Identification of an unstable 4-hydroxynoneal modification on the 20S proteasome subunit α7 by recombinant antibody technology

Free Radic Biol Med. 2015 Dec;89:786-92. doi: 10.1016/j.freeradbiomed.2015.10.405. Epub 2015 Oct 22.

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.

Keywords: 4-hydroxynonenal (HNE); Lipid peroxidation; Oxidative stress; Phage display; Post-translational modification; Proteasome; Recombinant antibodies; Unstable modification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldehydes / metabolism
  • Blotting, Western
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Immunoglobulin Variable Region / metabolism*
  • Oxidative Stress / physiology*
  • Proteasome Endopeptidase Complex / metabolism*
  • Recombinant Proteins / metabolism

Substances

  • Aldehydes
  • Immunoglobulin Variable Region
  • Recombinant Proteins
  • PSMA7 protein, human
  • Proteasome Endopeptidase Complex
  • 4-hydroxy-2-nonenal