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標題: The effect of ATP on the folding and function of human uridine phosphorylase 1
作者: Yu-Ting Huang
關鍵字: 人類尿苷磷酸化酶
human uridine phosphorylase 1 (hUP1)
adenosine triphosphate (ATP)
protein folding
protein function
apparent destabilization
引用: 1. D. Renck, et al., The Kinetic Mechanism of Human Uridine Phosphorylase 1: Towards the Development of Enzyme Inhibitors for Cancer Chemotherapy. Arch. Biochem. Biophys., 2010. 497(1-2): 35-42. 2. T.P. Roosild and S. Castronovo, Active Site Conformational Dynamics in Human Uridine Phosphorylase 1. PLoS One, 2010. 5(9): 6. 3. T.P. Roosild, 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 Struct. Biol., 2009. 9: 9. 4. M. Johansson, Identification of a Novel Human Uridine Phosphorylase. Biochem. Biophys. Res. Commun., 2003. 307(1): 41-46. 5. D. Renck, et al., Design of Novel Potent Inhibitors of Human Uridine Phosphorylase-1: Synthesis, Inhibition Studies, Thermodynamics, and in Vitro Influence on 5-Fluorouracil Cytotoxicity. J. Med. Chem., 2013. 56(21): 8892-8902. 6. D. Renck, et al., Human Uridine Phosphorylase-1 Inhibitors: A New Approach to Ameliorate 5-Fluorouracil-Induced Intestinal Mucositis. Invest. New Drugs, 2014. 32(6): 1301-7. 7. M.P. Liu, et al., Expression, Characterization, and Detection of Human Uridine Phosphorylase and Identification of Variant Uridine Phosphorolytic Activity in Selected Human Tumors. Cancer Res., 1998. 58(23): 5418-5424. 8. R. Yan, et al., Uridine Phosphorylase in Breast Cancer: A New Prognostic Factor? Front. Biosci., 2006. 11: 2759-66. 9. D. Cao, et al., Differential Expression of Uridine Phosphorylase in Tumors Contributes to an Improved Fluoropyrimidine Therapeutic Activity. Mol. Cancer Ther., 2011. 10(12): 2330-9. 10. Y. Qian, et al., Extracellular ATP is Internalized by Macropinocytosis and Induces Intracellular ATP Increase and Drug Resistance in Cancer Cells. Cancer Lett., 2014. 351(2): 242-51. 11. Y. Zhou, et al., Intracellular ATP Levels are a Pivotal Determinant of Chemoresistance in Colon Cancer Cells. Cancer Res., 2012. 72(1): 304-14. 12. X.Z. Chen, Y.R. Qian, and S.Y. Wu, The Warburg Effect: Evolving Interpretations of an Established Concept. Free Radic. Biol. Med., 2015. 79: 253-263. 13. C. Denise, et al., 5-Fluorouracil Resistant Colon Cancer Cells are Addicted to OXPHOS to Survive and Enhance Stem-Like Traits. Oncotarget, 2015. 6(39): 41706-41721. 14. G. Burnstock, Purinergic Signalling. Br. J. Pharmacol., 2006. 147: S172-S181. 15. P.F. Liu and C. Park, Selective Stabilization of a Partially Unfolded Protein by a Metabolite. J. Mol. Biol., 2012. 422(3): 403-413. 16. A. Fersht, Structure and Mechanism in Protein Science. Freeman, 1999. New York. 17. X. Du, et al., Insights into Protein-Ligand Interactions: Mechanisms, Models, and Methods. Int. J. Mol. Sci., 2016. 17(2): 34. 18. T.T. Caradoc-Davies, et al., Crystal Structures of Escherichia coli Uridine Phosphorylase in Two Native and Three Complexed Forms Reveal Basis of Substrate Specificity, Induced Conformational Changes and Influence of Potassium. J. Mol. Biol., 2004. 337(2): 337-54. 19. Y. Chang, et al., Simplified Proteomics Approach to Discover Protein-Ligand Interactions. Protein Sci., 2012. 21(9): 1280-7. 20. J.K. Myers, C.N. Pace, and J.M. Scholtz, Denaturant M-Values and Heat-Capacity Changes - Relation to Changes in Accessible Surface-Areas of Protein Unfolding. Protein Sci., 1995. 4(10): 2138-2148. 21. J.M. Ploeser and H.S. Loring, The Ultraviolet Absorption Spectra of the Pyrimidine Ribonucleosides and Ribonucleotides. J. Biol. Chem., 1949. 178(1): 431-7. 22. A.C. Saucier, et al., Ciliary Dynein Conformational Changes as Evidenced by the Extrinsic Fluorescent Probe 8-Anilino-1-Naphthalenesulfonate. Biochemistry, 1985. 24(26): 7581-5. 23. L. Stryer, The Interaction of a Naphthalene Dye with Apomyoglobin and Apohemoglobin. A Fluorescent Probe of Non-Polar Binding Sites. J. Mol. Biol., 1965. 13(2): 482-95. 24. T. Yoshimura, Monitoring Protein Conformational Changes During Membrane Fusion. Methods Enzymol., 1993. 221: 72-82. 25. R.A. Cardone, V. Casavola, and S.J. Reshkin, The Role of Disturbed pH Dynamics and the Na+/H+ Exchanger in Metastasis. Nat. Rev. Cancer, 2005. 5(10): 786-795. 26. C. Stock and S.F. Pedersen, Roles of pH and the Na+/H+ Exchanger NHE1 in Cancer: From Cell Biology and Animal Models to an Emerging Translational Perspective? Semin. Cancer Biol., 2017. 43: 5-16. 27. A.L. Mallam and S.E. Jackson, A Comparison of the Folding of Two Knotted Proteins: YbeA and YibK. J. Mol. Biol., 2007. 366(2): 650-665. 28. N. Wolff, et al., Histidine pK(a) Shifts and Changes of Tautomeric States Induced by the Binding of Gallium-Protoporphyrin IX in the Hemophore HasA(SM). Protein Sci., 2002. 11(4): 757-765. 29. C. Valery, et al., Atomic View of the Histidine Environment Stabilizing Higher-pH Conformations of pH-Dependent Proteins. Nat. Commun., 2015. 6: 8.
摘要: 人類的第一型尿苷磷酸化酶(human uridine phosphorylase 1, hUP1)是嘧啶回收路徑(Pyrimidine salvage pathway)中重要的酵素之一,為細胞內將核酸重新再利用所不可或缺的蛋白質,此酵素亦是催化化療前趨藥物5-fluorouracil的重要蛋白質。然而,近期發現對5-fluorouracil具有抗藥性的癌細胞,其細胞內通常累積了較高濃度的ATP,同時,本實驗室先前的研究也發現ATP會對E. coli的尿苷磷酸化酶造成去穩定化的現象。因此,我們假設高濃度的ATP也會對hUP1之蛋白質折疊及功能造成影響,並以此機制探討高濃度ATP與癌細胞產生5-FU抗藥性的可能關係。 由實驗結果顯示,ATP的確會對hUP1造成明顯的去穩定化現象,且去穩定化的程度同時受到ATP以及hUP1的濃度所影響;此外,ATP的加入可明顯累積hUP1的部分展開中介態(partially unfolded intermediate state)。更有趣的是,當我們逐步提高ATP的濃度時可同時觀察到hUP1的酵素活性降低至完全的被抑制。我們也發現ATP具有提升hUP1展開速率的作用,而此作用可能同時伴隨著hUP1的展開速率決定步驟之改變。綜合以上結果,我們推測癌細胞內高濃度ATP之所以導致癌細胞對5-FU具有抗藥性,可能與ATP抑制hUP1的酵素活性有關,進而降低了5-FU在癌細胞內的治療效果。
Human Uridine phosphorylase 1 (hUP1) is an important enzyme in pyrimidine salvage pathway. This enzyme catalyzes the reversible phosphorylysis of uridine to uracil and ribose-1-phosphate. Meanwhile, the function of this enzyme is critical in activating 5-fluorouracil for chemotherapy. Interestingly, it was recently found that ATP apparently destabilizes E. coli uridine phosphorylase. However, whether ATP induces similar effect on human uridine phosphorylase 1 is still unknown. Here, we carefully investigated the effect of ATP on the folding and function of human uridine phosphorylase 1. Our results showed that ATP apparently decreases the stability of hUP1, and this destabilizing effect is not only relevant to the concentration of ATP but also hUP1. ANS-binding experiment suggests that ATP possibly accumulates the partially unfolded intermediate state of hUP1 for this apparent destabilization. Moreover, activity assay indicates that increased concentration of ATP simultaneously decreases the protein stability and inhibits the enzymatic activity. We also observed that ATP accelerates the unfolding rate of hUP1 with specific two phases. These results imply that ATP changes the protein folding and inhibits the enzyme activity of hUP1. This effect is possibly related to the efficacy of 5-FU in cancer cells that is well known with high-level of ATP for unconstrained proliferation.
文章公開時間: 2017-07-21
Appears in Collections:食品暨應用生物科技學系



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