TY - JOUR
T1 - Differential redox proteomics allows identification of proteins reversibly oxidized at cysteine residues in endothelial cells in response to acute hypoxia
AU - Izquierdo-Álvarez, Alicia
AU - Ramos, Elena
AU - Villanueva, Joan
AU - Hernansanz-Agustín, Pablo
AU - Fernández-Rodríguez, Rubén
AU - Tello, Daniel
AU - Carrascal, Montserrat
AU - Martínez-Ruiz, Antonio
N1 - Funding Information:
This study was supported by the following grants from the Spanish Government to A.M.-R.: CP07/00143 (Miguel Servet program), CSD2007-00020 (RosasNet, Consolider-Ingenio 2010 program) and PS09/00101 . We thank Silvia Juárez and Sergio Ciordia from the Proteomics Facility (CNB-CSIC) and “ProteoRed-ISCIII” platform for their helpful technical support with the protein identification. We thank Lorena Vega Piris and Francisco Rodriguez Salvanés, from the Methodology Unit of the Instituto de Investigación Sanitaria Princesa, for help with statistical analysis. We also thank Drs Jesús Vázquez, Santiago Lamas, María José Calzada and Manuel O. Landázuri for helpful discussions of the results and the manuscript, and Mark Sefton (BiomedRed) for careful revision of the manuscript.
PY - 2012/9/18
Y1 - 2012/9/18
N2 - Adaptation to decreased oxygen availability (hypoxia) is crucial for proper cell function and survival. In metazoans, this is partly achieved through gene transcriptional responses mediated by hypoxia-inducible factors (HIFs). There is abundant evidence that production of reactive oxygen species (ROS) increases during hypoxia, which contributes to the activation of the HIF pathway. In addition to altering the cellular redox balance, leading to oxidative stress, ROS can transduce signals by reversibly modifying the redox state of cysteine residues in certain proteins. Using the "redox fluorescence switch" (RFS), a thiol redox proteomic technique that fluorescently labels reversibly oxidized cysteines, we analyzed endothelial cells subjected to acute hypoxia and subsequent reoxygenation. We observed a general increase in cysteine oxidation during hypoxia, which was reversed by reoxygenation, and two-dimensional electrophoresis revealed the differential oxidation of specific proteins. Using complementary derivatization techniques, we confirmed the modification of individual target proteins and identified specific cysteine residues that were oxidized in hypoxic conditions, thereby overcoming several limitations associated with fluorescence derivatization. These findings provide an important basis for future studies of the role of these modifications in HIF activation and in other acute adaptive responses to hypoxia.
AB - Adaptation to decreased oxygen availability (hypoxia) is crucial for proper cell function and survival. In metazoans, this is partly achieved through gene transcriptional responses mediated by hypoxia-inducible factors (HIFs). There is abundant evidence that production of reactive oxygen species (ROS) increases during hypoxia, which contributes to the activation of the HIF pathway. In addition to altering the cellular redox balance, leading to oxidative stress, ROS can transduce signals by reversibly modifying the redox state of cysteine residues in certain proteins. Using the "redox fluorescence switch" (RFS), a thiol redox proteomic technique that fluorescently labels reversibly oxidized cysteines, we analyzed endothelial cells subjected to acute hypoxia and subsequent reoxygenation. We observed a general increase in cysteine oxidation during hypoxia, which was reversed by reoxygenation, and two-dimensional electrophoresis revealed the differential oxidation of specific proteins. Using complementary derivatization techniques, we confirmed the modification of individual target proteins and identified specific cysteine residues that were oxidized in hypoxic conditions, thereby overcoming several limitations associated with fluorescence derivatization. These findings provide an important basis for future studies of the role of these modifications in HIF activation and in other acute adaptive responses to hypoxia.
KW - Cell signaling
KW - Cysteine oxidation
KW - Post-translational modifications
KW - Redox fluorescence switch (RFS)
KW - Thiol redox proteomics
KW - Two-dimensional electrophoresis (2-DE)
UR - http://www.scopus.com/inward/record.url?scp=84865540710&partnerID=8YFLogxK
U2 - 10.1016/j.jprot.2012.06.035
DO - 10.1016/j.jprot.2012.06.035
M3 - Article
C2 - 22800641
AN - SCOPUS:84865540710
SN - 1874-3919
VL - 75
SP - 5449
EP - 5462
JO - Journal of Proteomics
JF - Journal of Proteomics
IS - 17
ER -