Lipid peroxidation yields the aldehydes 4-hydroxynonenal (4HNE) and 4-oxononenal (4ONE). Protein adduction by 4HNE is thought to be involved in the pathogenesis of several diseases. Currently, the reactivity of 4ONE toward proteins is unknown. The purpose of this study was to identify amino acids that react with 4HNE and 4ONE, characterize the chemical structure of the adduct, and determine the preference for amino acid modification. Model peptides containing one or more nucleophilic residues (i.e., Arg, Cys, His, Met, and Lys) were reacted with 4HNE and 4ONE and analyzed using matrix-assisted laser desorption/ionization mass spectrometry. Post-source decay analysis was used to confirm peptide modification. The bimolecular rate constant for adduction of amino acids and peptides by 4HNE and 4ONE was measured. Results of this work indicate that Cys, His, and Lys are modified by 4HNE and 4ONE. In addition, Arg was adducted by 4ONE. The predominant adduct resulting from modification of peptides by 4HNE or 4ONE had a mass of 156 or 154 Da (respectively), indicating that adduction occurs via Michael addition. Reactivity of amino acids toward 4HNE and 4ONE was found to have the following order: Cys >> His > Lys (> Arg for 4ONE). The presence of an Arg on a Cys-containing peptide increased the reaction rate with 4HNE and 4ONE by a factor of approximately 5-6 compared to the Cys nucleophile alone. Rate constants determined for the modification of Cys by the lipid aldehydes demonstrated a >100-fold difference in reactivity between 4HNE and 4ONE toward Cys. Results of the present study indicate that both 4HNE and 4ONE modify amino acid nucleophiles; however, the reactivity between these two lipid aldehydes differs both qualitatively and quantitatively.

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Dopamine (DA) has been implicated as an endogenous neurotoxin to explain the selective neurodegeneration as observed for Parkinson's disease (PD). However, previous work demonstrated 3,4-dihydroxyphenylacetaldehyde (DOPAL) to be more toxic than DA. DOPAL is generated as a part of DA catabolism via the activity of monoamine oxidase and the mechanism of DOPAL toxicity is proposed to involve protein modification. Previous studies have demonstrated protein reactivity via the aldehyde moiety; however, DOPAL contains two reactive functional groups (catechol and aldehyde) both with the potential for protein adduction. The goal of this work was to determine whether protein modification by DOPAL occurs via a thiol-reactive quinone generated from oxidation of the catechol, which is known to occur for DA, or if the aldehyde forms adducts with amine nucleophiles. To accomplish this objective, the reactivity of DOPAL towards N-acetyl-lysine (NAL), N-acetyl-cysteine (NAC) and two model proteins was determined. In addition, several DOPAL analogues were obtained and used for comparison of reactivity. Results demonstrate that at pH 7.4 and 37°C, the order of DOPAL reactivity is NAL ≫ NAC and the product of NAL and DOPAL is stable in the absence of reducing agent. Moreover, DOPAL will react with model proteins, but in the presence of amine-selective modifiers citraconic anhydride and 2-iminothiolane hydrochloride, the reactivity of DOPAL towards the proteins is diminished. In addition, DOPAL-mediated protein cross-linking is observed when a model protein or a protein mixture (i.e. mitochondria lysate) are treated with DOPAL at concentrations of 5-100 μM. Protein cross-linking was diminished in the presence of ascorbate, suggesting the involvement of a quinone in DOPAL-mediated protein modification. These data indicate DOPAL to be highly reactive towards protein nucleophiles with the potential for protein cross-linking.

Lipid peroxidation during oxidative stress leads to increased concentrations of thiol-reactive ␣,␤-unsaturated aldehyde, including 4-hydroxy-2-nonenal (4-HNE) and 4-oxo-2-nonenal (4-ONE). These aldehydes have a documented ability to disrupt protein function following adduct formation with specific residues. Therefore, to identify 4-HNE-modified proteins in a model of ethanol-induced oxidative stress, a proteomic approach was applied to liver fractions prepared from rats fed a combination high-fat/ethanol diet. The results revealed that essential 90-kDa heat shock protein (Hsp90) was consistently modified by 4-HNE in the alcohol-treated animals. In vitro chaperoning experiments using firefly luciferase as a client protein were then performed to assess the functional effect of 4-HNE modification on purified recombinant human Hsp90, modified with concentrations of this aldehyde ranging from 23 to 450 M. Modification of Hsp90 with 4-ONE also led to significant inhibition of the chaperone. Because 4-HNE and 4-ONE react selectively with Cys, a thiol-specific mechanism of inhibition was suggested by these data. Therefore, thiol sensitivity was confirmed following treatment of Hsp90 with the specific thiol modifier N-ethylmaleimide, which resulted in more than 99% inactivation of the chaperone by concentrations as low as 6 M (1:1 M ratio). Finally, tryptic digest of 4-HNE-modified Hsp90 followed by liquid chromatography/tandem mass spectrometry peptide analysis identified Cys 572 as a site for 4-HNE modification. The results presented here thus establish that 4-HNE consistently modifies Hsp90 in a rat model of alcohol-induced oxidative stress and that the chaperoning activity of this protein is subject to dysregulation through thiol modification.

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 μM 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 μM 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 thiolunreactive 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 μM 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.

Persistent inflammation and the generation of reactive oxygen and nitrogen species play pivotal roles in tissue injury during disease pathogenesis and as a reaction to toxicant exposures. The associated oxidative and nitrative stress promote diverse pathologic reactions including neurodegenerative disorders, atherosclerosis, chronic inflammation, cancer, and premature labor and stillbirth. These effects occur via sustained inflammation, cellular proliferation and cytotoxicity and via induction of a proangiogenic environment. For example, exposure to the ubiquitous air pollutant ozone leads to generation of reactive oxygen and nitrogen species in lung macrophages that play a key role in subsequent tissue damage. Similarly, studies indicate that genes involved in regulating oxidative stress are altered by anesthetic treatment resulting in brain injury, most notable during development. In addition to a role in tissue injury in the brain, inflammation, and oxidative stress are implicated in Parkinson's disease, a neurodegenerative disease characterized by the loss of dopamine neurons. Recent data suggest a mechanistic link between oxidative stress and elevated levels of 3,4-dihydroxyphenylacetaldehyde, a neurotoxin endogenous to dopamine neurons. These findings have significant implications for development of therapeutics and identification of novel biomarkers for Parkinson's disease pathogenesis. Oxidative and nitrative stress is also thought to play a role in creating the proinflammatory microenvironment associated with the aggressive phenotype of inflammatory breast cancer. An understanding of fundamental concepts of oxidative and nitrative stress can underpin a rational plan of treatment for diseases and toxicities associated with excessive production of reactive oxygen and nitrogen species.

Recent work indicates that oxidative stress is a factor in Parkinson's disease (PD); however, it is unknown how this condition causes selective dopaminergic cell death. The neurotransmitter dopamine (DA) has been implicated as an endogenous neurotoxin to explain the selective neurodegeneration. DA undergoes catabolism by monoamine oxidase (MAO) to the reactive intermediate 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is further oxidized to 3,4-dihydroxyphenylacetic (DOPAC) acid via mitochondrial aldehyde dehydrogenase (ALDH). Previous studies found DOPAL to be more toxic than DA, and the major lipid peroxidation products, that is, 4-hydroxynonenal (4HNE) and malondialdehyde (MDA), potently inhibit ALDH. The hypothesis of this work is that lipid peroxidation products inhibit DOPAL oxidation, yielding aberrant levels of the reactive aldehyde intermediate. Treatment of striatal synaptosomes with 2-100 microM 4HNE or 2-50 microM MDA impaired DOPAL oxidation, resulting in elevated [DOPAL]. The aberrant concentration of DOPAL yielded an increase in protein modification by the DA-derived aldehyde, evident via staining of proteins with nitroblue tetrazolium (NBT). Pretreatment of synaptosomes with an MAO inhibitor significantly decreased NBT staining. On the basis of NBT staining, the order of protein reactivity for DA and metabolites was found to be DOPAL>>DOPAC>DA. Mass spectrometric analysis of a model peptide reacted with DOPAL revealed the adduct to be a Schiff base product. In summary, these data demonstrate the sensitivity of DA catabolism to the lipid peroxidation products 4HNE and MDA even at low, physiologic levels and suggest a mechanistic link between oxidative stress and generation of aberrant levels of an endogenous and protein reactive dopaminergic toxin relevant to PD.

A proteomic approach was applied to mitochondrial protein isolated from the livers of rats fed a combination high-fat and ethanol diet to identify proteins modified by 4-hydroxynonenal (4-HNE). Using this approach, the endoplasmic reticulum chaperone, protein disulfide isomerase (PDI), which participates in the maturation of newly synthesized proteins through promoting correct disulfide formation, was consistently found to be modified by 4-HNE. Further mass spectral analysis of PDI isolated from the animals revealed modification of an active site Cys residue thought to be involved in client protein binding. To test the hypothesis that 4-HNE inhibits the chaperone, purified bovine PDI was treated with concentrations of 4-HNE ranging from 20 to 200 microM (10-100-fold molar excess aldehyde), resulting in 14-56% inhibition, respectively. Similar treatments with the lipid peroxidation products acrolein (ACR) and 4-oxononenal (4-ONE) resulted in 60 and 100% inhibition, respectively, suggesting inactivation of the chaperone via Cys modification. Thiol sensitivity was confirmed through concentration-dependent inhibition of PDI by the Cys modifier N-ethylmaleimide (NEM). While some degree of sensitivity to these lipid aldehydes is suggested by the data, when compared to inactivation of other proteins by 4-HNE, PDI has demonstrated a relative resistance. It was also observed that physiologic (e.g., 4 mM) concentrations of GSH were capable of removing the 4-HNE adducts, likely serving as a protective mechanism against inactivation by 4-HNE and other lipid peroxidation products. However, because an active site Cys was found to be modified by 4-HNE on PDI in vivo, it is possible that the protective effect of GSH on the chaperone decreases under conditions of sustained oxidative stress, such as during chronic alcohol consumption, as GSH is depleted. The data presented here thus suggest potential impairment of an important molecular chaperone during oxidative stress.