Bioconjugate Chem. 2001, 12, 51−55
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Fluorescence-Labeled Octapeptides as Substrates for Histone Deacetylase Katharina Hoffmann,† Richard M. So¨ll,‡ Annette G. Beck-Sickinger,‡ and Manfred Jung*,† Department of Pharmaceutical Chemistry, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Hittorfstrasse 58-62, 48149 Mu¨nster, Germany and Institute of Biochemistry, University of Leipzig, Talstrasse 33, D 04103 Leipzig, Germany. Received May 9, 2000; Revised Manuscript Received September 15, 2000
The determination of histone deacetylase (HDAC) activity and the screening of potential inhibitors is gaining increasing importance due to the involvement of HDAC in transcription regulation. The level of histone acetylation can be modulated by HDAC inhibitors resulting in differentiation and/or apoptosis in cancer cells. We have previously reported the development of a nonisotopic assay for HDAC using a fluorescent derivative of -acetyl lysine. Here we report fluorescein-labeled octapeptides which are substrates for HDAC that bear closer resemblance to the native substrate. HPLC with fluorescence detection is successfully applied to the analysis of the time- and site-dependent deacetylation. LC-MS analyses are used to confirm the findings. The observed selectivity toward one of two possible deacetylation sites might result from steric hindrance by the label but the methodology presented here could be applied to similar larger peptides which might be improved tools to characterize HDAC site selectivity in vitro.
1. INTRODUCTION
The reversible acetylation of lysine residues near the N-termini of nucleosomal histones by histone deacetylases (HDACs) and histone acetyltransferases (HATs) regulates chromatin structure and transcriptional activity. The importance of the level of histone-acetylation for basic cellular functions such as DNA-replication, transcription, differentiation, and apoptosis is highlighted by a increasing number of experimental studies (1, 2). Most important, HDACs and HATs usually are recruited by other proteins in suppressing or activating transcriptional activity with HDACs usually being co-suppressors and HATs acting as co-activators (3-7). In several cases, deregulated histone acetylation has been correlated with malignant disease, and interactions of HDAC with cancer-related proteins such as mutated retinoic acid receptors PML-RARR and PLZF-RARR in acute promyelocytic leukemia (APL) have been identified as the basis of the disease on a molecular level (8, 9). Therefore, HDAC inhibitors are an attractive target for drug discovery and valuable tools for studying the impact of chromatin acetylation on gene regulation. One way toward a better understanding of the function of various deacetylases is the analysis of their reactivity in terms of time- and substrate-dependent turnover. This may also lead to the design of new inhibitors. The natural substrate, acetylated histones (10), or synthetic oligopeptides which consist of 8 (11) or 24 (12) amino acids, have traditionally been used in activity and inhibition assays. The latter are derived from sequences from the N-termini of histones. In either case, the substrate is labeled with [3H]acetic acid and the liberation of tritiated acid is quantitated by scintillation * To whom correspondence should be addressed. E-Mail:
[email protected]. Phone: +49-(0)251-83-33335. Fax: +49-(0)251-83-32144. † Westfa ¨ lische Wilhelms-Universita¨t Mu¨nster. ‡ University of Leipzig.
counting or a scintillation proximity assay (13). Thus, there are problems with the exposure of operating personnel and the disposal of radioactive waste. The labeled histones are obtained by a procedure involving treatment of chickens with phenylhydrazine over several days and the sacrifice of the animals is unavoidable. The acetylation degree of the prelabeled histones is difficult to standardize resulting in varying substrate properties. There is also a cell-based screening assay that relies on antibody-mediated detection of H4-hyperacetylation (14). The analysis of HDAC-site-selectivity in substrates with multiple lysines has so far been restricted to histones and requires gel electrophoresis and the use of several specific antibodies (15). Our studies deal with the development of fluorescence-based in vitro assays for the characterization of HDAC reactivity and its inhibition. We have previously reported on the first nonradioactive HDAC in vitro screening assay that relies on the use of a fluorescent cumarin-labeled acetyl-lysine 1 (see Figure 1). It can be used to analyze HDAC-activity in protein purification and for the screening of new inhibitors (16, 17) and has become commercially available. In this report, we describe the enzymatic conversion of an octapeptide 2a upon incubation with rat liver HDAC. It contains two acetyl-lysine residues and is labeled with fluorescein carboxylic acid at the N-terminus (see Figure 1) and we apply HPLC and fluorescence detection to the question of deacetylation-site preference. One of the lysines is deacetylated readily by a rat liver HDAC whereas only little of the bis-deacetylation product 2d can be found. 2. EXPERIMENTAL PROCEDURES
2.1. Chemicals. Trichostatin A was purchased from Wako. 5-Aminofluorescein was obtained from Sigma, tertbutyl alcohol, 4(5)-carboxyfluorescein, N,N-dimethylformamide (DMF), 1-hydroxybenzotriazole, thioanisole, and trifluoracetic acid from Fluka (Buchs, Switzerland). Fmoc-amino acids were obtained from Alexis (La¨ufelfin-
10.1021/bc000051l CCC: $20.00 © 2001 American Chemical Society Published on Web 12/28/2000
52 Bioconjugate Chem., Vol. 12, No. 1, 2001
Hoffmann et al.
Figure 1. Fluorescent HDAC-substrates.
gen, Switzerland) and 4-(2’,4’-dimethoxyphenyl-Fmocaminomethyl)phenoxy (Rink amide) resin from NovaBiochem (La¨ufelfingen, Switzerland). Diisopropylcarbodiimide and thiocresol were obtained from Aldrich (Buchs, Switzerland), and diethyl ether and acetonitrile from Romil (Cambridge, England). 3H-Labeled histones were a gift from Prof. Dr. P. Loidl (University of Innsbruck). 2.2. Octapeptides. Peptides were built up by Fmocstrategy on a Rink amide resin by using a robot system (Syro, MultiSynTech, Bochum) as described previously (18). Trt side-chain protection was used for His, Boc for Lys, and 2,2,5,7,8-pentamethyl-6-chromansulfonyl for Arg. Acetylated Lys was incorporated directly. The peptides were subsequently labeled at the N-terminus with 4(5)-carboxy-fluorescein prior to cleavage from the resin using a 10-fold excess of the acid, diisopropylcarbodiimide, and 1-hydroxybenzotriazol (all 0.25 mM in DMF) at 25 °C in DMF (30 min) (19). They were cleaved with trifluoroacetic acid/thioanisole/thiocresol (20:1:1; v/v) within 3 h, precipitated and washed with cold diethyl ether, collected by centrifugation and lyophilized from water/ tert-butyl alcohol (3:1). Cleavage products were investigated by reversed-phase HPLC (Merck-Hitachi, Darmstadt, Germany) on a Nucleosil RP-18 (5 µm) column (125 × 3 mm, Macherey and Nagel) at a flow rate of 0.6 mL/min. Absorbance was measured at 220 and 437 nm. The solvent system consisted of 0.1% trifluoracetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B). A linear gradient from 5 to 55% B in 30 min was applied. Furthermore, the peptides were analyzed by electrospray mass spectrometry (MS) (SSQ710, Finnigan, San Jose, CA). The following analytical data were obtained: (2a) HPLC retention time, 14.7 min; MS, Mexperimental ) 1392.3 amu and Mtheoretical ) 1392.5 amu; (2b) HPLC retention time, 13.6 min; MS, Mexperimental ) 1350.4 amu and Mtheoretical ) 1350.5 amu; (2c) HPLC retention time, 13.8 min; MS, Mexperimental ) 1350.3 amu and Mtheoretical ) 1350.5 amu; 2d: HPLC retention time, 13.0 min; MS, Mexperimental ) 1308.3 amu and Mtheoretical ) 1308.5 amu. 2.3. Fluorescence Spectra and Chromatography. Fluorescence spectra of 2 were recorded in a mixture of solvents C and D (80 + 20, v/v, see below) on a Shimadzu RF-540, and the strongest maxima (excitation 475 nm, emission 520 nm) were used for detection in HPLC. A Shimadzu RF 535 was used as fluorescence detector for HPLC (λex ) 475 nm, λem ) 520 nm). A Nucleosil RP18, 3 µm, 125 × 4 mm (Macherey-Nagel) was used for analysis of the incubation experiments and a column from the same brand (10 µm, 2 × 4 mm) served as guard column. The eluent consisted of solvent C (doubly distilled water with 0.06% v/v trifluoroacetic acid) and solvent D (acetonitrile/doubly distilled water 80/20 v/v). A gradient was run increasing the percentage of D from 20% (0 min) to 28% (50 min) and decreasing it back to 20% (70 min) with a flow of 0.8 mL/min. Retention times of the substrates were 26.84/29.33 min (2a), 14.69/15.60 min (2b), 17.07/19.63 min (2c), 7.82/9.03 min (2d), and 23.82 min for the internal standard (5-aminofluorescein).
2.4. Enzyme and Radioactive Assay. A partially purified rat liver histone deacetylase was used for the incubation experiments. It is prepared via separation on Q-sepharose big beads (Pharmacia) with an increased gradient of NaCl. The detailed procedure is described elsewhere (17). Radioactive assays were performed as described (20) previously. Shortly, 100 µL of rat enzyme preparation (at 37 °C) were incubated (30 min) with 10 µL of total [3H]acetate prelabeled chicken reticulocyte histones (1 mg/mL). Reaction was stopped by addition of 36 µL of stop solution (1 M HCl/0.4 M acetate) and 800 µL of ethyl acetate. After centrifugation (10000g, 5 min) an aliquot of 600 µL of the upper phase was counted for radioactivity in 3 mL of liquid scintillation cocktail. 2a showed no inhibition of HDAC at 80 µM. 2.5. Stock Solutions, Incubation, and Sample Preparation. For qualitative analysis, small amounts of the peptides were dissolved in water. The solutions are stable at -20 °C for about 2 weeks. For the quantitative measurements, solutions of the peptides in water were prepared in the following concentrations (mg/mL): 3.540 (2a), 9.740 (2c), and 9.469 (2d). As the peptides contain between 25 and 30% of salt, a salt content of 25% was assumed for all compounds and accordingly corrected concentrations were used for all calculations. 2b was not used for quantitative analysis as it is not formed upon incubation. The concentration of the internal standard stock solution was 5.021 mg/mL. For the calibration, different aliquots of 2a, 2c, and 2d were added to 110 µL of incubation buffer [15 mM tris-HCl, pH 7.9; 0.25 mM EDTA; 10 mM NaCl; 10% (v/v) glycerol; 10 mM 2-mercaptoethanol]. A total of 1.5 µL of internal standard solution was added, and incubation buffer was added to a final volume of 120 µL. The reaction was stopped immediately (200 µL stop solution) and vortexed (2 min). 200 µL of solvent C were added and the mixture was vortexed again (2 min). After centrifugation (10 000 rpm, 5 min) an aliquot was taken from the supernatant. A total of 20 µL was injected in the HPLC system via autosampler (n ) 2). Linear regression provided the following equations: f(x) ) 0.007 83 + 0.02242x, r ) 0.9992 (2a); f(x) ) 0.014 74 + 0.02188x, r ) 0.9997 (2c); f(x) ) - 0.099 28 + 0.02512x, r ) 0.9978 (2d). For the time-dependent analysis of conversion of 2a, 15 parallel reactions were set up as above. The mixture was incubated on ice for 15 min and at 37 °C afterward. The reaction was stopped and the samples were prepared as above after 1, 2, 3, 4, 6, 8, 10, 12, 14, 18, 22, 24, 36, and 42 h (n ) 3, except for t >18 h, n ) 2). 2.6. Mass Spectrometry. A Finnigan LCQ mass spectrometer with an electrospray interface was used for LC-MS coupling analyses. For identification of the incubation products, the column and solvent conditions were used as above (injection volume was changed to 50 µL), and the elution was monitored by UV detection (210 nm). Full-Scan and Zoom-Scan modes were used for analysis of the MS pattern of the standards and the incubation reactions (12 and 20 h). Spiking of the incubation
Octapeptides as Histone Deacetylase Substrates
Bioconjugate Chem., Vol. 12, No. 1, 2001 53 Table 1. LC-MS Signals for Octapeptides 2 standard mixture compd
Mr
2a 2a 2c 2c 2d 2d
1392.5 1392.5 1350.5 1350.5 1308.5 1308.5
a
Figure 2. HPLC chromatograms of a standard mixture of 2a-d and internal standard (IS) (left) and a mixture after incubation with rat liver HDAC for 20 h (right).
reactions with the standards did not lead to other signals. Solutions without substrates or enzyme did not result in signals in the observed range nor in peaks monitored at 210 nm with similar retention times. RESULTS
The peptides 2 were built up by Fmoc-strategy on a Rink amide resin by using a robot system and subsequently labeled with 4(5)-carboxy-fluorescein prior to cleavage from the resin. The latter reagent had been previously proven to be superior to established derivatization reagents 4(5)-fluoresceinisothiocyanate and 4(5)carboxyfluorescein-N-succinimidylester (19). The labeled peptides were obtained in amounts of 5-10 mg. A chromatographic system for the separation of the peptides 2a-d had to be established. Especially the separation of the two mono-acetylated species 2b and 2c proved to be difficult. We succeeded applying a system with an increasing gradient of trifluoroacetic acid in water/acetonitrile and the use of a 3 µm RP-material was crucial to the success. Each of the peptides is showing two peaks due to the isomerism in the commercial starting material used for the labeling procedure. It was not necessary to separate the respective isomers but all eight peptides could be discriminated with the gradient method. Throughout all experiments, there was no indication for a preference of the enzyme for one of the fluorescein isomers. To increase the accuracy and precision of the system, an internal standard was sought and 5-aminofluorescein proved to be suitable. Figure 2 is showing a standard chromatograph of the peptides 2a-d and the internal standard (IS). We then subjected the peptide 2a to incubation with a partially purified rat liver HDAC. At first only the mono-deacetylation product 2c could be detected, while after 3 h also some bis-deacetylation product 2d was monitored. There was no indication for formation of the mono-deacetylation product 2b even after 20 h (see Figure 2). The identity of the incubation products with the synthetic metabolites was not only demonstrated by coelution. LC-MS analyses were performed with the standards and incubation mixtures after 12, 20, and 30 h proved identity of the compounds. Table 1 is showing selected observed mass peaks for a standard mixture and a 30 h incubation mixture obtained by LC-MS coupling. Zoom scan analyses ascertained that monitored signals are indeed (M + n)/z (n ) z ) 2 or 3) peaks for the intact peptides (i.e., protonated species of the highly basic compounds) and not peaks resulting from degradation of the octapeptides by potential residual proteolytic activity in the partially purified rat liver preparation. Spiking of the incubation mixtures with the synthetic
30 h incubation mixture
tR [M + 2] [M + 3] tR [M + 2] [M + 3] (min) z ) 2 z ) 3 (min) z ) 2 z)3 27.06 26.53 18.92 16.81 10.37 9.44
697.0 697.0 675.9 676.0 a a
465.1 465.1 451.1 451.0 437.1 437.1
28.05 25.29 18.93 16.69 10.20 9.60
697.0 696.9 675.8 675.9 a a
465.1 465.0 451.0 451.1 437.1 437.1
Not observed.
reference materials did not lead to any new signals in the time range monitored. Analysis of samples without the substrate 2a or solvent blanks did not results in any signals with respective MS signals nor registration of peaks with the retention times of the standards with fluorescence or UV (210 nm) detection. For quantitative analysis calibrations with standard samples were performed for the substrate 2a and the two deacetylation products 2c and 2d. Figure 3 is showing the calibration curves for the respective peptides. No calibration was performed for 2b as it is not formed upon incubation with rat liver HDAC. To monitor the exact time course of the deacetylation reaction we set up 15 parallel experiments in which the enzyme incubation was terminated after increasing periods of time. The results are depicted in Figure 4. The total area under the curve (AUC) is obtained by multiplying the starting concentration of 2a and the total incubation time of 42 h and equals 3328.678 h µmol/L. The measured AUC-values for the three peptides are 384.496 ( 24.163 h µmol/L (2a), 2267.858 ( 140.571 h µmol/L (2c), and 684.861 ( 33.868 h µmol/L (2d). The AUC values in relation to the total AUC are 11.6 ( 0.7% (2a), 68.1 ( 4.2% (2c), and 20.6 ( 1.0% (2d). This adds up to 100.3 ( 5.9%. No additional peaks at 520 nm nor at 210 nm (UV detection) were detected in the time frame of 70 min. Thus, remaining 2a and all of its biotransformation products are quantitated by the presented method. DISCUSSION
Fluorescein-labeled octapeptide 2a is a substrate for histone deacetylase that is gradually deacetylated at the acetyl-lysine near its C-terminus and eventually formation of some bis-deacetylation product 2d can be observed. This pattern of reactivity may be due to steric hindrance by the N-terminal label but as some deacetylation near the label is monitored this might be an indication for a deacetylation-site preference. Our studies show for the first time that HPLC-chromatography in combination with fluorescence detection and mass spectroscopy is a powerful tool to address questions of regioselective deacetylation in HDAC substrates with several acetyl-lysine residues in-vitro. They may serve as a model for similarly labeled larger peptides that are known to be substrates of histone deacetylase (12) and might not suffer from steric constraint by the label. Similar investigations have just recently been presented with a 20mer-peptide fragment from the N-terminus of histone H3 and the histone acetyltransferase PCAF (p300/CBP-associated factor) (21). Here MALDI/TOF analysis of tryptic digests of the peptidic substrate was used to identify the single acetylation site. Deacetylation site preferences are important for the characterization of various deacetylases and might have an impact on the design of substrate-analogues as HDAC inhibitors. Finally, due to its excitation wavelength of 475 nm our
54 Bioconjugate Chem., Vol. 12, No. 1, 2001
Hoffmann et al. ACKNOWLEDGMENT
M. Jung likes to thank Prof. Unterhalt, Mu¨nster, for generous support. Funding by the Deutsche Forschungsgemeinschaft, Deutsche Pharmazeutische Gesellschaft and the Fonds der Chemischen Industrie is also gratefully acknowledged. LITERATURE CITED
Figure 3. Calibration curves for octapeptides 2a, 2c, and 2d.
Figure 4. Time-dependent conversion of 2a-2c and 2d.
substrate 2a can be used for applications that are not amenable to the existing cumarin-derived substrate, e.g., fluorescence correlation spectroscopy.
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