Inhibition of Hsp72-mediated protein refolding by 4-hydroxy-2-nonenal

Chem Res Toxicol. 2004 Nov;17(11):1459-67. doi: 10.1021/tx049838g.


A proteomic approach was applied to liver cytosol from rats fed a diet consisting of high fat and ethanol to identify 4-hydroxy-2-nonenal (4-HNE)-modified proteins in vivo. Cytosolic Hsp72, the inducible variant of the Hsp70 heat shock protein family, was consistently among the proteins modified by 4-HNE. Despite 1.3-fold induction of Hsp72 in the livers of ethanol-fed animals, no increase in Hsp70-mediated luciferase refolding in isolated heptocytes was observed, suggesting inhibition of this process by 4-HNE. A 50% and 75% reduction in luciferase refolding efficiency was observed in rabbit reticulocyte lysate (RRL) supplemented with recombinant Hsp72 which had been modified in vitro with 10 and 100 microM 4-HNE, respectively. This observation was accompanied by a 25% and 50% decrease in substrate binding by the chaperone following the same treatment; however, no effect on complex formation between Hsp72 and its co-chaperone Hsp40 was observed. Trypsin digest and mass spectral analysis of Hsp72 treated with 10 and 100 microM 4-HNE consistently identified adduct formation at Cys267 in the ATPase domain of the chaperone. The role of this residue in the observed inhibition was demonstrated through the use of DnaK, a bacterial Hsp70 variant lacking Cys267. DnaK was resistant to 4-HNE inactivation. Additionally, Hsp72 was resistant to inactivation by the thiol-unreactive aldehyde malondialdehyde (MDA), further supporting a role for Cys in Hsp72 inhibition by 4-HNE. Finally, the affinity of Hsp72 for ATP was decreased 32% and 72% following treatment of the chaperone with 10 and 100 microM 4-HNE, respectively. In a model of chronic alcoholic liver injury, induction of Hsp72 was not accompanied by an increase in protein refolding ability. This is likely the result of 4-HNE modification of the Hsp72 ATPase domain.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Aldehydes / chemistry
  • Aldehydes / toxicity*
  • Animals
  • Cross-Linking Reagents / chemistry
  • Cross-Linking Reagents / toxicity*
  • Cytosol / chemistry
  • Cytosol / drug effects
  • Cytosol / metabolism
  • Diet
  • Ethanol / administration & dosage
  • HSP72 Heat-Shock Proteins
  • Heat-Shock Proteins / pharmacology*
  • Hepatocytes / chemistry
  • Hepatocytes / drug effects
  • Hepatocytes / metabolism
  • Liver / chemistry
  • Liver / drug effects
  • Liver / metabolism
  • Male
  • Protein Folding*
  • Rats
  • Rats, Sprague-Dawley


  • Aldehydes
  • Cross-Linking Reagents
  • HSP72 Heat-Shock Proteins
  • Heat-Shock Proteins
  • Ethanol
  • 4-hydroxy-2-nonenal