臺灣博碩士論文加值系統

研究背景：許多物種之基因雖然陸續定序出爐，但註解錯誤仍然十分普遍。病原寄生蟲中基因的註解和鑑定仍然是一個挑戰。雖然一氧化氮已被證明在陰道滴蟲 (Trichomonas vaginalis, TV) 中作為一項生存因子，但到目前為止，一氧化氮合成酶 (Nitric oxide synthase, NOS) 尚未在 TV 基因組中被註解。
研究目的：為了發現陰道滴蟲(TV)中的一氧化氮合成酶，我們創建並展示「證人-嫌疑人」策略和實驗驗證。
研究材料及方法：為了證實此一鑑定，我們發展出混合序列比對的方式去發現可能的陰道滴蟲NOS基因。運用人工智慧技術，包括Google alphafold和RoseTTAfold模擬出蛋白質的結構，再將蛋白質結構和輔酶因子對接。此外，我們進行了TV NOS的基因表現、蛋白質合成與純化、質譜分析和酶活性測定。
結果：我們的數據證明，儘管 TV NOS 蛋白預測結構的同源性與其他物種的 NOS 同源性較低，但所有NOS輔酶因子都可以與TV NOS蛋白以高親和力相互作用。此外，純化的蛋白質顯示具NOS酶活性，並在體外試管中產生一氧化氮。
討論：這項研究提出了一種創新的策略，可以確定TV中被錯誤註解的基因，並辨別TV中一個新穎NOS並可能與生存因子相關。此外，我們發展出一種「證人-嫌疑人」對比策略，通過將 TV 的整個編碼序列與來自其他生物體的數千個已知蛋白質序列進行深度和重複比對，通過 Smith-Waterman 和 Needleman-Wunsch 算法的混合運行來識別 TV 中錯誤註解的基因。在這項研究中，一種新的 TV NOS被成功鑑定，該蛋白質最初被註解為水解酶，顯示出較高的證人對嫌疑人評分，並包含所有 NOS 輔因子結合位置（NADPH、BH4、血紅素和 FAD）。實驗證實之前是被註解為水解酶，但我們發現其實為一條新穎之陰道滴蟲一氧化氮合成酶，對於NCBI資料庫可做為輔助註解。
結論：藉由生醫資訊方法，使用創新的「證人-嫌疑人」對比策略，成功發現陰道滴蟲中的一氧化氮合酶蛋白質序列，並且成功進行電腦模擬鑑定和實驗功能驗證。

Background: Gene annotation and identification of pathogenic parasites remain a challenge. Although nitric oxide is a survival factor in Trichomonas vaginalis (TV), nitric oxide synthase (NOS) has not been annotated in the TV genome.
Objective: To discover whether nitric oxide synthase is present in TV, we create and demonstrate a witness-to-suspect strategy protocol of alignment and experimental functional assays.
Materials and methods: To confirm this identification, we developed a hybrid sequence alignment approach to discover possible NOS genes in TV. Artificial intelligence techniques, including Google alphafold and RoseTTAfold simulate proteins’ structures where docking sites dock with enzyme cofactors. Furthermore, we conduct, respectively, gene expression, protein synthesis and purification, functional assays, and mass spectrometry of TV NOS.
Results: Our data demonstrate that all cofactors can interact with TV NOS proteins with high affinity even though the homology of the TV NOS sequence has a low percentage of identity with NOSs of other species. In addition, the purified proteins show enzymatic activity and produce nitric oxide in vitro.
Discussion: These studies created an innovative strategy protocol to identify misannotated genes in TV and demonstrated a novel NOS that can be associated with survival factors in TV. In addition, we developed a witness-to-suspect ratio strategy by deeply and repeatedly comparing the entire coding sequence of TV with thousands of known protein sequences from other organisms by Smith-Waterman and Needleman-Wunsch algorithms were run in a hybrid to identify misannotated genes in TV. In this study, a new TV NOS was successfully identified; it was initially annotated as a hydrolase and contained all NOS cofactor binding domains (NADPH, BH4, heme, and FAD domains). Experiments confirmed that we identified a novel nitric oxide synthase from TV, previously annotated as a hydrolase, which can be used as a complementary annotation to the NCBI database.
Conclusion: The nitric oxide synthase protein sequence in TV was successfully discovered by a bioinformatic approach using an innovative witness-to-suspect strategy and was successfully simulated by artificial intelligence for identification and experimental functional enzyme activity validation.

正文目錄
正文目錄 I
『表』目錄 IV
『圖』目錄 VII
中文摘要 XII
英文摘要 XIV
第一章 緒論 1
第一節 研究背景與動機 1
第二節 研究目的 12
第二章 材料與方法 13
第一節 生醫資訊部分 13
一、基因註解的資料來源與搜尋 13
二、混合基因注釋方法(Hybrid Gene Annotation Method)：2個步驟 17
第二節 人工智慧蛋白質結構預測與蛋白質體學應用 25
一、陰道滴蟲TV NOS的蛋白質結構預測 25
二、使用生醫資訊學工具預測域蛋白質結構域 25
第三節 實驗驗證 26
一、重組蛋白製備 26
第三章 結果 29
第一節.結合Smith-Waterman演算法和Needle-man-Wunsch演算法的混合註解方法 29
第二節.用Discovery Studio模擬TV NOS和TV NOS2還原酶結構域 37
第三節.透過RoseTTAFold和AlpaFold2對TV 具NOS可能性之蛋白質結構進行預測和模擬 39
第四節. TV NOS, TV NOS2與輔因子和NOS抑制劑（L-NMMA）的分子對接 47
一、分子對接(確定結構的Mus musculus NOS結構來模擬血紅素結合) 50
二、TV NOS和TV NOS2的蛋白質模擬示意圖 52
第五節.模擬TV NOS與Calmodulin蛋白的蛋白質-蛋白質相互作用 92
第六節. TV NOS基因的實驗驗證 94
第四章 討論 114
第五章 結論與建議 129
第一節 結論 129
第二節 建議 129
第三節 展望 130
第七章 參考文獻 132
附錄 1
附錄 1
附錄 1 發表著作 1
附錄 2 獲獎榮譽 1
附錄 3 資格考文件 1
附錄 4 進度報告文件 9


 
『表』目錄
表1.Harris與長庚大學推測之NOS可能序列名稱整理表 6
表 2.NCBI 搜尋序列數量、identical protein groups（IPG）搜尋序列數量、MATLAB對比重複序列後剩下的序列數量 15
表 3.此表為不同胺基酸物理化學性質整理 20
表4. MATLAB 所採用之BLOSUM50 評分矩陣 21
表 5. 本研究被比對出有可能具有NOS活性的基因名稱與被非陰道滴蟲NOS序列識別之頻率次數與百分比之整理 30
表6 在RoseTTAFold和AlpaFold2中預測了TV NOS的3D蛋白結構模型 45
表7. ROC AUC值的解釋力整理表 46
表8. AlphaFold pLDDT分數代表意義表格 47
表9. Mus musculus NOS結構模擬血紅素結合Affinity (kcal/mol)等結果 50
表10. TV NOS結構模擬Heme結合Affinity (kcal/mol)等結果 52
表11. TV NOS結構模擬Heme結合氫鍵位置 54
表12. TV NOS2結構模擬Heme結合Affinity (kcal/mol)等結果 55
表13. TV NOS2結構模擬Heme結合氫鍵位置 57
表14. TV NOS結構模擬BH4結合Affinity (kcal/mol)等結果 58
表15. TV NOS2結構模擬BH4結合氫鍵位置 60
表16. TV NOS2結構模擬BH4結合Affinity (kcal/mol)等結果 61
表17. TV NOS2結構模擬BH4結合氫鍵位置 63
表18. TV NOS結構模擬FMN結合Affinity (kcal/mol)等結果 64
表19. TV NOS結構模擬FMN結合氫鍵位置 67
表20. TV NOS2結構模擬FMN結合Affinity (kcal/mol)等結果 68
表21. TV NOS2結構模擬FMN結合氫鍵位置 70
表22. TV NOS結構模擬FAD結合Affinity (kcal/mol)等結果 71
表23. TV NOS結構模擬FAD結合氫鍵位置 73
表24. TV NOS2結構模擬FAD結合Affinity (kcal/mol)等結果 74
表25. TV NOS2結構模擬FAD結合氫鍵位置 77
表26. TV NOS結構模擬NADP結合Affinity (kcal/mol)等結果 78
表27. TV NOS結構模擬NADP結合氫鍵位置 81
表28. TV NOS2結構模擬NADP結合Affinity (kcal/mol)等結果 82
表29. TV NOS2結構模擬NADP結合氫鍵位置 85
表30. TV NOS結構模擬L-NMMA結合Affinity (kcal/mol)等結果 86
表31. TV NOS結構模擬L-NMMA結合氫鍵位置 88
表32. TV NOS2結構模擬L-NMMA結合Affinity (kcal/mol)等結果 89
表33. TV NOS2結構模擬L-NMMA結合氫鍵位置 91
表34.藉由HDOCK預測TV NOS跟TV NOS2的模擬蛋白與Calmodulin蛋白質間的相互作用(Protein-protein interaction) 93
表35. LC/MS/MS鑑定的蛋白質清單使用Spodoptera frugiperda和TVAG資料庫 98
表36. 各一氧化氮合成酶輔因子結合位置序列與TV NOS 質譜儀蛋白質使用NCBI BLASTp比對結果 102
表37.Harris、長庚大學推測之NOS可能序列、本研究發現相關NOS序列整理表 128
 
『圖』目錄
圖 1.一氧化氮合成酶示意圖，包含兩大部分還原酶與氧化酶 4
圖 2.陰道滴蟲ADI酶位於hydrogenosome內會將Arginine代謝成citrulline與NH3 7
圖 3. 於NCBI網站上面之protein 資料庫搜尋是否有陰道滴蟲一氧化氮合成酶之截圖 8
圖 4. 本研究之生醫資訊部分(bioinformatic)流程圖 19
圖 5. 本實驗室所發展出之內部驗證和外部驗證生物資訊學方法 24
圖 6. 參與本研究搜索候選出TV NOS 序列之273次非陰道滴蟲NOS之蛋白序列鑑定次數 [i] 29
圖 7.共有 6 個陰道滴蟲序列被認為是 NOS 的候選者 31
圖 8.對陰道滴蟲NOS序列和非陰道滴蟲NOS進行親源分析 32
圖 9.兩個陰道滴蟲NOS可能序列和四個來自黃麴黴的NOS之間Heme binding domain的序列排列 33
圖 10.兩個陰道滴蟲NOS可能序列和四個來自黃麴黴的NOS之間BH4 binding domain及Calmodulin binding site的序列排列 34
圖 11.兩個陰道滴蟲NOS可能序列和四個來自黃麴黴的NOS之間FAD binding domain及NADPH binding domain的序列排列 35
圖 12.兩個陰道滴蟲NOS可能序列和四個來自黃麴黴的NOS之間NADPH binding domain的序列排列 36
圖13. 使用Discovery Studio軟體後， TV NOS和TV NOS2還原酶結構域的模擬結構示意圖 38
圖 14.Alphafold2預測出來之TV NOS結構 41
圖 15.RoseTTAFold 預測出來之TV NOS結構 42
圖 16.Alphafold2預測出來之TV NOS2結構 43
圖 17.RoseTTAFold 預測出來之TV NOS2結構 44
圖18. 小家鼠（Mus musculus）NOS蛋白（3DWJ）與Heme的蛋白質-輔因子交互作用結果 51
圖19.TV NOS與Heme的蛋白質-輔因子相互作用結果 53
圖20. TV NOS2與Heme的蛋白質-輔因子交互作用結果 56
圖21. TV NOS與BH4的蛋白質-輔因子交互作用結果 59
圖22.TV NOS2與BH4的蛋白質-輔因子交互作用結果 62
圖23. TV NOS與FMN的蛋白質-輔因子交互作用結果 67
圖24. TV NOS2與FMN的蛋白質-輔因子交互作用結果 69
圖25. TV NOS與FAD的蛋白質-輔因子交互作用結果 72
圖26. TV NOS2與FAD的蛋白質-輔因子交互作用結果 76
圖27. TV NOS與NADP的蛋白質-因數相互作用結果 80
圖28. TV NOS2與NADP的蛋白質-輔因子交互作用結果 85
圖29. TV NOS與NOS抑制劑（L-NMMA）的蛋白質-配體交互作用結果 87
圖30. TV NOS2與NOS抑制劑（L-NMMA）的蛋白質-配體交互作用結果 90
圖 31. 圖示為本研究TVNOS序列一氧化氮合成酶的實驗步驟 94
圖 32.本實驗之載體(pFastBac1 Vector of TV NOS) 95
圖 33.本研究實驗資料圖 96
圖34.質譜儀heme 結合位置之蛋白質序列 103
圖35. 質譜儀BH4 結合位置蛋白質序列 104
圖36. 質譜儀calmodulin 結合位置之蛋白質序列 105
圖37. 質譜儀FMN 結合位置之蛋白質序列 106
圖38. 質譜儀FAD pyrophosphate 結合位置之蛋白質序列 107
圖39. 質譜儀FAD isoaaloxazine 結合位置之蛋白質序列 108
圖40. 質譜儀NADPH ribose 結合位置之蛋白質序列 109
圖41. 質譜儀NADPH adenine 結合位置之蛋白質序列 110
圖42. TV NOS質譜鑑定之序列覆蓋率 111
圖43. 分析陰道滴蟲細胞的crude lysate、外源性過表達TV NOS的Sf昆蟲細胞的crude lysate以及純化後的TV NOS蛋白中的NOS活性 113
圖44. TV NOS於Uniprot網站之3D結構示意圖 120
圖45. TV NOS(淺藍色)與Uniport網站上之A2DJB5結構(綠色) 使用Pymol程式superimpose(疊合)之結果 121
圖46. TV NOS(藍色)與Uniport網站上之A2DJB5結構(紅色) 使用TM-score分析 superimpose(疊合)之結果 122
圖47. TV NOS2於Uniprot網站之3D結構示意圖 123
圖48. TV NOS2(粉紅色)與Uniport網站上之A2EA13結構(黃色)使用Pymol程式superimpose(疊合)之結果 124
圖49. TV NOS(藍色)與Uniport網站上之A2EA13結構(紅色) 使用TM-score分析 superimpose(疊合)之結果 125
 
『附錄』目錄
附錄 1 發表著作 1
附錄 2 獲獎榮譽 1
附錄 3 資格考文件 1
附錄 4 進度報告文件 9

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