Cytosolic and Nuclear Protein Targets of Thiol-Reactive Electrophiles

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Chem. Res. Toxicol. 2006, 19, 20-29

Chemical Profiles Cytosolic and Nuclear Protein Targets of Thiol-Reactive Electrophiles Michelle K. Dennehy,† Karolyn A. M. Richards,§ Gregory R. Wernke,† Yu Shyr,‡ and Daniel C. Liebler*,† Department of Biochemistry and Mass Spectrometry Research Center, Vanderbilt UniVersity School of Medicine, NashVille, Tennessee 37232, Department of Pharmacology and Toxicology, College of Pharmacy, UniVersity of Arizona, Tucson, Arizona 85721, and Department of Biostastics and Vanderbilt-Ingram Cancer Center, Vanderbilt UniVersity School of Medicine, NashVille, Tennessee 37232 ReceiVed NoVember 9, 2005

Reactive electrophiles formed from toxic drugs and chemicals and by endogenous oxidative stress covalently modify proteins. Although protein covalent binding is thought to initiate a variety of adaptive and toxic responses, the identities of the protein targets are generally unknown, as are protein structural features that confer susceptibility to modification. We have analyzed the protein targets in nuclear and cytoplasmic proteomes from HEK293 cells treated in vitro with two biotin-tagged, thiol-reactive electrophiles, (+)-biotinyl-iodoacetamidyl-3, 6-dioxaoctanediamine (PEO-IAB) and 1-biotinamido-4(4′-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC). Biotinylated peptides were captured by affinity enrichment using neutravidin beads, and the adducted peptides were then analyzed by multidimensional liquid chromatography-tandem mass spectrometry. A total of 897 adducts were mapped to different cysteine residues in 539 proteins. Adduction was selective and reproducible, and >90% of all adducted proteins were modified at only one or two sites. A core group of 125 cysteines (14% of the total) was consistently modified by both electrophiles. Selective modification of several protein domain structures and motifs indicates that certain protein families are particularly susceptible to alkylation. This approach can be extended to studies of other protein-damaging oxidants and electrophiles and can provide new insights into targets and consequences of protein damage in toxicity and disease. The biotransformation of drugs and environmental chemicals and the formation of endogenous products of cellular oxidative damage all generate reactive electrophiles that modify DNA and proteins (1-3). Although most attention to toxicity and carcinogenesis mechanisms has centered on DNA damage (2), proteins are major targets of reactive electrophiles due to the diverse nucleophilic chemistries they exhibit. Of these, cysteine thiol groups are particularly important both because of their high reactivity toward diverse oxidants and electrophiles and because of their prominence in regulating the activity of important cellular signaling proteins (4-7). Covalent protein modification may trigger changes in gene expression in response to many prototypical stressors such as UV light, hydrogen peroxide, arsenic, and alkylating agents (4, 8-11). Protein binding also is a critical event in target organ toxicity and hypersensitivity reactions (1, 12-14). Nevertheless, the identities of adducted proteins and the mechanisms by which protein damage elicits adaptive effects and toxicity remain largely unknown. * To whom correspondence should be addressed: Daniel C. Liebler, Department of Biochemistry, Vanderbilt University School of Medicine, 9110A Medical Research Building III, 465 21st Avenue South, Nashville, TN 37232-8575. Phone, 615 322 3063; fax, 615 343 8372; e-mail, [email protected]. † Department of Biochemistry and Mass Spectrometry Research Center, Vanderbilt University School of Medicine. § University of Arizona. ‡ Department of Biostastics and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine.

The specificity of protein targeting by electrophiles is an important problem. Identification of electrophile targets may reveal cellular mechanisms of injury and adaptive responses, as certain proteins appear to act as sensors for oxidants and electrophiles. For example, the thiol-rich protein Keap1 mediates activation of the transcription factor Nrf2 upon modification at Keap1 cysteine thiols by electrophiles, thus, allowing nuclear accumulation of Nrf2 and activation of numerous phase II and antioxidant genes (15-17). Other potential sensors include thiolcontaining proteins that that modulate signaling and include phosphatases, peroxiredoxins, and thioredoxin (18-20). The existence of redox- or alkylation-sensor proteins raises the general question of selectivity in modification of proteins by electrophiles. It is not clear what the degree of specificity of covalent modifications on a proteome-wide scale is. Are modifications random or more specific? A related question is whether certain protein structures confer susceptibility to damage. Previous work using immunochemical detection of adducts suggested that targeting is nonrandom, but few targets were identified (21-26). Several more recent studies have applied 2D gel-based proteomics approaches to identify protein targets of electrophiles (27-30). These studies typically have relied on the use or radiolabeled chemicals followed by autoradiography to detect modified proteins in the gels. MALDIMS1 analyses of peptides from in-gel digests of the spots identified putative protein targets but did not identify actual adducts. These previous studies typically have identified up to

10.1021/tx050312l CCC: $33.50 © 2006 American Chemical Society Published on Web 12/30/2005

Protein Targets of ReactiVe Electrophiles

Figure 1. Structures of PEO-IAB and BMCC.

about 25 proteins each as apparent electrophile targets, and these generally have been highly abundant proteins. The problem of identifying protein targets of reactive electrophiles is a difficult analytical challenge. Protein modification by electrophiles at physiologically or toxicologically relevant levels is probably substoichiometricsonly a fraction of proteins may be modified and only a small mole fraction of each protein target may be present in a modified form. Whole proteomes or even major proteome subfractions, such as the cytosolic and nuclear extracts, are highly complex and, once digested, contain only small amounts of modified peptides. Although affinity chromatography can enrich samples for peptides containing certain modifications (e.g., phosphorylation (31, 32)), affinity tools for selective capture of adducted proteins are not currently available. To circumvent this problem, we have employed the thiolreactive, biotin-tagged model electrophiles PEO-IAB and BMCC (Figure 1) as model electrophiles to label cytosolic and nuclear proteome samples under native conditions. Biotinavidin chromatography and multidimensional LC-MS-MS were used to identify the adducted peptides and map the adducts to specific sequence positions on the proteins. These studies identified nearly 900 nuclear and cytosolic cysteine thiol targets of PEO-IAB and/or BMCC. Protein adduction was selective and reproducible and involved targets in multiple functional classes. Some protein domain and motif structures displayed high sensitivity to modification. These studies serve as a prototype for investigations of protein damage by defined electrophile chemistries in complex proteomes.

Experimental Procedures Reagents. Mouse anti-biotin antibodies conjugated to horseradish peroxidase were obtained from Zymed (South San Francisco, CA). PEO-IAB, BMCC, TCEP, Neutravidin, and DTT were from Pierce (Rockford, IL). Cell Culture and Preparation of Subcellular Fractions. HEK293 cells were obtained frozen at low passage from Master Cell Bank cultures from GIBCO Life Technologies (Grand Island, NY). Cells were cultured as described previously, and cells between 1 Abbreviations: Ambic, ammonium bicarbonate; BCA, bicinchoninic acid; BMCC, 1-biotinamido-4-(4′-[maleimidoethylcyclohexane]-carboxamido)butane; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; IAM, iodoacetamide; KH, K-homology domain; LC-MS-MS, liquid chromatography-tandem mass spectrometry; MALDI-MS, matrixassisted laser desorption/ionization mass spectrometry; MS-MS, tandem mass spectrometry; PBS, phosphate-buffered saline; PEO-IAB, (+)biotinyl-iodoacetamidyl-3, 6-dioxaoctanediamine; RRM, RNA recognition motif; TCEP, tris(carboxyethyl)phosphine; TPR, tetratricopeptide; WD, WD40 or β-transducin repeat.

Chem. Res. Toxicol., Vol. 19, No. 1, 2006 21 passages 10 and 40 post-recovery from cryopreservation were used (33). Cells were lysed, and the cytosol was collected with the Clontech (Palo Alto, CA) TransFactor Extraction Kit according to manufacturer’s instructions. The nuclear extract was isolated as previously described with minor modifications (34). Briefly, the HEK293 cells were washed and harvested with cold PBS. The cells were pelleted at 450g for 8 min, then the buffer was aspirated. The ∼300 µL pellet was resuspended in 800 µL of lysis buffer (10 mM HEPES, pH 7.8, 10 mM KCl, 2 mM MgCl2, and 0.1 mM EDTA) and incubated on ice for 15 min. The protein suspension was mixed rapidly for 5 s after the addition of 75 µL of 10% NP-40. A 30 s centrifugation was followed by resuspension in extraction buffer (50 mM HEPES, pH 7.8, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, and 10% glycerol) and incubation on ice for 20 min. The nuclear extract was then centrifuged at 18 000g for 10 min. The cytosol and nuclear fractions were washed with Amicon Microcon centrifugal ultrafiltration devices (10 000 molecular weight cutoff; Millipore, Billerica, MA) to remove the fractionation buffers, and the protein was resuspended in 0.1 M ammonium bicarbonate (pH 7.4). Protein concentrations were determined using the bicinchoninic acid assay kit (Pierce, Rockford, IL) assay. Reaction of Protein Fractions with Reactive Electrophiles. Protein from the nuclear or cytosolic fractions (6 µg/µL) was reacted in 1 mL of 0.1 M ammonium bicarbonate buffer (pH 7.4) at 37 °C with PEO-IAB (200 µM) for 30 min or with BMCC (10 µM) for 10 min. The reactions were quenched with β-mercaptoethanol (for Western blot analysis) or DTT (for subsequent tryptic digest). Prior to enzymatic hydrolysis, the samples were washed with spin filters (10 000 molecular weight cutoff, Amicon Ultrafree-MC, Millipore, Billerica, MA) to remove excess DTT (35). For digestion, protein was resuspended in 300 µL of ammonium bicarbonate buffer containing 10 mM DTT and 4 mM TCEP in the spin filter upper reservoir and incubated at 50 °C for 15 min (35). IAM was added to a final concentration of 20 mM at 25 °C for 15 min. To quench any excess IAM, additional DTT was added to a final concentration of 20 mM. Trypsin (Trypsin Gold, mass spectrometry grade; Promega, Madison, WI) was added in a 1:50 trypsin/protein ratio, and the samples were incubated at 37 °C for 18-24 h. Tryptic peptides were collected by centrifugation of the filter at 5000g, and the filtrate was acidified with 0.5 µL of concentrated formic acid. Immunoblot Analysis. Cell lysate proteins were diluted 1:1 (v/ v) with Laemmli buffer containing 10% β-mercaptoethanol and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 12% Tris HCl Ready-Gels (Bio-Rad, Hercules, CA). Resolved proteins were electrophoretically transferred to polyvinylidene fluoride membranes, which were blocked with 5% milk in TBST buffer (20 mM Tris HCl, pH 7.5, 200 mM NaCl, and 0.1% Tween 20) for 2 h and then probed with anti-biotin antibody at a 1:1000 dilution for 2 h all at 25 °C. Immunostained proteins were detected by enhanced chemiluminescence with Western blotting luminal reagent (Santa Cruz, Santa Cruz, CA). Affinity Capture of Biotinylated Proteins. Immobilized Neutravidin on agarose beads (1 mL bead slurry/1 mg protein) was centrifuged at 5000g for 5 min followed by two washes with 1 mL of 0.1 M ammonium bicarbonate (pH 7.4); beads were centrifuged for 1 min at 1000g between each wash. Peptides were added to the beads, and the suspension was mixed by rotation at 25 °C for 30 min followed by centrifugation for 10 min at 5000g. The beads were washed three times with 0.1 M ammonium bicarbonate (pH 7.4), then three times with deionized water (1 mL of buffer or water/1 mL of beads). Elution buffer (500 mM formic acid in 25% acetonitrile in the PEO-IAB experiements and 70% formic acid in the BMCC experiments) was added to the beads (1 mL of buffer/1 mL of bead slurry), and the samples were rotated at 25 °C for 20 min. The beads were pelleted by centrifugation at 5000g for 5 min, and the supernatant was collected and evaporated to 60 µL in vacuo. Multidimensional LC-MS-MS Analyses and Protein Target Identification. Peptides were fractionated by strong cation exchange on a polysulfoethyl-A, 5 µm particle size, 100 mm × 2.1 mm

22 Chem. Res. Toxicol., Vol. 19, No. 1, 2006 column (Nest Group, Southboro, MA). Samples were eluted with 10 mM ammonium formate (pH 3.0)/acetonitrile (75:25, v/v) for 5 min at 0.2 mL/min followed by a 30 min linear gradient to 200 mM ammonium formate (pH 8.0)/acetonitrile (75:25, v/v) with a 10 min wash at the final mobile phase composition. Fifteen 3-min fractions were collected, and fractions 1-3 and 13-15 were combined. All fractions were evaporated in vacuo to 20-30 µL, desalted using Zip-Tips (Millipore C18, P10), and analyzed by LCMS-MS. The samples were analyzed on a Thermo LTQ linear trap instrument equipped with a Thermo microelectrospray source, and a Thermo Surveyor pump and autosampler (Thermo Electron Corporation, San Jose, CA). LC-MS-MS analyses were done by reverse-phase chromatography on an 11 cm fused silica capillary column (100 µm i.d.) packed with Monitor C-18 (5 µm) (Column Engineering, Ontario, CA) with the flow set at 700 nL/min. The mobile phase consisted of 0.1% formic acid in either HPLC grade water (A) or acetonitrile (B). Peptides were eluted initially with 99% A, then 95% A from 3 to 5 min, then a linear gradient to 72% A by 33 min, then to 20% A at 40 min and held to 45 min, and then to 99% A at 52 min and held until 60 min. MS-MS spectra were acquired using one data-dependent scan on the most intense precursor ion in the full scan. In some analyses, the full scan was followed by four data-dependent scans on the four most intense precursor ions. Precursors that were detected twice within 15 s were put on a dynamic exclusion list for a period of 60 s. MS-MS spectra were matched to human database sequences with TurboSequest (ThermoElectron, San Jose, CA) (36). S-Carboxymethylation of Cys (+57 amu), PEO-IAB adduction at Cys (+414 amu and +430 amu (oxidized adduct)), and/or BMCC adduction at Cys (+533 amu and +549 amu (oxidized adduct)) were specified as dynamic modifications. Sequest outputs were analyzed with a custom-designed software and database system called CHIPS (Complete Hierarchical Integration of Protein Searches), which enables filtering of Sequest output files based on Sequest output parameters for sequence-spectrum matches and other criteria. Sequence-spectrum assignments were accepted based on the following filtering criteria: (1) all peptide sequence assignments were required to result from fully tryptic cleavages; (2) all peptides possessed the appropriate reactive electrophile adduct mass; (3) adducted peptides needed to be present in at least three out of six sample sets in the PEO-IAB experiments and the cytosolic BMCC experiments or two out of two sample sets in the nuclear BMCC experiments to be accepted; (4) singly, doubly, and triply charged ions were accepted if their XCorr scores were greater than 2, 2.5, and 3, respectively; and (5) all putative matches were confirmed by visual inspection of the spectra. Peptide identifications for all 15 SCX fractions in each sample were recombined with CHIPS, and the lists of corresponding proteins were compared. Statistical Analysis of Influence of Sequence Context on Adduction Frequency. The effect of adjacent amino acids in primary sequence on cysteine adduction was evaluated by calculating the normalized frequency of appearance of each amino acid in five N-terminal positions (-1 through -5) and five C-terminal positions (+1 through +5) relative to cysteine (position zero). Normalized frequency for an amino acid at a specific position was calculated as the number of times that amino acid was observed in that position divided by the total number of observations (e.g., adducted sequences) in the entire dataset. To estimate baseline frequencies for the amino acids in each position, a similar calculation divided the number of occurrences of the amino acid at the specified position relative to all cysteines in the Uniref100 database (www.uniprot.org). Statistical analysis of influence of sequence context on adduction frequency is completed using the Weighted Generalized Linear Model to adjust the total number of possible peptide hits per protein ID. The goodness-of-fit of the model is examined by using the Scaled Deviance Method. The p-value of the goodness-of-fit of the reported model is greater than 0.35, which indicates the specified model fits the data reasonably well.

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Figure 2. Reactions of PEO-IAB and BMCC with cysteine thiols and examples of analogous chemistries for other toxicologically relevant electrophiles.

Results and Discussion Overview of Approach. The objective of this study was to map adducts of the two different thiol-reactive electrophiles PEO-IAB and BMCC on nuclear and cytosolic protein targets at the level of amino acid sequence. Although these compounds are both considered thiol-reactive electrophiles, they react by different mechanisms (Figure 2). PEO-IAB displays SN2 electrophilic chemistry analogous to toxicologically relevant electrophiles, such as aliphatic epoxides, alkyl halides, and episulfonium ions. BMCC reacts with cysteine thiols by a Michael addition mechanism, which is typical of quinones and R,β-unsaturated carbonyls, including many lipid peroxidation products (37, 38). Together, these two compounds mimic the reaction chemistry of many toxicologically relevant electrophiles. The use of a cell-free system eliminated effects on protein adduction selectivity due to differential access to the compounds to protein subcellular compartments. To maximize the identification of targets, nuclear and cytosolic fractions were treated and studied separately. Because subcellular fractions are less complex than whole cells or lysates, adducts on lower abundance proteins are more readily detected. Protein targets in crude subcellular fractions at neutral pH are present in the same relative concentrations found in intact cells. Moreover, proteins in these complex mixtures presumably maintain many proteinprotein associations that normally occur in vivo and may govern their reactivity toward electrophiles. The method used to detect and map PEO-IAB and BMCC protein adducts is depicted in Figure 3. Subcellular fractions were incubated under near-native conditions with electrophile. Optimal conditions for electrophile adduction were determined empirically by Western blotting in preliminary experiments. Preliminary studies of the time and concentration dependence of labeling indicated that incubation with 200 µM PEO-IAB or 10 µM BMCC for 30 min achieved detectable biotin labeling across the molecular weight range of proteins without appearing to saturate all targets (see Supporting Information Figure 1). After electrophile treatment, protein fractions were treated with excess DTT to neutralize unreacted electrophile and then digested with trypsin and enriched for adducted peptides using

Protein Targets of ReactiVe Electrophiles

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Figure 3. Schematic representation of protein adduct analysis (see text for discussion).

immobilized Neutravidin on agarose beads. Neutravidin capture of adducted peptides following protein digestion yielded almost exclusively adducted peptides for LC-MS-MS analysis and greatly increased the number of targets and adduction sites identified. Multidimensional LC-MS-MS was used to maximize the numbers of adducted peptides identified in the complex peptide-adduct mixtures. All peptide adduct assignments were based on database searches with the Sequest algorithm (36), in which cysteines were modified with expected masses for PEOIAB and BMCC adducts (see below). Only matches that met defined criteria were accepted (see Experimental Procedures). Several representative spectra are depicted in Supporting Information Figures 2-11. Replicate incubations and analyses were done, and only peptide adducts present in at least three of six experiments (PEO-IAB and cytosolic BMCC samples) or two of two (nuclear BMCC samples) experiments were reported. Cytosolic and Nuclear Protein Targets. A complete listing of the proteins adducted by PEO-IAB and BMCC is provided in Supporting Information Table 1. These studies identified a total of 897 modified peptides derived from 539 proteins. Of these, 343 were adducted by PEO-IAB and 554 by BMCC (Figure 4A). A total of 125 were adducted by both electrophiles, most often at the same cysteine. All identified peptide adducts contained cysteine modifications of +414 Da for PEO-IAB and +533 Da for BMCC or their corresponding S-oxidized products (+433 Da and +549 Da). These adducts correspond to the expected SN2 reaction product of the cysteine thiol with the iodoacetamido group in PEO-IAB or to the expected Michael addition product for BMCC. The first interesting observation from this dataset is that thiol modification selectivity by these two electrophiles differed significantly. Only 90 proteins (20% of the total) and 125 peptides (14% of the total) were modified by both electrophiles (Figure 4A, Table 1). This observation indicates that the chemistry of the electrophile is a more important determinant

Figure 4. (A) Overlap of adduct targets of PEO-IAB and BMCC. (B) Number of adduct sites on identified protein targets of PEO-IAB (filled bars) and BMCC (open bars).

of target selectivity than any intrinsic reactivity of individual cysteine thiols. On the other hand, those cysteine thiols that reacted with both electrophiles may constitute a small subset of targets with a high reactivity toward multiple electrophiles. Such targets may have significance as general biomarkers for electrophile stress.

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Table 1. Consensus Protein Targets of Reactive Electrophiles UniRef no.

protein

target cysteine(s)a

P23528 Q9ULV4 P21333 Q86XU5 P35579 O00151 P68366 P07437 Q8IWR2 P46013 P49321 P78527 P12004 Q9Y230 P25205 P11388 Q9HAP0 P09211 P04406 P49915 Q01581 O15067 P14618 P50991 P17987 P49368 P30050 P50914 Q07020 Q02543 Q862I1 Q9BQQ5 Q9NY85 P62910 P36578 P62424 P05388 P62280 P08708 Q8NI62 P60866 P62857 P23396 P46782 P62241 P13639 Q8TA90 P31948 P30041 P10599 Q92945 Q99729 P31943 O43390 Q00839 P55769 Q15365 Q13310 Q9Y383 Q8WVW9 O75533 Q13838 O75643 Q04917 P61981 P27348 Q10567 P08134 Q9Y3F4 Q15424 Q00610 P61289 O60526 P22314 P09936 P68036 Q6P391 Q8TDQ5 P51858 Q7Z4V5 Q9Y3E8 Q9NZ87

Cofilin-1 Coronin-1C Filamin A Myosin, heavy polypeptide 9 (MYH9) Myosin heavy chain-nonmuscle type A PDZ and LIM domain protein 1 Tubulin alpha-1 chain Tubulin beta-2 chain Tubulin beta class II Antigen KI-67 Nuclear autoantigenic sperm protein isoform 1 DNA-activated protein kinase, catalytic subunit Proliferating cell nuclear antigen RuvB-like 2 DNA replication licensing factor MCM3 DNA topoisomerase II - alpha isozyme Valosin-containing protein Glutathione S-transferase P1-1 Glyceraldehyde-3-phosphate dehydrogenase GMP synthase, glutamine-hydrolyzing Hydroxymethylglutaryl-CoA synthase Phosphoribosylformylglycinamidine synthase Pyruvate kinase - isozymes M1/M2 T-complex protein 1 - delta T-complex protein 1, alpha T-complex protein 1, gamma Ribosomal protein L12, 60S Ribosomal protein L14, 60S Ribosomal protein L18, 60S Ribosomal protein L18a, 60S Ribosomal protein L24, 60S Ribosomal protein L27a Ribosomal protein L3 Ribosomal protein L32, 60S Ribosomal protein L4, 60S Ribosomal protein L7a, 60S Ribosomal protein P0, 60S Ribosomal protein S11, 60S Ribosomal protein S17, 40S Ribosomal protein S2 Ribosomal protein S20, 40S Ribosomal protein S28 Ribosomal protein S3, 40S Ribosomal protein S5, 40S Ribosomal protein S8, 40S Elongation factor 2 Similar to Elongation factor 2b Stress induced phosphoprotein-1 Peroxiredoxin 6 Thioredoxin Far upstream element binding protein 2 Heterogeneous nuclear ribonucleoprotein A/B Heterogeneous nuclear ribonucleoprotein H Heterogeneous nuclear ribonucleoprotein R Heterogeneous ribonuclear particle protein U NHP2-like protein 1 Poly(rC) binding protein 1 Polyadenylate-binding protein 4 Putative RNA-binding protein Luc7-like 2 Similar to signal recognition particle 9kD Splicing factor 3B subunit 1 Spliceosome RNA helicase BAT1 U5 small nuclear ribonucleoprotein 200 kDa helicase 14-3-3 protein eta chain 14-3-3 protein gamma 14-3-3 protein theta Adapter-related protein complex 1 beta 1 Rho-related GTP-binding protein RhoC Serine-threonine kinase receptor-associated Scaffold attachment factor B Clathrin heavy chain 1 Proteasome activator complex subunit 3 Antigen NY-CO-7 Ubiquitin activating enzyme E1 Ubiquitin carboxyl-terminal esterase L1 Ubiquitin-conjugating enzyme E2 L3 PSIP1 protein Arsenite-resistant protein ASR2 Hepatoma Derived Growth Factor Hepatoma Derived Growth Factor-2 CGI-150 protein Uncharacterized bone marrow protein BM034

C138 C420, 456 C1017, 1157, 1402, 2543 C931, 988 C1379 C72, 262 C347 C303, 354 C12 C1285, 3014, 3199 C254, 708 C25, 111, 223, 491, 974, 1455, 1904 C81, 162 C226 C263 C392 C150 C47 C151, 246 C489, 523 C86, 406 C270, 512 C48 C294, 378, 409 C76, 147, 357, 397 C213 C17 C41, 53 C133 C109 C6, C26 C70 C198 C96 C96, 208 C199 C27, 119 C60 C34 C15 C36 C27 C97, 134 C65, 154, 171 C99, 173 C40 C226 C461 C90 C72 C434 C97 C21 C99 C561, 593, 606 C30 C109, 158 C132 C348 C7 C1035 C165 C428 C86 C112 C134 C95 C16 C305 C225, 362 C869 C92 C48, 199 C234 C90 C86 C204 C406, 556 C12 C631 C251 C86

a

classification cytoskeleton

DNA replication/repair

metabolism

protein synthesis/folding

protein synthesis/folding

redox regulation RNA processing

signal transduction

transcriptional regulation transport, intracellular ubiquitin/proteasome

unknown

Target cysteines are numbered according to the UniProt Database Entries (www.uniprot.org). Three targets without UniProt entries are not listed.

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Figure 5. Frequency plots for distribution of lysine (A), arginine (B), histidine (C), and cysteine (D) in sequences adducted by PEO-IAB (dashed line) and BMCC (dot-dash line). Solid lines indicate normalized frequency for appearance of the amino acid at all specified positions relative to cysteine in all entries in the Uniref100 database. The x-axis depicts the adduct position (zero) and N-terminal positions (-1 through -5) and C-terminal positions (+1 through +5).

Selectivity of Protein Adduction by PEO-IAB and BMCC. A striking feature of the data is the selectivity of protein modification by each electrophile. Of the identified protein targets, 66% of the PEO-IAB adducted proteins and 59% of the BMCC adducted proteins were adducted at a single cysteine (Figure 4B). An additional 23% of the PEO-IAB targets and 26% of the BMCC targets were adducted at only two cysteines. Thus, the most protein targets were modified at only one or two sites. A few proteins were modified at several cysteine residues. For example, DNA-activated protein kinase catalytic subunit (DNA-PKcs) was modified at 16 different cysteines with either PEO-IAB or BMCC, whereas fatty acid synthase had 11 different cysteines modified, but only by PEO-IAB. The reproducibility of cysteine modification patterns for individual protein-electrophile combinations indicates that adduction is selective and that certain protein targets are much more kinetically reactive than others. Effects of Protein Structure on Adduction. We hypothesized that protein structural features may confer susceptibility to modification. First, we asked whether neighboring residues in protein primary sequences affected the frequency of cysteine adduction by PEO-IAB and BMCC. We determined the frequency with which specific amino acids appear at positions from -5 (N-terminal direction) to +5 (C-terminal direction) relative to each cysteine residue in all proteins in the Uniref100 database (2 636 099 entries) (www.uniprot.org). This baseline frequency plot for lysine residues relative to cysteine residues is essentially level in both the N- and C-terminal directions, indicating that lysines are homogeneously distributed within (5 residues of cysteines in all database sequences (Figure 5A). However, lysine frequency was significantly elevated at positions -2, -3, and +5 relative to BMCC-adducted cysteines,

yet much less so near PEO-IAB-adducted cysteines. Adjacent lysines thus favor Michael addition by the BMCC N-alkylmaleimide while disfavoring SN2 alkylation by the PEO-IAB iodoacetamide. The effect of arginine was similar but much less pronounced (Figure 5B), and no effect was observed for histidine (Figure 5C). The baseline frequency of other cysteines relative to cysteines in database sequences was significantly elevated at positions -3 and +3 (Figure 5D). These adjacent cysteine pairs can stabilize helices by disulfide formation. However, cysteine residues were significantly less frequent within (4 residues of both PEO-IAB and BMCC adducts. These adjacent cysteine pairs would be disulfide-linked in most proteins and therefore unavailable to react with the electrophiles. Other amino acids did not occur in adducted sequences with frequencies significantly different from baseline values (Supporting Information Figures 13-16). Enhanced adduction by BMCC at cysteines with adjacent lysines may result from hydrogen-bonding interactions between the BMCC N-alkylmaleimide R,β-unsaturated carbonyls and the protonated lysine amine. Such hydrogen-bonding interactions between hydrogen donors and delocalized π systems stabilize protein structures (39, 40) and are optimal within a distance of approximately 5 Å, which is consistent with the distance between a cysteine and lysine 2-3 residues away in a helical or coiled structure. Protonated lysines also may catalyze BMCC adduction by acting as proton donors for the Michael reaction. Consistent with its lower adduction-directing effect, arginine has a higher side-chain pKa (12.5), which would reduce the hydrogen-bonding efficiency and retard proton transfer. The directing effect of adjacent lysine and arginine residues may govern thiol modification by many endogenous electrophiles formed by lipid and carbohydrate oxidation, which are R,β-

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Figure 6. Selective adduction of hnRNP proteins in RRM sequences. Filled arrows indicate positions of adduction in RRM; open arrows indicate adducts formed outside RRM.

unsaturated carbonyl compounds that react similarly to BMCC (37). A total of 163 adducted cysteines (18%) were located in protein domains or protein regions having a key feature (Supporting Information Table 2). These features included zinc fingers, WD repeats, filamin and tetratricopeptide (TPR) repeats, HEAT repeats, K-homology (KH) domains, and nucleotide phosphate binding regions. The most frequently modified targets were cysteines in RNA recognition motifs (RRM). Both RRM and KH domains are characteristic of a large family of RNA binding proteins, which includes heterogeneous nuclear ribonucleoproteins (hnRNPs), small nuclear ribonucleoproteins (snRNPs), and RNA splicing factors (41). The preference for RRM adduction was remarkable, as 88% of adducts in these proteins fell within RRM (Figure 6). Modification selectivity for RRM may reflect the occurrence of lysine and arginine residues adjacent to the modified thiol in 51% of the modified sites. Active site cysteines were modified in glyceraldehyde-3phosphate dehydrogenase, peroxiredoxin 6, creatine kinase, a ubiquitin carboxyl-terminal esterase (L1), and two ubiquitinconjugating enzymes (E2 L1 and E2 N) (Supporting Information Table 2). Other cysteine modifications may have functional consequences, even when the cysteines are not in active sites or do not participate directly in catalysis. For example, both PEO-IAB and BMCC alkylated Cys47 of GSTP1-1. Although this residue does not participate in catalysis, alkylation leads to collapse of the glutathione binding site and enzyme inhibition (42). Core Targets for Electrophile Adduction. Despite the differences in their overall alkylation selectivity, PEO-IAB and

BMCC both targeted a core group of 125 cysteine thiols (Table 1). The modified thiols may comprise a core group of redoxsensitive proteins that also are high-susceptibility targets for damage. This core group represents cellular processes and networks known to be affected by electrophile and oxidative stress. For example, the consensus targets cofilin1 and filamin A are actin cytoskeleton regulators (43, 44), whereas coronin 1-C regulates both actin and microtubule networks (45). Two tubulin isoforms also were consensus targets. High susceptibility of these targets may explain the ability of diverse oxidants and electrophiles to trigger cytoskeletal abnormalities (46). Moreover, these proteins exhibit other interactions that may regulate signaling networks. Ribosomal proteins comprise the largest consensus target group. Protein synthesis inhibition is a classic cytotoxicity endpoint, and alkylation of multiple ribosomal protein targets is consistent with this observed effect of electrophile damage. Another intriguing group of targets are proteins regulating DNA replication and repair, particularly in the context of DNA damage responses. The DNA-PKcs protein is unusually reactive toward electrophiles and was adducted at seven different sites by both electophiles (Table 1). DNA-PKcs participates in the sensing and repair of DNA damage, particularly double strand breaks (47). The proteins RuvB-like 2 and MCM3 participate in helicase complexes that regulate histone acetylation and replication forks, respectively (48, 49). Proliferating cell nuclear antigen (PCNA) is a sliding clamp accessory to DNA replication that organizes recruitment of proteins involved in DNA replication and repair (50). PCNA modifications by ubiquitin and sumo proteins differentially govern the processivity of replication in

Protein Targets of ReactiVe Electrophiles

damaged DNA (51). Valosin-containing protein recently was found to be associated with DNA damage responses (52). Although DNA damage responses are thought to be triggered by recognition of DNA damage itself, damage to these proteins may contribute to DNA damage-associated stress responses. Indeed, essentially all physical and chemical DNA damaging agents also damage proteins. RNA binding and processing proteins comprised one of the largest groups of targets identified. These proteins appear to be especially susceptible to adduction in RRM or KH domains, which may affect the fidelity of RNA recognition and the splicing and transport of mRNA (41). hnRNP proteins also have been identified as targets of antinuclear antibodies in several autoimmune diseases, which frequently involve inflammation (53, 54). Reaction of hnRNPs with electrophiles may generate modified protein forms with enhanced antigenicity or crossreactivity with antibodies to other proteins. Both electrophiles also targeted the essential redox regulatory proteins thioredoxin and peroxiredoxin 6. Surprisingly, the wellknown thioredoxin cysteine 32/cysteine 35 redox couple was not modified. Although cysteine 32 acts as a nucleophile for protein disulfide reduction, both electrophiles modified cysteine 73 instead, which mediates thioredoxin dimerization via intermolecular disulfide bonds (55). Since thioredoxin redox state regulates stress signaling through apoptosis signal-regulating kinase 1 (ASK-1), electrophile adduction may be a stress-sensing mechanism (19). Other proteins that are functionally related to the consensus targets were adducted at separate sites by one or both electrophiles (Supporting Information Table 1). This suggests that processes, networks, and functions represented by these targets are subject both to redox control and to damage by diverse electrophile chemistries. We denote damage in this context as irreversible thiol modification, which contrasts with the reversible oxidations characteristic of redox switches (56). Nevertheless, damage probably has diverse consequences. Modification of catalytic residues certainly can cause loss of enzyme function, but modification may also induce misfolding, including the stabilization of toxic protein forms (57, 58). Most interestingly, some modified protein forms may translocate to other cellular compartments to regulate processes unrelated to their known functions. For example, S-nitrosation of glyceraldehyde-3phosphate dehydrogenase (GAPDH) at cysteine 151 triggered nuclear translocation of the enzyme in neurons with concomitant induction of apoptosis (59). This same GAPDH cysteine is one of the consensus targets for PEO-IAB and BMCC. One of the most interesting questions to be addressed is whether members of the consensus target group we identified include other examples of stress sensors.

Conclusions These studies represent the first global survey of protein adduction by reactive electrophiles, in which modifications were mapped at the level of amino acid sequence. Our application of affinity-tagged electrophiles and shotgun proteomic analyses has increased the number of identified protein targets by over an order of magnitude. Moreover, all adducts have been mapped to specific sequences, which enables new insights into the chemistry and significance of covalent binding. Our approach is similar to the activity-based protein profiling strategy developed by Cravatt and colleagues (60, 61), in which fluorophore- or biotin-tagged probes displaying different functional chemistries and an electrophilic group were used to identify proteins sharing a common binding site or enzymatic

Chem. Res. Toxicol., Vol. 19, No. 1, 2006 27

activity. Related work by Sethuraman et al. employed thiolreactive ICAT reagents to identify 18 redox-sensitive cysteine residues in rabbit heart membranes exposed to hydrogen peroxide in vitro (62, 63). The proteins identified here as electrophile targets inevitably represent a fraction of those that are damaged in most cells and tissues. We did not study membrane-associated or mitochondrial proteins, and proteins with cell type-specific expression may not have been represented in our proteome fractions. Detection of thiol targets reflects thiol reactivity, abundance of the protein targets, and ability to detect modified peptides by the LC-MSMS instruments used in these studies. Thus, not all targets present in our cytosolic and nuclear fractions were identified. Nevertheless, these data indicate the essential characteristics of thiol reactivity and susceptibility to electrophile damage on a proteomic scale. A core group of protein thiols targeted by both electrophiles may serve multiple roles as switches and sensors for redox regulation and as targets for damage in inflammatory and degenerative diseases and chemical toxicity. Acknowledgment. Support for this research by NIH Grants ES010056, ES011811, and ES000267 is gratefully acknowledged. We thank Prof. F. P. Guengerich for helpful discussions of protein adduction chemistry. Supporting Information Available: Preliminary data of Western blots to determine the optimal conditions for electrophile adduction (Figure 1); several representative spectra of protein targets of PEO-IAB and BMCC (Figures 2-11); frequency plots for amino acids distribution in sequences adducted by PEO-IAB and BMCC (Figures 12-15); and tables showing the protein targets of (Table 1) and the protein domains and features targeted by (Table 2) PEO-IAB and BMCC. This material is available free of charge via the Internet at http://pubs.acs.org.

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