The Cytotoxic Lipid Peroxidation Product 4-Hydroxy-2-nonenal Covalently Modifies a Selective Range of Proteins Linked to Respiratory Function in Plant Mitochondria

Plants encounter a variety of environmental stresses that affect their cellular machinery and that they adapt to on a daily basis. Lipid peroxidation is one consequence, at the cellular level, of such stresses and yields cytotoxic lipid aldehydes, including 4-hydroxy-2-nonenal (HNE), that react with specific sites in proteins, leading to diverse changes in protein function and/or stability. We have assessed the sensitivity of plant mitochondrial proteins to HNE modification, using one-dimensional and two-dimensional denaturing PAGE and blue native-PAGE coupled to immunological detection and tandem mass spectrometry identification. A select range of proteins was modified by exogenous application of HNE to mitochondria isolated from Arabidopsis cell cultures. These included a number of proteins that directly interact with the ubiquinone pool, as well as a number of soluble matrix proteins. Mitochondria isolated from cell cultures following hydrogen peroxide, antimycin A, or menadione treatment had significantly reduced respiratory capacity and elevated levels of HNE adduction to specific subsets of proteins. Targets identified included the proteins affected by direct application of HNE but also some new proteins, including a number of matrix dehydrogenases, the inner membrane adenine nucleotide translocator, and the outer membrane voltage-dependent anion channel. Degradation products of some proteins were also found to be HNE adducted, suggesting a link between HNE adduction and protein turnover. Some of the major enzyme complexes that were HNE adducted did not show demonstrable changes in their maximal activity measured with artificial acceptors, but changes did occur in associations between respiratory chain complexes following stress treatments. Plants encounter a variety of environmental stresses that affect their cellular machinery and that they adapt to on a daily basis. Lipid peroxidation is one consequence, at the cellular level, of such stresses and yields cytotoxic lipid aldehydes, including 4-hydroxy-2-nonenal (HNE), that react with specific sites in proteins, leading to diverse changes in protein function and/or stability. We have assessed the sensitivity of plant mitochondrial proteins to HNE modification, using one-dimensional and two-dimensional denaturing PAGE and blue native-PAGE coupled to immunological detection and tandem mass spectrometry identification. A select range of proteins was modified by exogenous application of HNE to mitochondria isolated from Arabidopsis cell cultures. These included a number of proteins that directly interact with the ubiquinone pool, as well as a number of soluble matrix proteins. Mitochondria isolated from cell cultures following hydrogen peroxide, antimycin A, or menadione treatment had significantly reduced respiratory capacity and elevated levels of HNE adduction to specific subsets of proteins. Targets identified included the proteins affected by direct application of HNE but also some new proteins, including a number of matrix dehydrogenases, the inner membrane adenine nucleotide translocator, and the outer membrane voltage-dependent anion channel. Degradation products of some proteins were also found to be HNE adducted, suggesting a link between HNE adduction and protein turnover. Some of the major enzyme complexes that were HNE adducted did not show demonstrable changes in their maximal activity measured with artificial acceptors, but changes did occur in associations between respiratory chain complexes following stress treatments. The polyunsaturated fatty acids of membrane phospholipids are highly susceptible to peroxidation by reactive oxygen species (ROS), 4The abbreviations used are: ROSreactive oxygen speciesANTadenine nucleotide translocatorAOXalternative oxidaseDCPIPdichlorophenylindophenolFeCN[Fe(CN)6]3–HNE4-hydroxy-2-nonenalIEFisoelectric focusingKGDCα-ketoglutarate dehydrogenase complexn-PGn-propyl gallatePDCpyruvate dehydrogenase complexTEMEDN′,N′,N′,N′-tetramethylethylenediamineUQubiquinoneUQH2reduced ubiquinoneBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolCIcomplex IHAEhydroxyalkenalMS/MStandem mass spectrometryLCliquid chromatographyTES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acidCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineBNblue nativeNLnonlinear 4The abbreviations used are: ROSreactive oxygen speciesANTadenine nucleotide translocatorAOXalternative oxidaseDCPIPdichlorophenylindophenolFeCN[Fe(CN)6]3–HNE4-hydroxy-2-nonenalIEFisoelectric focusingKGDCα-ketoglutarate dehydrogenase complexn-PGn-propyl gallatePDCpyruvate dehydrogenase complexTEMEDN′,N′,N′,N′-tetramethylethylenediamineUQubiquinoneUQH2reduced ubiquinoneBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolCIcomplex IHAEhydroxyalkenalMS/MStandem mass spectrometryLCliquid chromatographyTES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acidCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineBNblue nativeNLnonlinear and a self-propagating chain of free radical reactions can produce various aldehydes, alkenals, and hydroxyalkenals (1Schneider C. Tallman K.A. Porter N.A. Brash A.R. J. Biol. Chem. 2001; 276: 20831-20838Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 2Moller I.M. Jensen P.E. Hansson A. Annu. Rev. Plant Biol. 2007; 58: 459-481Crossref PubMed Scopus (1260) Google Scholar). These aldehydes are cytotoxic, generally more stable than ROS, and can cause extensive damage to proteins and other cellular constituents. HNE is the most abundant and toxic aldehyde generated through ROS-mediated peroxidation of abundant lipids in plants such as linoleic acid (1Schneider C. Tallman K.A. Porter N.A. Brash A.R. J. Biol. Chem. 2001; 276: 20831-20838Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 3Esterbauer H. Schaur R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5821) Google Scholar). HNE is a highly reactive electrophile, with the primary reactivity of the molecule lying at the unsaturated bond of the C-3 atom. HNE has been shown to form Michael adducts via this C-3 atom with the sulfhydryl group of Cys residues, the imidazole group of His residues, and the ϵ-amino group of Lys residues on a large number of proteins (3Esterbauer H. Schaur R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5821) Google Scholar). Recently it has been proposed that HNE can also modify Arg residues of proteins (4Isom A.L. Barnes S. Wilson L. Kirk M. Coward L. Darley-Usmar V. J. Am. Soc. Mass Spectrom. 2004; 15: 1136-1147Crossref PubMed Scopus (123) Google Scholar). In addition to Michael adduct formation, Lys residues also form Schiff bases and pentylpyrrole adducts with HNE via the C-1 aldehyde group (5Sayre L.M. Arora P.K. Iyer R.S. Salomon R.G. Chem. Res. Toxicol. 1993; 6: 19-22Crossref PubMed Scopus (156) Google Scholar). HNE has also been shown to react via the C-3 position with the sulfhydryl groups of lipoic acid moieties on proteins (6Humphries K.M. Szweda L.I. Biochemistry. 1998; PubMed Scopus Google Scholar). of proteins by HNE has the to have in a cell of of acids and the to form in proteins (1Schneider C. Tallman K.A. Porter N.A. Brash A.R. J. Biol. Chem. 2001; 276: 20831-20838Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 2Moller I.M. Jensen P.E. Hansson A. Annu. Rev. Plant Biol. 2007; 58: 459-481Crossref PubMed Scopus (1260) Google Scholar, 3Esterbauer H. Schaur R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5821) Google Scholar, A.L. Barnes S. Wilson L. Kirk M. Coward L. Darley-Usmar V. J. Am. Soc. Mass Spectrom. 2004; 15: 1136-1147Crossref PubMed Scopus (123) Google Scholar, L.M. Arora P.K. Iyer R.S. Salomon R.G. Chem. Res. Toxicol. 1993; 6: 19-22Crossref PubMed Scopus (156) Google Scholar). reactive oxygen species adenine nucleotide 4-hydroxy-2-nonenal dehydrogenase dehydrogenase ubiquinone reduced ubiquinone tandem mass spectrometry acid acid blue reactive oxygen species adenine nucleotide 4-hydroxy-2-nonenal dehydrogenase dehydrogenase ubiquinone reduced ubiquinone tandem mass spectrometry acid acid blue of the of HNE in mitochondria show that HNE can proteins in the respiratory activity is by HNE in a in J. J. 2001; Full Text PDF PubMed Scopus (123) Google Scholar, J. S. 1998; PubMed Scopus Google by to of the A. Biochemistry. PubMed Scopus Google Scholar). (4Isom A.L. Barnes S. Wilson L. Kirk M. Coward L. Darley-Usmar V. J. Am. Soc. Mass Spectrom. 2004; 15: 1136-1147Crossref PubMed Scopus (123) Google and the of the dehydrogenase V. J. PubMed Scopus Google Scholar, Szweda Szweda L.I. Free Radic. Biol. Med. 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HNE of Mitochondria and of from Arabidopsis cell cultures were with in by antimycin A in or menadione in mitochondria were with of HNE at with HNE treatment of mitochondria by oxygen was by of HNE with mitochondrial protein in of oxygen in the oxygen at of oxygen were using a oxygen to a of the was by to the in at or and by to the addition of to the to oxygen in the and are as Arabidopsis cell was in cell to a of of was measured at following the addition of the through the or the in was measured following the of or mitochondria of were in of oxygen at isolated mitochondrial was measured at in the of various as and and A via the by isolated mitochondria was measured in the of the the and the via the was measured in the of the was to PubMed Scopus Google Scholar). were with or to membrane was using of range or were by addition of to to a of were at to of proteins. were at and in or of or or of in was to were used to or at was using a at were and in The were by the on of and following native-PAGE was on the by and H. 1991; PubMed Scopus Google Scholar). were at at The was in with at of protein to the were on and at to The was to a new at of The was on using the The of a and a The were with a in the and in the of the The was at a of and with a of with at used were of and the was to a on the were with or and were with mitochondrial by direct of to of the inner dehydrogenase of the respiratory was measured using a modified from Plant PubMed Google Scholar). of protein was to of dehydrogenase The was by the addition of to the dehydrogenase activity was measured as at of of reduced was using of activity was on the in L. 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