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.
Highlights
Proteins exposed to oxidative stress are degraded via proteolytic pathways
The present investigation addresses the question of whether protein oxidation is followed by amino acid side chain modifications or changes in secondary and tertiary protein structure and whether these effects correlate with increased proteolytic susceptibility
To obtain new insights into the recognition process of oxidized proteins by the proteasome, we combined two different techniques: structural changes of the model protein RNase A upon oxidation by hydrogen peroxide were followed by Fourier transform infrared (FT-IR) spectroscopy, while the changes of susceptibility to proteasome degradation were tested by biochemical methods
Summary
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. It was concluded that the oxidative modification of amino acid side chains disrupts, at least locally, the tertiary protein structure, which is, in turn, accompanied by exposure of hydrophobic moieties to the surface of the protein. This increase in surface hydrophobicity seems to be the recognition signal for the 20 S proteasome for binding and degradation of the substrate protein (14, 16, 18, 20 –22). The present investigation addresses the question of whether protein oxidation is followed by amino acid side chain modifications or changes in secondary and tertiary protein structure and whether these effects correlate with increased proteolytic susceptibility. PH*, glass electrode pH reading in D2O solutions without correction for isotope effects; ASA, accessible surface area(s)
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