Abstract
The lipid peroxidation product 4-hydroxynonenal (4-HNE) has been shown to interfere with protein function. The goal of this study was to determine the effects of substrate modification by 4-HNE on protein degradation. Equine liver alcohol dehydrogenase (ADH, EC 1.1.1.1) treated with 2-fold molar excess 4-HNE was degraded by a rabbit reticulocyte lysate (RRL) system approximately 1.5-fold faster than control, while treatment with concentrations up to 100-fold molar excess aldehyde were inhibitory to degradation. Involvement of the 26S proteasome (EC 3.4.99.46) was demonstrated through the use of specific proteasome and ATPase inhibitors, and confirmed by measuring the extent of ADH polyubiquitination. Tryptic digestion and LC/MS analysis of 4-HNE-treated ADH identified modification of two zinc chelating Cys residues. Through molecular modeling experiments a conformational shift in both zinc-containing regions was predicted, with an approximate doubling of the distance between the structural zinc and its respective chelating residues. Modification of residues in the active site zinc binding motif resulted in less pronounced alteration in protein structure. The data presented here demonstrate accelerated ubiquitination and proteasomal degradation of ADH modified with 4-HNE, and suggest a conformational change after 4-HNE docking as a mechanism behind these observations.
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