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.

Oxidative stress and lipotoxicity

“…Proteomic analysis of lysates from a rodent model of ALD confirms the susceptibility of chaperones HSP72, HSP90, and GRP78 to adduction by lipid alde-including in neurodegenerative disease (72,77,104,109), cerebral ischemia-reperfusion injury (77,110), cardiac ischemia-reperfusion injury (111)(112)(113), and in lymphocytes from aged humans (57,106). In-depth study of proteasome oxidation revealed that specific subunits are preferentially modified by reactive lipids and that, depending on which subunit is modified, catalytic activity of some, or all three, of the catalytic peptidase activities of the 20S proteasome can be inhibited (111,114,115). Although the precise mechanisms behind such inhibition of proteolysis remain complex, it is clear that protein carbonylation is a dynamic regulatory process in proteolysis.…”

Oxidative damage in naturally aged mouse oocytes is exacerbated by dysregulation of proteasomal activity

“…In somatic cells, core elements of the proteasomal complex are vulnerable to direct 4-HNE adduction leading to a consequential reduction in the activity of this proteolytic pathway (71)(72)(73)(74)(75)(76)(77)(78). Key among these are the proteasome subunits ␣2, ␣5, and ␣6 as well as proteasome subunits ␤1, ␤3, and ␤7.…”

“…In seeking to link these data, it has been shown that several components of the proteasome are themselves targeted for 4-HNE modification. Indeed, with the exception of ␤2, all other ␣ and ␤ subunits represented in the proteasome core have been reported as being vulnerable to 4-HNE adduction and have been associated with an attendant reduction in the trypsin, chymotrypsin, and peptidylglutamyl peptide hydrolase activities of the proteasome (71)(72)(73)(74)(75)(76)(77)(78), ultimately suppressing the cell's ability to resolve alternative oxidatively-damaged proteins. In seeking to determine whether similar mechanisms contribute to declining oocyte quality, here we have investigated the efficacy of proteasomal clearance of 4-HNE-modified proteins in aged oocytes.…”

Precise assembly and regulation of 26S proteasome and correlation between proteasome dysfunction and neurodegenerative diseases

“…Based on analyses of 20S CPs purified from HME-treated liver, Farout et al reported that seven proteasome subunits (α2-α5, β3, β4, and β1i) are HNE modified (81). In addition, based on phage display studies using recombinant antibodies, Just et al recently identified an unstable HNE modification on the human α7-subunit (82).…”