Abstract
Numerous cellular functions rely on an active proteasome allowing degradation of damaged or misfolded proteins. Therefore changes in the proteasomal activity have important physiological consequences. During oxidative stress the production of free radicals can result in the formation of 4-hydroxynonenal (HNE) following lipid peroxidiation. The HNE moiety is highly reactive and via a nucleophilic attack readily forms covalent links to cysteine, histidine and lysine side chains. However, as the chemical properties of these amino acids differ, so does the kinetics of the reactions. While covalent linkage through Michael addition is well established, reversible and unstable associations have only been indicated in a few cases. In the present study we have identified an unstable HNE adduct on the α7 subunit of the 20S proteasome using phage display of recombinant antibodies. This recombinant antibody fragment recognized HNE modified proteasomes in vitro and showed that this epitope was easily HNE modified, yet unstable, and influenced by experimental procedures. Hence unstable HNE-adducts could be overlooked as a regulatory mechanism of proteasomal activity and a participating factor in the decreased proteasomal activity associated with oxidative stress.