Abstract
A target for protein oxidation by reactive oxygen species (ROS) is tyrosine, which may form the age- and/or disease-related biomarkers dityrosine, chlorotyrosine and nitrotyrosine. It was already recognized that immunochemical assays should facilitate studies on the role of oxidative stress in aging and chronic disease and simplify the evaluation of
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therapeutic approaches. This thesis describes a contribution to the field of measuring protein oxidation by introducing and utilising a novel oxidation sensitive probe. Upon oxidation the fluorescein-labelled tyramine (hence called TyrFluo) may couple to oxidized proteins carrying a tyrosyl radical by forming a dityrosine bond. Exposure of the cells to hydrogen peroxide (H2O2) in the presence of either TyrFluo or (the acetylated cellmembrane permeable) acTyrFluo gave a cellular labelling characteristic for each probe (immunodetection was performed with an HRP-conjugated polyclonal antibody against the fluorescein moiety) independent of the abundance of a particular protein. In order to identify those intracellular proteins that are most susceptible to oxidation by H2O2 we have used acTyrFluo to label proteins in normal human dermal fibroblasts (NHDF). 2D-PAGE and subsequent Western-blotting were performed on a mixed sample of acTyrFluo-labelled and 35S-methionine-labelled proteins of NHDF. The identification of TyrFluo-labelled proteins was achieved by matching the obtained immunoblot and the autoradiogram to our reference database of the human MRC-5 fibroblasts. This revealed that the labelled proteins were associated with the endoplasmic reticulum (ER; i.e. BiP/GRP78, calnexin, GRP94, PDI, and GRP58/Erp57). In agreement, fluorescence microscopy showed a co-localization of the acTyrFluo-labelled proteins with the KDEL-receptor ERD2 on the ER membrane. Since H2O2 is a weak oxidant we infer that it must be converted first into a hydroxyl radical (OH.) in the ER (possibly. by a Fenton reaction using transition metal ions available in the ER) for proteins and acTyrFluo to become oxidized. In addition, by altering the anti-oxidative capacities of the cells (e.g. GSH content) the extent of protein labelling could be modulated. To investigate whether ER-related protein folding/maturation is impaired after subjecting cells to oxidative stress the maturation of a [35S]-methionine labelled model protein, the low-density lipoprotein receptor (LDLr), was followed in pulse-chase experiments under oxidative conditions. Under the same conditions the ratio of GSH versus GSSG was measured and related to the degree of maturation of the LDLr. The velocity of LDLr-maturation was reduced or even abolished in an H2O2-dependent manner. This impairment may result from oxidized folding proteins, sulfhydryls of LDLr, glycosylating enzymes in the ER and/or Golgi, or of eradicated vesicular transport towards the Golgi complex. However, when cells were allowed to recover after the oxidation maturation could be restored. This restoration depended protein synthesis, since blocking of translation by cycloheximide prevented restoration of maturation. In addition, the low GSH/GSSG balance could not be restored during this recovery period. This impaired maturation cannot be put on the account of the ER solely. The transport towards the Golgi complex may also be reduced dramatically, after the H2O2 treatment. The 'free radical theory of aging' describes the age-related accumulation of radical-induced damage to biomolecules and has been extended to the oxidative "garbage catastrophe theory". According to this theory, aging may derive from imperfect clearance of oxidatively damaged, relatively indigestible material, the accumulation of which further hinders cellular catabolic and anabolic functions and mainly affects postmitotic non-proliferating cells. Damage to the ER, and its subsequent impaired functionality may be involved in the process of aging. There are several phenomena that suggest its involvement. The contribution to the age-related accumulation of damaged protein as a consequence of the oxidation of ER resident proteins is discussed.
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