Previous work showed that from all cellular proteins, the endoplasmic reticulum (ER) resident proteins are most sensitive to oxidative stress [hydrogen peroxide (H(2)O(2))], as determined using the oxidation-sensitive, membrane-permeable, acetylTyrFluo probe. Because of the importance of these proteins in proper cellular functioning, we studied (a) whether modifying the cellular redox state/antioxidant status alters the susceptibility of those proteins toward H(2)O(2) oxidative stress and (b) whether H(2)O(2) affects ER function with regard to protein folding. The cellular redox and/or antioxidative capacity was modified in several ways. Lowering the capacity increased H(2)O(2)-induced protein oxidation, and increasing the capacity lowered H(2)O(2)-induced protein oxidation. The effect of H(2)O(2) on ER-related protein maturation was investigated, using the maturation of the low-density lipoprotein receptor as a model. Its maturation was not affected at low concentrations of H(2)O(2) (< or = 400 micro M), which do result in oxidation of ER resident proteins. Maturation was slowed down or reversibly inhibited at higher concentrations of H(2)O(2) (1.5-2.0 mM). These results might be caused by several events, including oxidation of the low-density lipoprotein receptor itself or ER resident proteins resulting in decreased folding (capacity). Alternatively, oxidation of cytosolic proteins involved in ER Golgi transport might attenuate transport and maturation. Clearly, the mechanism(s) responsible for the impairment of maturation need further investigation.

No abstract

Recently, we demonstrated that hydrogen peroxide (H2O2) inhibits the internalization of the epidermal growth factor (EGF) receptor and the EGF-induced mono-ubiquitination of EGF receptor pathway substrate clone #15 (Eps15) in fibroblasts. In addition, it was suggested that EGF receptor internalization might be inhibited by H2O2 by inhibition of ubiquitination of proteins involved in endocytosis. Here, we show that H2O2 also inhibits the poly-ubiquitination of the EGF receptor in fibroblasts. Furthermore, recovery of the cells resulted in re-establishment of ubiquitination of both the EGF receptor and Eps15 and coincided with restoration of internalization of those receptors that had bound EGF in the presence of H2O2. In addition, EGF receptor internalization was inhibited by the sulphydryl reagent N-ethylmaleimide (NEM), indicating that intact SH groups might be required for receptor-mediated endocytosis. Furthermore, H2O2 rapidly induced an increase in the cellular ratio of GSSG:GSH (oxidized glutathione:reduced glutathione) and removal of H2O2 resulted in a fast restoration of the ratio of GSSG:GSH. Therefore, these results suggest a relation between the inhibition of internalization ubiquitination and an increase in GSSG:GSH ratio, which strengthens the hypothesis that H2O2 inhibits EGF receptor internalization by an inhibition of ubiquitination of proteins involved in EGF receptor-mediated endocytosis.

No abstract

No abstract