A combined in vitro/bioinformatic investigation of redox regulatory mechanisms governing cell cycle progression

Abstract

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 (H₂DCFDA), and DRAQ5, respectively. GSH content was significantly greater in G₂/M compared with G₁phase cells, whereas GSH was intermediate in S phase cells. ROS content was similar among phases. Together, these data demonstrate that G₂/M cells are more reduced than G₁cells. 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 G₂/M phase. As the G₂/M phase is more reduced than the G₁phase, 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.