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Inhibition of Hsp72-mediated protein refolding by 4-hydroxy-2-nonenal
Journal article   Open access   Peer reviewed

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

David L Carbone, Jonathan A Doorn, Zachary Kiebler, Brante P Sampey and Dennis R Petersen
Chemical research in toxicology, Vol.17(11), pp.1459-1467
11/2004
DOI: 10.1021/tx049838g
PMCID: PMC2956495
PMID: 15540944
url
https://doi.org/10.1021/tx049838gView
Published (Version of record) Open Access

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

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