The Octapeptidic End of the C-Terminal Tail of Histone H2A Is

Nov 21, 2003 - Van M. Hoang,§ Robert J. Fisher,§ and Kazimierz S. Kasprzak*,†. Laboratory of Comparative Carcinogenesis, NCI at Frederick, Frederi...
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Chem. Res. Toxicol. 2003, 16, 1555-1559

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The Octapeptidic End of the C-Terminal Tail of Histone H2A Is Cleaved Off in Cells Exposed to Carcinogenic Nickel(II) Aldona A. Karaczyn,† Wojciech Bal,‡ Susan L. North,† Robert M. Bare,† Van M. Hoang,§ Robert J. Fisher,§ and Kazimierz S. Kasprzak*,† Laboratory of Comparative Carcinogenesis, NCI at Frederick, Frederick, Maryland 21702, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland, and Protein Chemistry Laboratory, SAIC Frederick, Frederick, Maryland 21702 Received May 27, 2003

We have demonstrated previously that Ni(II) binds to the C-terminal -TESHHKAKGK motif of isolated bovine histone H2A. At physiological pH, the bound Ni(II) assists in hydrolysis of the E-S peptide bond in this motif that results in a cleavage of the terminal octapeptide SHHKAKGK off the histone’s C-tail. To test if the hydrolysis could also occur in living cells, we cultured CHO (Chinese hamster ovary), NRK-52 (rat renal tubular epithelium), and HPL1D (human lung epithelium) cells with 0.1-1 mM Ni(II) for 3-7 days. As found by gel electrophoresis, Western blotting, and liquid chromatography/mass spectrometry, histones extracted from the cells contained a new fraction of histone H2A lacking the terminal octapeptide (q-H2A). The abundance of q-H2A increased with Ni(II) concentration and exposure time. It can be anticipated that the truncation of histone H2A may alter chromatin structure and affect gene expression. The present results provide evidence for novel mechanisms of epigenetic effects of Ni(II) that may be involved in nickel toxicity and carcinogenesis.

Introduction

different mammalian cell lines without and with Ni(II) (introduced to the culture media as nickel(II) acetate) and analyzed their histone fractions by gel electrophoresis, Western blotting, and LC/MS.

Nickel compounds are well-established as human carcinogens (1), but the underlying molecular events remain to be unveiled. The mechanistic concepts in nickel carcinogenesis include promutagenic DNA damage (2) and epigenetic effects in chromatin (3, 4) resulting from Ni(II) binding to the cell nucleus (5). Full understanding of intracellular Ni(II) reactivity is crucial for the design of preventative and therapeutic measures. The relatively weak binding of Ni(II) to DNA leaves nuclear proteins, especially histones and protamines, as the major possible targets for this metal (6, 7). In a study on oligopeptide models, we have determined that Ni(II) can bind to amino acid residues of the C-terminal tail of mammalian histone H2A. This binding at physiological pH leads to a hydrolytic cleavage of the peptide bond between the Glu121 and the Ser122 residues (the N-terminal Met not counted), as demonstrated using two partial peptides and the whole isolated bovine H2A. The cleaved off octapeptide forms a strong square-planar Ni(II)-SHHKAKGK complex capable of promoting oxidative damage to DNA by hydrogen peroxide (8, 9). Following those findings, we hypothesized that if the Ni(II)-assisted hydrolysis of H2A occurred in living cells, it could lead to both genetic and epigenetic toxicity (8, 9). In the present study, constituting the first step in testing the above hypothesis and aimed at verification of the histone H2A truncation in cells, we cultured

Cell Culture. Chinese hamster ovary cells (CHO; ATCC-CCL 61), normal rat kidney epithelial-like cells (NRK-52E; ATCCCRL 1571), and human pulmonary lung cells (HPL1D; provided by T. Takahashi, Nagoya University, Nagoya, Japan) were used. The cells were cultured at 37 °C, under 5% CO2-containing air, for 3, 5, or 7 days, in the Ham’s F-12 or DMEM media (Biofluids Inc., Rockville, MD) to which nickel(II) acetate (Sigma-Aldrich, St. Louis, MO) was added, starting at 70-90% of confluence in 162 cm2 flasks, to make 0.1-1 mM final concentrations. After the Ni(II)-containing media were discarded, the cultures were rinsed twice with ice-cold PBS, pH 7.4, and only cells attached to the plates were collected for histone analysis. As found by the Trypan blue exclusion method and proliferation after Ni(II) exposure, the viability of the attached cells after a 7 day treatment ranged from over 95% for 0.1 mM Ni(II) (same as in the control cultures) to below 70% at higher concentrations. Histones from the control cells were extracted when the cultures approached 95% confluence.

* To whom correspondence should be addressed. Tel: 301-846-5738. Fax: 301-846-5946. E-mail: [email protected]. † NCI at Frederick. ‡ Polish Academy of Sciences. § SAIC Frederick.

One-Dimensional (1D) Gel Electrophoresis. The histones extracted as above were separated by 1D gel electrophoresis, using the NuPage SDS Bis-Tris1 10% or 4-12% gradient gels (Invitrogen, Carlsbad, CA) under reducing conditions provided

Materials and Methods

Extraction of Histones. Cellular histones were isolated from cells and cell nuclei by acid extraction with 0.5 M HCl as described by Bonner (10). The reference bovine histone standards were purchased from Roche Applied Science (Indianapolis, IN). The q-H2A standard was prepared by in vitro incubation of 0.07 mM histone H2A with 0.25 mM Ni(II) acetate in PBS, pH 7.4, for 3 days at 37 °C.

10.1021/tx0300277 CCC: $25.00 © 2003 American Chemical Society Published on Web 11/21/2003

1556 Chem. Res. Toxicol., Vol. 16, No. 12, 2003 by 10% v/v β-mercaptoethanol. Total protein concentration in the extracts was determined using the Bradford method with albumin calibration (Pierce, Rockford, IL). Samples containing 0.2-5 µg of total protein were loaded on the gels and separated at 70 V/100 V for 2.5-3 h in 2-(N-morpholino)ethanosulfonic acid (MES)/SDS running buffer. For larger amounts of total proteins, Bis-Tris gels were casted according to Strating and Clarke (11); the running gels, consisting of 10% T, with C being held constant at 2.67%, were prepared in 0.4 M Tris-Bis buffer, pH 6.5-7.0, and 0.22 mM ammonium persulfate; stacking gels were prepared with 5% acrylamide in 0.2 M Tris-Bis buffer, pH 6.3, and 0.22 mM ammonium persulfate (final concentrations). The protein bands in gels were visualized with silver or Simple Blue staining (Invitrogen). Two-Dimensional (2D) Gel Electrophoresis. The 2D electrophoresis was applied for fine separation of H2A fractions using the AU/AUT system as described by Dimitrov and Wolffe (12). The first dimension separations were accomplished with the use of 12 and 15% acetic acid-urea minislab gels at 80 V/gel for 9 h. For the second dimension separation, 15% 18/20 cm or 10/10 cm minislab gels were run at 8 mA/gel for up to 21 h. Before they were loaded, the histone extracts for analysis were dissolved in 25 µL of the loading buffer (8 M urea, 1 M acetic acid, and 1 M β-mercaptoethanol) at a total amount of about 50-200 µg and incubated for 2-3 h at 37 °C. The gels were stained as described above. Western Blotting. Proteins separated by 1D or 2D electrophoresis were transferred at 4 °C onto a Bio-Trace PVDF (Pall Gelman Laboratory, Ann Arbor, MI) or nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ) in 25 mM 3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer, pH 9.5, at 30 V for 1.5 h. The AU gels were equilibrated prior to transfer as described earlier (13, 14). The histone H2A bands were visualized with the use of a series of primary rabbit antibodies, including anti-H2A N terminus (Cell Signaling Technology, Inc., Beverly, MA) and anti-H2A acidic patch (Upstate Biotechnology, Lake Placid, NY). Anti-rabbit antibodies labeled with horseradish peroxidase (Cell Signaling Technology) served as the secondary antibodies together with LumiGLO (Cell Signaling Technology) or Super Signal (Pierce) reagents to detect the luminescent signal. LC/MS Analysis. Histone extracts and commercial histone standards were separated by gel electrophoresis in amounts of 5-20 µg, and bands were visualized by Simple Blue staining (Invitrogen). The bands were excised from the gel and destained in 50% methanol containing 10% acetic acid for 4-6 h at 25 °C with gentle vortexing. After they were rinsed in deionized water, the excised protein bands were dried and then fully immersed in the extraction solution (ca. 30 µL per band) consisting of formic acid/water/2-propanol (1:3:2, v/v/v) (15). The resulting mixture was vortexed overnight at room temperature. Prior to analysis, the supernatant was ultrafiltered through 30 kDa filters (Millipore Corp., Bedford, MA) at 2000g for 15 min. The HP 1100 Series LC/MSD instrument (Hewlett-Packard Company, Palo Alto, CA) was used for on-line LC/MS analysis of the protein fractions. For the LC separation, a reverse phase column, ZORBAX 300SB-C3 (Agilent Technologies, Palo Alto, CA) was used with a linear gradient of 5% v/v acetic acid and 100% acetonitrile (0-100% acetonitrile within 30 min). The protein fractions were eluted at a flow rate of 0.2 mL/min, detected at 280 nm, and introduced on-line to the ESI source. Mass spectral analysis was performed using software provided by the manufacturer (Agilent Technologies). MALDI-MS Analysis. Sequencing grade-modified trypsin was obtained from Promega (Madison, WI), and R-cyano-4hydroxycinnamic acid was from Agilent Technologies. All other reagents were acquired from Sigma-Aldrich. After gels were 1 Abbreviations: AU/AUT, acetic acid-urea-triton; Bis-Tris, 2,2bis-(hydroxymethyl)-2,2′,2′′-nitrilotriethanol; ESI, electrospray ionization; MALDI, matrix-assisted laser desorption ionization; PBS, 50 mM phosphate-buffered 150 mM NaCl, pH 7.4; PVDF, polyvinylidene fluoride; TOF, time-of-flight.

Karaczyn et al. destained in 10% acetic acid, protein spots were excised and macerated with a scalpel. In-gel trypsin digests were performed as previously described (16). Briefly, the gel pieces were transferred to a microfuge tube and washed with 50% acetonitrile/25 mM ammonium bicarbonate. Protein gel spots were digested overnight with 100 ng of trypsin at 37 °C, and tryptic peptides were eluted with 50% acetonitrile/5% formic acid in deionized water. After concentration under vacuum in a Speed Vac Concentrator (Savant, Holbrook, NY), portions (typically 1/20th) of the unseparated tryptic digests were cocrystallized in a matrix of R-cyano-4-hydroxycinnamic acid and analyzed by using a PerSeptive Biosystems DE-PRO mass spectrometer (Foster City, CA) equipped with delayed extraction operated in the reflector mode. Spectra were internally calibrated using trypsin autoproteolysis peaks, and the accuracy of mass measurements of all peptides was in the range of (0.05 Da. MS/ MS data were acquired as described before (17) on a MALDI TOF/TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems, Foster City, CA). MS and MS/MS spectra were interpreted to yield protein identities using the MS-Fit and MSTag programs (http://prospector.ucsf.edu) (18). ESI-MS Analysis. For the elctrospray analysis, samples were reduced, alkylated, and digested with trypsin (sequencing grade; Promega, Madison, WI) following a standard protocol (19). A sample was applied to RP-18 precolumn (LC Packings, Amsterdam, The Netherlands) using the 0.1% TFA mobile phase and then transferred to nano-HPLC RP-18 column (LC Packings) using the linear acetonitrile gradient of 0-45% acetonitrile in water, over 30 min, in the presence of 0.05% formic acid, at the flow rate of 40 nL/min. Column outlet was directly coupled to nano-Z-spray ion source of Q-Tof electrospray mass spectrometer (Micromass, Manchester, U.K.) working in the regime of datadependent MS to MS/MS switch, allowing for a 3 s sequencing scan for each detected peptide. The data were analyzed using the MassLynx program (www.micromass.co.uk).

Results Separation and Western Blotting. As shown in Figure 1, the treatment of CHO cells with Ni(II) resulted in the advent of a new protein band showing the same electrophoretic mobility as that of the truncated histone H2A (q-H2A) obtained by in vitro incubation of bovine H2A with Ni(II), as described previously (8, 9). The density of this new band increased with Ni(II) concentration and the duration of exposure, similar to our previous model studies (9). The emergence of the q-H2A-like band could first be detected when cells were grown with at least 0.1 mM Ni(II) for 7 days. For higher concentrations of Ni(II), the new band could be spotted earlier, e.g., at day 5 for 0.5 mM Ni(II). The q-H2A-like protein band could also be observed in gels following electrophoresis of histone extracts from rat kidney (NRK-52) and human lung (HPL1D) cells cultured with Ni(II). The results were similar to those obtained for the hamster CHO cells, although relative intensities of the q-H2A bands varied (Figure 2). Attempting to verify the identity of the observed q-H2A-like protein, we hybridized Western blots of cellular histone extracts with commercial anti-H2A antibodies specific for the N-terminal part of mammalian histone H2A. The results were positive (Figure 3) indicating that the H2A and q-H2A-like bands contained proteins having the same epitopes and might thus be related to each other. To eliminate possible artifactual contribution of other proteins to this result, we employed the AU/AUT system of 2D gel electrophoresis designed for better separation of histone variants and other proteins (12). Following the mobility patterns of the reference histones

Exposure of Histone H2A to Carcinogenic Nickel(II)

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Figure 4. Two-dimensional gel electrophorogram (18/20 cm slab gel) of histone extracts from CHO cells cultured alone or with 1 mM Ni(II) for 7 days. The standard H2A and q-H2A samples were obtained as described in the Materials and Methods. Silver stain; untr, untreated cells; Ni, Ni(II)-treated cells. Table 1. LC/MS Analysis of Histone H2A and q-H2A Bands Eluted from Polyacrylamide Gelsa

Figure 1. Time of emergence and staining intensity of the q-H2A band depend on Ni(II) concentration and the duration of treatment. (A) CHO cells cultured with 0-1 mM Ni(II) for 7 days; the staining intensity increases with Ni(II) concentration. (B) CHO cells cultured with 0.25 or 0.5 mM Ni(II) for 3, 5, and 7 days; the q-H2A band appears earlier in cells exposed to the higher concentration of Ni(II). Standards were prepared as described in the Materials and Methods. Silver stain; untr, untreated; Ni, Ni(II)-treated.

Figure 2. Generation of q-H2A is also observed in human HPL1D (A) and rat NRK-52E (B) cells. The cells were cultured with 1 mM Ni(II) for 7 days. Standards were prepared as described in the Materials and Methods. Silver stain; untr, untreated; Ni, Ni(II)-treated.

Figure 3. Western blots obtained with anti-histone H2A antibody. Histones extracted from CHO and NRK-52E cells, cultured with 1 mM Ni(II) for 7 days, were separated in 1D gels. Standards were prepared as described in the Materials and Methods; untr, untreated; Ni, Ni(II)-treated.

H2A and q-H2A in this system, we found the presence of the q-H2A-like protein among histones extracted from Ni(II)-treated cells (Figure 4). Its identity with q-H2A was again strongly suggested by the positive reaction with the anti-H2A antibody (not shown). On large 2D

band H2A q-H2A

molecular mass (Da)

relative abundance (%)

13 920.05b 13 894.08c 13 045.89b 13 020.10c

100 73 7 16

a The histones were separated by gel electrophoresis following isolation from CHO cells cultured for 7 days with 1 mM Ni(II). b,c The difference of 874 Da in molecular mass between proteins in the H2A and q-H2A bands marked with the same superscript is equal to the change in molecular mass expected for hydrolytic loss of the SHHKAKGK end from histone H2A’s C-terminal tail.

gels, the H2A band was split into two spots of similar intensities. Also, the staining intensities of the q-H2Alike bands, in both the gels and the Western blots, were lower in the 2D gels than in comparable 1D gels. This indicated that the corresponding 1D bands were not homogeneous (see below). Mass Spectral Analysis. To ultimately prove the identity of the q-H2A-like protein, various mass spectral and sequencing techniques were used. The LC/MS analysis (Table 1) of the respective bands excised from gels revealed two major mass fractions in each band, in agreement with the pattern observed in 2D gels. The two fractions within the H2A band differed from each other in molecular mass by 26 Da, most likely representing two variants of this histone. Likewise, the q-H2A electrophoretic band contained two peaks with virtually the same mass difference. Assuming that the two q-H2A fractions of higher and lower molecular masses originate from the respective higher and lower mass H2A variants, each of the q-H2As is smaller by 874 Da than its respective parental molecule. Thus, the calculated values match the expected mass difference of 874 Da between the full-size histone H2A and the same histone lacking the octapeptidic SHHKAKGK end of its C-terminal tail. The identity of q-H2A was also confirmed by tryptic digests of proteins present in the respective electrophoretic band. As shown in Table 2, the MALDI-MS and ESI-MS analyses, followed by peptide mapping, yielded four peptides with molecular masses of 850.53, 944.50, 1274.69, and 1931.16 Da, representing the highly conserved amino acid sequences unique for all major variants of mammalian histone H2A. The identities of the

1558 Chem. Res. Toxicol., Vol. 16, No. 12, 2003 Table 2. Peptides Found in Tryptic Digests of Histone q-H2A Electrophoretic Bandsa peptideb

molecular mass (Da)

HLQLAIRc,d AGLQFPVGRc,d,e SSRAGLQFPVGRc VTIAQGGVLPNIQAVLLPKd,e

850.53 944.52 1274.69 1931.16

a The histones were separated by gel electrophoresis following isolation from CHO cells cultured with 1 mM Ni(II) for 7 days. b Amino acid sequences matching the above peptides in the hamster histone H2A.2 (the only variant published; accession no. I48091), in bold: sgrgkqggk arakaksrss raglqfpvgr vhrllrkgny aervgagapv ymaavleylt aeilelagna ardnkktrii prhlqlairn deelnkllgk vtiaqggvlp niqavllpkk te-shhkakgk; the Ni(II)-assisted truncation site (8, 9) is hyphenated. c MALDI-MS. d ESI-MS. e Sequences confirmed by MS/MS.

second and the last peptides were confirmed by MALDIMS/MS and ESI-MS/MS, respectively. In addition to the above, the LC/MS and tryptic digest analyses of the q-H2A histone bands eluted from 1D gels revealed the presence of other proteins comigrating with q-H2A, thus confirming the observations of nonhomogeneity of these bands, mentioned above. The nature of those proteins has not been established and will be the subject of future investigations.

Discussion The results of the present study clearly indicate that Ni(II) can indeed assist in the truncation of histone H2A’s C-terminal tail in living cells. Because this truncation requires site specific binding of Ni(II) (8, 9), the results also demonstrate that the histone tail is accessible to Ni(II) and binds this metal even in the presence of other competing ligands in the cellular environment, as we proposed previously (7). This accessibility is consistent with the known sensitivity of this tail to proteolytic enzymes and cross-linking agents (20). What remains to be determined is the biological relevance of the truncation. On the basis of the published data on the role of mammalian histone H2A’s C-terminal tail in chromatin, we can anticipate that generally, Ni(II) binding to and truncation of this tail have the potential of disturbing its interaction with DNA that may lead to alterations in chromatin structure and derangement of gene expression. Decondensation and fragmentation of the long arm of chromosome X in nickel-treated cells, reported before (21), would be indicative of such a possibility. Indeed, it has been shown by several authors that the C-terminal tail of histone H2A is one of the structural elements stabilizing nucleosome particles and participating in the assembly of higher order chromatin structures in the chromosomes (22). This flexible tail is located at the region of the nucleosome where the DNA enters and leaves the histone octamer core (23, 24). It allows the H2A-H2B histone dimers to interact with core histone tetramer (20), linker DNA, and histone H1 (23). Its cleavage at Val114 by a H2A specific protease releases the terminal pentadecapeptide off the C-tail and results in dissociation of the histone H2A/H2B dimer from the core octamer (25). The histone-DNA cross-linking studies demonstrated the ability of histone H2A’s C-terminal domain to rearrange (switch DNA binding sites) and take part in the processes of nucleosome disassembly and reassembly, which allow for the passage of polymerases through the nucleosome (23). The entire tail is 34 amino acid residues long and contains eight positively charged

Karaczyn et al.

amino acid residues, five of them being located in the SHHKAKGK octapeptide cleaved by Ni(II). Removal of these charges together with shortening of the tail will undoubtedly weaken attraction of the tail to DNA’s phosphate groups and may destabilize chromatin in a way similar to that produced by the H2A specific protease, mentioned above. In addition, the Lys119 residue is subject to monoubiquitination, a process thought to serve as a recognition tag for chromatin remodeling complexes (26). The close proximity of this residue to the tail-cutting site allows for speculation that the truncation may alter the reversible H2A ubiquitination processes and their signaling importance, as well. Finally, it is also possible that loss of the octapeptidic end of H2A will deprive chromatin of other potential regulatory sites, i.e., amino acid residues, such as Ser, His, and Lys, prone to acetylation, methylation, or phosphorylation. Although there are no published data to support this notion in regard to mammalian cells, phosphorylation of Ser122 in histone H2A was observed in the protozoan Tetrahymena pyriformis (27). Carcinogenic Ni(II) has been associated with a variety of epigenotoxic events in cells, including changes in chromosome morphology, usually ascribed to indirect effects of the metal on DNA methylation (condensation of chromatin) or histone H4 acetylation (chromatin decondensation) pathways (reviewed in ref 28). The present experimental data provide evidence for a more direct molecular mechanism that may lead to the same final result: derangement of gene expression that has been proclaimed as the major cause of nickel-induced carcinogenesis. In addition, through generation of the cutoff redox active Ni(II)-SHHKAKGK complex at the chromatin site, the very same mechanism may enhance the production of reactive oxygen species (e.g., from metabolic H2O2), known to cause promutagenic oxidative DNA damage, and thus initiate carcinogenesis (2). Evaluation of the possible contribution of both effects to the latter requires further extensive studies.

Acknowledgment. We are grateful to T. Takahashi, Nagoya University, Nagoya, Japan, for the generous gift of HPL1D cells; J. Oledzki and E. Kopera, Laboratory of Mass Spectrometry and Proteomics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, for skillful technical assistance; T. Guszczynski, Protein Chemistry Core Facility, National Cancer Institute at Frederick, for mass spectral analysis advice; and A. Byrd and S. Tarasov, Structural Biophysics Laboratory, National Cancer Institute at Frederick, for making the LC/ MS instrument available to us. Critical comments of A. Maciag and M. Dadlez on this paper and editorial help of K. Breeze are also gratefully acknowledged.

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