Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/98005
標題: 探討在三磷酸腺苷存在下配體對第一型人類尿苷磷酸化酶功能狀態的影響
Study the effect of ligands to the functional state of human uridine phosphorylase 1 in the presence of ATP
作者: 葉佩珒
Pei-Chin Yeh
關鍵字: 人類尿苷磷酸化酶
三磷酸腺苷
蛋白質功能狀態
human uridine phosphorylase 1 (hUP1)
adenosine triphosphate (ATP)
protein functional state
Thermal shift assay (TSA)
Pulse proteolysis
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Liu, Y.K., T.H. Lin, and P.F. Liu, ATP alters protein folding and function of Escherichia coli uridine phosphorylase. Archives Biochemistry Biophysics, 2017. 634: p. 11-20. 17. Knowles, J.R., Enzyme-catalyzed phosphoryl transfer reactions. Annual Review of Biochemistry, 1980. 49: p. 877-919. 18. Bhattacharya, B., M.F. Mohd Omar, and R. Soong, The Warburg effect and drug resistance. British Journal of Pharmacology, 2016. 173(6): p. 970-9. 19. Chen, X., Y. Qian, and S. Wu, The Warburg effect: Evolving interpretations of an established concept. Free Radical Biology and Medicine, 2015. 79: p. 253- 263. 20. Schuster, S., et al., Mathematical models for explaining the Warburg effect: a review focussed on ATP and biomass production. Biochemical Society Transactions, 2015. 43: p. 1187-1194. 21. Zhou, Y., et al., Intracellular ATP levels are a pivotal determinant of chemoresistance in colon cancer cells. Cancer Research, 2012. 72(1): p. 304- 14. 22. Qian, Y., et al., Extracellular ATP is internalized by macropinocytosis and induces intracellular ATP increase and drug resistance in cancer cells. Cancer Letters, 2014. 351(2): p. 242-51. 23. Qian, Y.R., et al., Extracellular ATP a New Player in Cancer Metabolism: NSCLC Cells Internalize ATP In Vitro and In Vivo Using Multiple Endocytic Mechanisms. Molecular Cancer Research, 2016. 14(11): p. 1087-1096. 24. Renck, D., et al., Design of novel potent inhibitors of human uridine phosphorylase-1: synthesis, inhibition studies, thermodynamics, and in vitro influence on 5-fluorouracil cytotoxicity. Journal of Medicinal Chemistry, 2013.56(21): p. 8892-902. 25. Ploeser, J.M. and H.S. Loring, The ultraviolet absorption spectra of the pyrimidine ribonucleosides and ribonucleotides. The Journal of Biological Chemistry, 1949. 178(1): p. 431-7. 26. Mateus, A., T.A. Maatta, and M.M. Savitski, Thermal proteome profiling: unbiased assessment of protein state through heat-induced stability changes. Proteome Science, 2017. 15: p. 7. 27. Patel, A., ATP as a biological hydrotrope. Biochemistry, 2017. 第二部分 1. Roosild, T.P., et al., Implications of the structure of human uridine phosphorylase 1 on the development of novel inhibitors for improving the therapeutic window of fluoropyrimidine chemotherapy. Bmc Structural Biology, 2009. 9: p. 14. 2. Liu, Y.K., T.H. Lin, and P.F. Liu, ATP alters protein folding and function of Escherichia coli uridine phosphorylase. Archives Biochemistry Biophysics, 2017. 634: p. 11-20. 3. Sam Hopkins and Verne Schirch, Properties of a Serine Hydroxymethyltransferase in Which an Active Site Histidine Has Been Changed to an Asparagine by Site-directed Mutagenesis. The Journal of Biological Chemistry, 1986 4. Y. Chang, et al., Simplified Proteomics Approach to Discover Protein-Ligand Interactions. Protein Science, 2012. 21(9): 1280-7. 5. 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摘要: 人類第一型尿苷磷酸化酶(human uridine phosphorylase 1, hUP1)是嘧啶回收 路徑(Pyrimidine salvage pathway)中的重要酵素,同時此酵素也負責活化治療癌症 的輔助性化療藥物 5-氟尿嘧啶(5-fluorouracil, 5-FU)。本實驗室先前的研究發現增 加 ATP 的濃度會同時降低 hUP1 的蛋白質穩定度和酵素活性。有趣的是,癌症 細胞為了不斷的增生通常會比一般的健康細胞累積更高濃度的細胞內 ATP,而 此高濃度的 ATP 是否會對癌細胞內的 hUP1 造成類似的影響,進一步改變 5-FU 在癌症化學療法的功效則是未知。 為了進一步研究 ATP 對細胞內 hUP1 的影響,我們首次將 Thermal shift assay (TSA)與 Pulse proteolysis (Pulse)兩種實驗方式進行結合,我們將此新方法稱為 TSA-Pulse,基於這種新方法,我們明確的觀察到 ATP 的存在改變了純化的 hUP1 的功能狀態。接著,我們選擇人類結腸癌細胞株(HCT116)作為測試模組,研究細 胞內hUP1的功能狀態,利用Cellular thermal shift assay (CETSA)實驗測定顯示 hUP1 在 HCT116 內部分呈現展開之狀態,我們的結果第一次證明在 HCT116 中 僅有一部分 hUP1 具有完整的折疊狀態。此外,由於細胞內的數種代謝物具有與 ATP 相似的化學結構,因此我們使用 TSA 和 TSA-Pulse 測試了多種配體對 hUP1 的影響,其中,我們發現尿苷擁有增加 hUP1 蛋白質穩定度之效能,而此作用能 減少 ATP 降低 hUP1 穩定度的現象,此外其代謝物 UTP (uridine triphosphate)則 同時擁有與折疊態、中介態作用之特性,綜合以上,此研究成果可提供 5-FU 功 效研究上新的參考信息。
Human uridine phosphorylase 1 (hUP1) is an important enzyme responsible for the activation of 5-fluorouracil (5-FU) in chemotherapy. Our previous experiments demonstrated that increased concentration of ATP simultaneously decreases the stability and activity of hUP1. Because cancer cells are well known with high-level of ATP for unconstrained proliferation, this ATP effect is possibly related to the efficacy of 5-FU for chemotherapy. In order to study the effect of ATP to hUP1 in cancer cells, we combined thermal shift assay (TSA) with pulse proteolysis as a new method called TSA-pulse. Based on this new method, we observed that ATP clearly changes the functional states of purified hUP1. Furthermore, we choose the human colon cancer cell line (HCT116) to investigate the functional state of hUP1 in cells. The cellular thermal shift assay showed that hUP1 is partially unfolded in HCT116. Together, our results first demonstrated that only a fraction of hUP1 processes folded state in HCT116 cell line. Because several metabolites possess similar chemical structure as ATP, we also tested the effect of multiple ligands to hUP1 with TSA and TSA-Pulse. Among them, we found that uridine has the effect of increasing the stability of hUP1, and it reduces the effect of ATP on the stability of hUP1. In addition, uridine triphosphate (UTP) has the characteristics of interacting with both folded state and intermediate state of hUP1. Our results indicate that several metabolites influence the functional state of hUP1. These metabolites may play a role in the efficacy of 5-FU. Further experiments will be needed to understand the ligand-binding effect to hUP1 in colon cancer cells.
URI: http://hdl.handle.net/11455/98005
文章公開時間: 2020-08-13
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