Mechanisms of Nrf2 protection in astrocytes as identified by quantitative proteomics and siRNA screening.
The Nrf2 (NF-E2 related factor 2)-ARE (antioxidant response element) pathway controls a powerful array of endogenous cellular antioxidant systems and is an important pathway in the detoxification of reactive oxygen species (ROS) in the brain. Using a combination of quantitative proteomics and siRNA screening, we have identified novel protective mechanisms of the Nrf2-ARE pathway against oxidative stress in astrocytes. Studies from our lab and others have shown Nrf2 overexpression protects astrocytes from oxidative stress. However, the exact mechanisms by which Nrf2 elicits these effects are unknown. In this study, we show that induction of Nrf2 reduces levels of reactive oxygen species (ROS) produced by various oxidative stressors and results in robust cytoprotection. To identify the enzymes responsible for these effects, we used stable isotope labeling by amino acids in cell culture (SILAC) and quantitative shotgun proteomics to identify 72 Nrf2-regulated proteins in astrocytes. We hypothesized a subset of these proteins might play a critical role in Nrf2 protection. In order to identify these critical proteins, we used bioinformatics to narrow our target list of proteins and then systematically screened each candidate with siRNA to assess the role of each in Nrf2 protection. We screened each target against H2O2, tert-butyl hydroperoxide, and 4-hydroxynonenal and subsequently identified three enzymes-catalase, prostaglandin reductase-1, and peroxiredoxin-6-that are critical for Nrf2-mediated protection in astrocytes.
Division of Pharmaceutical Science, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
James A
JA
Dowell
Jeffrey A
JA
Johnson
D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000447:Aldehydes / Q000378:metabolism; D000818:Animals; D061745:Antioxidant Response Elements; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D002374:Catalase / Q000378:metabolism; D005786:Gene Expression Regulation; D005978:Glutathione / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D051267:NF-E2-Related Factor 2 / Q000235:genetics* / Q000378:metabolism*; D018384:Oxidative Stress / Q000235:genetics; D054465:Peroxiredoxin VI / Q000378:metabolism; D040901:Proteomics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D020122:tert-Butylhydroperoxide / Q000378:metabolism
D000447:Aldehydes; D051267:NF-E2-Related Factor 2; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D000426:Alcohol Dehydrogenase; D054465:Peroxiredoxin VI; D002374:Catalase; D005978:Glutathione; C027576:4-hydroxy-2-nonenal