Protein Electrophile-Binding Motifs: Lysine-Rich Proteins Are Preferential Targets of Quinones

Abstract

ABSTRACT:Quinones represent an important class of endogenous compounds such as neurotransmitters and coenzyme Q10, electrophilic xenobiotics, and environmental toxicants that have known reactivity based on their ability to redox cycle and generate oxidative stress, as well as to alkylate target proteins. It is likely that topological, chemical, and physical features combine to determine which proteins become targets for chemical adduction. Chemical-induced post-translational modification of certain critical proteins causes a change in structure/function that contributes to the toxicological response to chemical exposure. In this study, we have identified a number of proteins that are modified by quinone-thioethers after administration of 2-(glutathion-S-yl)HQ. Parallel one-dimensional gel electrophoresis was performed, and the Coomassie-stained gel was aligned with the corresponding Western blot, which was probed for adductions. Immunopositive bands were then subjected to trypsin digestion and analyzed via liquid chromatography/tandem mass spectrometry. The proteins that were subsequently identified contained a higher than average (9.7 versus 5.5%) lysine content and numerous stretches of lysine run-ons, which is a presumed electrophile binding motif. Approximately 50% of these proteins have also been identified as targets for electrophilic adduction by a diverse group of chemicals by other investigators, implying overlapping electrophile adductomes. By identifying a motif targeted by electrophiles it becomes possible to make predictions of proteins that may be targeted for adduction and possible sites on these proteins that are adducted. An understanding of proteins targeted for adduction is essential to unraveling the toxicity produced by these electrophiles.Proteins have long been recognized as being critical targets of chemicals that produce acute tissue toxicities and cancer. Moreover, the adverse effects of many drugs are frequently mediated via their metabolic activation to reactive electrophilic metabolites, which subsequently bind covalently to proteins. Such reactive metabolites usually have low electron density and react with molecular centers of high electron density (i.e., nucleophiles). Target proteins for adduction usually contain strong nucleophilic sites, including cysteine thiols, lysine amines, histidine imidazoles, and protein N-terminal amines, which are readily attacked by reactive species (Guengerich et al., 2001;Casini et al., 2002). Other proteins contain weaker nucleophilic sites, including methionine sulfur, arginine guanidinium, tyrosine phenols, serine and threonine hydroxyls, and aspartate and glutamate carboxyls.Attempts to identify protein targets of reactive electrophilic metabolites have been complicated because 1) proteins have tremendously diverse structures and properties, 2) unmodified proteins are frequently present in excess, and 3) radiolabeled chemicals of sufficiently high specific activity are often unavailable or prohibitively expensive. Antibody-based approaches to a...

Chlorinated Biphenyl Quinones and Phenyl-2,5-benzoquinone Differentially Modify the Catalytic Activity of Human Hydroxysteroid Sulfotransferase hSULT2A1

“…This, however, was not surprising due to the fact that the sequence of this enzyme does not contain any of the lysine motifs previously identified in other proteins as being reactive with quinones. 62,63 …”

Detection of electrophile-sensitive proteins

“…Recently, Labenski et al showed that lysine-rich regions of proteins promote adduct formation with electrophilic quinones [55]. As more is discovered about specific electrophile-responsive proteomes, additional electrophile binding motifs may be discovered.…”

A New Look on Protein-Polyphenol Complexation during Honey Storage: Is This a Random or Organized Event with the Help of Dirigent-Like Proteins?

“…However, phenoxyl radicals can also be formed in non-enzymatic reactions via polyphenol auto-oxidation in the presence of oxygen radicals, H 2 O 2 and metal ions [29], [30]. The phenoxyl radicals (semi-quinones and quinone radicals) can spontaneously react with nucleophilic groups of amino acids and proteins, such as the epsilon amino group of lysine or the sulfhydryl group of cysteine, forming stable, covalent bonds [31], [32]. The examples of protein complexation by quinones relate to melanin formation [33], melanization and sclerotization of insect cuticle [34], [35] and lignin biosynthesis in plants [36]–[38].…”