Chemical Research on Pathological Role of Nitric Oxide

一氧化氮病理作用的化学研究

• 批准号: 12480173
• 负责人: MAKINO Keisuke
• 金额: $ 9.09万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12480173/
• 关键词: Nitric Oxide; Cancer Promotion; Mechanism for Reaction with Gene; Deoxyoxanosine; Diazoate Intermediates; Mechanism of Oxidative Deamination; Repair Enzymes; Isomeric Four-Stranded Structures of Telomere; 塩基損傷; 腫瘍学; 遺伝子損傷; 発ガンと細胞死; デオキシオキザノシン(

Exploration of cancer promotion mechanism caused by NO overproduced in inflammation is of great importance. Before this project, we demonstrated that NO converts dGuo to deoxyoxanosine (dOxo) with high yield and dCyd to dCyd-diazoate, a reaction intermediate. In this project, we have revealed the followings.(1) We have identified dGuo-diazoate as an intermediate for the dGuo conversion to dOxo and established the complete reaction mechanism. Based on the mechanism, oxydative deamination which has been thought to be responsible for the base damages by NO, has been found to be not true for dGuo, and ring-opening initiated by NO attack to NH_3 followed by the diazoate formation, water molecule addition to the resulting cations, and ring-closure has been found to be involved.(2) Closs-linking reactions between dOxo and diazoates have been identified, indicative of promotion to cancer and cellular death by NO. Also it has been determined that 5-methycytidine, major component in the CpG site … More of inhibitory gene p53 which is known as a hot spot for cancer promotion, is converted to ^<5me>dCyd-diazoate.(3) Detection methods for dOxo have been developed based on capillary electrophoresis and HPLC combined with fluorescent labeling. Using the HPLC method with the detection limit of 2-3 fmol, the amount of dOxo generated in E. coli exposed to HNO_2 was estimated to be less than the detection limit.(4) To explore structural change of DNA caused by dOxo formation and the following misreading mechanism, we tried to prepare phosphoroamidite monomer. Because of the low reactivity of 5'-OH, the yield of DMTr-dOxo was 30%.(5) We explored repair enzymes recognizing dOxo among known repair enzymes and found that AlkA and Endo VIII show a very slight deletion activity.(6) A DNA oligomer, dCCCTAA which is a sequence repeated in human telomere susceptible to NO attack, has been investigated by 2D-NMR and the three isomeric four-stranded structures have been found, implying that such a complex structural diversity of such a simple sequence may play a role controlling cell death and cancer promotion. Less

探索炎症过程中NO过量产生的促癌机制具有重要意义。在这个项目之前，我们证明了 NO 可以高产率地将 dGuo 转化为脱氧氧核苷 (dOxo)，并将 dCyd 转化为反应中间体 dCyd-重氮酸盐。在这个项目中，我们揭示了以下内容：(1)我们确定了dGuo-重氮酸酯作为dGuo转化为dOxo的中间体，并建立了完整的反应机理。基于该机制，人们认为氧化脱氨作用是造成NO碱基损伤的原因，但发现dGuo并不成立，NO攻击NH_3引发开环，随后形成重氮酸盐，水分子加成到所得阳离子上，并闭环。(2)已经确定了dOxo和重氮酸盐之间的交联反应，表明促进作用 NO 导致癌症和细胞死亡。还确定了被称为促癌热点的抑制基因p53的CpG位点的主要成分5-甲基胞苷被转化为^<5me>dCyd-diazoate。(3)基于毛细管电泳和HPLC结合荧光标记，开发了dOxo的检测方法。采用检测限为2-3 fmol的HPLC方法，估计HNO_2作用下大肠杆菌中产生的dOxo量低于检测限。(4)为了探究dOxo形成引起的DNA结构变化以及随后的误读机制，我们尝试制备亚磷酰胺单体。由于5'-OH的反应性较低，DMTr-dOxo的产率为30%。(5)我们在已知的修复酶中探索了识别dOxo的修复酶，发现AlkA和Endo VIII表现出非常轻微的缺失活性。(6)DNA寡聚体dCCCTAA是人端粒中易受NO攻击的重复序列，已通过2D-NMR和三种方法进行了研究。 异构四链结构的发现，意味着如此简单的序列如此复杂的结构多样性可能起到控制细胞死亡和促进癌症的作用。较少的

项目成果

T.Suzuki, H.Ide, M.Yamada, N.Endo, K.Kanaori, K.Tajima, T.Morii, K.Makino: "Formation of 2'-deoxyoxanosine from 2'-deoxyguanosine and nitrous acid : mechanism and intermediates"Nucleic Acids Res.. 28. 544-551 (2000)

T.Suzuki、H.Ide、M.Yamada、N.Endo、K.Kanaori、K.Tajima、T.Morii、K.Makino：“从 2-脱氧鸟苷和亚硝酸形成 2-脱氧氧杂苷：机制和

Masaoka, H.Terato, A.Honsho, Y.Ohyama, T.Suzuki, M.Yamada, K.Makino, H.Ide: "Preparation and enzymatic recognition of guanine lesions induced by nitrogen oxide"Nucleic Acids Res. Symp. Ser.. 44. 87-88 (2000)

Masaoka、H.Terato、A.Honsho、Y.Ohyama、T.Suzuki、M.Yamada、K.Makino、H.Ide：“氮氧化物诱导的鸟嘌呤损伤的制备和酶促识别”核酸研究。

T.Morii, T.Tanaka, S.Sato, M.Hagiwara, Y.Aizawa, K.Makino: "A general strategy to determine a target DNA sequence of a short peptide : Application to a D-peptide"J. Am. Chem. Soc.. 124. 180-181 (2002)

T.Morii、T.Tanaka、S.Sato、M.Hagiwara、Y.Aizawa、K.Makino：“确定短肽目标 DNA 序列的一般策略：在 D 肽中的应用”J.

T.Suzuki, M.Yamada, T.Nakamura, H.Ide, K.Kanaori, K.Tajima, T.Morii, K.Makino: "Formation of a fairy stable diazoate intermediate of 5-methyl-2'-deoxycytidine by HNO_2 and NO, and its implication to a novel mutation mechanism in CpG site"Bioorg. & Med. Ch

T.Suzuki、M.Yamada、T.Nakamura、H.Ide、K.Kanaori、K.Tajima、T.Morii、K.Makino：“5-甲基-2-脱氧胞苷的稳定重氮酸酯中间体的形成

吉川敏一編: "一酸化窒素によるDNA傷害"酸化ストレス-フリーラジカル医学生物学の最前線 医歯薬出版). 4 (2001)

吉川敏和主编：“一氧化氮引起的DNA损伤”氧化应激-自由基医学生物学的最前沿石药出版4(2001)。