Endoplasmic reticulum resident proteins of normal human dermal fibroblasts are the major targets for oxidative stress induced by hydrogen peroxide.
The membrane-permeable fluorescein-labelled tyramine conjugate (acetylTyrFluo) was used to identify the proteins of normal human dermal fibroblasts most susceptible to oxidation by hydrogen peroxide [Van der Vlies, Wirtz and Pap (2001) Biochemistry 40, 7783-7788]. By exposing the cells to H(2)O(2) (0.1 mM for 10 min), TyrFluo was covalently linked to target proteins. TyrFluo-labelled and [(35)S]Met-labelled cell lysates were mixed and subjected to two-dimensional PAGE. After Western blotting the (35)S-labelled proteins were visualized by autoradiography and the TyrFluo-labelled proteins by using anti-fluorescein antibody. The TyrFluo-labelled proteins were matched with the (35)S-labelled proteins and identified by comparison with our mastermap of proteins. Protein disulphide isomerase (PDI), IgG-binding protein (BiP), calnexin, endoplasmin and glucose-regulated protein 58 (endoplasmic reticulum protein 57/GRP58) were identified as targets of oxidation. All these proteins reside in the endoplasmic reticulum and are part of the protein folding machinery. In agreement, confocal laser scanning microscopy showed co-localization of TyrFluo-labelled proteins and the KDEL receptor ERD-2, a marker for the endoplasmic reticulum.
van der Vlies
Dennis
D
Centre for Biomembranes and Lipid Enzymology, Department of Biochemistry of Lipids, Institute of Biomembranes, Utrecht University, The Netherlands.
Pap
Eward H W
EH
Post
Jan Andries
JA
Celis
Julio E
JE
Wirtz
Karel W A
KW
D015153:Blotting, Western; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D018613:Microscopy, Confocal; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism
D006861:Hydrogen Peroxide; D007770:L-Lactate Dehydrogenase; D010100:Oxygen