title: The H2O2-sensitive HyPer protein targeted to the endoplasmic reticulum as a mirror of the oxidizing thiol-disulfide milieu.
abstract: Oxidative protein folding in the endoplasmic reticulum (ER) is associated with the formation of native disulfide bonds, which inevitably results in the formation of hydrogen peroxide (H(2)O(2)). Particularly in pancreatic β-cells with their high secretory activity and extremely low antioxidant capacity, the H(2)O(2) molecules generated during oxidative protein folding could represent a significant oxidative burden. Therefore this study was conducted to elucidate the H(2)O(2) generation during disulfide bond formation in insulin-producing RINm5F cells by targeting and specifically expressing the H(2)O(2)-sensitive biosensor HyPer in the ER (ER-HyPer). In addition the influence of overexpression of the H(2)O(2)-metabolizing ER-resident peroxiredoxin IV (PRDXIV) on H(2)O(2) levels was examined. The ER-HyPer fluorescent protein was completely oxidized, whereas HyPer expressed in cytosol, peroxisomes, and mitochondria was prevalently in the reduced state, indicating a high basal H(2)O(2) concentration in the ER lumen. These results could also be confirmed in non-insulin-producing COS-7 cells. Overexpression of PRDXIV in RINm5F cells effectively protected against H(2)O(2)-mediated toxicity; however, it did not affect the fluorescence signal of ER-HyPer. Moreover, the inhibition of de novo protein synthesis and the associated H(2)O(2) generation by cycloheximide had no influence on the ER-HyPer redox state. Taken together, these findings strongly suggest that the H(2)O(2)-sensitive biosensor reflects exclusively the oxidative milieu in the ER and not the H(2)O(2) concentration in the ER lumen.
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School, 30623 Hannover, Germany.
authors.forename: Ilir
authors.initials: I
authors.lastname: Mehmeti
authors.forename: Stephan
authors.initials: S
authors.lastname: Lortz
authors.forename: Sigurd
authors.initials: S
authors.lastname: Lenzen
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005453:Fluorescence; D015870:Gene Expression; D017930:Genes, Reporter; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D015335:Molecular Probes; D010084:Oxidation-Reduction; D054464:Peroxiredoxins / Q000235:genetics / Q000378:metabolism; D020675:Peroxisomes / Q000187:drug effects / Q000378:metabolism; D017510:Protein Folding; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D004220:Disulfides; D008164:Luminescent Proteins; D015335:Molecular Probes; D013438:Sulfhydryl Compounds; D003513:Cycloheximide; D006861:Hydrogen Peroxide; D054464:Peroxiredoxins