1

Identification of a novel inhibitor of CARM1-mediated methylation of histone

H3R17

B. Ruthrotha Selvi1, Kiran Batta1, A. Hari Kishore1, K. Mantelingu1, Radhika A Varier1,

K. Balasubramanyam1, Suman Kalyan Pradhan2, Dipak Dasgupta2, S. Sriram3, Shipra Agrawal3, Tapas K. Kundu1#.

1Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, JNCASR, Jakkur, Bangalore 560 064 2 Saha Institute of Nuclear Physics, I/AF, Bidhannagar, Kolkata 700 064. 3 Institute of Bioinformatics and Applied Biotechnology, International Technology Park Bangalore (ITPB), Whitefield Road, Bangalore 560 066. #To whom correspondence should be addressed: Tapas K. Kundu, Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore- 560064, INDIA. Tel (0091) 80-2208-2841, fax (0091) 80 – 22082766 email: tapas@jncasr.ac.in Running title: TBBD (Ellagic acid), a site specific inhibitor of CARM1

http://www.jbc.org/cgi/doi/10.1074/jbc.M109.063933The latest version is at JBC Papers in Press. Published on December 17, 2009 as Manuscript M109.063933

Copyright 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

2

Methylation of the arginine residues

of histones by methyltransferases has

important consequences for chromatin

structure and gene regulation; however, the

molecular mechanism(s) of

methyltransferase regulation is still unclear,

as is the biological significance of

methylation at particular arginine residues.

Here, we report a novel specific inhibitor of

coactivator-associated arginine

methyltransferase 1 (CARM1; also known

as PRMT4) that selectively inhibits

methylation at arginine-17 of histone H3

(H3R17). Remarkably, this plant-derived

inhibitor, called TBBD (ellagic acid), binds

to the substrate (histone) preferentially at

the signature motif, ‘KAPRK’, where the

proline residue (P16) plays a critical role

for the interaction and subsequent enzyme

inhibition. In a promoter-specific context,

inhibition of H3R17 methylation represses

expression of p21, a p53-responsive gene,

thus implicating a possible role for H3R17

methylation in tumor suppressor function.

These data establish TBBD as a novel

specific inhibitor of arginine methylation

and demonstrate substrate sequence-

directed inhibition of enzyme activity by a

small molecule and its physiological

consequence.

Introduction

Arginine methylation of nonhistone proteins has been known for nearly four decades, regulating various cellular processes such as transcription and RNA processing, DNA replication and repair (1, 2). However, histone arginine methylation and its role in gene regulation were discovered much more recently (3). Protein arginine methyltransferases (PRMTs) are classified into class I and class II enzymes. Class I PRMTs catalyze the formation of asymmetric dimethylarginine (involved in transcriptional activation), whereas class II enzymes are responsible for generating symmetric dimethylarginine (involved in transcriptional repression) (4). The class I enzyme CARM1 (coactivator-associated arginine

methyltransferase 1, also known as PRMT4), was first identified as a p160 coactivator-interacting protein in a yeast two-hybrid screen, and later as a histone methyltransferase and transcriptional coactivator (5). All the PRMTs except CARM1, target a glycine arginine recognition (GAR) motif, whereas CARM1 recognizes XXPRX or XXRPX, where X is any amino acid (6).

CARM1 interacts with GRIP1 (glucocorticoid receptor-interacting protein) and is a secondary coactivator of several nuclear receptors (7-9). CARM1 also interacts with other chromatin-modifying enzymes such as p300/CBP and PRMT1 to bring about cooperative transcriptional activation of p53-responsive genes (10). CARM1 and PRMT1 interactions have also been shown to regulate gene expression in different contexts (11, 12). CARM1 is a positive regulator of both cyclin E1 (13) and NF-κB promoter activity (14). CARM1 also participates in various other cellular processes through its ability to methylate nonhistone substrates. Recently, CARM1 has been implicated in muscle (15) and T-cell development (6), stem cell differentiation (16), adipocyte differentiation (17), RNA processing (18) and tumorigenesis (19). In spite of such broad functional significance, the exact molecular mechanisms of the enzyme function are not understood, in part due to the unavailability of specific modulators. For example, in the case of lysine methyltransferases, only two specific inhibitors chaetocin (20) and BIX-01294 (21) are known. However, no specific inhibitors for CARM1 with proper characterization are known so far.

There is an intensive ongoing effort to identify specific arginine methylation inhibitors (22-24). Small-molecule inhibitors of protein function are powerful tools to probe the physiological roles of enzymes. Furthermore, such modulators are potential lead molecules for therapeutic purposes, as evidenced by the recent clinical trials of histone deacetylase inhibitors. Along with the latter, the recent discovery of specific and nontoxic small molecule modulators of histone acetyltransferases (HATs) and histone

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

3

methyltransferases (HMTases) may portend a new era of epigenetics-based drugs (25).

We have established a general screening procedure to identify small molecule modulators of chromatin-modifying enzymes present in plant extracts (obtained from bark, stem, root or fruit). Using this approach we discovered several small molecule modulators of HATs (25). The same extracts from 25 different plant sources were also screened for HMTase modulatory activity, which led to the identification of a molecule (TBBD) having specific activity towards CARM1, as reported here. This small molecule inhibitor, TBBD (ellagic acid) shows a substrate sequence dependence for enzyme specific inhibition. Furthermore, the inhibitor is also active physiologically with significant consequence on p53 dependent gene expression.

Methods:

Protein purifications:

The details of the protein purifications are provided in the supplementary information. Site-directed mutagenesis (SDM)

Histone H3 point mutants A25P and P16A were obtained by SDM. Histone H3 expression clone (Xenopus) was used as the template and the mutagenesis was done by using a Stratagene SDM kit according to the manufacturer’s instructions. Positive clones were sequenced and transformed into the BL21 strain of E.coli. Expression and purification of the mutant protein were done as detailed in the supplementary section. Histone methyltransferase (HMTase) Assay

HMTase assay has been performed as described elsewhere (26), also see supplementary information Histone acetyltransferase (HAT) assay

HAT assays were performed as described elsewhere (26). Isothermal titration calorimetry (ITC)

ITC experiments were carried out in a VP-ITC system (Microcal LLC, Northampton, MA) at 25ºC. Samples were centrifuged and degassed prior to titration. Titration of TBBD against protein (histone H3/CARM1) was carried out by injecting 0.14 mM TBBD in HMTase assay buffer against 0.007 mM histone H3/ CARM1. A two-minute interval was allowed between injections for equilibration, sufficient for the return of the heat signal to baseline. A total of 35 injections were carried out to ensure complete titration. The details of the analysis is provided in the supplementary information Kinetic characterization of TBBD-mediated

inhibition

The HMTase reaction was carried out with CARM1 in the presence of three concentrations of TBBD (10, 20 and 50 µM). The HMTase reaction consisted of two substrates, histone H3 and the tritiated methyl group donor of SAM. In the first assay, the concentration of histone H3 was kept constant at 1.467 µM and ³H-SAM was varied from 0.39 µM to 1.98 µM. In the second assay, the concentration of ³H-SAM was kept constant at 1.98 µM and histone H3 was varied from 0.026 µM to 1.467 µM. The incorporation of radioactivity was taken as a measure of the reaction velocity, which was recorded as counts per minute (cpm). The obtained values were plotted on a Lineweaver-Burk plot using GraphPad Prism software. Immunoblotting analysis

In vitro-modified histones were processed for immunoblotting analysis as described below. In vivo-modified histones were obtained from TBBD-treated cells. The maximum DMSO concentration used was 0.1%. The quantitated histones were resolved on a 12% SDS polyacrylamide gel. After electrophoresis, protein on the gel was electrotransferred onto an Immobilon membrane (Millipore Corp., Bedford, MA). The membranes were then blocked in 5% nonfat milk solution in 1X PBS containing 0.05% Tween 20 and immunoblotted with anti-dimethylated histone H3R17 (Abcam ab8284) and anti-dimethylated histone H3R26 (Upstate 07-215), acetylated H3K14 (Upstate 07-353) and

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

4

acetylated H3K18 (Upstate 07-354) polyclonal antibodies. Detection was carried out with goat anti-rabbit secondary antibody (Bangalore Genei), and bands were visualized with an ECL detection system (Pierce). Docking protocol

Atomic coordinates of histone H3 (chain A and E) from the X-ray structure of the nucleosome core particle complex (1KX5) were extracted from the PDB. The PDB ID of CARM1 structure used was 2V74. Polar hydrogens were added, nonpolar hydrogens were merged, AutoDock 4 atom types and Gasteiger charges were assigned to all atoms of the receptor molecule (i.e., histone H3). Rotatable bonds, AutoDock 4 atom types, and Gasteiger charges were also assigned to the ligand molecule (TBBD) according to AutoDock Tools (ADT). To target residues 14 to 18 (KAPRK) of the receptor, ‘P’ (proline 16) was assigned as the center of the grid, and an affinity grid box was made with default grid spacing 0.375Ǻ using AutoGrid 4. A Lamarckian genetic algorithm was selected to evaluate the ligand binding energies with the receptor using AutoDock 4 with default parameters except the number of energy evaluations (ga_num_evals, 25000000). Chromatin immmunoprecipitation assay

(ChIP)

ChIP was carried out for both HEK 293T and H1299 cell lines as described elsewhere (27). The pulldown was done using antibody against dimethyl histone H3R17 (Abcam ab8284), dimethyl histone H3R26 (Upstate 07-215), and the immunoprecipitated samples were deproteinized and ethanol-precipitated to recover the DNA. PCR analysis was performed using primers for p53-responsive site on the p21 promoter. Endogenous gene expression assay

Human Embryonic Kidney cell line HEK 293T was treated with doxorubicin to increase p53 levels and was later treated with TBBD for 24 h or left untreated. Following the stipulated treatment time, total RNA was isolated using Trizol reagent (Invitrogen).

cDNA was synthesized with oligo dT (28 mer) (Invitrogen) and MMLV reverse transcriptase (Sigma-Aldrich, Saint Louis, MO), and the expression analysis was carried out using iQTM SyBR green supermix (BioRad) and gene-specific primers of p21 and actin. Statistical analysis:

All experiments were performed atleast as triplicates. The statistical significance of the difference in the means was evaluated by two-tailed paired student’s t-test using Graph Pad Prism software, version 4.0. Means were considered significant if the p values of the paired t-test were less than 0.05.

Results:

TBBD is a specific inhibitor of the arginine

methyltransferase CARM1

Pomegranate (Punica gratum) extract has been known to possess anticancer activity since several decades, however the molecular target for these extract components have not yet been identified. The effect of this extract was tested on the chromatin modifying enzymes in an in vitro assay. The crude extract of pomegranate fruit skin, processed as described in Supplementary section, was found to inhibit the histone arginine methyltransferase activity of CARM1/PRMT4 as well as the acetyltransferase activity of p300/KAT3B (Supplementary Fig. S1, lane 2 vs lane 3). Further purification of the active components from the fraction yielded a highly purified compound, TBBD (Fig. 1A), which led to a dose dependent inhibition of CARM1 methyltransferase activity (Fig. 1B, lanes 3-6), as observed by a filterbinding assay. However, when a similar assay was performed with the histone acetyltransferase p300/KAT3B (which was inhibited by the crude extract), a minimal effect was observed even with increasing concentrations of TBBD (Fig. 1B, lanes 3-6). Furthermore, TBBD did not show any significant effect on the HMTase activity of lysine methyltransferase G9a, as well as on the HAT activity of PCAF (Fig. 1B, lanes 3-6). Although, the activity of all the four enzymes (CARM1, p300, G9a, PCAF) were normalized with the histone substrate (Fig. 1B, lane 2), the purified component, TBBD had significant

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

5

inhibitory effect on the arginine methyltransferase CARM1 alone, indicating its specificity towards CARM1 in an in vitro reaction. This inhibitory effect of TBBD on CARM1 was found to be partial, even at an inhibitor concentration tenfold above the IC50 of 25 µM (Supplementary Fig. S2). TBBD could inhibit CARM1-mediated methylation of recombinant histone H3 (Supplementary Fig. S3) as well as histone H3 in the nucleosomal context (data not shown), suggesting that the state (arrangement) of the histone H3 tail is not involved in the mechanism of inhibition by TBBD. This specificity of the inhibitor was further confirmed by subjecting the in vitro reaction to immunoblotting with site specific histone H3R17 dimethylation antibody. This residue is one of the modification sites of CARM1 on histone H3 tail. A dose dependent inhibition of H3R17 methylation was observed with increasing concentration of TBBD (Fig. 1C, Panel II, lanes 3-6). The same reaction was probed with antibody against histone H3, which indicated similar levels of histone H3 (Fig. 1C, Panel III, lanes 3-6), inspite of a drastic decrease in the H3R17 methylation levels. Thus, the small molecule TBBD, purified from pomegranate fruit skin crude extract, is a specific inhibitor of arginine methyltransferase CARM1 in vitro. TBBD is an uncompetitive inhibitor of

arginine methyltransferase CARM1

A detailed kinetic characterization of the inhibition was performed using recombinant histone H3 as substrate. The methyltransferase reaction consists of two substrates, histone H3 and the methyl cofactor donor, S-adenosyl methionine (SAM). Hence, the kinetic analysis was done by performing two sets of reactions, wherein once histone H3 concentration was kept constant and SAM concentration was varied and vice versa for the second set. It was observed that TBBD-mediated inhibition of CARM1 activity is uncompetitive in nature for both the substrates histone H3 and S-adenosyl-L-(methyl-3H)methionine (³H-SAM) (Fig. 2A and 2B respectively). These results suggest the involvement of a functional interaction

between TBBD and the enzyme–substrate (ES) complex in the process of inhibition. To understand the affinity of the inhibitor towards the two components of the ES complex, we investigated the nature of the interactions of TBBD with CARM1 and histone H3 by employing isothermal calorimetry (ITC) studies, Titration of the ligand (TBBD) with histone H3 showed a significant heat change, as expected (Fig. 2C). The heat change due to the buffer and buffer–ligand contribution was appropriately subtracted. The resultant heat change could be fitted to a single binding site (n = 1.43 ± 0.0372) which is enthalpy driven ((∆H = -18.3 kcal/mole) and Table 1). Interestingly, the ITC studies showed very minimal interaction between TBBD and the enzyme, CARM1 alone (Fig. 2D). This apparent lack of interaction between the enzyme and inhibitor was verified by surface-enhanced Raman spectroscopy (SERS) studies (data not shown). Together, the biophysical and kinetic data suggest that the partial inhibition of CARM1 by TBBD is mediated through its interaction with the ES complex, and predominantly through the substrate, histone H3 at a single site.

TBBD is a site-specific inhibitor of

CARM1-mediated methylation of histone

H3R17

CARM1 can methylate histone H3 at three sites: R2, R17 and R26 (28). Though R2 has so far been reported as an in vitro site of methylation by CARM1, the enzyme has been shown to methylate both R17 and R26 (Fig. 3A) in vivo with several functional consequences. Recent studies have implicated H3R2 as an in vivo site of methylation for PRMT6 (29). Since, TBBD-mediated inhibition of H3 methylation by CARM1 was found to be partial even at high concentrations of TBBD, and because the ITC results suggest that one site on histone H3 is favored for TBBD binding, we hypothesized that TBBD could be a site-specific inhibitor of CARM1. To investigate this phenomenon in the physiological condition, HeLa cells were treated with TBBD for 24 hours and the acid-extracted histones from the cells were probed with specific antibodies against methylated

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

6

H3R17 and H3R26. Significantly, it was observed that methylation of the H3R17 residue was inhibited by TBBD in a dose dependent manner (Fig. 3B, panel I, lanes 3-5). The lower concentration of 5 µM led to more than 50% reduction of H3R17 methylation. As the dose increased to 25 µM, a drastic reduction of almost 80% was observed. At the higher concentration of 100 µM, there was about 95% reduction in the H3R17 methylation levels, whereas, H3R26 methylation was not affected even with 100 µM TBBD (Fig. 3B, panel II, lanes 3-5). Closer examination of the histone H3 tail sequence revealed a characteristic pentapeptide consensus sequence, KAXRK, at the two CARM1 methylation sites, H3R17 and H3R26 (Fig. 3A). The pentapeptide motif differs by one amino acid between the two sites. In the case of the residue whose methylation is inhibited (R17), the amino acid (X) preceding R17 is P16. For residue R26 whose methylation is not affected by TBBD, R26 is preceded by A25. Because proline is known to be a conformational disrupter of polypeptide chains, we hypothesized that P16 may be acting as the docking site for TBBD and thus preventing methylation at R17. To confirm whether, the inhibition observed is a true enzyme inhibition or an artefact of nonspecific blocking of histone accessibility at a certain region, we examined the acetylation status of histone H3K14 and K18 which are also adjacent to the H3P16 site. When histones from TBBD treated HeLa cells were probed with antibodies against acetylated histone H3K14 and K18, interestingly, the acetylation status of both these residues was not affected by TBBD treatment (Fig. 3B, panel III, panel IV, lanes 4-5). The in vivo acetylation status of acetylated H3 K14 and acetylated H3 K18 were monitored by checking the steady state levels of the total histones. To exclude the possibility of TBBD affecting the total acetylation status by modulating acetyltransferase and deacetylase activities, an in vitro acetylation reaction in the presence of TBBD, using p300/KAT3B was performed using core histones as substrate. These in vitro modified histones were processed for immunoblotting using the acetylated H3K14

and K18 antibodies. It was observed that the histone acetylation was unaffected by TBBD treatment (Supplementary Fig. S4, panel III and IV, lanes 2-4) although the H3R17 methylation was affected at similar concentrations (Supplementary Fig. S4, panel I, lanes 2-4). H3R26 methylation was unaffected (Supplementary Fig. S4, panel II, lanes 2-4) as observed earlier. These observations clearly indicate that TBBD is a specific inhibitor of CARM1 even in the in vivo context with preference towards H3R17 methylation.

Docking studies of TBBD on CARM1-

Histone H3 complex To verify any differential interaction

of TBBD with the two motifs, KAPRK and KAARK, we resorted to molecular modeling and docking studies. We docked the pentapeptide sequence from amino acids 14 to 18, KAPRK of histone H3 into the active site of CARM1 (30) along with TBBD. Since, the reported crystal structure was solved with only the three residues ‘PRK’ of the above motif in the active site, the other two residues ‘KA’ were simulated in the active site of the enzyme (Fig. 4A). It was observed that TBBD exactly docks into the above ES active site (Fig. 4B). Interestingly, this arrangement yields a ligand conformation with ∆G=-5.05 kcal/mol, which is the lowest free energy of binding among the analyzed structures. However, to get a better understanding of TBBD interaction with the pentapeptide motif, the subsequent docking analysis was done using the pentapeptide motif and the inhibitor. As per the docking studies, TBBD forms three hydrogen bonds with A15, R17 and Q19 of histone H3, with ∆G=-5.36 kcal/mol (Fig. 4C, panels I and II). When the docking experiment was conducted by targeting the grid centered on the motif spanning amino acids 23 to 27, KAARK, of the same receptor molecule (histone H3), a different ligand conformation was obtained with a higher ∆G (Fig. 4D, panels I and II). The only difference between the two motifs is the presence of proline at the third position. This residue, P16, in histone H3 is predicted to form hydrophobic interactions with TBBD, thus giving this conformation the lowest free

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

7

energy of binding, indicating the possibility of the proline residue playing a critical role in determining the inhibition.

The proline residue of the histone H3 tail is

essential for TBBD-mediated inhibition of

arginine methyltransferase activity

Involvement of the P16 residue of histone H3 in bringing about the inhibition of CARM1 methylation at a single site was further validated by using site directed mutagenesis. As mentioned above, wild-type histone H3 (Fig. 5A) is methylated by CARM1 at two sites, R17 and R26. As revealed by the docking data, TBBD binds the KAPRK stretch of histone H3, preventing methylation of H3R17 (Fig. 5B, panel I, lanes 2-4). However, KAARK is not bound by TBBD, thus allowing R26 in this motif to be methylated (Fig. 5B, panel II, lanes 2-4). A mutant histone H3 (Fig. 5A) in which the alanine residue was mutated to proline (A25P) showed inhibition of H3R17 methylation by TBBD as expected (Fig. 5B, panel I, lanes 6-8). Significantly, we observed that the presence of proline at position 25 (A25P) led to inhibition of R26 methylation by TBBD (Fig. 5B, panel II, lanes 6-8). However, a point mutant P16A (Fig. 5A), in which proline at residue 16 was mutated to alanine, did not show any inhibition of methylation at both sites (Fig. 5B, panel I and panel II, lanes 10-12), establishing that it is indeed the proline residue which is responsible for TBBD-mediated inhibition of CARM1 methylation. Thus, in agreement with the mechanistic differences in the methylation of H3R17 and R26 as observed by X-ray crystallographic analysis (30), TBBD-mediated inhibition of R17 methylation is also uniquely specific.

ITC studies using these point mutants confirmed the above observation. Data for the point mutant A25P, which has two proline residues adjacent to the arginine residue, indicates the presence of two binding sites (n = 2.40 ± 0) for TBBD on histone H3 (Fig. 5C). The binding of TBBD to this mutant substrate is also enthalpy driven, ∆H = -15.8 kcal/mole (Table 1). Significantly, the P16A mutant, which lacks the proline residue preceding arginine, does not show any binding (n = 7.30

± 1.59E3) with TBBD (Fig. 5D). Taken together, these observations suggest that P16 of histone H3 is the docking site for TBBD, and thus this single amino acid determines the inhibitory effect of TBBD on CARM1 activity.

Histone H3R17 methylation regulates p53-

responsive gene expression

Methylation of R17 and R26 has been linked to transcriptional activation (31). However, the significance of R17 methylation alone has not yet been elucidated. Recent reports have established functional interactions of p300, CARM1 and PRMT1 in p53-responsive gene expression (10). We found that TBBD has minimal effect on p300 HAT activity and PRMT1 methyltransferase activity on histone H4R3 (data not shown); rather it inhibits CARM1-mediated histone H3R17 methylation only, as shown above. These unique properties of TBBD provided an opportunity to determine the effect of CARM1 methylation (especially of H3R17) on p53-responsive gene expression. We therefore investigated the effect of TBBD inhibition of H3R17 methylation of histones at the promoter of the p53-responsive gene, p21, in HEK293T cells which have endogenous wildtype p53. HEK293T cells were subjected to genotoxic insult by doxorubicin to induce p53 followed by TBBD treatment for 24 hours and processed for ChIP or gene expression analysis (Fig. 6A). H3R17 methylation at the p21 promoter was verified by ChIP analysis (Fig. 6B, lane 1 vs lane 2). However, the H3R26 methylation at this promoter region was minimally affected by the treatment (Fig. 6B, lane 1 vs lane 2). Strikingly, reduced H3R17 methylation (upon TBBD treatment) at the p21 promoter was significantly correlated with repression of p21 expression (Fig. 6C, lane 1 vs lane 2). The p21 expression upon TBBD treatment was found to be about two fold reduced as compared to the solvent control ((Fig. 6D, lane 1 vs lane 2). Similar results were obtained with H1299 cells transfected with p53 (data not shown). Taken together, these results suggest that H3R17 methylation by CARM1 is essential for p53-dependent regulation of p21 gene expression.

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

8

Discussion:

The novel CARM1-specific inhibitor reported here, selectively blocks methylation of H3R17 but not of H3R26. Remarkably, the sequence of the histone H3 tail (specifically the proline residue at position 16) determines the specificity of the inhibition brought about by TBBD. This report also demonstrates a role for H3R17 methylation in p21 gene expression dependent of p53; thus, TBBD could be used to investigate the roles of methylation in both apoptosis and tumor suppressor pathways. Most importantly, the data establish a new mechanism of specific enzyme inhibition determined by the amino acid sequence of the substrate.

The arginine methyltransferase, CARM1/PRMT4 has been shown to regulate important cellular processes such as pluripotency maintenance (16, 32), differentiation (6, 15, 17), splicing (18), transcriptional activation (10-14) as well as tumor manifestation and progression (33, 34). However, the methyltransferase activity of this coactivator has been linked only with few cases of transcriptional activation, muscle and thymocyte differentiation and tumor progression. In such a scenario, the identification of a specific small molecule modulator targeting the methyltransferase activity would be highly useful in further delineating the essential role of CARM1 methyltransferase in gene expression and other cellular processes.

The small molecule inhibitor, TBBD (ellagic acid), reported here is a major component of pomegranate crude extract, which has been used against various ailments such as parasitic diseases, diarrhoea, ulcers and most importantly as an anticancer agent (35). The antitumor activity has been shown for several cancers especially prostate cancer, breast cancer and also for colorectal cancer (36, 37). However, the active component from the crude extract and the exact molecular target within the physiological system has not been identified as yet. Possibly, the so far unidentified molecular target for TBBD is CARM1 activity. The crude extract could inhibit both the p300 acetyltransferase activity as well as the arginine methyltransferase

activity of CARM1. This could be because of the various tannins and polyphenolic components of the pomegranate crude extract.

The mechanism of action of TBBD on CARM1 activity is a novel enzyme inhibition effect. Although in classical biochemistry there are examples of the substrate being sequestered by inhibitor and thereby leading to inhibition (38), TBBD recognizes the substrate sequence and thus brings about inhibition at a single site of modification without affecting the other residue modification. The significance of the ES complex involving the KAPRK motif for R17 methylation inhibition was further confirmed by verifying the modification status of the flanking residues, K14 and K18 acetylation which were not affected on TBBD treatment. The sequence recognition and the subsequent enzyme inhibition seems to require an active site in the context of the residue being recognized. Although the characterization of the inhibition reported here, is with respect to histone H3, the protein arginine methyltransferase CARM1 also methylates a few nonhistone proteins including the acetyltransferase CBP, HuR, PABP1 and TARPP (4). In order to have a further understanding of the molecular mechanism of TBBD mediated inhibition of the arginine methyltransferase activity, these substrates also need to be subjected to similar studies.

CARM1 activity has been shown to be necessary for tumor progression as well as for p53 function (which is a tumor suppressor). The elucidation of the role of CARM1 in maintaining cellular homeostasis is essential. Such studies need to be characterized in the light of the various cellular signals. Importantly, TBBD treatment did not affect expression of p21 in HeLa cells, whereas TBBD treatment caused a significant reduction of p21 gene expression in H1299 and HEK293T cells (Fig. 6C). The latter cells were subjected to DNA damage which leads to activation of p53 downstream targets. Previous work showed that p53-dependent p21 expression is modulated by arginine methylation (39), and because H3R17 regulates the transcriptional activation of p53-responsive genes, our results also implicate

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

9

methylation of H3R17 in p53 dependent gene expression. FACS analysis of TBBD-treated HeLa cells (which lack functional p53) showed that there are no cell-cycle defects (Supplementary Fig. S5). Possibly, H3R17 methylation is an important modulator of tumor suppressor function. However, a more intensive analysis of CARM1-mediated H3R17 methylation in different cell lines under different conditions should be undertaken to elucidate the spatiotemporal importance of H3R17 methylation.

Apart from elucidating the role of H3R17 methylation in p53 downstream target

gene expression and other tumor suppression events, to our knowledge, the present report is the first to establish the molecular target of TBBD as the arginine methyltransferase CARM1, with important physiological consequences. Moreover, the inhibitory action of TBBD seems to be regulated by the substrate sequence itself, thereby highlighting a novel mechanism of enzyme inhibition. Thus, TBBD and its derivatives can be used, not only as probes for elucidating the role of H3R17 methylation in gene expression but also as therapeutic molecules targeting this modification in pathophysiological conditions.

References

1. Liu, Q., and Dreyfuss, G. (1995) Mol Cell Biol. 15: 2800-2808. 2. Kim, S., Park, G.H., and Paik, W.K. (1998) Amino acids. 15: 291-306.

3. Lee, D.Y., Teyssier, C., Strahl, B.D., and Stallcup, M.R. (2004). Endocr. Rev. 26: 147-

170.

4. Bedford, M.T., and Richard, S. (2005). Mol Cell. 18: 263-272. Review.

5. Schurter, B.T., Koh, S.S., Chen, D., Bunick, G.J., Harp, J.M., Hanson, B.L., Henschen-Edman, A., Mackay, D.R., Stallcup, M.R., and Aswad, D.W. (2001) Biochemistry. 40: 5747–5756.

6. Kim, J., Lee, J., Yadav, N., Wu, Q., Carter, C., Richard, S., Richie, E., and Bedford, M.T.

(2004). J Biol Chem. 279: 25339–25344. 7. Kang, Z., Ja¨nne, O., and Palvimo, J.J. (2004). Mol Endocrinol. 18: 2633–2648. 8. Chen, D., Huang, S.M., and Stallcup, M.R. (2000). J. Biol. Chem. 275: 40810–40816

9. Stallcup, M.R., Kim, J.H., Teyssier, C., Lee, Y.H., Ma, H., and Chen, D. (2003) J Steroid

Biochem Mol Biol. 85: 139-145.

10. An, W., Kim, J., and Roeder, R.G. (2004). Cell 117: 735-748.

11. Hassa, P.O., Covic, M., Bedford, M.T., and Hottiger, M.O. (2008). J Mol Biol. 377: 668-678

12. Kleinschmidt, M.A., Streubel, G., Samans, B., Krause, M., and Bauer, U.M. (2008)

Nucleic Acids Res. 36: 3202-3213.

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

10

13. El Messaoudi, S., Fabricio, E., Rodríguez, C., Chuchana, P., Fauquier, L., Cheng, D., Theillet, C., Vandel, L., Bedford, M.T., and Sardet, C. (2006). Proc Natl Acad Sci U S A. 103: 13351-13356.

14. Covic, M., Hassa, P.O., Saccani, S., Buerki, C., Meier, N.I., Lombardi, C., Imhof, R.,

Bedford, M.T., Natoli, G., and Hottiger, M.O. (2005). EMBO J. 24: 85–96.

15. Chen, S.L., Loffler, K.A., Chen, D., Stallcup, M.R., and Muscat, G.E. (2002). J Biol Chem 277: 4324–4333.

16. Torres-Padilla, M.E., Parfitt, D.E., Kouzarides, T., and Zemicka-Goetz, M. (2007)

Nature. 445: 214-218. 17. Yadav, N., Cheng, D., Richard, S., Morel, M., Iyer, V.R., Aldaz, C.M., and Bedford.

M.T. (2008) EMBO Rep. 9: 193-198.

18. Cheng, D., Cote, J., Shaaban, S., and Bedford, M.T. (2007). Mol Cell. 25: 71-83.

19. Majumder, S., Liu, Y., Ford, O.H. III., Mohler, J.L., and Whang, Y.E. (2006) Prostate 66: 1292–1301.

20. Greiner, D., Bonaldi, T., Eskeland, R., Roemer, E., and Imhof, A. (2005) Nat. Chem.

Biol. 1: 143-145. 21. Kubicek, S., O'Sullivan, R.J., August, E.M., Hickey, E.R., Zhang, Q., Teodoro, M.L.,

Rea, S., Mechtler, K., Kowalski, J.A., Homon, C.A., Kelly, T.A., and Jenuwein T. (2007) Mol Cell. 25: 473-481.

22. Krause, C.D., Yang, Z.H., Kim, Y.S., Lee, J.H., Cook, J.R., and Petska, S. (2007)

Pharmacol Ther. 113: 50-87. Review. 23. Spannhoff, A., Machmur, R., Heinke, R., Trojer, P., Bauer, I., Brosch, G., Schüle, R.,

Hanefeld, W., Sippl, W., and Jung, M. (2007) Bioorg Med Chem Lett. 17: 4150-4153.

24. Osborne, T., Roska, R.L., Rajski, S.R., and Thompson, P.R. (2008). J Am Chem Soc. 130: 4574-4575.

25. Swaminathan, V., Reddy, B.A., Selvi, B.R., Sukanya. M.S., and Kundu, T.K. (2007)

Chromatin and Disease, Kundu T K, Dasgupta, D. ed. (Springer Press), 397-428. 26. Selvi, B. R., Pradhan, S.K., Shandilya, J., Das, C., Sailaja, B.S., Shankar, G.N., Gadad,

S.S., Reddy, A., Dasgupta, D., and Kundu T.K. (2009) Chem Biol 16: 203-216. 27. Pokholok, D.K., Harbison, C.T., Levine, S., Cole, M., Hannett, N.M., Lee, T.I., Bell,

G.W., Walker, K., Rolfe, P.A., Herbolsheimer, E., Zeitlinger, J., Lewitter, F., Gifford, D.K., and Young, R.A. (2005) Cell. 122: 517-527.

28. Teyssier, C., Chen, D., and Stallcup, M.R. (2002). J Biol.Chem. 277: 46066-46072.

29. Guccione, E., Bassi, C., Casadio, F., Martinato, F., Cesaroni, M., Schuchlautz, H.,

Lüscher, B., and Amati, B. (2007). Nature 449: 933-937.

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

11

30. Yue, W.W., Hassler, M., Roe, S.M., Thompson-Vale, V., and Pearl, L.H. (2007) EMBO

J. 26: 4402-4412.

31. Bauer, U.M., Daujat, S., Nielsen, S.J., Nightingale, K., and Kouzarides, T. (2002). EMBO Rep. 3: 39-44.

32. Wu, Q., Bruce, A.W., Jedrusik, A., Ellis, P.D., Andrews, R.M., Langford, C.F., Glover,

D.M., and Zernicka-Goetz, M. (2009) Stem Cells 27: 2637-2645.

33. Frietze, S., Lupien, M., Silver, P.A., and Brown, M. (2008). Cancer Res. 68: 301-306. 34. Hong, H., Kao, C., Jeng, M.H., Eble, J.N., Koch, M.O., Gardner, T.A., Zhang. S., Li, L.,

Pan, C.X., Hu, Z., MacLennan, G.T., and Cheng, L. (2004) Cancer 101: 83-89. 35. Jurenka, J. (2008). Alt Med Rev 13: 128-144. 36. Dorai, T., and Aggarwal, B.B. (2004). Cancer Lett. 215: 129-140. Review. 37. Losso, J.N., Bansode, R.R., Trappey, A2nd., Bawadi, H.A., and Truax, R. (2004). J

Nutr.Biochem. 15: 672-678 38. McGovern, S.L., Helfand, B.T., Feng, B., and Shoichet, B.K. (2003). J. Med. Chem. 46:

4265-4272. 39. Li, P., Yao, H., Zhang, Z., Li, M., Luo, Y., Thompson, P.R., Gilmour, D.S., and Wang,

Y. (2008). Mol Cell Biol. 28: 4745-4758.

Footnotes: The authors declare no conflict of interest. Abbreviations used: CARM1- Coactivator associated arginine methyltransferase 1, TBBD- (2,3,7,8-tetrahydroxy[1] benzopyrano [5,4,3(de)[1] benzopyran5,10 dione], ES complex- enzyme-substrate complex. ACKNOWLEDGMENTS

This work was supported by funding from JNCASR and the Department of Biotechnology, Government of India. We acknowledge Prof. M.R.S. Rao, President, JNCASR for his constant support and encouragement, Prof. Yoichi Shinkai for the G9a baculovirus and A. Mangaiarkarasi, Molecular Virology Laboratory, JNCASR, for help with the FACS analysis. RS is a CSIR, Government of India, Senior Research Fellow.

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

12

Figure Legends

Figure 1. TBBD is a specific inhibitor of the arginine methyltransferase CARM1

(A) Structure of TBBD. Structural formula representation (Panel I) and ball and stick model (Panel II). (B) Filter-binding assay for inhibition of histone modification. The HMTase assay was performed with CARM1 and G9a, and the HAT assay was performed with p300, PCAF in the presence or absence of TBBD by using highly purified HeLa core histones and processed for filter-binding assay. Lane 1, core histones without enzyme; lane 2, histones with enzyme in the presence of DMSO; lanes 3-6, histones with enzyme in the presence of 10 µM, 25 µM, 50µM or 100 µM TBBD. Error bars represent standard deviations of the means of duplicate reactions. (C) HMTase assay with CARM1 using core histones as substrate in the presence or absence of TBBD, processed for immunoblotting analysis. Lane 1, core histones without enzyme; lane 2, histones with enzyme in the presence of DMSO; lanes 3-6, histones with enzyme in the presence of 10 µM, 25 µM, 50 µM or 100 µM TBBD. Panel I represents coomassie staining, Panel II represents immunoblotting with dimethylated H3R17 antibody. Panel III represents the histone loading control using histone H3 antibody. Figure 2. TBBD is an uncompetitive inhibitor of CARM1 with preferential affinity to the

substrate Histone H3.

(A) Lineweaver-Burk plot representation of TBBD’s effect on CARM1 activity at a fixed concentration of (³H-SAM) (0.88 µM) and increasing concentrations of histone H3 in the presence (10, 20 or 50 µM) or absence of TBBD. The results were plotted using GraphPad Prism software. (B) Lineweaver-Burk plot representation of TBBD’s effect on CARM1 activity at a fixed concentration of histone H3 (1.467 µM) and increasing concentrations of (³H-SAM) in the presence (10, 20 or 50 µM) or absence of TBBD. The results were plotted using GraphPad Prism software. (C) Isothermal calorimetric (ITC) titration was carried out by titrating histone H3 (7 µM) against the ligand TBBD (140 µM) at 25°C. The one-site binding model of the isotherm is shown. (D) Isothermal calorimetric (ITC) titration was carried out by titrating CARM1 (7 µM) against the ligand TBBD (140 µM) at 25°C. Figure 3. TBBD is a site-specific inhibitor of CARM1-mediated methylation of histone H3.

(A) The possible sites of acetylation and arginine methylation on histone H3 tail sequence are indicated. (B) HeLa cells were treated as indicated for 24 h; histones isolated from untreated cells (lane 1); DMSO-treated cells (lane 2); TBBD-treated cells (lanes 3-5). Histone modifications were probed by western blotting using the indicated site specific antibodies. Figure 4. Molecular modeling studies of TBBD with histone H3.

(A) H3 (P-R-L) peptide has already been modeled into CARM1 active site. The H3 pentapeptide KAPRK has been modeled in the active site of CARM1. The figure shows the H3 pentapeptide near the active site residues of CARM1 (TYR417, HIS415, ASP166 & TYR 262). (B) TBBD is modeled into this docked complex of CARM1 and H3 pentapeptide. (Green – CARM1, Magenta – H3 pentapeptide, Red – TBBD) (C) Figure showing the docked conformation of TBBD near the KAPRK motif of histone H3. The free energy of binding for this ligand conformation is ∆G=-5.36 kcal/mol.

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

13

(D) Figure showing the docked conformation of TBBD near the KAARK motif of histone H3. The free energy of binding for this ligand conformation is ∆G=-3.72 kcal/mol, which is higher than the free energy of binding at the KAPRK motif. Figure 5. The proline residue (P16) of the histone H3 tail is responsible for TBBD-mediated

inhibition of arginine methyltransferase activity. (A) Sequence details of the histone H3 proteins used for the assay. (B) In vitro histone methyltransferase assays were performed with CARM1 in the presence or absence of TBBD by using histone H3 (lanes 1-4), mutant A25P histone H3 (lanes 5-8), and

mutant P16A histone H3 (lanes 9-12) as substrate. Histone H3 in the absence of enzyme (lane 1); or in the presence of enzyme and DMSO (lane 2); or in the presence of 25 µM and 100 µM TBBD (lanes 3-4). Histone H3 was probed with antibodies against dimethylated H3R17 (Panel I) or dimethylated H3R26 (Panel II). As a loading control, histones were probed with antibody against histone H3 (Panel III). (C) Isothermal calorimetric (ITC) titration was carried out by titrating histone H3 mutant A25P (7 µM) against the ligand TBBD (140 µM) at 25°C. The one-site model with two binding sites is shown. (D) Isothermal calorimetric (ITC) titration was carried out by titrating histone H3 mutant P16A (7 µM) against the ligand TBBD (140 µM) at 25°C. No interaction was observed. Figure 6. Histone H3R17 methylation regulates p53-responsive gene expression.

(A) Scheme representing the experimental procedure. The HEK293T cells were treated as indicated and processed for either ChIP or gene expression analysis. (B) State of histone H3R17/H3R26 methylation in the p53-responsive element (p53 RE) of the p21 promoter analyzed by ChIP. ChIP by input ratio in cells treated with DMSO (lane1) or TBBD (lane 2). (n=3, p<0.005). (C) Repression in p21 expression was observed on TBBD treatment in the HEK293T cell line. Reverse transcriptase PCR amplification of p21 expression in cells treated with DMSO (lane 1) or TBBD (lane 2). (D) The expression of p21 is repressed on TBBD treatment. Realtime PCR quantification of fold change in p21 gene expression in cells treated with DMSO (lane 1) and treated with TBBD (lane 2). (n=3, p<0.001). Table 1:

Thermodynamic parameters and stoichiometry of binding for the association of TBBD with Histone H3 wildtype, CARM1, Histone H3 A25P and histone H3P16A in 10 mM Tris-HCl, pH 8.0, at 25°C. Kd, µM n (no.of binding

sites) ∆H (kcal)/mole

∆S (e.u)

Histone H3 wt 4 ± 0.736 1.43 ± 0.0372 -18.3 -35.4 CARM1 - - - - Histone H3A25P 7.9 ± 1.24 2.4 ± 0 -15.8 -26 HistoneH3 P16A - 7.30 ± 1.59 103 - -

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

14

CARM1

G9a

p300

PCAF

Figure 1

A

O

O

O

OH

OH

O

OH

OH

C14H6O8

(2,3,7,8-tetrahydroxy[1] benzopyrano [5,4,3(de)[1]benzopyran5,10-dione)

C

B

cp

m

DMSO

TBBD (µM)

enz

1 2 3 4 5 6

-

-

-

100502510-

+++++

+++++

-

-

-

100502510-

+++++

+++++CARM1

DMSO

TBBD (µM)

Coomassie

staining

αααα H3R17(me)2

αααα H3

Panel I

Panel II

Panel III

-

-

-

100502510-

+++++

+++++

-

-

-

100502510-

+++++

+++++

1 2 3 4 5 6

I II

0

1000

2000

3000

4000

5000

6000

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

15

-3 -2 -1 0 1 2 3

0.001

0.002

0.003 TBBD

50µµµµM

20µµµµM

10µµµµM

0µµµµM

[3H-SAM]

-1 (µµµµM

-1)

cp

m-1

-30 -20 -10 0 10 20 30 40

0.001

0.002

0.003

0.004

-1 -1

TBBD

50µµµµM

20µµµµM10µµµµM

0µµµµM

cp

m-1

[Histone-H3]-1

(µµµµM-1 )

-30 -20 -10 0 10 20 30 40

0.001

0.002

0.003

0.004

-1 -1

TBBD

50µµµµM

20µµµµM10µµµµM

0µµµµM

cp

m-1

[Histone-H3]-1

(µµµµM-1 )

Figure 2

0 1 2 3

-15

-10

-5

0

ExH3TBBD211_NDHExH3TBBD211_Fit

Molar Ratio

kc

al/m

ole

of

inje

cta

nt

0 1 2 3

-15

-10

-5

0

ExH3TBBD211_NDHExH3TBBD211_Fit

Molar Ratio

kc

al/m

ole

of

inje

cta

nt

A B

C

1/v

1/v

-1 0 1 2 3 4 5 6 7 8-1 0 1 2 3 4 5 6 7 8

CARM1TBBD1_NDHCARM1TBBD1_Fit

Molar Ratio

Kc

al/m

ole

of

inje

cta

nt

D

-2

-3

-4

-5

-1 0 1 2 3 4 5 6 7 8-1 0 1 2 3 4 5 6 7 8

CARM1TBBD1_NDHCARM1TBBD1_Fit

Molar Ratio

Kc

al/m

ole

of

inje

cta

nt

D

-2

-3

-4

-5

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

16

A

B

αααα H3

αααα AcH3 K18

αααα AcH3 K14

αααα H3R26(me)2

αααα H3R17(me)2

1 2 3 4 5

Panel I

Panel II

Panel III

Panel IV

Panel V

DMSO

TBBD 100255--

++++-

100255--

++++-

Figure 3

A-R-T-K-Q-T-A-R-K-S-T-G-G-K-A-P-R-K-Q-L-A-T-K-A-A-R-K-S-A-P

4 9 10 14 17 18 23 26 27

H3

Acetyl methyl

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

17

Figure 4

A B

C

D

Panel I Panel II

Panel I Panel II

ASP 166

HIS 415

TYR 262TYR 417

GLY 12

GLY 13

LYS 14ALA 15

PRO 16ARG 17

LYS 18

GLN 19

THR 22LYS 23

ALA 24

ALA 25

ARG 26

LYS 27

ALA 29

SER 28

THR 32ALA 31

GLY 33

GLY 34PRO 30

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

18

A

B

A R T K Q T A R K S T G G K A P R K Q L A T K A A R K S A PHistone H3 wt :

1 14 15 16 17 18 23 24 25 26 27 30

Histone H3 A25P : A R T K Q T A R K S T G G K A P R K Q L A T K A P R K S A P

Histone H3 P16A : A R T K Q T A R K S T G G K A A R K Q L A T K A A R K S A P

D

Figure 5

0.0 0.5 1.0 1.5 2.0 2.5 3.0

-40

-30

-20

-10

ExP16ATBBD2_Fit

Molar Ratio

0.0 0.5 1.0 1.5 2.0 2.5 3.0

ExP16ATBBD2_NDH

-50

αααα H3R17 (me)2

αααα H3R26 (me)2

αααα H3

1 2 3 4 5 6 7 8 9 10 11 12

10025--10025--10025--

+++-+++-+++-

+++-+++-+++-

++++++++++++

10025--10025--10025--

+++-+++-+++-

+++-+++-+++-

++++++++++++Histone H3

CARM1

DMSO

TBBD(µM)

Histone H3 wt Histone H3 A25P Histone H3 P16A

C

0 1 2 3 4 5 6

-10

0 ExA25PTBBD2_NDH

0 1 2 3 4 5 6

-15

ExA25PTBBD2_Fit

Molar Ratio

-5

kc

al/m

ole

of

inje

cta

nt

C

0 1 2 3 4 5 6

-10

0 ExA25PTBBD2_NDH

0 1 2 3 4 5 6

-15

ExA25PTBBD2_Fit

Molar Ratio

-5

kc

al/m

ole

of

inje

cta

nt

kc

al/m

ole

of

inje

cta

nt

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

19

Figure 6

A

C

p21

actin

1 2

HEK293T Doxorubicin (0.5µg/ml) treatment TBBD (100µM) treatment

Cells for 6 hours for 24 hours

Processed for ChIP with H3R17

and H3R26 antibodies followed by

PCR amplification with

p21(p53RE1) specific primers

Processed for RNA isolation and

cDNA synthesis followed by PCR

amplification with p21 gene specific

primers

B

DMSO TBBD0.000

0.025

0.050

0.075

0.100

0.125R17

R26

**

Treatment

Ch

IP /

In

pu

t R

ati

o

** p < 0.005

D

**p < 0.001

0.0

0.5

1.0

1.5

DMSO

TBBD

**

Ex

pre

ss

ion

ov

er

DM

SO

1 2

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

and Tapas K. KunduBalasubramanyam, Suman Kalyan Pradhan, Dipak Dasgupta, S. Sriram, Shipra Agrawal B. Ruthrotha Selvi, Kiran Batta, A. Hari Kishore, K. Mantelingu, Radhika A. Varier, K.

Identification of a novel inhibitor of CARM1-mediated methylation of histone H3R17

published online December 17, 2009J. Biol. Chem. 

  10.1074/jbc.M109.063933Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

  http://www.jbc.org/content/suppl/2009/12/16/M109.063933.DC1

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from