In vitro and in silico characterization of peroxiredoxin 6 modified by 4-hydroxynonenal and 4-oxononenal

James R Roede; David L Carbone; Jonathan A Doorn; Oleg V Kirichenko; Philip Reigan; Dennis R Petersen

doi:10.1021/tx800244u

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In vitro and in silico characterization of peroxiredoxin 6 modified by 4-hydroxynonenal and 4-oxononenal

Journal article

Peer reviewed

In vitro and in silico characterization of peroxiredoxin 6 modified by 4-hydroxynonenal and 4-oxononenal

James R Roede, David L Carbone, Jonathan A Doorn, Oleg V Kirichenko, Philip Reigan and Dennis R Petersen

Chemical research in toxicology, Vol.21(12), pp.2289-2299

12/2008

DOI: 10.1021/tx800244u

PMID: 19548352

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Abstract

Peroxiredoxin 6 (PRX6) belongs to the 1-Cys class of peroxiredoxins and is recognized as an important antioxidant protein in tissues such as cardiac muscle, skin, and lung. Preliminary in vivo proteomic data have revealed that PRX6 is adducted by 4-hydroxynonenal (4HNE) in the livers of rats chronically fed an ethanol-containing diet. The goals of this study were to evaluate the in vitro effect of aldehyde adduction on PRX6 peroxidase activity, identify specific sites of aldehyde modification using mass spectrometry, and predict conformational changes due to adduction using molecular modeling. PRX6 was found to be resistant to inactivation via aldehyde modification; however, Western blots of adducted protein revealed that both 4HNE and 4-oxononenal (4ONE) caused extensive cross-linking, resulting in high molecular mass species. Tandem mass spectrometry (ESI-LC-MS/MS) analysis demonstrated multiple sites of modification, but adduction of the active site Cys47 was not observed. Molecular modeling simulations indicated that adduction at Cys91 results in a change in protein active site conformation, which potentially restricts access of 4-HNE to Cys47. The Cys91-Lys209 cross-linked adducts could provide the conformational changes required to inactivate the protein by either restricting access to electrophiles or preventing important amino acid interactions within the catalytic triad.

Computational Biology - methods

Cross-Linking Reagents - chemistry

Oxidation-Reduction

Liver - metabolism

Molecular Conformation

Ethanol - administration & dosage

Models, Molecular

Rats

Male

Aldehydes - metabolism

Rats, Sprague-Dawley

Antioxidants

Cross-Linking Reagents - metabolism

Peroxiredoxin VI - metabolism

Peroxiredoxin VI - chemistry

Tandem Mass Spectrometry

Animals

Spectrometry, Mass, Electrospray Ionization

Liver - drug effects

Aldehydes - chemistry

Animal Feed

Details

Title: Subtitle: In vitro and in silico characterization of peroxiredoxin 6 modified by 4-hydroxynonenal and 4-oxononenal
Creators: James R Roede - Department of Pharmaceutical Sciences, School of Pharmacy, The University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Campus Box C238, Denver, Colorado 80262, USA
David L Carbone
Jonathan A Doorn
Oleg V Kirichenko
Philip Reigan
Dennis R Petersen
Resource Type: Journal article
Publication Details: Chemical research in toxicology, Vol.21(12), pp.2289-2299
Publisher: United States
DOI: 10.1021/tx800244u
PMID: 19548352
ISSN: 0893-228X
eISSN: 1520-5010
Grant note: F31 AA014308 / NIAAA NIH HHS R01ES09410 / NIEHS NIH HHS R01AA09300 / NIAAA NIH HHS F32 ES11937 / NIEHS NIH HHS F31 AA016710 / NIAAA NIH HHS
Language: English
Date published: 12/2008
Academic Unit: Iowa Neuroscience Institute; Pharmaceutical Sciences and Experimental Therapeutics; Medicinal and Natural Products Chemistry
Record Identifier: 9984070517402771

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