Division of Neurofunctional Genomics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan.
Mutat Res. 2010 Nov 28;703(1):37-42. doi: 10.1016/j.mrgentox.2010.06.005. Epub 2010 Jun 11.
To identify novel nucleotide pool sanitizing enzymes, we have established a comprehensive screening system for damaged nucleotide-binding proteins based on proteomics technology. In the screening system, affinity chromatography with resins carrying various damaged nucleotides is used for the purification of binding proteins, and the purified proteins are identified by mass-spectrometry. Inosine triphosphate (ITP) is a deleterious damaged nucleotide, and can be generated by nitrosative deamination of ATP or phosphorylation of inosine monophosphate (IMP). Using the above system, we performed screens for ITP-binding proteins from mouse and human cell extracts, and identified several ITP-binding enzymes. We identified both mouse inosine triphosphatase (ITPA) and human ITPA, well-known ITP hydrolyzing enzymes, as ITP-binding proteins. These results support the validity of this screening system. In addition to ITPA, we identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an ITP-binding protein. Biochemical analysis revealed that NUDT16 selectively hydrolyzes deoxyinosine diphosphate (dIDP) and IDP to deoxyinosine monophosphate (dIMP) and IMP, respectively. dITP and ITP are also hydrolyzed by NUDT16 to a lesser extent. The knockdown of NUDT16 in HeLa MR cells suppressed cell proliferation, and was accompanied by a significantly increased accumulation of strand breaks in nuclear DNA, suggesting that NUDT16 has an essential role in the maintenance of genome stability. RS21-C6, another ITP-binding protein identified in our screen, binds not only to ITP, but also to ATP. RS21-C6 hydrolyzes dCTP and 5-halo-dCTP, but does not hydrolyze ITP or ATP. It is likely that RS21-C6 may control dCTP levels or eliminate 5-halo-dCTP in the nucleotide pools. In conclusion, the results of these studies show that our screening system is applicable in studying the health effects of damaged nucleotides and cellular sanitizing systems for nucleotide pools.
为了鉴定新的核苷酸池净化酶，我们基于蛋白质组学技术建立了一个全面的损伤核苷酸结合蛋白筛选系统。在该筛选系统中，采用携带各种损伤核苷酸的树脂进行亲和层析来纯化结合蛋白，并用质谱法鉴定纯化蛋白。肌苷三磷酸（ITP）是一种有害的损伤核苷酸，可由 ATP 的硝化脱氨或肌苷单磷酸（IMP）的磷酸化产生。我们使用上述系统，从鼠和人细胞提取物中筛选出 ITP 结合蛋白，并鉴定出几种 ITP 结合酶。我们鉴定出两种 ITP 水解酶，即鼠肌苷三磷酸酶（ITPA）和人 ITPA，都是 ITP 水解酶，为 ITP 结合蛋白。这些结果支持了该筛选系统的有效性。除了 ITPA，我们还鉴定出人核苷酸二磷酸连接部分 X 基序 16（NUDT16）蛋白为 ITP 结合蛋白。生化分析表明，NUDT16 选择性水解脱氧肌苷二磷酸（dIDP）和 IDP 分别生成脱氧肌苷单磷酸（dIMP）和 IMP。dITP 和 ITP 也被 NUDT16 以较小的程度水解。在 HeLa MR 细胞中敲低 NUDT16 会抑制细胞增殖，并伴随着核 DNA 链断裂的显著增加，表明 NUDT16 在维持基因组稳定性方面具有重要作用。在我们的筛选中鉴定出的另一种 ITP 结合蛋白 RS21-C6，不仅与 ITP 结合，还与 ATP 结合。RS21-C6 水解 dCTP 和 5-卤代-dCTP，但不水解 ITP 或 ATP。RS21-C6 可能控制 dCTP 水平或消除核苷酸池中的 5-卤代-dCTP。总之，这些研究结果表明，我们的筛选系统适用于研究损伤核苷酸的健康影响和核苷酸池的细胞净化系统。
