Endoplasmic reticulum resident proteins of normal human dermal fibroblasts are the major targets for oxidative stress induced by hydrogen peroxide
| Autor: | E.H.W. Pap, Julio E Celis, Dennis van der Vlies, Jan A. Post, Karel W. A. Wirtz |
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| Rok vydání: | 2002 |
| Předmět: |
KDEL
Blotting Western Immunoblotting Biology Endoplasmic Reticulum Biochemistry Calnexin Humans Electrophoresis Gel Two-Dimensional Protein disulfide-isomerase Endoplasmin Molecular Biology Cells Cultured Microscopy Confocal Dose-Response Relationship Drug L-Lactate Dehydrogenase Endoplasmic reticulum STIM1 Hydrogen Peroxide Cell Biology Fibroblasts Molecular biology Oxygen Blot Oxidative Stress Microsome Research Article |
| Zdroj: | Biochemical Journal. 366:825-830 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj20020618 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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