In vitro and in silico characterization of peroxiredoxin 6 modified by 4-hydroxynonenal and 4-oxononenal.
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.
Roede
James R
JR
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.
Carbone
David L
DL
Doorn
Jonathan A
JA
Kirichenko
Oleg V
OV
Reigan
Philip
P
Petersen
Dennis R
DR
D000447:Aldehydes / Q000737:chemistry / Q000378:metabolism*; D000821:Animal Feed; D000818:Animals; D000975:Antioxidants; D019295:Computational Biology / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism*; D000431:Ethanol / Q000008:administration & dosage; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008958:Models, Molecular; D008968:Molecular Conformation; D010084:Oxidation-Reduction; D054465:Peroxiredoxin VI / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
C403894:4-oxo-2-nonenal; D000447:Aldehydes; D000975:Antioxidants; D003432:Cross-Linking Reagents; D000431:Ethanol; D054465:Peroxiredoxin VI; C515984:Prdx6 protein, rat; C027576:4-hydroxy-2-nonenal