 1968, 243 (24), 6361-6.
10. Yang, X. J.; Seto, E., HATs and HDACs: from structure, function and regulation to novel strategies for therapy and prevention. Oncogene 2007, 26 (37), 5310-8.
11. Gu, W.; Roeder, R. G., Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 1997, 90 (4), 595-606.
12. Anderson, K. A.; Hirschey, M. D., Mitochondrial protein acetylation regulates metabolism. Essays Biochem 2012, 52, 23-35.
13. Glozak, M. A.; Sengupta, N.; Zhang, X.; Seto, E., Acetylation and deacetylation of non-histone proteins. Gene 2005, 363, 15-23.
14. Brownell, J. E.; Allis, C. D., Special HATs for special occasions: linking histone acetylation to chromatin assembly and gene activation. Curr Opin Genet Dev 1996, 6 (2), 176-84.
15. Brown, C. E.; Lechner, T.; Howe, L.; Workman, J. L., The many HATs of transcription coactivators. Trends Biochem Sci 2000, 25 (1), 15-9.
16. Kalkhoven, E., CBP and p300: HATs for different occasions. Biochem Pharmacol 2004, 68 (6), 1145-55.
17. Blander, G.; Guarente, L., The Sir2 family of protein deacetylases. Annu Rev Biochem 2004, 73, 417-35.
18. Kouzarides, T., Acetylation: a regulatory modification to rival phosphorylation? EMBO J 2000, 19 (6), 1176-9.
19. Bordone, L.; Guarente, L., Calorie restriction, SIRT1 and metabolism: understanding longevity. Nat Rev Mol Cell Biol 2005, 6 (4), 298-305.
20. Brunet, A.; Sweeney, L. B.; Sturgill, J. F.; Chua, K. F.; Greer, P. L.; Lin, Y.; Tran, H.; Ross, S. E.; Mostoslavsky, R.; Cohen, H. Y.; Hu, L. S.; Cheng, H. L.; Jedrychowski, M. P.; Gygi, S. P.; Sinclair, D. A.; Alt, F. W.; Greenberg, M. E., Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 2004, 303 (5666), 2011-5.
21. Kim, S. C.; Sprung, R.; Chen, Y.; Xu, Y.; Ball, H.; Pei, J.; Cheng, T.; Kho, Y.; Xiao, H.; Xiao, L.; Grishin, N. V.; White, M.; Yang, X. J.; Zhao, Y., Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol Cell 2006, 23 (4), 607-18.
22. Thiagalingam, S.; Cheng, K. H.; Lee, H. J.; Mineva, N.; Thiagalingam, A.; Ponte, J. F., Histone deacetylases: unique players in shaping the epigenetic histone code. Ann N Y Acad Sci 2003, 983, 84-100.
23. Dokmanovic, M.; Clarke, C.; Marks, P. A., Histone deacetylase inhibitors: overview and perspectives. Mol Cancer Res 2007, 5 (10), 981-9.
24. Giannini, G.; Cabri, W.; Fattorusso, C.; Rodriquez, M., Histone deacetylase inhibitors in the treatment of cancer: overview and perspectives. Future Med Chem 2012, 4 (11), 1439-60.
25. Marks, P. A.; Miller, T.; Richon, V. M., Histone deacetylases. Curr Opin Pharmacol 2003, 3 (4), 344-51.
26. Hassig, C. A.; Tong, J. K.; Fleischer, T. C.; Owa, T.; Grable, P. G.; Ayer, D. E.; Schreiber, S. L., A role for histone deacetylase activity in HDAC1-mediated transcriptional repression. Proc Natl Acad Sci U S A 1998, 95 (7), 3519-24.
27. Grozinger, C. M.; Chao, E. D.; Blackwell, H. E.; Moazed, D.; Schreiber, S. L., Identification of a class of small molecule inhibitors of the sirtuin family of NAD-dependent deacetylases by phenotypic screening. J Biol Chem 2001, 276 (42), 38837-43.
28. Yoon, S.; Eom, G. H., HDAC and HDAC Inhibitor: From Cancer to Cardiovascular Diseases. Chonnam Med J 2016, 52 (1), 1-11.
29. Mattson, M. P.; Duan, W.; Guo, Z., Meal size and frequency affect neuronal plasticity and vulnerability to disease: cellular and molecular mechanisms. J Neurochem 2003, 84 (3), 417-31.
30. Wilson, A. J.; Byun, D. S.; Popova, N.; Murray, L. B.; L'Italien, K.; Sowa, Y.; Arango, D.; Velcich, A.; Augenlicht, L. H.; Mariadason, J. M., Histone deacetylase 3 (HDAC3) and other class I HDACs regulate colon cell maturation and p21 expression and are deregulated in human colon cancer. J Biol Chem 2006, 281 (19), 13548-58.
31. Zhang, Z.; Yamashita, H.; Toyama, T.; Sugiura, H.; Omoto, Y.; Ando, Y.; Mita, K.; Hamaguchi, M.; Hayashi, S.; Iwase, H., HDAC6 expression is correlated with better survival in breast cancer. Clin Cancer Res 2004, 10 (20), 6962-8.
32. Kook, H.; Lepore, J. J.; Gitler, A. D.; Lu, M. M.; Wing-Man Yung, W.; Mackay, J.; Zhou, R.; Ferrari, V.; Gruber, P.; Epstein, J. A., Cardiac hypertrophy and histone deacetylase-dependent transcriptional repression mediated by the atypical homeodomain protein Hop. J Clin Invest 2003, 112 (6), 863-71.
33. Montgomery, R. L.; Davis, C. A.; Potthoff, M. J.; Haberland, M.; Fielitz, J.; Qi, X.; Hill, J. A.; Richardson, J. A.; Olson, E. N., Histone deacetylases 1 and 2 redundantly regulate cardiac morphogenesis, growth, and contractility. Genes Dev 2007, 21 (14), 1790-802.
34. Elsakkar, M. G.; Eissa, M. M.; Hewedy, W. A.; Nassra, R. M.; Elatrebi, S. F., Sodium valproate, a histone deacetylase inhibitor, with praziquantel ameliorates Schistosoma mansoni-induced liver fibrosis in mice. Life Sci 2016, 162, 95-101.
35. Eom, G. H.; Cho, Y. K.; Ko, J. H.; Shin, S.; Choe, N.; Kim, Y.; Joung, H.; Kim, H. S.; Nam, K. I.; Kee, H. J.; Kook, H., Casein kinase-2alpha1 induces hypertrophic response by phosphorylation of histone deacetylase 2 S394 and its activation in the heart. Circulation 2011, 123 (21), 2392-403.
36. Kee, H. J.; Sohn, I. S.; Nam, K. I.; Park, J. E.; Qian, Y. R.; Yin, Z.; Ahn, Y.; Jeong, M. H.; Bang, Y. J.; Kim, N.; Kim, J. K.; Kim, K. K.; Epstein, J. A.; Kook, H., Inhibition of histone deacetylation blocks cardiac hypertrophy induced by angiotensin II infusion and aortic banding. Circulation 2006, 113 (1), 51-9.
37. Lu, X.; Wu, S.; Blackwell, C. E.; Humphreys, R. E.; von Hofe, E.; Xu, M., Suppression of major histocompatibility complex class II-associated invariant chain enhances the potency of an HIV gp120 DNA vaccine. Immunology 2007, 120 (2), 207-16.
38. Vega, R. B.; Harrison, B. C.; Meadows, E.; Roberts, C. R.; Papst, P. J.; Olson, E. N.; McKinsey, T. A., Protein kinases C and D mediate agonist-dependent cardiac hypertrophy through nuclear export of histone deacetylase 5. Mol Cell Biol 2004, 24 (19), 8374-85.
39. Singh, B. N.; Zhang, G.; Hwa, Y. L.; Li, J.; Dowdy, S. C.; Jiang, S. W., Nonhistone protein acetylation as cancer therapy targets. Expert Rev Anticancer Ther 2010, 10 (6), 935-54.
40. Sadoul, K.; Wang, J.; Diagouraga, B.; Khochbin, S., The tale of protein lysine acetylation in the cytoplasm. J Biomed Biotechnol 2011, 2011, 970382.
41. Choudhary, C.; Kumar, C.; Gnad, F.; Nielsen, M. L.; Rehman, M.; Walther, T. C.; Olsen, J. V.; Mann, M., Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 2009, 325 (5942), 834-40.
42. Imai, S.; Armstrong, C. M.; Kaeberlein, M.; Guarente, L., Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 2000, 403 (6771), 795-800.
43. Bheda, P.; Swatkoski, S.; Fiedler, K. L.; Boeke, J. D.; Cotter, R. J.; Wolberger, C., Biotinylation of lysine method identifies acetylated histone H3 lysine 79 in Saccharomyces cerevisiae as a substrate for Sir2. Proc Natl Acad Sci U S A 2012, 109 (16), E916-25.
44. Luo, J.; Nikolaev, A. Y.; Imai, S.; Chen, D.; Su, F.; Shiloh, A.; Guarente, L.; Gu, W., Negative control of p53 by Sir2alpha promotes cell survival under stress. Cell 2001, 107 (2), 137-48.
45. Garcia, B. A.; Mollah, S.; Ueberheide, B. M.; Busby, S. A.; Muratore, T. L.; Shabanowitz, J.; Hunt, D. F., Chemical derivatization of histones for facilitated analysis by mass spectrometry. Nat Protoc 2007, 2 (4), 933-8.
46. Roberts, J. D., Determination of Organic Structures by Physical Methods. 1955.
47. Zenkevich, Acids: Derivatization for GC Analysis Encyclopedia of Chromatography. 2009.
48. Liao, R.; Wu, H.; Deng, H.; Yu, Y.; Hu, M.; Zhai, H.; Yang, P.; Zhou, S.; Yi, W., Specific and efficient N-propionylation of histones with propionic acid N-hydroxysuccinimide ester for histone marks characterization by LC-MS. Anal Chem 2013, 85 (4), 2253-9.
49. Cheng, H. L.; Mostoslavsky, R.; Saito, S.; Manis, J. P.; Gu, Y.; Patel, P.; Bronson, R.; Appella, E.; Alt, F. W.; Chua, K. F., Developmental defects and p53 hyperacetylation in Sir2 homolog (SIRT1)-deficient mice. Proc Natl Acad Sci U S A 2003, 100 (19), 10794-9.
50. Anderson, R. M.; Bitterman, K. J.; Wood, J. G.; Medvedik, O.; Sinclair, D. A., Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae. Nature 2003, 423 (6936), 181-5.
51. Li, T.; Song, B.; Wu, Z.; Lu, M.; Zhu, W. G., Systematic identification of Class I HDAC substrates. Brief Bioinform 2014, 15 (6), 963-72.
52. Guo, X.; Kesimer, M.; Tolun, G.; Zheng, X.; Xu, Q.; Lu, J.; Sheehan, J. K.; Griffith, J. D.; Li, X., The NAD(+)-dependent protein deacetylase activity of SIRT1 is regulated by its oligomeric status. Sci Rep 2012, 2, 640.
53. Anderson, G. W., Tonsillectomy and poliomyelitis. JAMA 1963, 184, 80.
54. Wang, L.; Du, Y.; Lu, M.; Li, T., ASEB: a web server for KAT-specific acetylation site prediction. Nucleic Acids Res 2012, 40 (Web Server issue), W376-9.