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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 徐駿森(Chun-Hua Hsu) | |
dc.contributor.author | Ming-Ying Lee | en |
dc.contributor.author | 李明穎 | zh_TW |
dc.date.accessioned | 2021-06-15T16:09:23Z | - |
dc.date.available | 2020-10-16 | |
dc.date.copyright | 2015-10-16 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2015-08-19 | |
dc.identifier.citation | 1. 中華民國103年國人死因統計結果。行政院衛生福利部104年6月。 2. Beer, T.M., et al., Enzalutamide in metastatic prostate cancer before chemotherapy. New England Journal of Medicine, 2014. 371(5): p. 424-433. 3. Neal, D.E., et al., Prostate specific antigen and prostatitis I. Effect of prostatitis on serum PSA in the human and nonhuman primate. The Prostate, 1992. 20(2): p. 105-111. 4. Brawer, M.K., et al., Screening for prostatic carcinoma with prostate specific antigen. The Journal of Urology, 1992. 147(3 Pt 2): p. 841-845. 5. Kumaresan, K., et al., Diagnostic utility of α-methylacyl CoA racemase (P504S) HMWCK in morphologically difficult prostate cancer. Diagnostic Pathology, 2010. 5(1): p. 83. 6. Jiang, Z. and B.A. Woda, Diagnostic utility of α-methylacyl CoA racemase (P504S) on prostate needle biopsy. Advances in Anatomic Pathology, 2004. 11(6): p. 316-321. 7. Zhou, M., et al., Alpha-Methylacyl-CoA racemase: a novel tumor marker over-expressed in several human cancers and their precursor lesions. The American Journal of Surgical Pathology, 2002. 26(7): p. 926-931. 8. Luo, J., et al., Alpha-methylacyl-CoA racemase: a new molecular marker for prostate cancer. Cancer Research, 2002. 62(8): p. 2220-6. 9. Rubin, M.A., et al., α-Methylacyl coenzyme A racemase as a tissue biomarker for prostate cancer. Journal of the American Medical Association, 2002. 287(13): p. 1662-1670. 10. Lloyd, M.D., et al., alpha-Methylacyl-CoA racemase (AMACR): metabolic enzyme, drug metabolizer and cancer marker P504S. Progress in Lipid Research, 2013. 52(2): p. 220-30. 11. Kumar-Sinha, C., et al., Elevated α-methylacyl-CoA racemase enzymatic activity in prostate cancer. The American Journal of Pathology, 2004. 164(3): p. 787-793. 12. Mukherji, M., et al., The chemical biology of branched-chain lipid metabolism. Progress in Lipid Research, 2003. 42(5): p. 359-376. 13. Wanders, R.J.A., R.B.H. Schutgens, and P.G. Barth, Peroxisomal disorders: a review. Journal of Neuropathology Experimental Neurology, 1995. 54(5): p. 726-739. 14. Lloyd, M.D., et al., Alpha-methylacyl-CoA racemase--an 'obscure' metabolic enzyme takes centre stage. Federation of European Biochemical Societies, 2008. 275(6): p. 1089-102. 15. Qu, X., et al., Hydrolysis of ibuprofenoyl-CoA and other 2-APA-CoA esters by human acyl-CoA thioesterases-1 and-2 and their possible role in the chiral inversion of profens. Biochemical Pharmacology, 2013. 86(11): p. 1621-1625. 16. Reichel, C., et al., 2-Arylpropionyl-CoA epimerase: partial peptide sequences and tissue localization. Biochemical Pharmacology, 1995. 50(11): p. 1803-1806. 17. Darley, D.J., et al., Synthesis and use of isotope-labelled substrates for a mechanistic study on human alpha-methylacyl-CoA racemase 1A (AMACR; P504S). Organic Biomolecular Chemistry, 2009. 7(3): p. 543-52. 18. Ferdinandusse, S., et al., Subcellular localization and physiological role of alpha-methylacyl-CoA racemase. The Journal of Lipid Research, 2000. 41(11): p. 1890-6. 19. Ferdinandusse, S., et al., Mutations in the gene encoding peroxisomal alpha-methylacyl-CoA racemase cause adult-onset sensory motor neuropathy. Nature Genetics, 2000. 24(2): p. 188-91. 20. Takahara, K., et al., Conversion of prostate cancer from hormone independency to dependency due to AMACR inhibition: involvement of increased AR expression and decreased IGF1 expression. Anticancer Research, 2009. 29(7): p. 2497-2505. 21. Zha, S., et al., α-Methylacyl-CoA racemase as an androgen-independent growth modifier in prostate cancer. Cancer Research, 2003. 63(21): p. 7365-7376. 22. Savolainen, K., et al., A mouse model for α-methylacyl-CoA racemase deficiency: adjustment of bile acid synthesis and intolerance to dietary methyl-branched lipids. Human Molecular Genetics, 2004. 13(9): p. 955-965. 23. Carnell, A.J., et al., Design, synthesis, and in vitro testing of α-methylacyl-CoA racemase inhibitors. Journal of Medicinal Chemistry, 2007. 50(11): p. 2700-2707. 24. Morgenroth, A., et al., New Molecular Markers for Prostate Tumor Imaging: A Study on 2‐Methylene Substituted Fatty Acids as New AMACR Inhibitors. Chemistry-A European Journal, 2011. 17(36): p. 10144-10150. 25. Wilson, B.A.P., et al., High-throughput screen identifies novel inhibitors of cancer biomarker α-methylacyl coenzyme A racemase (AMACR/P504S). Molecular Cancer Therapeutics, 2011. 10(5): p. 825-838. 26. Parnham, M.J. and H. Sies, The early research and development of ebselen. Biochem Pharmacol, 2013. 86(9): p. 1248-53. 27. Linsky, T., Y. Wang, and W. Fast, Screening for dimethylarginine dimethylaminohydrolase inhibitors reveals ebselen as a bioavailable inactivator. ACS Medicinal Chemistry Letters, 2011. 2(8): p. 592-596. 28. Azad, G.K. and R.S. Tomar, Ebselen, a promising antioxidant drug: mechanisms of action and targets of biological pathways. Molecular Biology Reports, 2014. 41(8): p. 4865-79. 29. Lass, A., et al., Inhibition of copper-and peroxyl radical-induced LDL lipid oxidation by ebselen: antioxidant actions in addition to hydroperoxide-reducing activity. Biochimica et Biophysica Acta (BBA)-Lipids and Lipid Metabolism, 1996. 1303(2): p. 111-118. 30. Savolainen, K., et al., Alpha-methylacyl-CoA racemase from Mycobacterium tuberculosis. Mutational and structural characterization of the active site and the fold. The Journal of Biological Chemistry, 2005. 280(13): p. 12611-20. 31. Bhaumik, P., et al., The catalysis of the 1,1-proton transfer by alpha-methyl-acyl-CoA racemase is coupled to a movement of the fatty acyl moiety over a hydrophobic, methionine-rich surface. Journal of Molecular Biology, 2007. 367(4): p. 1145-61. 32. Mullins, E.A., et al., Formyl-coenzyme A (CoA):oxalate CoA-transferase from the acidophile Acetobacter aceti has a distinctive electrostatic surface and inherent acid stability. Protein Science, 2012. 21(5): p. 686-96. 33. Gruez, A., et al., The crystal structure of the Escherichia coli YfdW gene product reveals a new fold of two interlaced rings identifying a wide family of CoA transferases. The Journal of Biological Chemistry 2003. 278(36): p. 34582-6. 34. Ricagno, S., et al., Formyl-CoA transferase encloses the CoA binding site at the interface of an interlocked dimer. EMBO Journal, 2003. 22(13): p. 3210-9. 35. Mullins, E.A., K.L. Sullivan, and T.J. Kappock, Function and X-ray crystal structure of Escherichia coli YfdE. PLOS One, 2013. 8(7): p. e67901. 36. Lee, K.S., et al., Crystal structure of fatty acid-CoA racemase from Mycobacterium tuberculosis H37Rv. Proteins, 2006. 64(3): p. 817-22. 37. Kim, B.J., et al., Expression and characterization of alpha-methylacyl CoA racemase from Anisakis simplex larvae. The Korean Journal of Parasitology, 2012. 50(2): p. 165-71. 38. Tamura, K., et al., MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 2013. 30(12): p. 2725-2729. 39. Pei, J., B.-H. Kim, and N.V. Grishin, PROMALS3D: a tool for multiple protein sequence and structure alignments. Nucleic Acids Research, 2008. 36(7): p. 2295-2300. 40. Abramoff, M.D., P.J. Magalhaes, and S.J. Ram, Image processing with ImageJ. Biophotonics International, 2004 11: 36–42.. 41. Garman, E.F. and S. Doublie, Cryocooling of macromolecular crystals: optimization methods. Methods Enzymol, 2003. 368: p. 188-216. 42. Garman, E.F. and R.L. Owen, Cryocooling and radiation damage in macromolecular crystallography. Acta Crystallographica Section D: Biological Crystallography, 2006. 62(Pt 1): p. 32-47. 43. McFerrin, M.B. and E.H. Snell, The development and application of a method to quantify the quality of cryoprotectant solutions using standard area-detector X-ray images. Journal of Applied Crystallography, 2002. 35: p. 538-545. 44. Taylor, G.L., Introduction to phasing. Acta Crystallographica Section D: Biological Crystallography, 2010. 66(4): p. 325-338. 45. Emsley, P. and K. Cowtan, Coot: model-building tools for molecular graphics. Acta Crystallographica Section D: Biological Crystallography, 2004. 60(Pt 12 Pt 1): p. 2126-32. 46. Krissinel, E.B., et al., The new CCP4 Coordinate Library as a toolkit for the design of coordinate-related applications in protein crystallography. Acta Crystallographica Section D: Biological Crystallography, 2004. 60(Pt 12 Pt 1): p. 2250-5. 47. Potterton, L., et al., Developments in the CCP4 molecular-graphics project. Acta Crystallographica Section D: Biological Crystallography, 2004. 60(Pt 12 Pt 1): p. 2288-94. 48. Woodman, T.J., et al., Chiral inversion of 2-arylpropionyl-CoA esters by human alpha-methylacyl-CoA racemase 1A (P504S)--a potential mechanism for the anti-cancer effects of ibuprofen. Chemical Communications, 2011. 47(26): p. 7332-4. 49. Favrot, L., D.H. Lajiness, and D.R. Ronning, Inactivation of the Mycobacterium tuberculosis Antigen 85 complex by covalent, allosteric inhibitors. Journal of Biological Chemistry, 2014. 289(36): p. 25031-25040. 50. Berthold, C.L., et al., Reinvestigation of the catalytic mechanism of formyl-CoA transferase, a class III CoA-transferase. Journal of Biological Chemistry, 2008. 283(10): p. 6519-6529. 51. Li, C.-F., et al., AMACR amplification in myxofibrosarcomas: a mechanism of overexpression that promotes cell proliferation with therapeutic relevance. Clinical Cancer Research, 2014. 20(23): p. 6141-6152. 52. Joice, A.C., et al., Exploring the mode of action of ebselen in Trypanosoma brucei hexokinase inhibition. International Journal for Parasitology: Drugs and Drug Resistance, 2013. 3: p. 154-60. 53. Lieberman, O.J., et al., High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases. ACS Chemical Biology, 2014. 9(1): p. 183-92. 54. Mukherjee, S., et al., Ebselen inhibits hepatitis C virus NS3 helicase binding to nucleic acid and prevents viral replication. ACS Chemical Biology, 2014. 9(10): p. 2393-403. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52197 | - |
dc.description.abstract | α-甲基醯基輔酶A 消旋酶(α-methylacyl-CoA racemase, AMACR)被發現在前列腺癌細胞中大量表現,並已作為診斷的生物標記。此酵素參與人體中支鏈脂肪酸的代謝途徑,負責支鏈脂肪酸中Cα的手性反轉,其受質包含攝食紅肉中富含的支鏈脂肪酸、藥物(如布洛芬, ibuprofen)或人體內源性的膽酸前驅物。目前已知降低AMACR 表現量有助於減緩癌細胞增生速率,因此被認為是有潛力的藥物標靶。此外,AMACR 的單一核苷酸變異(SNP)造成S52P 和L107P 的突變也使得酵素功能缺失,而導致神經性病變的症狀。由於缺乏蛋白質結構的資訊,SNP 與蛋白功能的相關性也尚未明瞭。 透過蛋白質晶體學實驗,我們已順利地解出人類AMACR 結構,及其與受質類似物Isobutytryl CoA (IBCoA)與酵素共結晶的複合體結構,藉此暸解受質結合區域及酵素催化機制。結構分析可發現,H122 和D152 為活性殘基,而受質的thioester oxygen 可與酵素D123 殘基骨幹的氮形成氫鍵。而AMACR 的SNP 研究發現的兩個致病突變S52P、︑L107P,實驗分析發現可能會影響蛋白質穩定性,同時S52P、 L107P 位置接近受質結合位置,可能也會影響受質的結合。我們也進行兩個已發表之非競爭型AMACR 抑制劑Ebselen、Ebselen oxide 的研究,實驗顯示Ebselen和Ebselen oxide 都會降低蛋白質的穩定性。從晶體結構觀察Ebselen、Ebselen oxie都會使C117 附近殘基的結構失序(disorder),推測其作用機制為選擇性共價性修飾,進而造成蛋白質穩定性下降並失去活性。本篇研究提供人類AMACR 結構,探討其反應機制及疾病相關突變S52P 和L107P 的結構闡述,並推測非競爭型抑制劑的作用機制,希望能提供未來藥物設計的結構依據。 | zh_TW |
dc.description.abstract | AMACR (α-methylacyl-CoA racemase), catalyzing a key chiral inversion step in metabolism of branched-chain fatty acid, ibuprofen and related drugs, was overproduced in prostate cancer and has been used as a prostate cancer biomarker. Previous studies shows that decreasing the expression of AMACR through using short interfering RNA constructs can slow the growth of prostate cancer cell lines, indicating that AMACR may be a new treatment for cancers. In addition, AMACR is associated with human diseases because of function deficiency caused by single base mutation S52P and L107P. Due to the lack of any experimentally determined structure of the human AMACR, the roles of SNP correlated to protein functions remains unclear. Here, the crystal structure of human AMACR and the complex structure with Isobutytryl CoA (IBCoA), an structure analogue, were determined, showing that the catalytic residues are H122 and D152, also showing the thioester oxygen hydrogen bonded to the backbone amide proton of D123. We fine that pathogenic mutants L107P and S52P are unstable than the wild type, and both mutants located very closed to the binding pocket. Ebselen and ebslene oxide, two reported non-competitive AMACR inhibitors, decrease the Tm and disordered the residues structure near C117, probably specific covalent bond to C117 then make structure change. The structures of human AMACR provide an attractive structural basis of substrate recognition for further drug design. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T16:09:23Z (GMT). No. of bitstreams: 1 ntu-103-R02623010-1.pdf: 30981574 bytes, checksum: f4d22742b89aead4b7133e3228c549a4 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 目錄 致謝 i 中文摘要 iii Abstract v 目錄 vii 圖目錄 xiii 表目錄 xvi 縮寫表 xvii 壹、前言 1 一、前列腺癌的診斷 1 二、AMACR參與支鏈脂肪酸的代謝途徑 2 三、AMACR突變導致酵素功能缺失 8 四、藥物開發潛力目標 8 五、研究目的 11 貳、材料與方法 12 一、實驗材料 12 1. 蛋白質表現載體之構築 12 2. 蛋白質點突變表現載體之構築 12 3. 菌株及培養系統 13 二、實驗方法 13 1. 轉型作用 13 2. 質體DNA抽取 13 3. DNA定序比對 14 4. SDS 蛋白質膠體電泳分析 14 5. 蛋白質表現 14 6. 蛋白質純化 15 7. 膠體過濾層析法(Gel Filtration Chromatography) 15 8. 蛋白質透析與濃縮 16 9. 蛋白質濃度測定 17 10. 胺基酸序列比對 17 11. 親緣關係圖 18 12. 蛋白質結構比對 18 13. 圓二色光譜儀測試蛋白質熱穩定性 19 14. 示差掃描螢光法蛋白質熱穩定性測試 19 15. 恆溫滴定微量熱儀 20 16. 西方墨點法 21 三、X-ray晶體繞射技術解析蛋白質結構 22 四、X-ray 晶體繞射實驗方法 23 1. 蛋白質結晶測試 23 2. 蛋白質結晶條件篩選 23 3. 蛋白質晶體形成條件微調 24 4. 適合抗凍劑之篩選 24 5. X-ray 晶體繞射數據收集及處理 (Data collection and processing) 25 6. 晶格中所包含的分子數分析 26 7. 蛋白質晶體結構建立 26 8. 蛋白質結構模型修正 26 9. 蛋白質結構確校 (Ramachandran plot) 27 參、實驗結果 29 一、AMACR WT 表現與純化 29 二、利用膠體過濾層析法分析AMACR WT的分子量 29 三、AMACR WT蛋白質晶體培養 29 四、AMACR WT X-ray繞射數據分析與單位晶格判斷 30 五、AMACR WT晶格中分子數分析 31 六、以SAD解AMACR WT 相位角 31 七、AMACR WT蛋白質結構建立 32 八、AMACR WT蛋白質結構折疊 32 九、AMACR WT 與 Class III CoA-transferase superfamily members 結構比較 33 1. AMACR WT與CoA-transferase結構比較 33 2. AMACR WT與racemase結構比較 34 十、物種間AMACR WT 胺基酸序列比對 34 十一、受質結構類似物(substrate analogue) IBCoA及MMCoA對AMACR WT 熱穩定性的影響 34 十二、受質結構類似物IBCoA及MMCoA結合能力測試 35 十三、IBCoA-AMACR WT複合體晶體培養 35 十四、IBCoA-AMACR WT X-ray繞射數據分析與單位晶格判斷 37 十五、IBCoA-AMACR WT晶格中分子數分析 37 十六、IBCoA-AMACR WT結構建立 37 十七、apoAMACR WT與IBCoA-AMACR WT 結構比較 38 十八、AMACR WT的IBCoA結合區域 38 十九、AMACR 反應機制 39 二十、R-結合區域、S-結合區域組成分析 39 二十一、疾病相關突變S52P、L107P位置 40 二十二、S52P、L107P表現及純化 40 二十三、S52P、L107P與AMACR WT 穩定性探討 40 二十四、抑制劑EO、EBS對AMACR WT 的影響具專一性 41 二十五、EO對AMACR WT 穩定性的影響 42 1. 不同反應時間對AMACR WT熱穩定性的影響 42 2. 不同反應劑量對AMACR WT熱穩定性的影響 42 二十六、EBS對AMACR WT 穩定性的影響 42 1. 不同反應時間對AMACR WT 熱穩定性的影響 42 2. 不同反應劑量對AMACR WT 熱穩定性的影響 43 二十七、以EO浸潤AMACR WT晶體 43 二十八、EO-soaked-AMACR WT繞射數據分析與晶格判斷 44 二十九、比較EO-soaked-AMACR WT與AMACR WT B-factor 值 44 三十、以EBS浸潤AMACR WT晶體 45 三十一、EBS-soaked-AMACR WT繞射數據分析與晶格判斷 45 三十二、比較EBS-soaked-AMACR WT與AMACR WT-2 B-factor 值 46 三十三、EO-AMACR WT複合體晶體培養 47 三十四、EO-AMACR複合體繞射數據分析與晶格判斷 48 三十五、EO-AMACR複合體結構中Cysteine 284被修飾 48 肆、討論 49 一、AMACR WT與O. formigene FCOCT結構與反應機制的差異 49 二、疾病相關突變S52P及L107P結構與穩定性的探討 50 三、抑制劑EO、EBS對AMACR WT可能的作用機制 51 伍、圖表 55 陸、參考文獻 121 柒、附錄 128 附錄一、免疫組織染色AMACR 128 附錄二、AMACR抑制劑的結構 129 附錄三、受質類似物IBCoa、MMCoA結構 130 附錄四、O. formigenes FRC的反應機制 131 附錄五、Ebselene共價鍵修飾的可能機制 132 附錄六、Ebselen oxide抑制腫瘤增大 133 附錄七、溶液配方 134 附錄八、蛋白質載體資訊和構築示意圖 139 | |
dc.language.iso | zh-TW | |
dc.title | 人類α-甲基醯基輔酶A消旋酶之催化機制及疾病相關突變的結構闡述 | zh_TW |
dc.title | Structural elucidation of human α-methylacyl-CoA racemase provides insight into its catalytic mechanism and disease-associated mutations | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳基旺(Ji-Wang Chern),詹迺立(Nei-Li Chan) | |
dc.subject.keyword | α-甲基醯基輔?A消旋?,蛋白質結晶學,前列腺癌,藥物標靶, | zh_TW |
dc.subject.keyword | AMACR,protein crystallography,prostate cancer,drug target, | en |
dc.relation.page | 141 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-08-19 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 農業化學研究所 | zh_TW |
顯示於系所單位: | 農業化學系 |
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