雙特異性磷酸酶26 (DUSP26) 被歸類在基於半胱胺酸進行催化的蛋白質酪胺酸磷酸酶 (Cys-based PTPs) 家族。除了磷酸化酪胺酸，DUSP26還可以對磷酸化的絲胺酸和蘇胺酸進行去磷酸化。根據研究， DUSP26在不同的N端截短型態時，各有不同的蛋白質結構和活性。我們首先比較DUSP26-N (殘基39-211) 和DUSP26-C (殘基 61-211) 這兩種不同截短型態的活性 (藉由pNPP試驗)。結果顯示DUSP26-N具有磷酸酶的活性而DUSP26-C不具此活性。
在我們實驗室先前的研究中，我們發現到DUSPs家族裡具有一種重要的氫鍵作用網絡 (DPN-三環交互作用)，由三個保守的殘基組成，分別是D環中的天門冬胺酸，P環中的絲胺酸和N環中的天門冬醯胺來穩定活化位構造。結構上的分析揭示了在無活性的DUSP26-C (PDB ID: 4HRF) 中缺少了DPN-三環交互作用，而有活性的DUSP26-N (PDB ID: 5GTJ) 則維持了此交互作用。為了進一步研究DPN-三環交互作用，我們建構了關鍵殘基的丙胺酸取代突變體，分別為DUSP26-N S157A、DUSP26-N N191A，並對其進行活性測試。活性試驗結果顯示DUSP26-N S157A和DUSP26-N N191A的催化效率 (Kcat/KM) 分別比DUSP26-N WT下降了200倍和36倍。接著以圓二色旋光儀 (CD) 分析結果顯示，突變體與野生型在二級結構上並無明顯差異。已知的DUSP26-C (PDB ID: 4HRF) 結構是同源二聚體並且具有域交換 (domain swapping)，而DPN-三環交互作用中的重要殘基N191坐落於交換的α8螺旋。為了驗證這個結構，我們以 1.78 Å 的解析度確定了 DUSP26-C 的晶體結構。從我們解出的DUSP26-C晶體結構中也觀察到了域交換及喪失了DPN-三環交互作用，這個結果也再一次證明了DPN-三環交互作用對於維持蛋白活性的重要性。雖然在本篇研究中沒有得到DUSP26-N和兩個突變體的晶體，但我們使用電腦模擬的結構來幫助我們探討當DPN-三環交互作用力被破壞時，結構上可能產生的變化。從DUSP26-N N191A的結構模型中我們觀察到了D-loop的外翻，使催化的殘基D120離開了催化位點。綜合這些數據，我們提出DPN-三環交互作用的作用是將催化的天門冬胺酸錨定在催化位點來維持磷酸酶的活性。
除了上述的實驗，我們也對DUSPs的別位抑制劑 (allosteric inhibitor) 的發展有興趣，藉由DrugCentral數據庫的藥物資料，我們進行了針對DUSP26-N同源二聚體的分子對接篩選。分子對接的結果顯示大多數藥物都只結合在DUSP26-N二聚體的其中一條蛋白鏈，這是因為DUSP26-N二聚體的兩條蛋白鏈在藥物結合位點的結構並不完全相同。未來這些藥物還需要對DUSP26-N和其他的DUSPs成員分別進行活性試驗來確認其抑制效果及專一性。

Dual-specificity phosphatase 26 (DUSP26) is a member of the cysteine-based protein tyrosine phosphatases (Cys-based PTPs) superfamily. In addition to phosphotyrosine, DUSPs have additional phosphatase activity that can cleave the phosphate bond on serine or threonine residues in the substrate. DUSP26 in different N-terminal truncated forms have been reported to have different activities and structures. We first compared the activities of two truncated forms, DUSP26-N (residues 39-211) and DUSP26-C (residue 61-211), and confirmed that DUSP26-N has the phosphatase activity but DUSP26-C loses it (by pNPP assay).
Previous studies in our laboratory had shown that DUSPs possess an important hydrogen-bonding network (DPN-triloop interaction) formed by three conserved residues, aspartic acid in D-loop, serine in P-loop and asparagine in N-loop, to maintain the correct conformation of the active site. Structural analysis revealed that inactive DUSP26-C (PDB ID: 4HRF) lacks the DPN-triloop interaction, while functional DUSP26-N (PDB ID: 5GTJ) maintains the interaction. These results explain why the two truncated DUSP26 have different activities. The DPN-triloop interaction was further studied by mutagenesis and functional assay. The results showed that the catalytic efficiencies (Kcat/KM) of DUSP26-N S157A and N191A were 200-fold, and 36-fold lower than that of DUSP26-N WT, respectively. Analysis their structures by circular dichroism spectroscopy revealed that DUSP26-N WT and its mutants had similar secondary structure compositions. The reported crystal structure of DUSP26-C (PDB ID: 4HRF) is a homo-dimer with domain swapping, while the critical residue N191 in DPN-triloop interaction locates on the swapping α8-helix. To validate this structure, we determined the crystal structure of DUSP26-C at the resolution of 1.78 Å. Our structure also has domain swapping and loses the hydrogen-bonding network between D-loop, P-loop, and N-loop, once again suggests that the loss of activity in DUSP26-C is due to the loss of the DPN-triloop interaction. Because the crystals of the two mutants of DUSP26-N could not be obtained, the modeling structures were constructed to understand the changes in the active site upon disruption of the DPN-triloop interaction. The modeling structure of DUSP26-N N191A mutant showed that the D-loop flapped out to make catalytic aspartic acid leave the catalytic site. All these data revealed that the role of the DPN-triloop interaction is to anchor the catalytic aspartic acid in the correct position to maintain phosphatase activity. We are also interested in the development of allosteric inhibitor for DUSPs. We survey the possible allosteric site in DUSP26-N dimer with the compounds in the DrugCentral database for molecular docking. The docking results showed that most compounds only docked at the one chain in DUSP26-N dimer, and the docking site was near the active site. This is because these two protein chains in DUSP26-N dimer have different structures in the docking site. The further functional assay is needed to confirm the inhibitory efficiency of these compounds. The inhibition assay for others DUSPs also should be processed to check the specificity of these drug candidates.
v

中文摘要 I
ABSTRACT III
CONTENTS V
ABBREVIATIONS 7
CHAPTER 1. INTRODUCTION 8
1.1 Cysteine-based protein tyrosine phosphatases (Cys-based PTPs) and classical PTPs 8
1.2 Dual-specificity phosphatases (DUSPs) 10
1.3 DUSP26 12
1.4 Aim 13
Figures of Chapter 1 15
CHAPTER 2. MATERIALS AND METHODS 26
2.1 Construction of expression plasmids of DUSP26 and its mutants 26
2.2 Expression and Purification of DUSP26 and its mutants 27
2.3 Quantification of protein 29
2.4 SDS-PAGE 29
2.5 Circular dichroism (CD) spectroscopy 30
2.6 pNPP phosphatase activity assay 31
2.7 X-ray crystallography 32
2.8 Protein structural modeling 33
2.9 Allosteric drug screen by molecular docking 33
2.10 Software tools for protein visualization and analysis 34
Tables and Figures of Chapter 2 35
3.1 Comparison of DUSP26-F, DUSP26-N, and DUSP26-C 39
3.2 Production and characterization of DUSP26-N WT and mutants 40
3.3 The enzyme activity of DUSP26-N WT and its mutants 42
3.4 Structural analysis of DUSP26-N mutants 42
3.5 Drug virtual screening for DUSP26-N 45
Tables and figures of Chapter 3 49
CHAPTER 4. CONCLUSION 91
APPENDIX 93
1. The protein mutant structures modeled by SWISS-MODEL 93
2. The protein mutant structures modeled by AlphaFold 2 that used DUSP26-N (PDB ID: 5GTJ) as template 93
Figures of Chapter Appendix 94
REFERENCE 96

1. Alonso, A. and R. Pulido, The extended human PTPome: a growing tyrosine phosphatase family (vol 283, pg 1404, 2016). Febs Journal, 2016. 283(11): p. 2197-2201.
2. ThomasFriedrich, K.H.a., Voltage sensitive phosphatases: emerging kinship to protein tyrosine phosphatases from structure-function research. Pharmacology, 10 February 2015.
3. Tonks, N.K., Protein tyrosine phosphatases - from housekeeping enzymes to master regulators of signal transduction. Febs Journal, 2013. 280(2): p. 346-378.
4. Mustelin, T., T. Vang, and N. Bottini, Protein tyrosine phosphatases and the immune response. Nat Rev Immunol, 2005. 5(1): p. 43-57.
5. Kolmodin, K. and J. Aqvist, The catalytic mechanism of protein tyrosine phosphatases revisited. Febs Letters, 2001. 498(2-3): p. 208-213.
6. Pannifer, A.D.B., et al., Visualization of the cysteinyl-phosphate intermediate of a protein-tyrosine phosphatase by X-ray crystallography. Journal of Biological Chemistry, 1998. 273(17): p. 10454-10462.
7. Tonks, N.K., Protein tyrosine phosphatases: from genes, to function, to disease. Nature Reviews Molecular Cell Biology, 2006. 7(11): p. 833-846.
8. Meeusen, B. and V. Janssens, Tumor suppressive protein phosphatases in human cancer: Emerging targets for therapeutic intervention and tumor stratification. International Journal of Biochemistry & Cell Biology, 2018. 96: p. 98-134.
9. Hsu, F. and Y. Mao, The Sac domain-containing phosphoinositide phosphatases: structure, function, and disease. Front Biol (Beijing), 2013. 8(4): p. 395-407.
10. Nitzsche, A., Paladin is a PI(4,5)P2 phosphoinositide phosphatase that regulates endosomal signaling and angiogenesis. bioRxiv, 2020.
11. Nakada-Tsukui, K., et al., Phosphatidylinositol Kinases and Phosphatases in Entamoeba histolytica. Front Cell Infect Microbiol, 2019. 9: p. 150.
12. Lydia Tabernero, A.R.A., E. Yvonne Jones and Stefan E. Szedlacsek Protein tyrosine phosphatases: structure–function relationships. Febs Journal, 18 December 2007.
13. Zhao, Y., et al., Altering the nucleophile specificity of a protein-tyrosine phosphatase-catalyzed reaction - Probing the function of the invariant glutamine residues. Journal of Biological Chemistry, 1998. 273(10): p. 5484-5492.
14. Brandao, T.A.S., A.C. Hengge, and S.J. Johnson, Insights into the Reaction of Protein-tyrosine Phosphatase 1B CRYSTAL STRUCTURES FOR TRANSITION STATE ANALOGS OF BOTH CATALYTIC STEPS. Journal of Biological Chemistry, 2010. 285(21): p. 15874-15883.
15. ZHONG-YIN ZHANG, Y.W., AND JACK E. DIXON, Dissecting the catalytic mechanism of proteintyrosine phosphatases. Proc. Nati. Acad. Sci., 1994.
16. Andres Alonso, A.R., Adam Godzik, Tomas Mustelin, The dual-specific protein tyrosine phosphatase family. Topics in Current Genetics, 2004.
17. Ala, P.J., et al., Structural insights into the design of nonpeptidic isothiazolidinone-containing inhibitors of protein-tyrosine phosphatase 1B. J Biol Chem, 2006. 281(49): p. 38013-21.
18. Keyse, S.M., Protein phosphatases and the regulation of mitogen-activated protein kinase signalling. Current Opinion in Cell Biology, 2000. 12(2): p. 186-192.
19. Lang, R. and F.A.M. Raffi, Dual-Specificity Phosphatases in Immunity and Infection: An Update. Int J Mol Sci, 2019. 20(11).
20. Wishart, M.J. and J.E. Dixon, PTEN and myotubularin phosphatases: from 3-phosphoinositide dephosphorylation to disease. Trends Cell Biol, 2002. 12(12): p. 579-85.
21. Ohta, Y., et al., Differential activities, subcellular distribution and tissue expression patterns of three members of Slingshot family phosphatases that dephosphorylate cofilin. Genes Cells, 2003. 8(10): p. 811-24.
22. Yu, Y., et al., Aberrant splicing of cyclin-dependent kinase-associated protein phosphatase KAP increases proliferation and migration in glioblastoma. Cancer Res, 2007. 67(1): p. 130-8.
23. Manzano-Lopez, J. and F. Monje-Casas, The Multiple Roles of the Cdc14 Phosphatase in Cell Cycle Control. Int J Mol Sci, 2020. 21(3).
24. Zeng, Q., W. Hong, and Y.H. Tan, Mouse PRL-2 and PRL-3, two potentially prenylated protein tyrosine phosphatases homologous to PRL-1. Biochem Biophys Res Commun, 1998. 244(2): p. 421-7.
25. Denu, J.M. and J.E. Dixon, A Catalytic Mechanism for the Dual-Specific Phosphatases. Proceedings of the National Academy of Sciences of the United States of America, 1995. 92(13): p. 5910-5914.
26. Lai, C.H., et al., Structural Insights into the Active Site Formation of DUSP22 in N-loop-containing Protein Tyrosine Phosphatases. International Journal of Molecular Sciences, 2020. 21(20).
27. 陳重佑, Study on the structure and function of DPN-triloop interaction in human dual-specificity phosphatase 10. 2022.
28. Won, E.Y., et al., High-resolution crystal structure of the catalytic domain of human dual-specificity phosphatase 26. Acta Crystallogr D Biol Crystallogr, 2013. 69(Pt 6): p. 1160-70.
29. Thompson, E.M. and A.W. Stoker, A Review of DUSP26: Structure, Regulation and Relevance in Human Disease. International Journal of Molecular Sciences, 2021. 22(2).
30. Ravi Kumar Lokareddy, A.B., and Gino Cingolani, Atomic structure of DUSP26, a novel p53 phosphatase. Biochemistry, 2013 February 5.
31. Ren, J.X., et al., Identification of novel dual-specificity phosphatase 26 inhibitors by a hybrid virtual screening approach based on pharmacophore and molecular docking. Biomed Pharmacother, 2017. 89: p. 376-385.
32. Won, E.Y., et al., Structural Insight into the Critical Role of the N-Terminal Region in the Catalytic Activity of Dual-Specificity Phosphatase 26. Plos One, 2016. 11(9).
33. Afonine, P.V., et al., Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallographica Section D-Structural Biology, 2012. 68: p. 352-367.
34. Emsley, P., et al., Features and development of Coot. Acta Crystallographica Section D-Biological Crystallography, 2010. 66: p. 486-501.
35. Jumper, J., et al., Highly accurate protein structure prediction with AlphaFold. Nature, 2021. 596(7873): p. 583-589.
36. Gautam, B., Energy Minimization. IntechOpen, 2022.
37. Gannam, Z.T.K., et al., An allosteric site on MKP5 reveals a strategy for small-molecule inhibition. Science Signaling, 2020. 13(646).
38. Ursu, O., et al., DrugCentral: online drug compendium. Nucleic Acids Res, 2017. 45(D1): p. D932-D939.
39. Ben Chorin, A., et al., ConSurf-DB: An accessible repository for the evolutionary conservation patterns of the majority of PDB proteins. Protein Sci, 2020. 29(1): p. 258-267.
40. Wang, Z.J., L. Sun, and T. Heinbockel, Resibufogenin and cinobufagin activate central neurons through an ouabain-like action. PLoS One, 2014. 9(11): p. e113272.
41. Grinchii, D. and E. Dremencov, Mechanism of Action of Atypical Antipsychotic Drugs in Mood Disorders. Int J Mol Sci, 2020. 21(24).
42. Bixel, K. and J.L. Hays, Olaparib in the management of ovarian cancer. Pharmgenomics Pers Med, 2015. 8: p. 127-35.