Pronounced Toxicity Differences between Homobifunctional Protein

Article pubs.acs.org/crt

Pronounced Toxicity Differences between Homobifunctional Protein Cross-Linkers and Analogous Monofunctional Electrophiles Matthew K. Spencer,† Nikolai P. Radzinski,† Susmit Tripathi, Sreyan Chowdhury, Rachelle P. Herrin, Naveeshini N. Chandran, Abigail K. Daniel, and James D. West* Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry, The College of Wooster, Wooster, Ohio 44691, United States S Supporting Information *

ABSTRACT: Bifunctional electrophiles have been used in various chemopreventive, chemotherapeutic, and bioconjugate applications. Many of their effects in biological systems are traceable to their reactive properties, whereby they can modify nucleophilic sites in DNA, proteins, and other cellular molecules. Previously, we found that two different bifunctional electrophiles diethyl acetylenedicarboxylate and divinyl sulfoneexhibited a strong enhancement of toxicity when compared with analogous monofunctional electrophiles in both human colorectal carcinoma cells and baker’s yeast. Here, we have compared the toxicities for a broader panel of homobifunctional electrophiles bearing diverse electrophilic centers (e.g., isothiocyanate, isocyanate, epoxide, nitrogen mustard, and aldehyde groups) to their monofunctional analogues. Each bifunctional electrophile showed at least a 3-fold enhancement of toxicity over its monofunctional counterpart, although in most cases, the differences were even more pronounced. To explain their enhanced toxicity, we tested the ability of each bifunctional electrophile to crosslink recombinant yeast thioredoxin 2 (Trx2), a known intracellular target of electrophiles. The bifunctional electrophiles were capable of cross-linking Trx2 to itself in vitro and to other proteins in cells exposed to toxic concentrations. Moreover, most cross-linkers were preferentially reactive with thiols in these experiments. Collectively, our results indicate that thiol-reactive protein cross-linkers in general are much more potent cytotoxins than analogous monofunctional electrophiles, irrespective of the electrophilic group studied.

■

INTRODUCTION A number of organic molecules, including various phytochemicals, industrial chemicals, and lipid peroxidation byproducts, contain electron-deficient centers that render them reactive with biological nucleophiles and capable of altering cellular homeostasis.1−3 Many electrophiles have been advanced as chemopreventive or chemotherapeutic agents for age-associated diseases (e.g., cancer, neurodegenerative disease, heart disease),4−6 whereas others are known acute toxins, contact allergens, or mutagenic substances.7,8 Electrophiles elicit their diverse biological effects by covalently modifying cellular macromolecules, including DNA and/or numerous target proteins.3,8 Such damage can lead to pronounced alterations in cell signaling, gene expression, cytoskeletal organization, and cell cycle progression, which may ultimately influence cellular survival, depending on the duration and level of exposure.3,6,9,10 In addition to having a broad range of cellular effects, there is vast structural diversity among the electrophilic groups found within various naturally occurring and synthetic toxins. Such structural variability allows for differences in cellular uptake and reactivity with nucleophiles.10 In the present study, we focused on molecules bearing α,β-unsaturated dicarbonyl, vinyl sulfone, isothiocyanate, isocyanate, epoxide, nitrogen mustard, and aldehyde electrophilic centers. While several of these groups © 2013 American Chemical Society

are capable of reacting with nitrogenous bases in DNA (e.g., epoxides and nitrogen mustards),11,12 others (e.g., isothiocyanates) are thought to react exclusively with nucleophilic sites in proteins.13 Within proteins, “soft” electrophiles like α,β-unsaturated dicarbonyls undergo nucleophilic additions primarily with thiol groups in cysteine residues, whereas “harder” electrophilic groups can readily react with the primary and secondary amine groups in lysine and histidine, respectively.10 Although bifunctional electrophiles have been routinely used as biochemical tools to stabilize transient protein−protein interactions,14,15 there has been a renewed interest in developing chemotherapeutic agents with multiple electrophilic centers in recent years.16,17 Moreover, bifunctional electrophiles can afford enhanced properties when activating stressresponsive gene expression pathways, allowing for the design of more effective chemopreventive agents.5,18 While many groups have studied electrophiles that are heterobifunctional, a broad survey that relates the toxicity of bifunctional molecules containing duplicate electrophilic centers to similar monofunctional electrophiles has not been performed. Previously, we found that two homobifunctional electrophilesdiethyl Received: August 5, 2013 Published: October 18, 2013 1720

dx.doi.org/10.1021/tx400290j | Chem. Res. Toxicol. 2013, 26, 1720−1729

Chemical Research in Toxicology

Article

Dulbecco’s minimal essential medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotic-antimycotic (Life Technologies) at 37 °C and 5% CO2. Me2SO concentrations were ≤0.1% (v/v) in all experiments. Yeast Cell Culture. The Saccharomyces cerevisiae strain BY4741 (MATa his3Δ0 leu2Δ0 met15Δ0 ura3Δ0) was originally obtained from Open Biosystems and was a kind gift of Kevin Morano (University of Texas Medical School at Houston). BY4741 cultures were maintained in YPD medium (1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) glucose) at 30 °C. Me2SO concentrations in acute toxicity assays were ≤1% (v/v) and in long-term exposures were ≤0.1% (v/v). Cultures expressing FLAG-Trx2 were maintained in uracil-deficient medium as previously described.21 Toxicity Assays. Methods for determining toxicity of electrophiles in RKO cells and acute toxicity of electrophiles in yeast are described elsewhere.19,20 For monitoring each electrophile’s long-term toxicity in S. cerevisiae, overnight cultures of BY4741 were grown to stationary phase in YPD medium at 30 °C. Subsequently, cultures were diluted to OD600 of 0.2 in YPD. Fifty microliters of the diluted yeast was added to 50 μL of YPD containing 2× electrophile or 0.2% (v/v) Me2SO in a sterile 96-well plate. Cultures were grown on a shaking platform at 30 °C for 16 h. The OD600 for each well was measured using a Thermoskan Ascent plate reader (Hudson, NH). All treatments were done in quadruplicate for individual experiments. IC50 values for each electrophile were calculated from nonlinear regression analysis of toxicity data using GraphPad Prism (La Jolla, CA). Curves were fit with a variable slope and a minimum for each treatment set to 0% percent of the control (i.e., 100% cell death). Expression and Purification of Recombinant Trx2. Procedures for cloning the TRX2 gene into the bacterial expression vector pET45b have been described previously.20 Cloning of the TRX2 gene bearing the C31,34A mutation into pET45b was accomplished by using PCR to amplify the mutant TRX2 gene out of the yeast expression vector p416-GPD(21) and ligating this product into pET45b via its HindIII and XhoI sites. Expression plasmids for proteins were transformed into Escherichia coli BL21(DE3) cells. Proteins were expressed by induction with 1 mM isopropyl β-D-1-thiogalactopyranoside for 6 h at 37 °C and purified using Ni-nitrilotriacetate affinity chromatography according to previously published procedures.20 Wild-type and C31,34A-Trx2 were greater than 95 and 80% pure, respectively, by gel analysis. Modification of Thioredoxin by Bifunctional Electrophiles. For in vitro experiments, purified Trx2 (∼500 μM) was incubated for 1 h at 37 °C with 20 mM DTT to reduce thiol groups in the protein. Subsequently, DTT was removed using a Micro Biospin6 column (Bio-Rad, Hercules, CA) equilibrated in 50 mM HEPES (pH 8). Cross-linking reactions (20 μL) contained 50 μM Trx2, 50 mM HEPES (pH 8), 150 mM NaCl, 2.5 mM EDTA (pH 8), and varying concentrations of electrophiles (or Me2SO solvent). Reactions were incubated at 37 °C for 24 h prior to resolution via SDS-PAGE. To determine cross-linking in the C31,34A Trx2 mutant, proteins from cross-linking reactions were transferred to PVDF membrane for immunoblot analysis. Membranes were blocked in a modified Trisbuffered saline (TBS-T; 100 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Tween 20) containing 5% (w/v) nonfat dry milk, incubated with a mouse monoclonal antibody against polyhistidine (Sigma), washed three times with TBS-T, incubated with anti-mouse secondary antibody conjugated to horseradish peroxidase (Cell Signaling, Danvers, MA), and washed four times with TBS-T. Protein complexes were visualized with enhanced chemiluminescence detection. The procedure for monitoring intermolecular cross-linking of FLAG-Trx2 to other proteins following treatment of yeast cells with bifunctional electrophiles has been described previously.21

acetylenedicarboxylate (DAD) and divinyl sulfone (DVSF) show considerably enhanced toxicity in baker’s yeast and mammalian cells when compared with their monofunctional analogues.19,20 Here, we build upon our previous work by comparing the relative enhancement of toxicity for a diverse panel of aliphatic homobifunctional electrophiles with their monofunctional electrophiles. We report that bifunctional electrophiles range in their toxicity enhancement from 3-fold to over 500-fold, depending on the type of electrophilic center and the model system where toxicity was measured.

■

EXPERIMENTAL PROCEDURES

Chemicals. The following electrophiles were purchased from Sigma-Aldrich (St. Louis, MO): diethyl maleate (DEM), DAD, ethyl vinyl sulfone (EVSF), DVSF, n-butyl isothiocyanate (BITC), butane1,4-diisothiocyanate (BDITC), n-butyl isocyanate (BIC), butane-1,4diisocyanate (BDIC), propylene oxide (PROX), 1,2,3,4-diepoxybutane (DEB), mechlorethamine hydrochloride (HN2), acetaldehyde (AcCO), and glyoxal (GLO). 2-Diethylaminoethyl chloride (HN1) was purchased from Acros Organics (Pittsburgh, PA). Structures of electrophiles used are depicted in Figure 1. All other chemicals were purchased from Sigma-Aldrich.

■

RESULTS Enhanced Toxicity of Diverse Bifunctional Electrophiles Across Model Systems. Previously, we reported that DAD and DVSF showed approximately 5- to 6-fold greater toxicity than DEM and EVSF in human colon cancer cells and noted the presence of two electrophilic centers within these

Figure 1. Structures of molecules used in this study. Mammalian Cell Culture. The RKO cell line (a human colorectal carcinoma cell line) was originally obtained from the American Type Culture Collection and was a kind gift of Lawrence J. Marnett (Vanderbilt University School of Medicine). Cells were grown in 1721



Article

Figure 2. Enhanced toxicity of diverse bifunctional electrophiles in mammalian cells. RKO cells were treated with varying concentrations of the indicated monofunctional and bifunctional electrophiles (red and blue curves, respectively) for 24 h at 37 °C prior to determining cell viability with the dye WST-1. Results are the average of two to three independent experiments, each conducted in quadruplicate, ±SEM. Percentage of WST-1 conversion in each treatment was normalized to the untreated control (set at 100%) in each experiment.

Table 1. Comparison of Toxicities between Various Classes of Aliphatic Monofunctional and Bifunctional Electrophiles class of electrophile

molecule name

abbreviation

CAS no.

RKO cell IC50 (μM)a

yeast IC50 (μM)b

α,β-unsaturated dicarbonyl diethyl maleate diethyl acetylenedicarboxylate

DEM DAD

141-05-9 762-21-0

138 ± 8c 30 ± 1c

>5000 65 ± 4

ethyl vinyl sulfone divinyl sulfone

EVSF DVSF

1889-59-4 77−77−0

204 ± 12d 34 ± 3d

649 ± 50 136 ± 10

n-butyl isothiocyanate butane-1,4-diisothiocyanate

BITC BDITC

592-82-5 4430-51-7

83 ± 15 4.7 ± 0.4

>5000 7.6 ± 0.5

n-butyl isocyanate butane-1,4-diisocyanate

BIC BDIC

111-36-4 4538-37-8

3940 ± 130e 1280 ± 90e

>5000 1860 ± 130

propylene oxide 1,2,3,4-diepoxybutane

PROX DEB

75-56-9 1464-53-5

>5000 67 ± 7

>5000 984 ± 53

2-diethylaminoethyl chloride mechlorethamine

HN1 HN2

869-24-9 55-86-7

66 ± 12 5.4 ± 1.3

>2500 278 ± 26

acetaldehyde glyoxal

AcCO GLO

75-07-0 107-22-2

>5000 1340 ± 200

>5000 >5000

vinyl sulfone

isothiocyanate

isocyanate

epoxide

nitrogen mustard

aldehyde

a

IC50 values were calculated based on conversion of WST-1 by RKO cell cultures treated with compounds for 24 h, following comparison to the vehicle control. Results are the average ± SEM of two to three experiments, each performed in quadruplicate. bIC50 values were calculated from the OD600 values for cultures of S. cerevisiae treated for 16 h with varying doses of molecules as compared with the vehicle control. Results are the average ± SEM of three to five replicate experiments, each performed in quadruplicate. cResults from West et al. (2011).19 dResults from West et al. (2011).20 e Isocyanates were diluted in serum-free medium prior to addition to cultures, due to high response variability observed in medium containing serum (data not shown).

molecules.19,20 To explore whether other bifunctional electrophiles exhibited a similar enhancement in toxicity, we compared the relative toxicities of various bifunctional molecules to their monofunctional analogues in RKO cells (Figure 1). In RKO

cells, side-by-side comparison of electrophile pairs revealed that all bifunctional electrophiles were considerably (i.e., greater than 3-fold) more toxic than their monofunctional counterparts (Figure 2, Table 1). The greatest enhancement of toxicity was 1722



Article

this assay were treated with electrophiles, while initially in the stationary phase a growth state where cells typically have enhanced stress resistance.22 All bifunctional electrophiles exhibited significantly greater toxicity than structurally related monofunctional electrophiles when measurable, albeit at doses that were somewhat higher than that in mammalian cells (Figure 3, Table 1). Of the electrophile classes tested, highly pronounced differences in toxicity (i.e., differences of 50-fold or greater) were observed for isothiocyanate- and α,β-unsaturated dicarbonyl-based electrophiles (Table 2). Unlike the mammalian cell experiments, neither aldehyde-containing electrophile exhibited toxicity in this assay up to 5 mM (Supporting Information Figure S1A). Similar results were obtained when actively dividing yeast cultures were treated with electrophiles. Bifunctional electrophiles in each class, with the exception of aldehydes, were at least 5- to 25-fold more toxic than the corresponding monofunctional electrophiles in these acute toxicity experiments (Figures S1B and S2). The most pronounced differences in toxicity in these acute toxicity assays were between electrophiles in the α,β-unsaturated dicarbonyl class, where DAD was approximately 3 orders of magnitude more toxic than DEM. Collectively, our results indicate that bifunctional electrophiles are considerably more toxic than analogous monofunctional electrophiles in actively dividing and stationary phase yeast as well as cultured colon cancer cells. Intermolecular Protein Cross-Linking by Bifunctional Electrophiles. Based upon our toxicity results, we hypothesized that each of the bifunctional electrophiles could cross-link proteins as a means of enhancing its toxicity. To test this hypothesis, we performed cross-linking experiments with Trx2, a cytosolic thioredoxin from S. cerevisiae. Thioredoxins from a

Table 2. Relative Fold Enhancement in Toxicity in RKO Cells and S. cerevisiaea class of electrophile α,β-unsaturated dicarbonyl vinyl sulfone isothiocyanate isocyanate epoxide nitrogen mustard aldehyde

RKO cells

S. cerevisiae

4.6 6 17.7 3.1 >74.6 12.2 >3.7

>75 4.8 >650 >2.5 >5 >9 ndb

a

The overall fold enhancement for each molecule was calculated by dividing the IC50 for the monofunctional electrophile in each class by the IC50 for the bifunctional electrophile. IC50 values for all molecules are reported in Table 1. bIC50 values exceeded 5 mM for both the monofunctional and bifunctional aldehyde and, therefore, could not be determined in S. cerevisiae.

observed in electrophiles of the isothiocyanate and epoxide classes, with BDITC and DEB each showing at least a 15-fold increase in toxicity over their respective monofunctional counterparts BITC and PROX (Table 2). In contrast, electrophiles harboring isocyanate electrophilic centers BDIC and BICexhibited the most similar toxicities (Figure 2, Tables 1 and 2). Taken together, these results indicate that structurally diverse bifunctional electrophiles exhibit more than a 2-fold enhancement of toxicity in RKO cells, much more than would be expected by doubling the number of reactive centers. To quantify the toxicities of electrophiles in S. cerevisiae, we monitored the optical density of liquid cultures of yeast following continuous exposure to electrophiles for 16 h. Cells in

Figure 3. Enhanced long-term toxicity of diverse bifunctional electrophiles in yeast. Yeast cultures in the stationary phase were diluted in nonselective growth medium containing the indicated monofunctional and bifunctional electrophiles. Cells were grown for 16 h at 30 °C prior to measurement of culture optical densities at 600 nm. Results are the average of three to five independent experiments, each done in quadruplicate, ±SEM. Treated samples were normalized to the untreated control (set at 100%) in each experiment. 1723



Article

Figure 4. Cross-linking of recombinant yeast Trx2 by bifunctional electrophiles. Reduced recombinant Trx2 (50 μM) was incubated for 24 h at 37 °C with varying concentrations of the indicated electrophiles. The extent of intermolecular protein cross-linking was determined by SDS-PAGE and detection with Coomassie blue staining. The position of molecular weight standards (in kDa) is indicated on the left side of the gel image. Results are representative of three independent experiments.

reactions to determine whether they prevent Trx2 oligomerization. With the exception of GLO (where pronounced crosslinking was difficult to detect), cross-linking of Trx2 by all bifunctional molecules was inhibited in the presence of increasing β-ME concentrations (Figure 6). In contrast, marked inhibition of Trx2 cross-linking by EA was only observed in reactions containing DVSF (Figure 6). Similar competition trends were observed with Tris, a primary amine-containing molecule with a lower pKa than EA (Figure S3). Moreover, a decrease in free thiol content was observed in Trx2 following exposure to each of the electrophiles tested, as assessed by reaction of denatured Trx2 with 5,5′-dithiobis(2-nitrobenzoic acid) (Figure S4). Collectively, our results imply that each bifunctional electrophile possesses reactivity with thiols, although reactivity with amines is more dependent on the type of electrophilic functional group. To probe the preferential reactivity of each electrophile in a different manner, we compared cross-linking of wild-type (wt) Trx2 to a mutant Trx2 where the nucleophilic, active site Cys residues had been mutated to Ala (C31,34A). From these experiments, decreased levels of cross-linking were observed for Trx2 bearing the C31,34A mutant when treated with DAD, DEB, and HN2 (Figure 7). However, comparable levels of crosslinking were observed between wt Trx2 and mutant Trx2 treated with DVSF, BDITC, BDIC, and GLO, suggesting that amine-containing residues (e.g., His, Lys) are also targeted in

variety of species utilize nucleophilic Cys residues to facilitate disulfide reduction in various substrate proteins.23 The presence of redox active Cys residues in the thioredoxin active site renders it susceptible to electrophilic modification.24,25 In addition, we have recently identified thioredoxins as targets of bifunctional electrophiles in treated yeast and mammalian cells.20,21 Purified Trx2 was incubated with either the solvent (Me2SO) or increasing doses of the monofunctional or bifunctional electrophile in each pair. Dose-dependent oligomerization of Trx2 was observed with each bifunctional electrophile tested, as indicated by a mobility change in Trx2 following separation via SDS-PAGE (Figure 4). However, the monofunctional electrophile in each group of molecules did not cause significant intermolecular cross-linking of Trx2. Side-by-side comparison of cross-linking efficacy revealed that BDITC and DVSF induced the most pronounced intermolecular cross-linking among the bifunctional electrophiles tested (Figure 5). Taken together, these results suggest that the bifunctional electrophiles studied are capable of cross-linking proteins together. Determining the Preferential Reactivity of Protein Cross-Linkers. To determine which nucleophilic group(s) are potentially involved in promoting cross-linking by bifunctional electrophiles, we used both competition- and mutagenesisbased approaches. In the competition experiments, excess thiol or primary amine competitorsβ-mercaptoethanol (β-ME) or ethanolamine (EA), respectivelywere added to cross-linking 1724



Article

The majority of the electrophiles studied modify thiol groups in proteins preferentially, indicating that adduct formation in targets with highly nucleophilic Cys residues may play a key role in their toxicity. In recent years, a number of groups have performed proteome profiling studies to identify electrophilesensitive proteins that harbor nucleophilic Cys residues.30−32 The proteins identified in these surveys commonly utilize Cys residues to facilitate redox catalysis, bind metals to impart structural properties, and/or perform sensory/signaling roles.33,34 Modification by electrophiles and other thiol-reactive species often leads to the inactivation of such proteins,3,8,35 and the compound effects of inhibiting numerous susceptible targets by adducting functional Cys residues could ultimately trigger cell death. This may be especially true in cases where two hyper-reactive Cys residues are cross-linked to one another in the same or different proteins following exposure to a bifunctional electrophile. Indeed, intra- and intermolecular cross-linking that involves proteins is likely to be a strong contributing factor that accounts for the enhanced toxicity of bifunctional electrophiles over structurally similar monofunctional electrophiles. The types of intermolecular cross-links formed by bifunctional electrophiles in cells vary, making the study of their toxicity complex. In certain cases, some of the molecules tested form highly toxic DNA−protein cross-links. As an example, cross-links formed between the DNA repair protein O6-alkylguanine DNA alkyltransferase and DNA bases are proposed to contribute significantly to the toxicity of DNA-reactive bifunctional electrophiles, including DEB.36−38 Despite this, some of the electrophiles studied react nominally with amines in general, particularly those within DNA, and/or show limited mutagenicity in cell-based assays.13,19,39,40 We propose that, in these cases, protein−protein cross-linking plays a key role in mediating toxicity through a number of possible mechanisms, as noted below. In addition to inhibiting numerous proteins with functionally important Cys residues, cross-linking of protein targets may exacerbate the protein misfolding that is triggered by alkylation, ultimately overwhelming cellular protein folding and clearance machineries and leading to enhanced accumulation of toxic misfolded polypeptides.5 In support of this, monofunctional and heterobifunctional electrophiles can modify metastable proteins and promote their aggregation in vitro.41,42 Alternatively, electrophilic modification of a nonpathogenic prion protein with nitrogen mustard has been proposed as a means of preventing the structural transition to its pathogenic form, suggesting that electrophiles influence protein folding dynamics.43 Likewise, epicatechin gallate can cross-link and promote stabilization of amyloid beta fibrils, a potential way that this molecule protects against or delays neurodegeneration.44 Moreover, cross-linked proteins may promote aggregation of other unstable proteins as a consequence of their impairment of proteasome activity.45,46 Still, relatively few studies have been performed to probe the impact of bifunctional electrophiles on protein folding and aggregation. Given that disulfide formation between Cys residues can alter a protein’s tertiary and quaternary structure,33,47 it is likely that similar thiol-bridging events carried out by bifunctional electrophiles significantly alter protein folding, structural dynamics, and stability, having a pronounced impact on cellular physiology and viability. Modification of protein thiols by some electrophiles notably isothiocyanate-containing electrophilesmay be labile. In this scenario, the electrophilic modification can undergo

Figure 5. Side-by-side comparison of intermolecular cross-linking of Trx2 by bifunctional electrophiles. Recombinant Trx2 (50 μM) was incubated for 24 h at 37 °C with indicated electrophiles, with each at 5 mM. Protein cross-linking was determined by SDS-PAGE and detection with Coomassie blue staining. The position of molecular weight standards (in kDa) is indicated on the left. Results are representative of three independent experiments.

the absence of Cys nucleophiles. Taken together, our results suggest that each electrophile is preferentially thiol-reactive, but in the absence of thiols, nucleophilic amines may become modified by electrophiles to mediate cross-linking in proteins. Formation of Intermolecular Cross-Links between Trx2 and Other Proteins in Cells Treated with Bifunctional Electrophiles. To determine whether Trx2 is an intracellular target of these electrophiles, we treated yeast expressing a FLAG-tagged Trx2 with concentrations of cross-linkers that were approximately 2- to 3-fold higher than the IC50 for each molecule. Cross-linking of FLAG-Trx2 to other proteins was observed with all bifunctional electrophiles tested (Figure 8), although the levels and types of cross-linked complexes that formed in the presence of these agents varied. In contrast, limited cross-linking of other proteins to Trx2 occurred in Trx2 where its active site Cys residues were mutated to Ala, indicating that each of these molecules modify thiol groups preferentially in cells. In addition, these results indicate that the molecules tested are capable of promoting intermolecular crosslinking between proteins at toxic doses.

■

DISCUSSION Here, we report that structurally diverse bifunctional electrophiles are considerably more toxic than their monofunctional analogues in both mammalian cells and S. cerevisiae. These trends largely mirror our previous studies and those using other electrophiles in different model systems,16,19,20,26−29 where the toxicity enhancement afforded by doubling the number of electrophilic centers ranges from 3- to more than 500-fold. Each of the bifunctional molecules tested in our study has the propensity to promote intermolecular cross-linking of the yeast thioredoxin Trx2 in vitro and in cells exposed to toxic concentrations. Therefore, we propose that cross-linking reactions involving proteins contribute significantly to the enhanced toxicity observed with these molecules. 1725



Article

Figure 6. Intermolecular cross-linking of Trx2 in the presence of thiol and amine competitors. Recombinant Trx2 (50 μM) was incubated for 24 h at 37 °C with indicated electrophiles in reactions containing no competitor or increasing concentrations of either β-mercaptoethanol (β-ME) or ethanolamine (EA). Protein cross-linking was determined by SDS-PAGE and detection with Coomassie blue staining. The position of molecular weight standards (in kDa) is indicated on the left. Results are representative of two-three independent experiments.

electrophiles studied. The differences between the predicted partition coefficient (logP) for each monofunctional−bifunctional electrophile pair are nominal in most cases (Table S1). Moreover, structure−activity studies in mammalian cells with DEM and EVSF analogues reveal that there is only a slight enhancement of toxicity associated with increasing an electrophile’s hydrophobic character.19,20 In addition, altering the length of the linear aliphatic group attached to an isothiocyanate functional group from a butyl group to either a methyl or n-propyl group does not impact the overall toxicity of these molecules in mammalian cells (Supporting Information Figure S7 and Table S2). Similar results showing modest effects of increasing hydrophobicity on the toxicity of electrophiles have been observed by other groups,28,53,54 indicating that small differences in hydrophobicity marginally influence electrophile toxicity in a wide array of model systems. In contrast, the relative bulk of the molecule nearby the electrophilic center can exert considerable influence on toxicity. In support of this, tert-butyl isothiocyanate was not toxic in RKO cells at doses up to 1 mM (Figure S7 and Table S2). This result implies that the increased bulk of the branched aliphatic group disrupts interaction of the electrophilic center with cellular nucleophiles, thus limiting toxicity. Our current studies lend new insights into the cellular effects of bifunctional electrophiles. They demonstrate a general enhancement of toxicity observed for bifunctional electrophiles

group transfers to nearby Cys residues or dissociate through electronic rearrangements. For instance, isothiocyanate− thiol adducts can be transferred directly to free thiols via a trans-thiocarbamoylation reaction,48 and the thiol conjugates of aliphatic isothiocyanates often exhibit similar biological properties as the free forms.49−51 In our studies, we were unable to detect high levels of Trx2 cross-linked complexes from yeast cultures treated with toxic concentrations of BDITC (Figure S5), despite the fact that thioredoxins are known targets of isothiocyanate electrophiles in other organisms.52 In contrast, Trx2 forms stable complexes with its main redox partners in cells treated with other irreversible thiol cross-linkers.21 These results suggest that, if cross-links form between Trx2 and other proteins in cells treated with BDITC, they are dynamic and short-lived. However, cross-links formed between Trx2 monomers by BDITC in vitro were not reversible by treatment with excess thiols after BDITC treatment (Figure S6). Under these conditions, other isothiocyanate adducts (e.g., adducts with primary amines) that are irreversible likely form. Nonetheless, a factor that may influence the overall toxicity for some types of electrophiles is their ability to undergo reversible addition to thiols. Further work in this area with electrophiles prone to group transfer and retro-additions is needed to determine the impact of reversibility on toxicity. Unlike the factors mentioned earlier, altered hydrophobicity only exerts a minor influence on the toxicity of many of the 1726



Article

Figure 7. Alteration in intermolecular cross-linking in Trx2 lacking active site Cys residues. Wild-type (wt) or mutant (C31,34A) Trx2 (10 μM) was incubated for 24 h at 37 °C with indicated electrophiles or Me2SO. Protein cross-linking was visualized by immunoblot analysis using an antibody against the His tag. The position of molecular weight standards (in kDa) is indicated on the left. Results are representative of two-three independent experiments.

over monofunctional analogues that greatly exceeds the 2-fold effect anticipated by doubling the number of reactive centers. Indeed, natural and synthetic bifunctional electrophiles have been re-examined in a variety of recent studies in the search for new pharmaceuticals that induce cytoprotective gene expression, selective death of cancer cells, and alterations in neurodegenerative disease protein aggregation.18,43,44,55 A general theme that emerges from these studies is that molecules with bifunctional characteristics show greater promise as chemotherapeutic and chemopreventive agents,56 an effect that potentially results from their ability to form cross-links involving one or more proteins.

■

ASSOCIATED CONTENT

S Supporting Information *

Supplementary toxicity and protein cross-linking data; measurement of free thiol content following cross-linking. This material is available free of charge via the Internet at http://pubs.acs.org.

■

Figure 8. Intermolecular cross-links formed between Trx2 and other proteins in yeast treated with bifunctional electrophiles. Cultures of yeast expressing FLAG-Trx2 (either wt or C31,34A) were treated with the indicated concentrations of bifunctional electrophiles for 1 h. Protein lysates (5 μg) were resolved via SDS-PAGE and examined for the extent of intermolecular cross-links formed with Trx2 using an immunoblot against the FLAG tag. Pgk1 levels were monitored as a loading control. The position of molecular weight standards (in kDa) is indicated on the left. Results are representative of three independent experiments.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: (330) 263-2368. Fax: (330) 263-2378. Author Contributions †

These authors contributed equally to this work.

1727



Article

Funding

(12) Loeber, R. L., Michaelson-Richie, E. D., Codreanu, S. G., Liebler, D. C., Campbell, C. R., and Tretyakova, N. Y. (2009) Proteomic analysis of DNA−protein cross-linking by antitumor nitrogen mustards. Chem. Res. Toxicol. 22, 1151−1162. (13) Mi, L., Wang, X., Govind, S., Hood, B. L., Veenstra, T. D., Conrads, T. P., Saha, D. T., Goldman, R., and Chung, F. L. (2007) The role of protein binding in induction of apoptosis by phenethyl isothiocyanate and sulforaphane in human non-small lung cancer cells. Cancer Res. 67, 6409−6416. (14) Hermanson, G. T. (2008) Bioconjugation in the study of protein interactions. Bioconjugate Techniques, 2nd ed., pp 1003−1039, Academic Press, London, UK. (15) Tang, X., and Bruce, J. E. (2009) Chemical cross-linking for protein−protein interaction studies. Methods Mol. Biol. 492, 283−293. (16) Adams, D. J., Dai, M., Pellegrino, G., Wagner, B. K., Stern, A. M., Shamji, A. F., and Schreiber, S. L. (2012) Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs. Proc. Natl. Acad. Sci. U.S.A. 109, 15115−15120. (17) Das, S., Das, U., Varela-Ramirez, A., Lema, C., Aguilera, R. J., Balzarini, J., De Clercq, E., Dimmock, S. G., Gorecki, D. K., and Dimmock, J. R. (2011) Bis[3,5-bis(benzylidene)-4-oxo-1-piperidinyl]amides: a novel class of potent cytotoxins. ChemMedChem 6, 1892− 1899. (18) Dinkova-Kostova, A. T., Talalay, P., Sharkey, J., Zhang, Y., Holtzclaw, W. D., Wang, X. J., David, E., Schiavoni, K. H., Finlayson, S., Mierke, D. F., and Honda, T. (2010) An exceptionally potent inducer of cytoprotective enzymes: elucidation of the structural features that determine inducer potency and reactivity with Keap1. J. Biol. Chem. 285, 33747−33755. (19) West, J. D., Stamm, C. E., and Kingsley, P. J. (2011) Structure− activity comparison of the cytotoxic properties of diethyl maleate and related molecules: identification of diethyl acetylenedicarboxylate as a thiol cross-linking agent. Chem. Res. Toxicol. 24, 81−88. (20) West, J. D., Stamm, C. E., Brown, H. A., Justice, S. L., and Morano, K. A. (2011) Enhanced toxicity of the protein cross-linkers divinyl sulfone and diethyl acetylenedicarboxylate in comparison to related monofunctional electrophiles. Chem. Res. Toxicol. 24, 1457− 1459. (21) Naticchia, M. R., Brown, H. A., Garcia, F. J., Lamade, A. M., Justice, S. L., Herrin, R. P., Morano, K. A., and West, J. D. (2013) Bifunctional electrophiles cross-link thioredoxins with redox relay partners in cells. Chem. Res. Toxicol. 26, 490−497. (22) Werner-Washburne, M., Braun, E., Johnston, G. C., and Singer, R. A. (1993) Stationary phase in the yeast Saccharomyces cerevisiae. Microbiol. Rev. 57, 383−401. (23) Holmgren, A. (1995) Thioredoxin structure and mechanism: conformational changes on oxidation of the active-site sulfhydryls to a disulfide. Structure 3, 239−243. (24) Shibata, T., Yamada, T., Ishii, T., Kumazawa, S., Nakamura, H., Masutani, H., Yodoi, J., and Uchida, K. (2003) Thioredoxin as a molecular target of cyclopentenone prostaglandins. J. Biol. Chem. 278, 26046−26054. (25) Fang, J., and Holmgren, A. (2006) Inhibition of thioredoxin and thioredoxin reductase by 4-hydroxy-2-nonenal in vitro and in vivo. J. Am. Chem. Soc. 128, 1879−1885. (26) Schultz, T. W., Sinks, G. D., and Miller, L. A. (2001) Population growth impairment of sulfur-containing compounds to Tetrahymena pyriformis. Environ. Toxicol. 16, 543−549. (27) Schultz, T. W., and Comeaux, J. L. (1996) Structure−toxicity relationships for aliphatic isothiocyanates to Tetrahymena pyriformis. Bull. Environ. Contam. Toxicol. 56, 638−642. (28) Schultz, T. W., Yarbrough, J. W., and Pilkington, T. B. (2007) Aquatic toxicity and abiotic thiol reactivity of aliphatic isothiocyanates: Effects of alkyl-size and -shape. Environ. Toxicol. Pharmacol. 23, 10−17. (29) Schultz, T. W., Netzeva, T. I., Roberts, D. W., and Cronin, M. T. (2005) Structure−toxicity relationships for the effects to Tetrahymena pyriformis of aliphatic, carbonyl-containing, α,β-unsaturated chemicals. Chem. Res. Toxicol. 18, 330−341.

This work was supported through a Cottrell College Science Award from Research Corporation for Science Advancement and through start-up funds, the Luce Fund for Distinguished Scholarship, the Copeland Fund for Independent Study, and William H. Wilson Research Funds provided by The College of Wooster. M.K.S. and N.N.C. were supported through the College’s Sophomore Research Program. R.P.H. was supported by a Clare Boothe Luce Fellowship from The College of Wooster. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Larry Marnett for providing RKO cells and Kevin Morano for providing the yeast strain BY4741 used in our studies.

■

ABBREVIATIONS DEM, diethyl maleate; DAD, diethyl acetylenedicarboxylate; EVSF, ethyl vinyl sulfone; DVSF, divinyl sulfone; BITC, n-butyl isothiocyanate; BDITC, butane-1,4-diisothiocyanate; BIC, nbutyl isocyanate; BDIC, butane-1,4-diisocyanate; PROX, propylene oxide; DEB, 1,2,3,4-diepoxybutane; HN1, 2-diethylaminoethyl chloride; HN2, mechlorethamine; AcCO, acetaldehyde; GLO, glyoxyal; Trx2, thioredoxin 2 from S. cerevisiae; WST-1, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate; TBS-T, Tris-buffered saline with 0.1% Tween 20; β-ME, β-mercaptoethanol; EA, ethanolamine

■

REFERENCES

(1) Drahl, C., Cravatt, B. F., and Sorensen, E. J. (2005) Proteinreactive natural products. Angew. Chem., Int. Ed. 44, 5788−5809. (2) Liebler, D. C. (2008) Protein damage by reactive electrophiles: targets and consequences. Chem. Res. Toxicol. 21, 117−128. (3) Rudolph, T. K., and Freeman, B. A. (2009) Transduction of redox signaling by electrophile−protein reactions. Sci. Signaling 2, re7. (4) Calabrese, V., Cornelius, C., Dinkova-Kostova, A. T., Calabrese, E. J., and Mattson, M. P. (2010) Cellular stress responses, the hormesis paradigm, and vitagenes: novel targets for therapeutic intervention in neurodegenerative disorders. Antioxid. Redox Signaling 13, 1763−1811. (5) West, J. D., Wang, Y., and Morano, K. A. (2012) Small molecule activators of the heat shock response: chemical properties, molecular targets, and therapeutic promise. Chem. Res. Toxicol. 25, 2036−2053. (6) Mi, L., Di Pasqua, A. J., and Chung, F. L. (2011) Proteins as binding targets of isothiocyanates in cancer prevention. Carcinogenesis 32, 1405−1413. (7) Smith Pease, C. K. (2003) From xenobiotic chemistry and metabolism to better prediction and risk assessment of skin allergy. Toxicology 192, 1−22. (8) Marnett, L. J., Riggins, J. N., and West, J. D. (2003) Endogenous generation of reactive oxidants and electrophiles and their reactions with DNA and protein. J. Clin. Invest. 111, 583−593. (9) West, J. D., and Marnett, L. J. (2006) Endogenous reactive intermediates as modulators of cell signaling and cell death. Chem. Res. Toxicol. 19, 173−194. (10) Lopachin, R. M., Gavin, T., Decaprio, A., and Barber, D. S. (2012) Application of the hard and soft, acids and bases (HSAB) theory to toxicant−target interactions. Chem. Res. Toxicol. 25, 239− 251. (11) Park, S., Anderson, C., Loeber, R., Seetharaman, M., Jones, R., and Tretyakova, N. (2005) Interstrand and intrastrand DNA-DNA cross-linking by 1,2,3,4-diepoxybutane: role of stereochemistry. J. Am. Chem. Soc. 127, 14355−14365. 1728



Article

(30) Weerapana, E., Wang, C., Simon, G. M., Richter, F., Khare, S., Dillon, M. B., Bachovchin, D. A., Mowen, K., Baker, D., and Cravatt, B. F. (2010) Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468, 790−795. (31) Marino, S. M., Li, Y., Fomenko, D. E., Agisheva, N., Cerny, R. L., and Gladyshev, V. N. (2010) Characterization of surface-exposed reactive cysteine residues in Saccharomyces cerevisiae. Biochemistry 49, 7709−7721. (32) Vila, A., Tallman, K. A., Jacobs, A. T., Liebler, D. C., Porter, N. A., and Marnett, L. J. (2008) Identification of protein targets of 4hydroxynonenal using click chemistry for ex vivo biotinylation of azido and alkynyl derivatives. Chem. Res. Toxicol. 21, 432−444. (33) Pace, N. J., and Weerapana, E. (2013) Diverse functional roles of reactive cysteines. ACS Chem. Biol. 8, 283−296. (34) Marino, S. M., and Gladyshev, V. N. (2012) Analysis and functional prediction of reactive cysteine residues. J. Biol. Chem. 287, 4419−4425. (35) Paulsen, C. E., and Carroll, K. S. (2010) Orchestrating redox signaling networks through regulatory cysteine switches. ACS Chem. Biol. 5, 47−62. (36) Liu, L., Pegg, A. E., Williams, K. M., and Guengerich, F. P. (2002) Paradoxical enhancement of the toxicity of 1,2-dibromoethane by O6-alkylguanine-DNA alkyltransferase. J. Biol. Chem. 277, 37920− 37928. (37) Kalapila, A. G., Loktionova, N. A., and Pegg, A. E. (2008) Alkyltransferase-mediated toxicity of 1,3-butadiene diepoxide. Chem. Res. Toxicol. 21, 1851−1861. (38) Tretyakova, N. Y., Michaelson-Richie, E. D., Gherezghiher, T. B., Kurtz, J., Ming, X., Wickramaratne, S., Campion, M., Kanugula, S., Pegg, A. E., and Campbell, C. (2013) DNA-reactive protein monoepoxides induce cell death and mutagenesis in mammalian cells. Biochemistry 52, 3171−3181. (39) Heimann, K. G., Jung, R., and Kieczka, H. (1991) Maleic acid dimethylester: evaluation of dermal toxicity and genotoxicity. Food Chem. Toxicol. 29, 575−578. (40) Thompson, V. R., and Decaprio, A. P. (2013) Covalent adduction of nitrogen mustards to model protein nucleophiles. Chem. Res. Toxicol. 26, 1263−1271. (41) Siegel, S. J., Bieschke, J., Powers, E. T., and Kelly, J. W. (2007) The oxidative stress metabolite 4-hydroxynonenal promotes Alzheimer protofibril formation. Biochemistry 46, 1503−1510. (42) Kuhla, B., Haase, C., Flach, K., Luth, H. J., Arendt, T., and Munch, G. (2007) Effect of pseudophosphorylation and cross-linking by lipid peroxidation and advanced glycation end product precursors on tau aggregation and filament formation. J. Biol. Chem. 282, 6984− 6991. (43) Zhou, X., Bi, H., Wong, J., Shimoji, M., Wang, Y., Yuan, J., Xiao, X., Wang, G. X., and Zou, W. Q. (2011) Alkylating antitumor drug mechlorethamine conceals a structured PrP domain and inhibits in vitro prion amplification. J. Toxicol. Environ. Health, Part A 74, 1493− 1503. (44) Palhano, F. L., Lee, J., Grimster, N. P., and Kelly, J. W. (2013) Toward the molecular mechanism(s) by which EGCG treatment remodels mature amyloid fibrils. J. Am. Chem. Soc. 135, 7503−7510. (45) Friguet, B., Stadtman, E. R., and Szweda, L. I. (1994) Modification of glucose-6-phosphate dehydrogenase by 4-hydroxy-2nonenal: formation of cross-linked protein that inhibits the multicatalytic protease. J. Biol. Chem. 269, 21639−21643. (46) Friguet, B., and Szweda, L. I. (1997) Inhibition of the multicatalytic proteinase (proteasome) by 4-hydroxy-2-nonenal crosslinked protein. FEBS Lett. 405, 21−25. (47) Reichmann, D., and Jakob, U. (2013) The roles of conditional disorder in redox proteins. Curr. Opin. Struct. Biol. 23, 436−442. (48) Shibata, T., Kimura, Y., Mukai, A., Mori, H., Ito, S., Asaka, Y., Oe, S., Tanaka, H., Takahashi, T., and Uchida, K. (2011) Transthiocarbamoylation of proteins by thiolated isothiocyanates. J. Biol. Chem. 286, 42150−42161.

(49) Bruggeman, I. M., Temmink, J. H., and van Bladeren, P. J. (1986) Glutathione- and cysteine-mediated cytotoxicity of allyl and benzyl isothiocyanate. Toxicol. Appl. Pharmacol. 83, 349−359. (50) Jiao, D., Conaway, C. C., Wang, M. H., Yang, C. S., Koehl, W., and Chung, F. L. (1996) Inhibition of N-nitrosodimethylamine demethylase in rat and human liver microsomes by isothiocyanates and their glutathione, L-cysteine, and N-acetyl-L-cysteine conjugates. Chem. Res. Toxicol. 9, 932−938. (51) Jiao, D., Smith, T. J., Yang, C. S., Pittman, B., Desai, D., Amin, S., and Chung, F. L. (1997) Chemopreventive activity of thiol conjugates of isothiocyanates for lung tumorigenesis. Carcinogenesis 18, 2143−2147. (52) Mi, L., Hood, B. L., Stewart, N. A., Xiao, Z., Govind, S., Wang, X., Conrads, T. P., Veenstra, T. D., and Chung, F. L. (2011) Identification of potential protein targets of isothiocyanates by proteomics. Chem. Res. Toxicol. 24, 1735−1743. (53) Haynes, R. L., Szweda, L., Pickin, K., Welker, M. E., and Townsend, A. J. (2000) Structure−activity relationships for growth inhibition and induction of apoptosis by 4-hydroxy-2-nonenal in RAW264.7 cells. Mol. Pharmacol. 58, 788−794. (54) Chan, K., Poon, R., and O’Brien, P. J. (2008) Application of structure−activity relationships to investigate the molecular mechanisms of hepatocyte toxicity and electrophilic reactivity of α,βunsaturated aldehydes. J. Appl. Toxicol. 28, 1027−1039. (55) Raj, L., Ide, T., Gurkar, A. U., Foley, M., Schenone, M., Li, X., Tolliday, N. J., Golub, T. R., Carr, S. A., Shamji, A. F., Stern, A. M., Mandinova, A., Schreiber, S. L., and Lee, S. W. (2011) Selective killing of cancer cells by a small molecule targeting the stress response to ROS. Nature 475, 231−234. (56) Corson, T. W., Aberle, N., and Crews, C. M. (2008) Design and applications of bifunctional small molecules: why two heads are better than one. ACS Chem. Biol. 3, 677−692.

1729


Pronounced Toxicity Differences between Homobifunctional Protein

Recommend Documents