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Phalen, Timothy J.

Cell and Molecular Biology Program

2007

Ph. D.

Reactive oxygen species (ROS) have been shown to play an important and unique role in mitogenic signaling pathways that govern cell proliferation in response to growth factors. The production of hydrogen peroxide (H₂O₂) by mitochondria and NADPH oxidases participate in mitogenic signaling by altering the fhnction of specific protein components along the transduction pathway, including kinases, phosphatases, and transcription factors. The levels of H₂O₂ and other ROS are regulated in part by a variety of small molecule antioxidants and antioxidant enzyme systems which are incorporated into an intricate redox signaling network that controls diverse cell processes. Two-Cys peroxiredoxins (2-Cys Prxs) are abundantly expressed, highly conserved peroxidase enzymes that actively regulate several cell signaling pathways by modulating H₂O₂ levels, and by altering the activity of signaling components via direct protein-protein interactions. Although eukaryotic 2-Cys Prxs have a high affinity for hydroperoxides, they can become temporarily inactivated through hyperoxidation of one of the active site cysteines, thereby forming a sulfinic acid (Prx-SO₂H). 2-Cys Prxs undergo redox-coupled conformational changes that govern oligomeric organization. Hyperoxidation and other factors promote the formation of high molecular weight (HMW) complexes consisting of multiple 2-Cys Prx decamers. The role of 2-Cys Prxs in cell cycle reentry and recovery from cell cycle arrest was investigated using a cell culture model in which quiescent cells are induced to reenter the cell cycle by stimulation with medium containing 10% fetal bovine serum (FBS). Serum stimulation of quiescent mouse C10 lung epithelial cells induced the rapid formation of PrxI and PrxII homodimers, indicating that the PrxI and II catalytic cycles are engaged by mitogenic H₂O₂ production. Hyperoxidation of PrxI and I1 in response to serum was not observed, indicating that 2-Cys Prx inactivation is not required for mitogenic signaling. An H₂O₂ generating system, glucose oxidase (GOx), was used to examine the dose-dependent effects of H₂O₂ on cell cycle progression and arrest as measured by cyclin D1 expression. Doses of H₂O₂ that induced quantitative hyperoxidation of PrxI did not block expression of cyclin D1. Inhibition of cyclin D1 expression and cell cycle arrest did not occur until cells were treated with a threshold dose of H₂O₂ that caused formation of HMW PrxII-SO₂H complexes that appeared to associate with the actin cytoskeleton. When GOx was removed and cells were allowed to recover, cyclin Dl expression and resumption of cell cycle progression correlated with retroreduction of hyperoxidized PrxII and disruption HMW PrxII-SO₂H complexes. Ectopic expression of PrxI and II did not rescue C10 cells from H₂O₂ induced cell cycle arrest, but rather increased the total cellular burden of hyperoxidized PrxI and II, and delayed recovery of cyclin Dl expression and cell cycle progression. These results indicate that hyperoxidation of PrxII may serve as a warning signal for perturbations in H₂O₂ metabolism during mitogenic signaling. Here it is proposed that 2-Cys Prxs act as peroxide dosimeters in which oxidation-coupled structural and oligomeric transitions serve as the mechanism by which they sense and relay changes in subcellular redox stahls to cell signaling machinery.