建構人類雙重特異性去磷酸酵素22之C-端截斷突變及其結構與功能探討

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研究生：	張恪誌 Chang, Co-Chih
論文名稱：	建構人類雙重特異性去磷酸酵素22之C-端截斷突變及其結構與功能探討 Construction of C-terminal Truncated Human Dual-Specificity Phosphatase 22 for Structural and Functional Studies
指導教授：	呂平江 Lyu, Ping-Chiang
口試委員:	蘇士哲 Sue, Shih-Che 莊懷佳 Chuang, Huai-Chai
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	89
中文關鍵詞：	雙重特異性去磷酸酵素22
外文關鍵詞：	DUSP22
相關次數：	點閱：2 下載：0
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人類雙重特異性去磷酸酵素22 (human dual-specificity phosphatase 22, DUSP22)可以針對兩類磷酸化 (絲氨酸/蘇胺酸與酪氨酸) 位點進行去磷酸化，並且被發現到可以針對T細胞內Lck之酪氨酸394位點進行去磷酸化，進而調降T細胞的免疫活化反應。在紅斑性狼瘡病人檢體上發現到，病人T細胞中的人類雙重特異性去磷酸酵素22的蛋白表現量明顯下降顯示其有潛力成為診斷紅斑性狼瘡的生物標記。我們想建立人類雙重特異性去磷酸酵素22的核磁共振研究系統來解析出人類雙重特異性去磷酸酵素22的水溶液結構，並且利用實驗室現有的技術研究其催化機制。我們首先使用已被發表之人類雙重特異性去磷酸酵素22截斷突變結晶結構 (tDUSP221-163)1的蛋白來進行1H-15N二維核磁共振圖譜分析，發現到此圖譜還有改進的空間。我們將其未形成結構的區域刪除，建構出更多C-端截斷突變人類雙重特異性去磷酸酵素22 (tDUSP221-155)。此C-端截斷突變人類雙重特異性去磷酸酵素22 (tDUSP221-155) 保留相似的二級結構、三級結構，並且在1H-15N二維核磁共振圖譜有更多的訊號被發現到。並且我們解出C-端截斷突變人類雙重特異性去磷酸酵素22 (tDUSP221-155)之結晶結構，與已發表之結晶結構(tDUSP221-163)近乎相同，更利於我們後續C-端截斷突變人類雙重特異性去磷酸酵素22的水溶液結構之探討
人類雙重特異性去磷酸酵素22帶有序列高度保留之蛋白酪胺酸去磷酸酶迴圈 (PTP loop, CXXXXXRS/T)，我們針對其高度保留之催化位點進行突變研究，使用C-端截斷突變人類雙重特異性去磷酸酵素22 (tDUSP221-155)作為模板建構出C88S、R94K、R94A、D57E、D57A之點突變體。活性測試發現到相對於野生型，這些突變體的活性都有明顯的下降的趨勢，進一步利用等溫滴定量熱儀分析這些突變體與磷酸抑制劑:釩酸的結合能力。實驗結果發現到，D57E與D57A雖然保留與釩酸的親和力但是相較於野生型還是有下降，C88S、R94K與R94A則是都喪失了與釩酸結合的能力。我們在C88S的結晶結構發現到一個磷酸鑲嵌在其活性位，此磷酸可能阻礙的C88S的受質入口並且影響其等溫滴定量熱儀實驗分析。
綜合我們的實驗結果，C-端截斷突變人類雙重特異性去磷酸酵素22 (tDUSP221-155) 保留了類似的結構與活性，其1H-15N二維核磁共振圖譜被發現到有更多的訊號且穩定的維持七天，更利於後續相關水溶液結構的研究。針對人類雙重特異性去磷酸酵素22的活性位突變體研究結果類似於其他研究透徹之去磷酸酶例如蛋白酪胺酸去磷酸酶1B (PTP1B) 的突變研究，暗示著人類雙重特異性去磷酸酵素22的催化機制類似於其他典型之蛋白酪胺酸去磷酸酶。

Human dual-specificity phosphatase 22 (DUSP22) can dephosphorylate both phosphoserine/phosphothreonine and phosphotyrosine residues within one substrate. DUSP22 can directly dephosphorylate Lck-Y394 in T cells to attenuate T-cell receptor activation. An analysis of the T cells isolated from patients with systemic lupus erythematosus (SLE) revealed that the protein level of DUSP22 was decreased, indicating that DUSP22 could serve as a potential biomarker for SLE diagnosis. We established a system to study the nuclear magnetic resonance (NMR) solution structure and the catalysis mechanism of DUSP22. 1H-15N heteronuclear single quantum coherence (HSQC) spectra of different truncated forms of DUSP22 were used to screen the suitable target protein to investigate the solution structure and protein–protein interaction. tDUSP221-155 had structure and activity similar to those of tDUSP221-163; however, the 1H-15N HSQC spectrum of tDUSP221-155 exhibited more peaks and could remain stable for 7 days. We also ascertained the crystal structure of tDUSP221-155, which was almost identical to the known structure of tDUSP22 (protein data bank code: 1WRM). The results revealed that tDUSP221-155 is a suitable material for future NMR studies.
DUSP22 contains the highly conserved protein tyrosine phosphatase loop (PTP loop) as other protein-tyrosine phosphatases. Site-directed mutagenesis was conducted on the highly conserved residues to investigate their roles in catalysis. tDUSP221-155 served as a template to generate the following mutants: tDUSP221-155-C88S, R94K, R94A, D57E, and D57A. The activities of these mutants drastically decreased compared with those of tDUSP221-155 (as WT) during the p-NPP phosphatase activity assay. The binding affinities between these mutants and the phosphatase inhibitor vanadate were determined through isothermal titration calorimetry (ITC). The ITC results indicated that D57E and D57A retained the binding ability to vanadate; however, their affinity decreased. C88S, R94A, and R94K all lost their binding affinity to vanadate. We further observed a phosphate contaminant in the active site of C88S from the crystal structure. We propose that this intrinsic phosphate blocks the substrate entrance of C88S and causes C88S’s substrate binding inability. The mutants of the catalytic residues of DUSP22 exhibited similar characteristics to the mutations of the thoroughly studied PTP1B, suggesting that the catalysis mechanism of DUSP22 is similar to that of these typical protein tyrosine phosphatases.

Abbreviations    1
Chapter 1. Introduction    2
1.1 Dual-specificity phosphatases    2
1.2 DUSP22    3
1.3 The crystal structure of DUSP22    4
1.4 The catalytic mechanism of cysteine-based phosphatase    5
1.5 Aim    6
Chapter 2. Materials and methods    8
2.1 Construction of tDUSP22 and its mutants    8
2.2 Expression and Purification of tDUSP22 and its mutants    9
2.3 Tricine-SDS-PAGE    11
2.4 Quantification of protein concentration by Braford assay    12
2.5 Circular dichroism (CD) spectroscopy    12
2.6 Fluorescence spectroscopy    13
2.7 p-NPP phosphatase activity assay    13
2.8 NMR Data collection    13
2.9 X-ray crystallography    14
2.10 Determine the substrate binding affinity by isothermal titration calorimetry (ITC)    15
2.11 Software tools for protein visualization and analysis    16
Chapter 3. Results and Discussion    18
3.1 Design of tDUSP221-155    18
3.2 Production and characterization of tDUSP221-155    19
3.4 The crystal structure of tDUSP221-155    21
3.5 Design mutants of critical residues in tDUSP22 catalysis    22
3.6 Production and characterization of tDUSP221-155-C88S, R94K, R94A, D57E and D57A    22
3.7 The ITC studies of tDUSP221-155 and its mutants    23
3.8 The crystal structure of tDUSP221-155-C88S, R94K, R94A, D57E and D57A    25
3.9 Discussion    27
Chapter 4. Conclusion    29
References    84

 
List of Tables
Table 1: Oligonucleotide sequence for PCR    32
Table 2: The NMR M9 culture    33
Table 3: Buffer screen for tDUSP221-163    34
Table 4: The molecular weight of tDUSP221-163, tDUSP221-155 and mutants    35
Table 5: Comparison of tDUSP221-163 and tDUSP221-155 by ProtParam tool    36
Table 6: Data collection and refinement statistics for tDUSP221-155 and C88S    37
Table 7: The study of the protein-ligand interaction between vanadate and tDUSP221-155 and its mutants by ITC    38

 
List of Figures
Figure 1: The scheme of T-cell activation regulated by DUSP22.    40
Figure 2: The protein secondary structure and protein sequence of tDUSP221-163 (PDB ID: 1WRM)    41
Figure 3: The first crystal structure of DUSP22 (tDUSP221-163, PDB ID: 1WRM)    42
Figure 4: Illustrating of the active site of DUSP22:    43
Figure 5: Illustrating of the active site of DUSP22-C88S/ E24A/K28A/K30A.    44
Figure 6: The catalysis of cysteine-based phosphatase    45
Figure 7: Construction of pET21b-tDUSP221-155    46
Figure 8: The PCR results of tDUSP221-163, tDUSP221-155 and mutants’ construction    47
Figure 9: The flow charts of protein expression and purification    48
Figure 10: The reaction of p-NPP phosphatase activity assay    49
Figure 11: Protein crystals of tDUSP221-155 and C88S    50
Figure 12: The expression of (A) tDUSP221-163 and (B) tDUSP221-155    51
Figure 13: Purification of tDUSP221-163    52
Figure 14: The 1H-15N HSQC spectrum of 100 µM of tDUSP221-163    53
Figure 15: The buffer screen for tDUSP221-163    54
Figure 16: Purification of tDUSP221-155    55
Figure 17: The CD spectra of tDUSP221-163 and tDUSP221-155    56
Figure 18: The CD Tm curves of tDUSP221-163 and tDUSP221-155    57
Figure 19: Fluorescent spectra of intrinsic tryptophan between tDUSP221-163 and tDUSP221-155.    58
Figure 20: The phosphatase activities of tDUSP221-163 and tDUSP221-155    59
Figure 21: The overliad 1H-15N HSQC spectra of tDUSP221-163 and tDUSP221-155    60
Figure 22: The superimposition of 1H-15N HSQC spectra of tDUSP221-163 and tDUSP221-155    61
Figure 23: The 1H-15N HSQC spectrum stability of tDUSP221-155:    62
Figure 24: The topology and crystal structure of tDUSP221-155    63
Figure 25: The network of hydrogen bonds formed by PTP loop in tDUSP221-155    64
Figure 26: Investigation into the ligand binding site of tDUSP221-163 and tDUSP221-155 by Ligplot+    65
Figure 27: The sequence alignment of PTP1B and DUSP22    66
Figure 28: The superimposition of the catalytic domain between DUSP22 and PTP1B.    67
Figure 29: The expression of tDUSP221-155-C88S, R94K, R94A, D57E and D57A    68
Figure 30: Purification of tDUSP221-155-C88S    69
Figure 31: Purification of tDUSP221-155-R94K    70
Figure 32: Purification of tDUSP221-155-R94A.    71
Figure 33: Purification of tDUSP221-155-D57E    72
Figure 34: Purification of tDUSP221-155-D57A    73
Figure 35: The CD spectra of tDUSP221-155 and mutants    74
Figure 36: Fluorescent spectra of intrinsic tryptophan of tDUSP221-155 and its mutants    75
Figure 37: The phosphatase activities of tDUSP221-155 and mutants    76
Figure 38: The study of the protein-ligand interaction between MES and tDUSP221-155 by ITC    77
Figure 39: The study of the protein-ligand interaction between p-NPP and mutants of tDUSP221-155 by ITC    78
Figure 40: The study of the protein-ligand interaction between vanadate and tDUSP221-155 and mutants by ITC    79
Figure 41: The crystal structure of tDUSP221-155-C88S.    80
Figure 42: Investigation into the ligand binding site of tDUSP221-155 and tDUSP221-155-C88S by Ligplot+    81
Figure 43: Combining the backbone assignment result to our 1H-15N HSQC spectrum of tDUSP221-155    82
Figure 44: The locations of the extra peaks of residues of tDUSP221-155    83


                                

References
1. Yokota, T. et al. Crystal structure of human dual specificity phosphatase, JNK stimulatory phosphatase-1, at 1.5 Å resolution. Proteins: Structure, Function, and Bioinformatics 66, 272-278 (2007).
2. Patterson, Kate I., Brummer, T., O'brien, Philippa M. & Daly, Roger J. Dual-specificity phosphatases: critical regulators with diverse cellular targets. Biochemical Journal 418, 475-489 (2009).
3. Stoker, A.W. Protein tyrosine phosphatases and signalling. Journal of Endocrinology 185, 19-33 (2005).
4. Denu, J.M. & Dixon, J.E. A catalytic mechanism for the dual-specific phosphatases. Proceedings of the National Academy of Sciences of the United States of America 92, 5910-5914 (1995).
5. Ohta, Y. et al. Differential activities, subcellular distribution and tissue expression patterns of three members of Slingshot family phosphatases that dephosphorylate cofilin. Genes to Cells 8, 811-824 (2003).
6. Bessette, D.C., Qiu, D. & Pallen, C.J. PRL PTPs: mediators and markers of cancer progression. Cancer and Metastasis Reviews 27, 231-252 (2008).
7. Zeng, Q. et al. PRL-3 and PRL-1 Promote Cell Migration, Invasion, and Metastasis. Cancer Research 63, 2716-2722 (2003).
8. Yu, Y. et al. Aberrant Splicing of Cyclin-Dependent Kinase–Associated Protein Phosphatase KAP Increases Proliferation and Migration in Glioblastoma. Cancer Research 67, 130-138 (2007).
9. Wishart, M.J. & Dixon, J.E. PTEN and myotubularin phosphatases: from 3-phosphoinositide dephosphorylation to disease. Trends Cell Biol 12, 579-585 (2002).
10. Keyse, S.M. Protein phosphatases and the regulation of mitogen-activated protein kinase signalling. Curr Opin Cell Biol 12, 186-192 (2000).
11. Aoki, N., Aoyama, K., Nagata, M. & Matsuda, T. A Growing Family of Dual Specificity Phosphatases with Low Molecular Masses. The Journal of Biochemistry 130, 133-140 (2001).
12. Alonso, A. et al. Inhibition of T Cell Antigen Receptor Signaling by VHR-related MKPX (VHX), a New Dual Specificity Phosphatase Related to VH1 Related (VHR). Journal of Biological Chemistry 277, 5524-5528 (2002).
13. Aoyama, K., Nagata, M., Oshima, K., Matsuda, T. & Aoki, N. Molecular Cloning and Characterization of a Novel Dual Specificity Phosphatase, LMW-DSP2, That Lacks the Cdc25 Homology Domain. Journal of Biological Chemistry 276, 27575-27583 (2001).
14. Ríos, P., Nunes-Xavier, C.E., Tabernero, L., Köhn, M. & Pulido, R. Dual-Specificity Phosphatases as Molecular Targets for Inhibition in Human Disease. Antioxidants & Redox Signaling 20, 2251-2273 (2013).
15. Shen, Y. et al. Activation of the Jnk signaling pathway by a dual-specificity phosphatase, JSP-1. Proceedings of the National Academy of Sciences 98, 13613-13618 (2001).
16. Chen, A.J. et al. The Dual Specificity JKAP Specifically Activates the c-Jun N-terminal Kinase Pathway. Journal of Biological Chemistry 277, 36592-36601 (2002).
17. Li, J.P. et al. The phosphatase JKAP/DUSP22 inhibits T-cell receptor signalling and autoimmunity by inactivating Lck. Nat Commun 5, 3618 (2014).
18. Filipp, D., Ballek, O. & Manning, J. Lck, Membrane Microdomains, and TCR Triggering Machinery: Defining the New Rules of Engagement. Frontiers in Immunology 3, 155 (2012).
19. Chuang, H.-C. et al. Downregulation of the phosphatase JKAP/DUSP22 in T cells as a potential new biomarker of systemic lupus erythematosus nephritis. Oncotarget 7, 57593-57605 (2016).
20. Ju, A. et al. Scaffold Role of DUSP22 in ASK1-MKK7-JNK Signaling Pathway. PLOS ONE 11, e0164259 (2016).
21. Lountos, G.T., Cherry, S., Tropea, J.E. & Waugh, D.S. Structural analysis of human dual-specificity phosphatase 22 complexed with a phosphotyrosine-like substrate. Acta Crystallographica. Section F, Structural Biology Communications 71, 199-205 (2015).
22. Hobiger, K. & Friedrich, T. Voltage sensitive phosphatases: emerging kinship to protein tyrosine phosphatases from structure-function research. Frontiers in Pharmacology 6, 20 (2015).
23. Mullis, K. et al. Specific Enzymatic Amplification of DNA In Vitro: The Polymerase Chain Reaction. Cold Spring Harbor Symposia on Quantitative Biology 51, 263-273 (1986).
24. Schagger, H. Tricine-SDS-PAGE. Nat. Protocols 1, 16-22 (2006).
25. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254 (1976).
26. Purdie, N. Circular Dichroism and the Conformational Analysis of Biomolecules Edited by Gerald D. Fasman (Brandeis University). Plenum Press: New York. 1996. x + 738 pp. $125.00. ISBN 0-306-45142-5. Journal of the American Chemical Society 118, 12871-12871 (1996).
27. Royer, C.A. Probing Protein Folding and Conformational Transitions with Fluorescence. Chemical Reviews 106, 1769-1784 (2006).
28. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6, 277-293 (1995).
29. Johnson, B.A. Using NMRView to visualize and analyze the NMR spectra of macromolecules. Methods Mol Biol 278, 313-352 (2004).
30. Otwinowski, Z. & Minor, W. [20] Processing of X-ray diffraction data collected in oscillation mode. Methods in Enzymology 276, 307-326 (1997).
31. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D 66, 213-221 (2010).
32. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallographica Section D 66, 486-501 (2010).
33. Lam, W.W. & Siu, S.W. PyMOL mControl: Manipulating molecular visualization with mobile devices. Biochem Mol Biol Educ 45, 76-83 (2017).
34. Wallace, A.C., Laskowski, R.A. & Thornton, J.M. LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng 8, 127-134 (1995).
35. Laskowski, R.A. et al. PDBsum: a Web-based database of summaries and analyses of all PDB structures. Trends Biochem Sci 22, 488-490 (1997).
36. Zhang, Z.-Y. Protein Tyrosine Phosphatases: Structure and Function, Substrate Specificity, and Inhibitor Development. Annual Review of Pharmacology and Toxicology 42, 209-234 (2002).
37. Hoff, R.H., Hengge, A.C., Wu, L., Keng, Y.F. & Zhang, Z.Y. Effects on general acid catalysis from mutations of the invariant tryptophan and arginine residues in the protein tyrosine phosphatase from Yersinia. Biochemistry 39, 46-54 (2000).
38. Hengge, A.C., Sowa, G.A., Wu, L. & Zhang, Z.Y. Nature of the transition state of the protein-tyrosine phosphatase-catalyzed reaction. Biochemistry 34, 13982-13987 (1995).
39. Zhang, Y.-L. et al. Thermodynamic Study of Ligand Binding to Protein-tyrosine Phosphatase 1B and Its Substrate-trapping Mutants. Journal of Biological Chemistry 275, 34205-34212 (2000).
40. Hoff, R.H., Hengge, A.C., Wu, L., Keng, Y.-F. & Zhang, Z.-Y. Effects on General Acid Catalysis from Mutations of the Invariant Tryptophan and Arginine Residues in the Protein Tyrosine Phosphatase from Yersinia. Biochemistry 39, 46-54 (2000).
41. Flint, A.J., Tiganis, T., Barford, D. & Tonks, N.K. Development of “substrate-trapping” mutants to identify physiological substrates of protein tyrosine phosphatases. Proceedings of the National Academy of Sciences of the United States of America 94, 1680-1685 (1997).
42. Wu, L., Buist, A., den Hertog, J. & Zhang, Z.-Y. Comparative Kinetic Analysis and Substrate Specificity of the Tandem Catalytic Domains of the Receptor-like Protein-tyrosine Phosphatase α. Journal of Biological Chemistry 272, 6994-7002 (1997).
43. Denu, J.M., Lohse, D.L., Vijayalakshmi, J., Saper, M.A. & Dixon, J.E. Visualization of intermediate and transition-state structures in protein-tyrosine phosphatase catalysis. Proceedings of the National Academy of Sciences of the United States of America 93, 2493-2498 (1996).

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