Plitzko Birte, Havemeyer Antje, Kunze Thomas, Clement Bernd
From the Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-Albrechts-University of Kiel, 24118 Kiel, Germany.
From the Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-Albrechts-University of Kiel, 24118 Kiel, Germany
J Biol Chem. 2015 Apr 17;290(16):10126-35. doi: 10.1074/jbc.M115.640052. Epub 2015 Feb 23.
N-Hydroxylated nucleobases and nucleosides as N-hydroxylaminopurine (HAP) or N-hydroxyadenosine (HAPR) may be generated endogenously in the course of cell metabolism by cytochrome P450, by oxidative stress or by a deviating nucleotide biosynthesis. These compounds have shown to be toxic and mutagenic for procaryotic and eucaryotic cells. For DNA replication fidelity it is therefore of great importance that organisms exhibit effective mechanisms to remove such non-canonical base analogs from DNA precursor pools. In vitro, the molybdoenzymes mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) have shown to be capable of reducing N-hydroxylated base analogs and nucleoside analogs to the corresponding canonical nucleobases and nucleosides upon reconstitution with the electron transport proteins cytochrome b5 and NADH-cytochrome b5 reductase. By RNAi-mediated down-regulation of mARC in human cell lines the mARC-dependent N-reductive detoxication of HAP in cell metabolism could be demonstrated. For HAPR, on the other hand, the reduction to adenosine seems to be of less significance in the detoxication pathway of human cells as HAPR is primarily metabolized to inosine by direct dehydroxylamination catalyzed by adenosine deaminase. Furthermore, the effect of mARC knockdown on sensitivity of human cells to HAP was examined by flow cytometric quantification of apoptotic cell death and detection of poly (ADP-ribose) polymerase (PARP) cleavage. mARC2 was shown to protect HeLa cells against the apoptotic effects of the base analog, whereas the involvement of mARC1 in reductive detoxication of HAP does not seem to be pivotal.
N-羟基化核碱基和核苷，如N-羟基氨基嘌呤（HAP）或N-羟基腺苷（HAPR），可能在细胞代谢过程中由细胞色素P450、氧化应激或偏离的核苷酸生物合成内源性产生。这些化合物已被证明对原核细胞和真核细胞具有毒性和致突变性。因此，对于DNA复制保真度而言，生物体展现出从DNA前体池中去除此类非规范碱基类似物的有效机制至关重要。在体外，钼酶线粒体偕胺肟还原成分1和2（mARC1和mARC2）在与电子传递蛋白细胞色素b5和NADH-细胞色素b5还原酶重组后，已被证明能够将N-羟基化碱基类似物和核苷类似物还原为相应的规范核碱基和核苷。通过RNA干扰介导的人细胞系中mARC的下调，可以证明细胞代谢中mARC依赖的HAP的N-还原解毒作用。另一方面，对于HAPR，还原为腺苷在人细胞解毒途径中的重要性似乎较低，因为HAPR主要通过腺苷脱氨酶催化直接脱羟基化代谢为肌苷。此外，通过流式细胞术定量凋亡细胞死亡和检测聚（ADP-核糖）聚合酶（PARP）裂解，研究了mARC敲低对人细胞对HAP敏感性的影响。结果表明，mARC2可保护HeLa细胞免受碱基类似物的凋亡作用，而mARC1参与HAP的还原解毒作用似乎并非关键因素。
