Covalent Modification Of Cysteine Research Articles

Molecular Determinants of Species-Specific Activation or Blockade of TRPA1 Channels

TRPA1 is an excitatory, nonselective cation channel implicated in somatosensory function, pain, and neurogenic inflammation. Through covalent modification of cysteine and lysine residues, TRPA1 can be activated by electrophilic compounds, including active ingredients of pungent natural products (e.g., allyl isothiocyanate), environmental irritants (e.g., acrolein), and endogenous ligands (4-hydroxynonenal). However, how covalent modification leads to channel opening is not understood. Here, we report that electrophilic, thioaminal-containing compounds [e.g., CMP1 (4-methyl-N-[2,2,2-trichloro-1-(4-nitro-phenylsulfanyl)-ethyl]-benzamide)] covalently modify cysteine residues but produce striking species-specific effects [i.e., activation of rat TRPA1 (rTRPA1) and blockade of human TRPA1 (hTRPA1) activation by reactive and nonreactive agonists]. Through characterizing rTRPA1 and hTRPA1 chimeric channels and point mutations, we identified several residues in the upper portion of the S6 transmembrane domains as critical determinants of the opposite channel gating: Ala-946 and Met-949 of rTRPA1 determine channel activation, whereas equivalent residues of hTRPA1 (Ser-943 and Ile-946) determine channel block. Furthermore, side-chain replacements at these critical residues profoundly affect channel function. Therefore, our findings reveal a molecular basis of species-specific channel gating and provide novel insights into how TRPA1 respond to stimuli.

Why the Taste of Some Spicy Foods Lingers

TRPA1 is a member of the transient receptor potential (TRP) family of ion channels and is involved in the sensation of noxious stimuli, including chemicals in food that are perceived as pungent or spicy. Macpherson et al . report that structurally diverse chemicals that can covalently interact with cysteine residues activate mouse TRPA1 channels. Using alkyne-modified agonists, they detected the alkyne-tagged residues on the channels using a fluorophore that reacted with the alkyne. Preincubation of the channel with cysteine-modifying agonists lacking the alkyne tag decreased the fluorescent signal (prevented access to the cysteines for the alkyne-tagged agonist). However, agonists, such as icilin, that cannot modify cysteines did not alter the labeling of the TRPA1 by the alkyne-tagged cysteine-modifying agonist. Thus, there is more than one way to activate TRPA1. Mass spectrometry analysis showed that 14 cysteines in the N-terminal cytosolic region were sites of modification. However, only three cysteines (Cys 415 , Cys 422 , Cys 622 ) appeared to have an effect on channel activity based on mutagenesis analysis. Although mutation of any of these three cysteines reduced channel activity, normalized current analysis showed that channels with any of these mutations were selectively defective in response to cysteine-reactive agonists and not to icilin, with Cys 622 showing the strongest decrease in responsiveness. The persistent stimulation predicted to occur in response to a covalent modification was validated by experiments in which an increase in calcium concentrations or single-channel activity was observed following washout of cysteine-modifying agonists. Caterina discusses this work and that of Hinman et al ., who previously reported this mechanism of action. L. J. Macpherson, A. E. Dubin, M. J. Evans, F. Marr, P. G. Schultz, B. F. Cravatt, A. Patapoutian, Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines. Nature 445 , 541-545 (2007). [PubMed] M. J. Caterina, Sticky spices. Nature 445 , 491-492 (2007). [PubMed] A. Hinman, H.-h. Chuang, D. M. Bautista, D. Julius, TRP channel activation by reversible covalent modification. Proc. Natl. Acad. Sci. U.S.A. 103 , 19564-19568 (2006). [Abstract] [Full Text]

Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines

The nervous system senses peripheral damage through nociceptive neurons that transmit a pain signal. TRPA1 is a member of the Transient Receptor Potential (TRP) family of ion channels and is expressed in nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent natural compounds, and environmental irritants. How such diverse stimuli activate TRPA1 is not known. We observed that most compounds known to activate TRPA1 are able to covalently bind cysteine residues. Here we use click chemistry to show that derivatives of two such compounds, mustard oil and cinnamaldehyde, covalently bind mouse TRPA1. Structurally unrelated cysteine-modifying agents such as iodoacetamide (IA) and (2-aminoethyl)methanethiosulphonate (MTSEA) also bind and activate TRPA1. We identified by mass spectrometry fourteen cytosolic TRPA1 cysteines labelled by IA, three of which are required for normal channel function. In excised patches, reactive compounds activated TRPA1 currents that were maintained at least 10 min after washout of the compound in calcium-free solutions. Finally, activation of TRPA1 by disulphide-bond-forming MTSEA is blocked by the reducing agent dithiothreitol (DTT). Collectively, our data indicate that covalent modification of reactive cysteines within TRPA1 can cause channel activation, rapidly signalling potential tissue damage through the pain pathway.

Characterization of S-(N,N-Dialkylaminocarbonyl)cysteine Adducts and Enzyme Inhibition Produced by Thiocarbamate Herbicides in the Rat.

Thiocarbamates are a major class of herbicides used extensively in the agricultural industry. It has been shown that thiocarbamates can form reactive sulfoxide and sulfone intermediates, which may be involved in the toxicity of thiocarbamates through covalent modification of cysteine and serine active sites of enzymes. Molinate has been shown to generate an S-hexahydro-1H-azepine-1-carbonyl adduct on the Cys-125 residue of the beta2- and beta3-chains of rat globin analogous to that reported for disulfiram and to inhibit aldehyde dehydrogenase and nonspecific esterase activity. The present study examined whether other thiocarbamate herbicides produce similar covalent protein modifications and enzyme inhibition to that reported for molinate and whether S-(N,N-dialkylaminocarbonyl)cysteine adduct levels are correlated to enzyme inhibition or the structure of thiocarbamate herbicides. Additionally, the potential of molinate to act as a peripheral demyelinating agent similar to disulfiram was evaluated. To address these aims, rats were exposed ip to molinate, vernolate, ethiolate, EPTC, or butylate for 5 days after which hemogloblin was isolated and analyzed for protein adducts using HPLC and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In addition, brain, liver, and testes mitochondrial and microsomal fractions were assayed for nonspecific esterase, low Km ALDH, or total ALDH activities, and S-(N,N-dialkylaminocarbonyl)cysteine adducts were measured by LC/MS/MS. For the neurotoxicity assessments, rats were administered molinate parenterally for subchronic periods and morphological evaluations performed on peripheral nerves. All of the thiocarbamates except butylate produced S-(N,N-dialkylaminocarbonyl)cysteine adducts on globin and the quantity of adducts detected decreased with increasing size of the nitrogen substituents. In contrast, a clear relationship between cysteine modification in mitochondrial and microsomal samples to nitrogen substituents was not evident, and although molinate produced relatively high levels of adducts and esterase inhibition and butylate low levels of adducts and esterase inhibition for most samples, in general, the level of S-(N,N-dialkylaminocarbonyl)cysteine adducts did not appear to be related to enzyme inhibition. Molinate did not produce segmental demyelination in peripheral nerve, suggesting that molinate and possibly other thiocarbamates do not share the neurotoxic potential of dithiocarbamates.

Covalent modification of cysteine 193 impairs ATPase function of nucleotide-binding domain of a Candida drug efflux pump

N-ethylmaleimide (NEM) impairs the ATPase function of N-terminal NBD of Candida drug resistance gene product Cdr1p. To identify the reactive cysteine(s) for such a contribution, we adopted a three-arm approach that included covalent modification, cysteine mutagenesis, and structure homology modeling. The covalent modification results clearly indicate the ability of NEM and iodoacetic acid (IAA) to potently inhibit the ATPase activity of N-terminal NBD. Since this domain contains five cysteine residues in its sequence, we mutated each and found four of these (C325A, C363A, C402A, and C462A) to stay sensitive to NEM/IAA modification and influence ATPase activity, while C193A mutation completely abrogated the catalytic function. The structural homology modeling data further validate these biochemical findings by ruling out any plausible interactions within the cysteine residues, and deriving the importance of Cys-193 in lying at a bond length clearly feasible to interact with ATP and divalent cation to critically influence ATP hydrolysis.

Kaurane Diterpene, Kamebakaurin, Inhibits NF-κB by Directly Targeting the DNA-binding Activity of p50 and Blocks the Expression of Antiapoptotic NF-κB Target Genes

Kaurane diterpenes have been identified from numerous medicinal plants, which have been used for treatment of inflammation and cancer, however, their molecular mechanism of action remains unclear. We have previously shown that kamebakaurin and other three kaurane diterpenes selectively inhibit activation of transcription factor NF-kappaB, a central mediator of apoptosis and immune responses. We here demonstrate that kamebakaurin is a potent inhibitor of NF-kappaB activation by directly targeting DNA-binding activity of p50. Kamebakaurin prevented the activation of NF-kappaB by different stimuli in various cell types. Kamebakaurin did not prevent either stimuli-induced degradation of IkappaB-alpha or nuclear translocation of NF-kappaB, however, it significantly interfered DNA binding activity of activated NF-kappaB in cell and in vitro and preferentially prevented p50-mediated DNA-binding activity of NF-kappaB rather than that of RelA as measured using in vitro translated p50 and RelA proteins. Moreover, a p50 mutant with a Cys-62 --> Ser mutation was not inhibited with kamebakaurin, indicating that the effect of kamebakaurin was probably due to its interaction with cysteine 62 in p50. The covalent modification of p50 by kamebakaurin was further demonstrated by mass spectrometry analysis that showed an increase in the molecular mass of kamebakaurin-treated p50, and this modification was not reverted by addition of dithiothreitol. These results suggested that kamebakaurin exhibited its inhibitory activity by a direct covalent modification of cysteine 62 in the p50. Also, treatment of cells with kamebakaurin prevented the tumor necrosis factor-alpha (TNF-alpha)-induced expression of antiapoptotic NF-kappaB target genes encoding c-IAP1 (hiap-2) and c-IAP2 (hiap-1), members of the inhibitor of apoptosis family, and Bfl-1/A1, a prosurvival Bcl-2 homologue, and augmented the TNF-alpha-induced caspase 8 activity, thereby resulting in sensitizing MCF-7 cells to TNF-alpha-induced apoptosis. Taken together, kamebakaurin is a valuable candidate for the intervention of NF-kappaB-dependent pathological conditions such as inflammation and cancer.

Acrolein causes transcriptional induction of phase II genes by activation of Nrf2 in human lung type II epithelial (A549) cells

Acrolein, an α,β-unsaturated aldehyde, is by far the strongest electrophile present in cigarette smoke which is involved in several lung pathophysiological conditions. Acrolein depletes glutathione and creates thiol imbalance. Acrolein due to thiol imbalance as well as covalent modification of cysteine is known to inhibit the activity of redox sensitive transcription factors such as NF-κB and AP-1. Exposure of human type II lung epithelial (A549) cells to non-lethal dose of acrolein (150 fmol/cell for 1 h) depletes 80% of intracellular glutathione and increases the transcription of γ-glutamylcysteine synthetase (γ-GCS) at 6–12 h post-treatment, which helps in replenishing the glutathione to normal level. Acrolein treatment activates transcription of phase II genes in general, as indicated by an increase in mRNA for NAD (P) H:quinone oxidoreductase (NQO1). Western blot analysis revealed the increased level of the transcription factor, Nrf2 in the nuclear extract from acrolein treated cells. Electrophoretic mobility shift assay shows increased binding of nuclear proteins to human antioxidant response element (ARE) consensus sequence after treatment with acrolein. The involvement of Nrf2 in ARE mediated transcriptional activation in response to acrolein exposure has been confirmed by human NQO1-ARE reporter assay. The ability of acrolein to transcriptionaly activate genes responsible for phase II enzymes may form the basis of resistance against cell death and can have implications in cigarette smoke related lung carcinogenesis.

Liddle's syndrome mutations disrupt cAMP-mediated translocation of the epithelial Na(+) channel to the cell surface.

The epithelial Na(+) channel (ENaC) plays a critical role in Na(+) absorption, and mutations in this channel cause diseases of Na(+) homeostasis, including a genetic form of hypertension (Liddle's syndrome). To investigate cAMP-mediated stimulation of ENaC, alpha, beta, and gammaENaC were coexpressed in Fischer rat thyroid epithelia to generate apical Na(+) channels and transepithelial Na(+) current. cAMP agonists stimulated Na(+) current by 70%. Following covalent modification of cysteines introduced into ENaC, cAMP increased the rate of appearance of unmodified channels at the cell surface. In addition, cAMP increased the fluorescent labeling of ENaC at the apical cell surface. Inhibition of vesicle trafficking by incubating epithelia at 15 degrees C prevented the cAMP-mediated stimulation of ENaC. These results suggest that cAMP stimulates Na(+) absorption in part by increasing translocation of ENaC to the cell surface. Stimulation of ENaC by cAMP was dependent on a sequence (PPPXY) in the COOH terminus of each subunit. This sequence is the target for mutations that cause Liddle's syndrome, suggesting that cAMP-mediated translocation of ENaC to the cell surface is defective in this genetic form of hypertension.

Amino acid exchange and covalent modification by cysteine and glutathione explain isoforms of fatty acid-binding protein occurring in bovine liver

A unique property of the liver-type member of the family of fatty acid-binding proteins is the heterogeneic pattern observed upon isolation, which can only partly be ascribed to the state of lipidation. Here we unraveled the structural basis of the heterogeneity of delipidated liver-type fatty acid-binding protein (L-FABP). Charge fractions of L-FABP focusing at pH 6.0 and at pH 7.0/7.1 were first isolated from bovine liver. Upon reduction, however, two distinct isoforms, namely pI 6.0 L-FABP and pI 7.0 L-FABP, were observed. From these isoforms peptides were generated enzymically and chemically by four independent methods. Peptides were separated by reverse phase high performance liquid chromatography and analyzed by Edman degradation and plasma desorption mass spectrometry. The complete amino acid sequences of the isoforms were established; they consist of 127 amino acids and each is N-terminally blocked with an acetyl group. The difference between pI 6.0 L-FABP and pI 7.0 L-FABP was attributed to an asparagine-aspartate exchange at position 105. When tryptic peptides of the pH 7.0/7.1 fraction were analyzed, discrepancies between sequence and mass data of the peptides containing at position 69 the sole cysteine of L-FABP led to the disclosure of a cysteinylation occurring at this position and giving rise to the slightly more basic pH 7.1 species. Moreover, chemical modification studies revealed that a part of the pH 6.0 fraction was pI 7.0 L-FABP that was glutathionylated at Cys69. Neither modification, however, prevented the binding of fatty acids. Together amino acid exchange and covalent modification of cysteine entirely explain the heterogeneity of L-FABP from bovine liver.

Inhibition of ADP-induced platelet shape change and aggregation by o-phthalaldehyde: Evidence for covalent modification of cysteine and lysine residues

Platelets play a major role in the hemostatic process following vascular injury. Chemical modification of cysteine and/or lysine residues in platelet proteins has been shown to cause loss of platelet aggregation induced by diverse agonists; however, these investigations have not addressed the identity of the specific proteins affected. o-Phthalaldehyde (OPTH) is a unique chemical modification reagent that forms and permits the identification of fluorescent isoindole derivatives with proteins by covalently and simultaneously modifying closely spaced cysteine and lysine residues. We found that OPTH inhibited platelet aggregation induced by ADP, collagen, and U46619 (an analog of prostaglandin H 2), but had minimal effect on platelet aggregation induced by thrombin, plasmin, chymotrypsin, A23187 (a calcium ionophore), PMA (phorbol 12-myristate 13-acetate), and PMA + A23187. Since platelet aggregation induced by ADP, collagen, and U46619 has been shown to involve binding of endogenous or exogenous ADP to the platelet receptor, our further studies focused on platelet aggregation induced by ADP. OPTH inhibited ADP-induced shape change and aggregation in a concentration-dependent manner. The second-order rate constant for the inhibition of ADP-induced platelet shape change ( K sc = 1.0 × 10 3 M −1s −1) was lower than that for aggregation ( K agg = 5.4 × 10 3 M −1s −1). Fluorescence excitation and emission spectra of OPTHplatelet adduct exhibited maxima at 346 and 437 nm, respectively, consistent with the formation of an isoindole derivative(s). The nonpenetrating thiol-specific reagent, p-chloromercuribenzenesulfonate (pCMBS) (0.8 m m), is known to block the inhibition of stimulated adenylate cyclase induced by ADP but not the ADP-induced platelet shape change. The inhibition of ADP-induced platelet shape change ( K sc = 1.5 × 10 3 M −1s −1) by OPTH was not affected by pCMBS. OPTH, at concentrations (15–50 μ m) that inhibited ADP-induced platelet aggregation and shape change did not raise the intracellular levels of adenosine cyclic 3′,5′-monophosphate (cAMP) in platelets nor did it impair the ability of iloprost (a stable analog of prostaglandin I 2) to raise the platelet cAMP level. Thus, OPTH under these conditions did not interact with platelet adenylate cyclase. 5′- p-fluorosulfonylbenzoyladenosine (FSBA) has been previously shown to inhibit ADP-induced platelet shape change and aggregation by covalently modifying aggregin ( M r = 100 kDa), a putative ADP receptor on platelet surface. N-Ethylmaleimide (NEM) also inhibits ADP-induced platelet shape change and aggregation. Preincubation of platelets with OPTH prevented labeling of platelets by [ 3H]FSBA and [ 3H]NEM. The results show that OPTH inhibited ADP-induced platelet shape change and aggregation and chemically modifies closely spaced cysteine and lysine residues in aggregin, thus preventing binding of ADP to its receptor.

Activity changes in lac repressor with cysteine oxidation.

The effects of prior covalent cysteine modification or nonspecific DNA presence on the reaction of lac repressor protein with N-bromosuccinimide have been investigated. At low excesses, N-bromosuccinimide oxidation causes loss of operator DNA binding activity with simultaneous retention of inducer and nonspecific DNA binding activities. Cysteine and methionine are oxidized under the conditions utilized. Covalent modification of the cysteines of repressor prior to reaction decreased the observed loss of operator DNA binding capacity; the presence of nonspecific DNA partially prevented oxidation of the cysteines by N-bromosuccinimide, and concurrent protection of operator binding ability was observed. Methionine oxidation was observed in the cases where protection of the operator DNA binding capacity of repressor was seen. The region surrounding cysteine 107 was found to be influential in maintaining intact operator DNA binding function in repressor. This observation provides chemical evidence for the contribution of the core region of repressor in determining specificity of the protein in binding the lac operator. The protection from oxidation of cysteine residues in the core region by the presence of nonspecific DNA suggests that this binding influences the core region of the protein.

Methionyl Residue Critical for Activity and Regulation of Bovine Liver Glutamate Dehydrogenase

Abstract Glutamate dehydrogenase is inactivated by incubation with 4-iodoacetamidosalicylic acid at 37° and pH 6.0. The reaction obeys pseudo-first order kinetics, with a rate constant which is linearly proportional to the 4-iodoacetamidosalicylic acid concentration. A 4- to 5-fold decrease in the rate of inactivation is provided by the substrates α-ketoglutarate and glutamate when added to the incubation mixture with ADP and DPN, but no appreciable decrease in the inactivation rate is produced by the coenzymes themselves, either alone or when combined with ADP or GTP. These results suggest that 4-iodoacetamidosalicylic acid reacts in the region of the active site. 4-Iodoacetamidosalicylic acid produces a change in the response of the enzyme to the allosteric inhibitor GTP, as exemplified by an increase in the kinetic dissociation constant of the enzyme-coenzyme-GTP complex. Although the reagent causes inactivation of the enzyme in the absence of GTP, it does not influence the small residual activity (4 to 8%) seen when a saturating concentration of GTP is present. Only methionyl and cysteinyl residues of glutamate dehydrogenase are altered by 4-iodoacetamidosalicylic acid at periods up to 10 times the half-life for inactivation; lysyl and histidyl residues are not alkylated during this time. As determined by the release of iodide ion from 4-iodoacetamidosalicylic acid, approximately 4 amino acids per peptide chain are modified when the enzyme loses 90% of its activity: about 2 cysteinyl and 2 methionyl residues are alkylated. Several lines of evidence suggest that covalent modification of cysteine is not the primary cause of inactivation. The rate of alkylation of sulfhydryl groups (K-sh = 0.0015 min-1) is only one-sixth that of inactivation (Ki = 0.0102 min-1) for the enzyme in the absence of ligands. These two rate constants can be varied independently by the addition of substrates and allosteric modifiers. In contrast, a good correlation is observed between the degree of inactivation of the enzyme and the extent of modification of approximately 1 methionyl residue per peptide chain. Furthermore, treatment of 75% inactivated enzyme with dithiothreitol produces a 3-fold increase in the enzymatic activity, accompanied by the regeneration of methionine but no appreciable alteration of the number of modified cysteine residues. It is concluded that the integrity of a methionine residue is critical for the function of glutamate dehydrogenase.