Hydrogen Peroxide-induced Structural Alterations of RNase A

Abstract

Proteins exposed to oxidative stress are degraded via proteolytic pathways. In the present study, we undertook a series of in vitro experiments to establish a correlation between the structural changes induced by mild oxidation of the model protein RNase A and the proteolytic rate found upon exposure of the modified protein toward the isolated 20 S proteasome. Fourier transform infrared spectroscopy was used as a structure-sensitive probe. We report here strong experimental evidence for oxidation-induced conformational rearrangements of the model protein RNase A and, at the same time, for covalent modifications of amino acid side chains. Oxidation-related conformational changes, induced by H 2 O 2 exposure of the protein may be monitored in the amide I region, which is sensitive to changes in protein secondary structure. A comparison of the time-and H 2 O 2 concentration-dependent changes in the amide I region demonstrates a high degree of similarity to spectral alterations typical for temperature-induced unfolding of RNase A. In addition, spectral parameters of amino acid side chain marker bands (Tyr, Asp) revealed evidence for covalent modifications. Proteasome digestion measurements on oxidized RNase A revealed a specific time and H 2 O 2 concentration dependence; at low initial concentration of the oxidant, the RNase A turnover rate increases with incubation time and concentration. Based on these experimental findings, a correlation between structural alterations detected upon RNase A oxidation and proteolytic rates of RNase A is established, and possible mechanisms of the proteasome recognition process of oxidatively damaged proteins are discussed.

The proteasome and its role in the degradation of oxidized proteins

“…Since it is assumed that various oxidants are involved in the formation of various forms of oxidized proteins, it seems to be clear that there should be a common recognition mechanism. It was proposed by the groups of Stadtman and Davies (118)(119)(120) and later confirmed by our group (121)(122)(123)(124) that this common recognition motive is the exposure of hydrophobic amino acids to the surface of the protein, which means at least a partial unfolding of the substrate. The arings of the 20S ''core'' proteasome are responsible for substrate recognition and binding.…”

Proteasome Inhibition in Glyoxal-treated Fibroblasts and Resistance of Glycated Glucose-6-phosphate Dehydrogenase to 20 S Proteasome Degradation in Vitro

“…Oxidized protein degradation by the 20 S proteasome has been studied extensively by different groups (20,(52)(53)(54)(55). Mild oxidation leads to an increased proteolytic susceptibility, whereas highly oxidized protein becomes resistant to degradation by the 20 S proteasome.…”

“…It has been shown that mildly oxidized proteins exhibit decreased thermal and thermodynamic stability (56,57) and bind more efficiently the hydrophobic fluorescent probe ANSA (40). Both the exposure of hydrophobic surface on modified proteins and the loss of secondary structure have been proposed to act as a recognition signal for binding and degradation of the substrate protein by the 20 S proteasome (19,54,55). Because glycated proteins are poor substrates of the 20 S proteasome, structural studies of the glycated forms of G6PDH aimed at assessing their secondary and tertiary structure as well as their conformational stability were performed.…”

Protein Oxidation in Aging: Does It Play a Role in Aging Progression?

“…In contrast to the 26S proteasome, the 20S proteasome is able to recognize unfolded proteins and, therefore, oxidized proteins, due to their increased surface hydrophobicity ( Fig. 2) (52,95,128). On the other hand, the 20S proteasome seems to be more resistant to oxidative stress than the 26S proteasome (137,138).…”