探究DUSP-16的三環相互作用與潛在藥物靶向位點

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研究生：	洪瑞廷 Hong, Ruei-Ting
論文名稱：	探究DUSP-16的三環相互作用與潛在藥物靶向位點 Exploring DPN Tri-loop Interaction and Potential Drug Targeting Sites of DUSP16
指導教授：	呂平江 Lyu, Ping-Chiang
口試委員:	鄭惠春 Cheng, Hui-Chun 蕭乃文 Hsiao, Nai-Wan
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：	2024
畢業學年度：	112
語文別：	英文
論文頁數：	86
中文關鍵詞：	雙特異性蛋白磷酸酶16 、三環相互作用、潛在藥物靶向位點
外文關鍵詞：	DUSP16, DUSP16 dual specificity phosphatase 16, DPN Tri-loop Interaction, Potential Drug Targeting Sites
相關次數：	點閱：16 下載：0
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在我們實驗室的先前研究中，首先透過序列分析確定了DUSPs中普遍存在的N-loop，並進一步發現N-loop上的天門冬胺酸會與另外兩個保守胺基酸：D-loop上的天冬氨酸與P-loop上的絲氨酸形成氫鍵網絡。在DUSP22 和 DUSP10中個別突變這三個保守的胺基酸會導致活性喪失。突變體的晶體結構顯示了因為氫鍵網絡被破壞導致D-loop的位移，進而造成活性位點構型改變，所以活性顯著降低。我們將這個由三環作用的氫鍵網絡命名為 ”DPN三環交互作用”。本論文首先要證實除了DUSP22 和 DUSP10，DPN三環交互作用也存在於DUSP16中。我們只成功生產出DUSP16-D56A和N127A，它們的活性如同預期有顯著下降，但是二級結構組成和熱穩定性則與DUSP16-WT無顯著差異。因為蛋白質晶體無法順利獲得，所以我們用AlphaFold2和SWISS-MODEL來建DUSP16突變體的模擬結構。雖然大部分結果都與DUSP16-WT無差異，但是仍有出現D-loop位移的組別。因此我們推論DPN三環交互作用在DUSP16中的作用和在DUSP22 和 DUSP10中相似，都是形成氫鍵網絡維持活性位點構型和活性。本論文的第二部分在於探討DUSPs的潛在抑制劑靶向位點。關於DUSP10別構抑制劑的文獻提供了N-loop區域可能是別構抑制劑靶向位點的訊息。我們利用分子對接找出了8個靶向N-loop區域的可能別構抑制劑化合物，但其對DUSP10和DUSP16有著不同的親和力。雖然實驗結果顯示無明顯抑制作用，但是這部分的研究仍提供了DUSP別構抑制劑開發之靶向位點的可能位置和篩選方法。

In previous studies in our laboratory, we first identified the ubiquitous N-loop in DUSPs through sequence analysis, and further found that asparagine in the N-loop interacts with two other conserved amino acids, aspartate in the D-loop and serine on P-loop, to form a hydrogen bonding network. Individual mutations of these three conserved amino acids in DUSP22 and DUSP10 result in loss of activity. The crystal structure of the mutants show that the activity is significantly reduced due to the disruption of the hydrogen bonding network, resulting in the shift of the D-loop, which in turn causes the conformational change of the active site. We named this hydrogen bonding network as “DPN-triloop interaction”. The first aim of this thesis is to confirm that in addition to DUSP22 and DUSP10, the DPN-triloop interaction also exists in DUSP16. We only successfully produced DUSP16-D56A and N127A. Their activity decreased significantly as expected, but their secondary structure composition and thermal stability were not significantly different from DUSP16-WT. Because protein crystals could not be obtained, we used AlphaFold2 and SWISS-MODEL to build the simulated structures of the DUSP16 mutants. Although most of the results were indistinguishable from DUSP16-WT, there were still some groups with D-loop shift. We proposed that the role of DPN-triloop interaction in DUSP16 is similar to that in DUSP22 and DUSP10, forming a hydrogen bond network to maintain the active site conformation and activity. The second part of this thesis is to explore the potential inhibitor target sites of DUSPs. The literature on allosteric inhibitors to DUSP10 provides information that the N-loop region may be a target site for allosteric inhibitors. We used molecular docking to screen out eight potential compounds targeting N-loop, which have different affinities for DUSP10 and DUSP16. Although the experimental results show no obvious inhibitory effect, this study still provides potential target site and screening method for the development of allosteric inhibitors for DUSPs.

Acknowledgements…………………………………………………………………………….....I
中文摘要……………………………………………………………………………….............IV
Abstract…………………………………………………………………………………………..VI
Abbreviation……………...……………………….…………………………………...….…….XII
Chapter 1. Introduction……………………………………………………………………………1
1 Cysteine-based protein tyrosine phosphatase (Cys-based PTPs) ………………………    …….1
2 Classical PTPs…………………………………………………………………………………1
3 Dual-specificity phosphatases (DUSPs) ……………………………………………………...3
3.1 The N-loop-containing PTPs ………………………………………………….............3
3.2 The DPN-triloop interaction …………………………………………………...............3
3.3 Typical and atypical DUSPs …………………………………………………...............4
4 Dual-specificity phosphatases16 (DUSP16) …………………………………………..............5
5 Potential Drug Targeting Sites of DUSPs …………………………………………….............5
6 Aim ………………………………………………….………………………………………...6
Tables and Figures of Chapter 1 …………………………………………………...………………8
Chapter 2. Materials and methods …………………………………………………......................19
1 Construction of DUSP16 wild type and mutants ………………………………………….....19
2 Purification of DUSP16 and its mutants ………………………………………………….19
3 Circular dichroism (CD) spectroscopy ………………………………………………...........20
4 pNPP phosphatase activity assay …………………………………………………............22
5 Malachite Green activity assay …………………………………………………....................23
6 Protein structural modeling …………………………………………………...……………23
7 Allosteric inhibitor docking and pNPP assay ………………………………………………24
8 Software tools for protein visualization and analysis ………………………………………25
8.1 PyMOL …………………………………………………...…………………………25
8.2 PyRx …………………………………………………...………………………..........26
Tables and Figures of Chapter 2 …………………………………………………...……………26
Chapter 3. Results and discussions …………………………………………………...………….30
1 Purification of DUSP16 …………………………………………………...…………...........31
1.1 Colony select and induction condition for DUSP16-WT and mutants ……………31
1.2  Ni-NTA agarose column …………………………………………………...…..........32
1.3 Anion exchange chromatography column ……………………………………...........33
1.4 Size exclusion chromatography column ……………………………………….........33
2 Comparison of secondary structure between DUSP16-WT and mutants……………………33
3 Identification of DPN-triloop interaction in DUSP16 ……………………………………34
3.1 Activity of DUSP16-WT and mutants by pNPP assay ……………………………34
3.2 Malachite green assay using phospho-p38-mimic peptide as substrate ……….............35
3.3 Simulation of DUSP16 mutants structures with AlphaFold2 and SWISS-MODEL…..36
4  DUSP16 inhibitor docking and enzymatic assay…………………………………………….37
Tables and Figures of Chapter 3…………………………………………………………………40
Chapter 4. Conclusion …………………………………………………………………………78
References ………………………………………………………………………………………81

                                

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