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Sensitivity of plant mitochondrial terminal oxidases to the lipid peroxidation product 4-hydroxy-2-nonenal (HNE).

ABSTRACT: We have investigated the effect of the lipid peroxidation product, HNE (4-hydroxy-2-nonenal), on plant mitochondrial electron transport. In mitochondria isolated from Arabidopsis thaliana cell cultures, HNE inhibited succinate-dependent oxygen consumption via the Aox (alternative oxidase), but had minimal effect on respiration via Cox (cytochrome c oxidase). Maximal Cox activity, measured with reduced cytochrome c as substrate, was only slightly inhibited by high concentrations of HNE, at which Aox was completely inhibited. Incubation with HNE prevented dimerization of the Aox protein, suggesting that one site of modification was the conserved cysteine residue involved in dimerization and activation of this enzyme (Cys(I)). However, a naturally occurring isoform of Aox lacking Cys(I) and unable to be dimerized, LeAox1b from tomato (Lycopersicon esculentum), was equally sensitive to HNE inhibition, showing that other amino acid residues in Aox also interact with HNE. The presence of HNE in vivo in Arabidopsis cell cultures was also investigated. Induction of oxidative stress in the cell cultures by the addition of hydrogen peroxide, antimycin A or menadione, caused a significant increase in hydroxyalkenals (of which HNE is the most prominent). Western blotting of mitochondrial proteins with antibodies against HNE adducts, demonstrated significant modification of proteins during these treatments. The implications of these results for the response of plants to reactive oxygen species are discussed.

SUBMITTER: Winger AM

PROVIDER: S-EPMC1135019 | biostudies-other | 2005 May

REPOSITORIES: biostudies-other

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Project description:Reactive oxygen species (ROS) are elevated in the heart in response to hemodynamic and metabolic stress and promote hypertrophic signaling. ROS also mediate the formation of lipid peroxidation-derived aldehydes that may promote myocardial hypertrophy. One lipid peroxidation by-product, 4-hydroxy-trans-2-nonenal (HNE), is a reactive aldehyde that covalently modifies proteins thereby altering their function. HNE adducts directly inhibit the activity of LKB1, a serine/threonine kinase involved in regulating cellular growth in part through its interaction with the AMP-activated protein kinase (AMPK), but whether this drives myocardial growth is unclear. We tested the hypothesis that HNE promotes myocardial protein synthesis and if this effect is associated with impaired LKB1-AMPK signaling. In adult rat ventricular cardiomyocytes, exposure to HNE (10 μM for 1h) caused HNE-LKB1 adduct formation and inhibited LKB1 activity. HNE inhibited the downstream kinase AMPK, increased hypertrophic mTOR-p70S6K-RPS6 signaling, and stimulated protein synthesis by 27.1 ± 3.5%. HNE also stimulated Erk1/2 signaling, which contributed to RPS6 activation but was not required for HNE-stimulated protein synthesis. HNE-stimulated RPS6 phosphorylation was completely blocked using the mTOR inhibitor rapamycin. To evaluate if LKB1 inhibition by itself could promote the hypertrophic signaling changes observed with HNE, LKB1 was depleted in adult rat ventricular myocytes using siRNA. LKB1 knockdown did not replicate the effect of HNE on hypertrophic signaling or affect HNE-stimulated RPS6 phosphorylation. Thus, in adult cardiac myocytes HNE stimulates protein synthesis by activation of mTORC1-p70S6K-RPS6 signaling most likely mediated by direct inhibition of AMPK. Because HNE in the myocardium is commonly increased by stimuli that cause pathologic hypertrophy, these findings suggest that therapies that prevent activation of mTORC1-p70S6K-RPS6 signaling may be of therapeutic value.

Project description:Peroxidation of cellular membrane lipids, rich in polyunsaturated fatty acids, generates electrophilic, ?, ?-unsaturated aldehydes such as 4-hydroxy-2-nonenal (HNE). HNE is a highly reactive and cytotoxic molecule that can react with the nucleophilic sites in proteins causing posttranslational modification. The identification of protein targets is an important first step; however, quantitative profiling of site-specific modifications is necessary to understand the biological impact of HNE-induced carbonylation. We report a method that uses light (H(12)CHO) and heavy (D(13)CDO) isotopic variant of formaldehyde to differentially label primary amines (N-termini and ?-amino group of lysines) in peptides through reductive methylation and, combined with selective enrichment of modified peptides, permits comparison of the extent of carbonylation in two samples after mixing for simultaneous liquid chromatography-mass spectrometry. Specifically, dimethyl-labeled peptide carbonyls were fractionated from unmodified peptides using solid-phase hydrazide chemistry to immobilize them to porous glass beads and, after removing the unmodified peptides by thoroughly washing the beads, subsequently recover them by acid-catalyzed hydrolysis. The method was developed using HNE-modified synthetic peptides and also showing enrichment from a complex matrix of digested human plasma proteins. Applicability was confirmed using apomyoglobin as an analyte, implicating thereby its potential value to proteome-wide identification and relative quantification of posttranslational protein carbonylation with residue-specific information. Because HNE attachment may not necessarily cause change in protein abundance, this modification-focused quantification should facilitate the characterization of accompanied changes in protein function and, also, provide important insights into molecular signaling mechanisms and a better understanding of cellular processes associated with oxidative stress.

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Sensitivity of plant mitochondrial terminal oxidases to the lipid peroxidation product 4-hydroxy-2-nonenal (HNE).

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