title: Inhibition of Hsp72-mediated protein refolding by 4-hydroxy-2-nonenal.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, The University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
authors.forename: David L
authors.initials: DL
authors.lastname: Carbone
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Doorn
authors.forename: Zachary
authors.initials: Z
authors.lastname: Kiebler
authors.forename: Brante P
authors.initials: BP
authors.lastname: Sampey
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Petersen
mesh_terms: D000447:Aldehydes / Q000737:chemistry / Q000633:toxicity*; D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000633:toxicity*; D003600:Cytosol / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D004032:Diet; D000431:Ethanol / Q000008:administration & dosage; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000494:pharmacology*; D022781:Hepatocytes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008099:Liver / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008297:Male; D017510:Protein Folding; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000447:Aldehydes; D003432:Cross-Linking Reagents; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D000431:Ethanol; C027576:4-hydroxy-2-nonenal