Selective Covalent Binding of Acrylonitrile to Cys 186 in Rat Liver Carbonic Anhydrase III In Vivo

Nerland, Donald E.; Cai, Jian; Benz, Frederick W.

doi:10.1021/tx0256883

Cited by 18 publications

(22 citation statements)

References 35 publications

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“…The preceding data suggest that the sulfhydryl thiolate state of Cys152 is the preferential site of adduct formation for ACR 9,10,13,14 . As also indicated, sulfhydryl groups exist mostly in the non-nucleophilic thiol state (0; Table 3) at intracellular pH ranges (7.0-7.4).…”

Section: Discussionmentioning

confidence: 90%

Molecular Mechanism of Glyceraldehyde-3-phosphate Dehydrogenase Inactivation by α,β-Unsaturated Carbonyl Derivatives

Martyniuk

Fang

Koomen

et al. 2011

Chem. Res. Toxicol.

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α,β-Unsaturated carbonyls are an important class of chemicals involved in environmental toxicity and disease processes. Whereas adduction of cysteine residues on proteins is a well-documented reaction of these chemicals, such a generic effect cannot explain the molecular mechanism of cytotoxicity. Instead, more detailed information is needed regarding the possible specificity and kinetics of cysteine targeting and the quantitative relationship between adduct burden and protein dysfunction. To address these datagaps, purified human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was incubated with acrylamide (ACR), acrolein or methylvinyl ketone (MVK). Results show that these α,β-unsaturated carbonyl toxicants inhibited GAPDH activity in a concentration-and time-dependent manner. The rank order of enzyme inhibition (KI); i.e., ACR << MVK < acrolein, was related to the calculated electrophilic reactivity of each compound and to the corresponding kinetics of cysteine adduct formation. Tandem mass spectrometry revealed that adduct formation was selective at lower concentrations; i.e., ACR preferentially formed adducts with Cys152 (residues 146-162). At higher concentrations, ACR also formed adducts with Cys156 and Cys247 (residues 235-248). Adduct formation at Cys152 was correlated to enzyme inhibition, which is consistent with the regulatory role of this residue in enzyme function and its location within the GAPDH active site. Further analyses indicated that Cys152 was present in a pKa-lowering microenvironment (pKa = 6.03) and, at physiological pH, the corresponding sulfhydryl group exists in the highly reactive nucleophilic thiolate-state. These data suggest a general cytotoxic mechanism where electrophilic α,β-unsaturated carbonyls selectively form adducts with reactive nucleophilic cysteine residues specifically associated with the active sites of proteins. These specialized cysteine residues are toxicologically relevant molecular targets, since chemical derivitization causes loss of protein function.

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Section: Discussionmentioning

confidence: 90%

Molecular Mechanism of Glyceraldehyde-3-phosphate Dehydrogenase Inactivation by α,β-Unsaturated Carbonyl Derivatives

Martyniuk

Fang

Koomen

et al. 2011

Chem. Res. Toxicol.

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“…Thus, selectivity of adduction is governed by both the recipient nucleophile and the chemistry of the adducting electrophile. Likewise, Cys-239 in tubulin is preferentially alkylated by anticancer drugs, including the colchicines, Vinca alkaloids, rhizoxin/maytansine, and taxanes (Jordan et al, 1998;Shan et al, 1999;Kavallaris et al, 2001;Casini et al, 2002;Scozzafava et al, 2002), and carbonic anhydrase III, which contains five cysteine residues, is selectively alkylated at Cys-186 by acrylonitrile (Nerland et al, 2003). Cysteine thiols are clearly common targets of reactive electrophilic metabolites and endogenous electrophiles, including lipid-derived ␣,␤-unsaturated aldehydes, such as 4-hydroxynonenal (4-HNE) and 4-oxononenal, and reactive oxygen species.…”

Section: Discussionmentioning

confidence: 99%

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

Labenski¹,

Fisher²,

Lo³

et al. 2009

Drug Metabolism and Disposition

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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...

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“…In vivo rat exposure and ex vivo studies on rat liver preparations highlighted binding to few specific proteins, identified as specific glutathione-S-transferase (Nerland et al, 2001) and carbonic anhydrase (Nerland, Cai, & Benz, 2003) isoenzymes. In these studies, derivatization of control and SCHEME 5.…”

Section: Reaction With Activated Olefinsmentioning

confidence: 99%

Toward an “omic” physiopathology of reactive chemicals: Thirty years of mass spectrometric study of the protein adducts with endogenous and xenobiotic compounds

Rubino

Pitton

Fabio

et al. 2009

Mass Spectrometry Reviews

109

170

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Cancer and degenerative diseases are major causes of morbidity and death, derived from the permanent modification of key biopolymers such as DNA and regulatory proteins by usually smaller, reactive molecules, present in the environment or generated from endogenous and xenobiotic components by the body's own biochemical mechanisms (molecular adducts). In particular, protein adducts with organic electrophiles have been studied for more than 30 [see, e.g., Calleman et al., 1978] years essentially for three purposes: (a) as passive monitors of the mean level of individual exposure to specific chemicals, either endogenously present in the human body or to which the subject is exposed through food or environmental contamination; (b) as quantitative indicators of the mean extent of the individual metabolic processing which converts a non-reactive chemical substance into its toxic products able to damage DNA (en route to cancer induction through genotoxic mechanisms) or key proteins (as in the case of several drugs, pesticides or otherwise biologically active substances); (c) to relate the extent of protein modification to that of biological function impairment (such as enzyme inhibition) finally causing the specific health damage. This review describes the role that contemporary mass spectrometry-based approaches employed in the qualitative and quantitative study of protein-electrophile adducts play in the discovery of the (bio)chemical mechanisms of toxic substances and highlights the future directions of research in this field. A particular emphasis is given to the measurement of often high levels of the protein adducts of several industrial and environmental pollutants in unexposed human populations, a phenomenon which highlights the possibility that a number of small organic molecules are generated in the human organism through minor metabolic processes, the imbalance of which may be the cause of "spontaneous" cases of cancer and of other degenerative diseases of still uncharacterized etiology. With all this in mind, it is foreseen that a holistic description of cellular functions will take advantage of new analytical methods based on time-integrated metabolomic measurements of a new biological compartment, the "adductome," aimed at better understanding integrated organism response to environmental and endogenous stressors.

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Selective Covalent Binding of Acrylonitrile to Cys 186 in Rat Liver Carbonic Anhydrase III In Vivo

Cited by 18 publications

References 35 publications

Molecular Mechanism of Glyceraldehyde-3-phosphate Dehydrogenase Inactivation by α,β-Unsaturated Carbonyl Derivatives

Molecular Mechanism of Glyceraldehyde-3-phosphate Dehydrogenase Inactivation by α,β-Unsaturated Carbonyl Derivatives

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

Toward an “omic” physiopathology of reactive chemicals: Thirty years of mass spectrometric study of the protein adducts with endogenous and xenobiotic compounds

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