In Vitro and in Silico Characterization of Peroxiredoxin 6 Modified by 4-Hydroxynonenal and 4-Oxononenal

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.

Evaluation of three simple direct or indirect carbonyl detection methods for characterization of oxidative modifications of proteins

“…The HNE-modified protein spots could be clearly visualized after immunoblotting. The number of spots that was obtained indicated that a large number of proteins can be modified by HNE in vitro, which confirms previous data by other authors (Roede et al 2008).…”

“…Immunoreactive extra bands migrating close to 35 and 68 kDa may represent the formation of dimers and tetramers, respectively, during the HNE-protein reaction in these experimental conditions. This generation of multimeric species is likely due to the formation of crosslinks between proteins because of the double reactivity of HNE, which can also undergo Michael additions with Cys, His, or Lys residues of one protein molecule and Schiff bases with the Lys residue of another (Roede et al 2008). The absence of trimeric forms suggests that specific spatial conformations may be required for aggregation.…”

Posttranslational modification and regulation of glutamate–cysteine ligase by the α,β-unsaturated aldehyde 4-hydroxy-2-nonenal

“…Together these reactions can lead to intra- and/or inter-molecular cross-linking of proteins via Michael addition at one site and subsequent Schiff base formation with a neighboring Lys residue [10-12]. Covalent modification by 4-HNE almost invariably leads to an impairment of protein function [2, 12-18] and 4-HNE-mediated adduction/inactivation of specific cellular proteins can have dramatic effects on cell function [19-21]. …”

Overexpression of Peroxiredoxin 6 Does Not Prevent Ethanol-Mediated Oxidative Stress and May Play a Role in Hepatic Lipid Accumulation

“…Using rodent models of chronic ethanol consumption, our laboratory has demonstrated previously that Prx6 is both down-regulated by chronic ethanol consumption and modified by 4-hydroxynonenal (Roede et al, 2008a). Using Prx6 knockout (KO) mice and transgenic (TG) Prx6 overexpressing mice, we sought to evaluate any mechanistic consequences that chronic ethanol consumption would have on these mice.…”