title: Identification of an unstable 4-hydroxynoneal modification on the 20S proteasome subunit α7 by recombinant antibody technology.
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.
authors.affiliation: Aarhus University, Department of Molecular Biology and Genetics, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark.
authors.forename: Jesper
authors.initials: J
authors.lastname: Just
authors.affiliation: German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE), Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Jung
authors.affiliation: Aarhus University, Department of Molecular Biology and Genetics, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark.
authors.forename: Niels Anton
authors.initials: NA
authors.lastname: Friis
authors.affiliation: Department of Clinical Medicine, Aarhus University, Aarhus, Denmark; Sino-Danish Centre for Education and Research (SDC), Aarhus, Denmark.
authors.forename: Simon
authors.initials: S
authors.lastname: Lykkemark
authors.affiliation: Department of Clinical Medicine, Aarhus University, Aarhus, Denmark.
authors.forename: Kim
authors.initials: K
authors.lastname: Drasbek
authors.affiliation: Aarhus University, Department of Molecular Biology and Genetics, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark.
authors.forename: Gunhild
authors.initials: G
authors.lastname: Siboska
authors.affiliation: German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE), Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany.
authors.forename: Tilman
authors.initials: T
authors.lastname: Grune
authors.affiliation: Aarthus Univeristy, Department of Engineering, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark. Electronic address: pk@eng.au.dk.
authors.forename: Peter
authors.initials: P
authors.lastname: Kristensen
mesh_terms: D000447:Aldehydes / Q000378:metabolism; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000447:Aldehydes; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; C488662:PSMA7 protein, human; D046988:Proteasome Endopeptidase Complex; C027576:4-hydroxy-2-nonenal
keywords: 4-hydroxynonenal (HNE); Lipid peroxidation; Oxidative stress; Phage display; Post-translational modification; Proteasome; Recombinant antibodies; Unstable modification