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 H2O2 (0.1mM for 10min), TyrFluo was covalently linked to target proteins. TyrFluo-labelled and [35S]Met-labelled cell lysates were mixed and subjected to two-dimensional PAGE. After Western blotting the 35S-labelled proteins were visualized by autoradiography and the TyrFluo-labelled proteins by using anti-fluorescein antibody. The TyrFluo-labelled proteins were matched with the 35S-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.
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September 2002
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Research Article|
September 15 2002
Endoplasmic reticulum resident proteins of normal human dermal fibroblasts are the major targets for oxidative stress induced by hydrogen peroxide
Dennis van der VLIES;
Dennis van der VLIES
∗Centre for Biomembranes and Lipid Enzymology, Department of Biochemistry of Lipids, Institute of Biomembranes, Utrecht University, P.O. Box 80.054, 3508 TB Utrecht, The Netherlands
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Eward H.W. PAP;
Eward H.W. PAP
∗Centre for Biomembranes and Lipid Enzymology, Department of Biochemistry of Lipids, Institute of Biomembranes, Utrecht University, P.O. Box 80.054, 3508 TB Utrecht, The Netherlands
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Jan Andries POST;
Jan Andries POST
†Department of Molecular Cell Biology, Institute of Biomembranes, Utrecht University, The Netherlands,
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Julio E. CELIS;
Julio E. CELIS
‡Institute of Cancer Biology and Danish Centre for Human Genome Research, Copenhagen O, Denmark
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Karel W.A. WIRTZ
Karel W.A. WIRTZ
1
∗Centre for Biomembranes and Lipid Enzymology, Department of Biochemistry of Lipids, Institute of Biomembranes, Utrecht University, P.O. Box 80.054, 3508 TB Utrecht, The Netherlands
1To whom correspondence should be addressed (e-mail K.W.A.Wirtz@chem.uu.nl).
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Publisher: Portland Press Ltd
Received:
April 18 2002
Revision Received:
June 10 2002
Accepted:
June 19 2002
Accepted Manuscript online:
June 19 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 366 (3): 825–830.
Article history
Received:
April 18 2002
Revision Received:
June 10 2002
Accepted:
June 19 2002
Accepted Manuscript online:
June 19 2002
Citation
Dennis van der VLIES, Eward H.W. PAP, Jan Andries POST, Julio E. CELIS, Karel W.A. WIRTZ; Endoplasmic reticulum resident proteins of normal human dermal fibroblasts are the major targets for oxidative stress induced by hydrogen peroxide. Biochem J 15 September 2002; 366 (3): 825–830. doi: https://doi.org/10.1042/bj20020618
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