Abstract
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.
Highlights
Investigations of the effects of HNE in mammalian mitochondria show that HNE can inhibit proteins involved in the respiratory process
We have previously shown during oxidative stress in Arabidopsis [21] and environmental stress in pea [23] that, in addition to these defense responses, specific mitochondrial proteins are degraded under oxidative stress by an unknown pathway
Commercial polyclonal antibodies raised in rabbit that react with HNE adducts on a variety of amino acid residues were used in Western blots of mitochondrial proteins (Fig. 1)
Summary
Investigations of the effects of HNE in mammalian mitochondria show that HNE can inhibit proteins involved in the respiratory process. Protein Identification by Tandem Mass Spectrometry—Samples from HNE-treated isolated mitochondria (Fig. 2 and supplemental Table S1) were analyzed using an Agilent 1100 series
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