A combined in vitro/bioinformatic investigation of redox regulatory mechanisms governing cell cycle progression
A combined in vitro/bioinformatic investigation of redox regulatory mechanisms governing cell cycle progression
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DOI：
10.1152/physiolgenomics.00058.2004
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发表时间：
2004-07-08
影响因子：
4.6
通讯作者：
Gaskins, HR
中科院分区：
文献类型：
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作者：
Conour, JE;Graham, WV;Gaskins, HR
细胞内的还原-氧化（氧化还原）环境影响细胞周期的进展，然而，基本机制知之甚少。为了研究潜在的机制，通过流式细胞术，通过分别用一氯二亚胺、2 '，7'-二氯氢荧光素二乙酸酯（H（2）DCFDA）和DRAQ 5测量还原型谷胱甘肽（GSH）、活性氧（ROS）和DNA含量，在中国仓鼠卵巢成纤维细胞中表征每个细胞周期阶段的细胞内氧化还原环境。GSH在G（2）/M期细胞中的含量显著高于G（1）期细胞，而在S期细胞中的含量则介于两者之间。各阶段活性氧含量相似。总之，这些数据表明G（2）/M细胞比G（1）细胞减少得更多。定义调控机制的常规方法本质上是主观的，并且集中于单个蛋白质/途径。蛋白质组数据库提供了克服这些固有局限性的手段。因此，开发了一种新的生物信息学方法来彻底鉴定含有氧化还原敏感蛋白基序的推定的氧化还原调节细胞周期蛋白。使用InterPro（http：//www.ebi.ac.uk/interpro/）数据库，我们将536个氧化还原敏感基序分类为：1）活性/功能位点半胱氨酸，2）电子传递，3）血红素，4）铁结合，5）锌结合，6）金属结合（非Fe/Zn），和7）二硫化物。将该列表与来自Swiss-Prot和SpTrEMBL（http：//us.expasy.org/sprot/）的1，634种细胞周期相关蛋白进行比较，揭示了92种候选蛋白。四分之三（69/92）的候选蛋白质在转录，核苷酸代谢，（去）磷酸化和（去）泛素化的中央细胞周期过程中发挥作用。大多数氧化剂敏感候选蛋白（68.9%）在G（2）/M期起作用。由于G（2）/M相比G（1）相还原得更多，氧化剂敏感性蛋白质可能受到细胞内氧化还原环境振荡的时间调节。结合细胞内氧化还原区室化的证据，我们提出了一个时空机制，在功能上将振荡的细胞内氧化还原环境与细胞周期进程联系起来。
The intracellular reduction-oxidation ( redox) environment influences cell cycle progression; however, underlying mechanisms are poorly understood. To examine potential mechanisms, the intracellular redox environment was characterized per cell cycle phase in Chinese hamster ovary fibroblasts via flow cytometry by measuring reduced glutathione (GSH), reactive oxygen species (ROS), and DNA content with monochlorobimane, 2', 7'-dichlorohydrofluorescein diacetate (H(2)DCFDA), and DRAQ5, respectively. GSH content was significantly greater in G(2)/M compared with G(1) phase cells, whereas GSH was intermediate in S phase cells. ROS content was similar among phases. Together, these data demonstrate that G(2)/M cells are more reduced than G(1) cells. Conventional approaches to define regulatory mechanisms are subjective in nature and focus on single proteins/pathways. Proteome databases provide a means to overcome these inherent limitations. Therefore, a novel bioinformatic approach was developed to exhaustively identify putative redox-regulated cell cycle proteins containing redox-sensitive protein motifs. Using the InterPro (http://www.ebi.ac.uk/interpro/) database, we categorized 536 redox-sensitive motifs as: 1) active/functional-site cysteines, 2) electron transport, 3) heme, 4) iron binding, 5) zinc binding, 6) metal binding (non-Fe/Zn), and 7) disulfides. Comparing this list with 1,634 cell cycle-associated proteins from Swiss-Prot and SpTrEMBL (http://us.expasy.org/sprot/) revealed 92 candidate proteins. Three-fourths ( 69 of 92) of the candidate proteins function in the central cell cycle processes of transcription, nucleotide metabolism, (de) phosphorylation, and (de)ubiquitinylation. The majority of oxidant-sensitive candidate proteins (68.9%) function during G(2)/M phase. As the G(2)/M phase is more reduced than the G(1) phase, oxidant-sensitive proteins may be temporally regulated by oscillation of the intracellular redox environment. Combined with evidence of intracellular redox compartmentalization, we propose a spatiotemporal mechanism that functionally links an oscillating intracellular redox environment with cell cycle progression.