DUSP10 是雙特異性蛋白磷酸酶 (DUSPs) 家族的一員，其功能為將磷酸化酪氨酸或磷酸化絲氨酸/蘇氨酸去磷酸化。 DUSP10 可以與絲裂原活化蛋白激酶 (MAPK) 結合，並通過去磷酸化使 MAPK 失活。據報導，DUSP10 在多種惡性腫瘤、自身免疫性疾病和炎症性疾病中有表達增強的現象。因此，了解 DUSP10在人類疾病中的作用並確定其構效關係，將為未來的藥物開發提供重要信息。
最近有文獻報導 DUSPs 活性位點中的氫鍵網絡對維持構象及磷酸酶活性有重要作用。由三個保守殘基形成此一氫鍵網絡，分別為D-環中的天冬氨酸，P-環中的絲氨酸和N-環中的天冬酰胺。我們稱此氫鍵網絡為DPN-三環相互作用。在本論文中，我們構建了關鍵殘基的丙氨酸取代突變體，檢測了突變體的磷酸酶活性，並確定了它們的晶體結構。 tDUSP10-D59A、tDUSP10-S95A 和 tDUSP10-N130A 的催化效率 (kcat/KM) 分別比 tDUSP10-WT 低 66 倍、46 倍和 18 倍。這些結果表明，P-環中的非催化用絲氨酸和N-環中的非催化用天冬酰胺可通過DPN-三環相互作用調節催化。晶體結構顯示，三個突變體中的 D-環發生了偏移，導致催化用天冬氨酸遠離催化用半胱氨酸。綜上所述，這些對突變體的研究確定了我們的理論：如果 DPN-三環相互作用（氫鍵網絡）被破壞，D-環移動並使催化用天冬氨酸的位置發生改變將會改變活性位點的構象，進而使磷酸酶活性喪失。
我們也對 DUSP 10 別位抑制劑的開發有興趣。利用 PubChem 數據庫中的化合物與 DUSP10 結構進行分子對接篩選。分子對接結果與其他和癌症進展有關的 DUSPs（DUSP6、DUSP16 和 DUSP26）比較表明，DUSP10 的高親和力候選抑製劑對其他 DUSPs 的親和力較低，而且它們的預測結合位點也不同。此外，這些候選抑製劑中有大多數都被預測會結合在 N-環附近的別構位點上。未來工作將是進行抑制動力學測定並解出化合物-DUSP10複合物結構以確認抑制特異性和機制。

DUSP10 is a member of the dual specificity protein phosphatases (DUSPs) family, which dephosphorylates both phosphor-tyrosine and phosphor-serine/threonine in its substrates. DUSP10 can bind with mitogen activated protein kinase (MAPK), and inactivate MAPK through dephosphorylation. Enhanced expression of DUSP10 has been reported in various malignancies, autoimmune diseases, and inflammatory diseases. Thus, to understand the roles of DUSP10 in human diseases and to identify the structure–activity relationships, which will provide important information for future drug development.
A hydrogen bonding network in DUSP active site was recently reported to play an important conformational role and support the phosphatase activity. Three conserved residues, the catalytic aspartate in the D-loop, serine in the P-loop, and asparagine in the N-loop to form the hydrogen bonding network, the DPN-triloop interaction. In this study, the alanine-substituted mutants of the critical residues were constructed, their phosphatase activities were detected, and their crystal structures were determined. The catalytic efficiencies (kcat/KM) of tDUSP10-D59A, tDUSP10-S95A, and tDUSP10-N130A were about 66-fold, 46-fold, and 18-fold lower than that of tDUSP10-WT, respectively. These results indicated that the non-catalytic serine in P-loop and non-catalytic asparagine in N-loop also regulated the catalysis through DPN-triloop interaction. Their crystal structures revealed that the D-loop in three mutants shifted, causing the catalytic aspartate moved far away from the catalytic cysteine. Taken together, these studies of mutants identified our proposal: if the DPN-triloop interaction (hydrogen bonding network) was disrupted, D-loop shifted and the position of catalytic aspartate was changed to alter the conformation of active site, thus the phosphatase activity was lost.
We are also interested in development of allosteric inhibitor for DUSP 10. The compounds in PubChem database have been screened through molecular docking with DUSP10 structure. The comparison of the molecular docking results with other cancer-progression DUSPs (DUSP6, DUSP16, and DUSP26) revealed that the high-affinity inhibitor candidates of DUSP10 showed lower affinity to other DUSPs, while their predicted binding sites were also different. In addition, most of these candidates were predicted to bind at an allosteric site near N-loop. The future work is performing the inhibition kinetic assay and determining compound-DUSP10 complex structure to confirm the inhibitory specificity and the mechanism.

ABBREVIATIONS...1
CHAPTER 1. INTRODUCTION...1
1.1 Cysteine-based protein tyrosine phosphatases (Cys-based PTPs)...1
1.2 The catalytic mechanism of Cys-based PTPs...1
1.3 The active site differences between classical PTPs and DUSPs...2
1.4 Dual-specificity phosphatase10 (DUSP10)...4
1.5 Aim...5
TABLES AND FIGURES OF CHAPTER 1...7
Figure 1.1 The classification of human Cys-based PTPs...7
Figure 1.2 The catalytic mechanism of Cys-based PTPs...8
Figure 1.3 The structural comparison of classical PTP and DUSPs....9
Figure 1.4 List of N-loop-containing PTPs....10
Figure 1.5 The expression in tissues, interactions with stimuli, and relationships to diseases of DUSP10...11
Figure 1.6 The crystal structures of DUSP10 before and after drug binding....12
CHAPTER 2. MATERIALS AND METHODS...13
2.1 Construction of tDUSP10 and its mutants...13
2.2 Expression and Purification of tDUSP10 and its mutants...14
2.3 SDS-PAGE...15
2.4 p-NPP phosphatase activity assay...16
2.5 Circular dichroism (CD)...16
2.6 X-ray crystallography...17
2.7 Molecular docking of allosteric inhibitors at tDUSP10-WT...19
TABLES AND FIGURES OF CHAPTER 2...20
Table 2.1 Primers for mutagenesis PCR...20
Table 2.2 The mutagenesis reaction compositions and thermocycling condition...21
Figure 2.1 Construction of pET28a-tDUSP10...22
Figure 2.2 The reaction of p-NPP phosphatase activity assay...23
CHAPTER 3. RESULTS AND DISCUSSION...24
3.1 Production and characterization of tDUSP10 and its mutants...24
3.2 The enzyme activity of tDUSP10 and its mutants...25
3.3 Crystal structure of tDUSP10 and its mutants...25
3.4 In silica drug screening...28
CHAPTER 4. CONCLUSION...30
TABLES AND FIGURES OF CHAPTER 3...31
Table 3.1 Kinetic parameters of tDUSP10-WT and mutants...31
Table 3.2 Crystallization conditions for tDUSP10-WT and mutants...32
Table 3.3 Data collection and refinement statistics for tDUSP10-WT and mutants...33
Table 3.4 The molecular docking results with DUSPs...34
Figure 3.5 The secondary structure compositions of tDUSP10-WT...39
Figure 3.6 The secondary structure compositions of tDUSP10-D59A...40
Figure 3.7 The secondary structure compositions of tDUSP10-S95A...41
Figure 3.8 The secondary structure compositions of tDUSP10-N130A...42
Figure 3.9 The thermo-stability of tDUSP10-WT and mutants....43
Figure 3.10 The phosphatase activities of tDUSP10-WT and mutants...44
Figure 3.11 The enzyme kinetics results of tDUSP10-WT and mutants...45
Figure 3.12 The determined crystal structures of tDUSP10-WT...46
Figure 3.13 The determined crystal structures of tDUSP10-D59A...47
Figure 3.14 The determined crystal structures of tDUSP10-S95A...48
Figure 3.15 The determined crystal structures of tDUSP10-N130A...49
Figure 3.16 The structural comparison of tDUSP10-WT and -D59A...50
Figure 3.17 The B-factor comparison of tDUSP10-WT and -D59A...51
Figure 3.18 The structural comparison of tDUSP10-WT and -S95A...52
Figure 3.19 The B-factor comparison of tDUSP10-WT and -S95A...53
Figure 3.20 The structural comparison of tDUSP10-WT and -N130A...54
Figure 3.21 The B-factor comparison of tDUSP10-WT and -N130A...55
Figure 3.22 The structural comparison of tDUSP10-WT and mutants...56
Figure 3.23 The electron density map of tDUSP10-WT and -D59A...57
Figure 3.24 The electron density map of tDUSP10-WT and -S95A...58
Figure 3.25 The electron density map of tDUSP10-WT and -N130A...59
Figure 3.26 The comparison of known structures of DUSP10 catalytic domain...60
Figure 3.27 The distances of critical residues in known structures of DUSP10 catalytic domain...61
Figure 3.28 The molecular docking results of DUSPs...62
REFERENCES...63

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