When the level of reactive oxygen species (ROS) in cells exceeds a genetically coded defense capacity, the cells experience damage to vital components such as DNA, proteins and lipids that leads to non-specific interactions and the production of a series of high molecular weight protein aggregates. The dynamics of oxidative stress induced aggregation were studied here using model proteins and yeast. Model proteins were oxidized at increasing ROS concentrations and analyzed using size exclusion chromatography (SEC). Changes in the SEC elution profile showed that aggregation happens in stages and protein fragments produced as a result of oxidation also give rise to aggregates. Yeast cells were stressed with hydrogen peroxide to investigate in vivo aggregation. Equal amounts from control and oxidized lysates were chromatographed on a size exclusion column and proteins of molecular weight exceeding 700 kDa were collected from both samples which were then differentially labeled using light and heavy isotope coded N-acetoxysuccinamide and mixed in a 1:1 ratio. The coded mixture was analyzed using LC/MS and peptides that appeared as singlets representing the proteins that aggregated with higher molecular mass protein complexes were identified. Twenty-five proteins were identified to be of this type. Fifteen members in this group were found to have been carbonylated. These proteins are part of the proteome known as the aggresome. The protein content of the aggresome may provide vital information for mechanistic studies targeting disease and aging.

• PMID: 18760979
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Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't

Authors

Mirzaei H, Regnier F

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