CARM1 Preferentially Methylates H3R17 over ... - ACS Publications

Feb 5, 2016 - ABSTRACT: CARM1 is a type I arginine methyltransferase involved in the regulation of transcription, pre-mRNA splicing, cell cycle progre...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/biochemistry

CARM1 Preferentially Methylates H3R17 over H3R26 through a Random Kinetic Mechanism Suzanne L. Jacques,* Katrina P. Aquino, Jodi Gureasko,† P. Ann Boriack-Sjodin, Margaret Porter Scott,‡ Robert A. Copeland, and Thomas V. Riera Epizyme Inc., Cambridge, Massachusetts 02139, United States S Supporting Information *

ABSTRACT: CARM1 is a type I arginine methyltransferase involved in the regulation of transcription, pre-mRNA splicing, cell cycle progression, and the DNA damage response. CARM1 overexpression has been implicated in breast, prostate, and liver cancers and therefore is an attractive target for cancer therapy. To date, little about the kinetic properties of CARM1 is known. In this study, substrate specificity and the kinetic mechanism of the human enzyme were determined. Substrate specificity was examined by testing CARM1 activity with several histone H3-based peptides in a radiometric assay. Comparison of kcat/KM values reveals that methylation of H3R17 is preferred over that of H3R26. These effects are KM-driven as kcat values remain relatively constant for the peptides tested. Shortening the peptide at the C-terminus by five amino acid residues greatly reduced binding affinity, indicating distal residues may contribute to substrate binding. CARM1 appears to bind monomethylated peptides with an affinity similar to that of unmethylated peptides. Monitoring of the CARM1-dependent production of monomethylated and dimethylated peptides over time by self-assembled monolayer and matrix-assisted laser desorption ionization mass spectrometry revealed that methylation by CARM1 is distributive. Additionally, dead-end and product inhibition studies suggest CARM1 conforms to a random sequential kinetic mechanism. By defining the kinetic properties and mechanism of CARM1, these studies may aid in the development of small molecule CARM1 inhibitors.

C

NCOA2,4 and EP300.5 In addition, CARM1 plays a role in the regulation of post-transcriptional RNA processing and turnover through the methylation of proteins such as PABP1 and SmB.6−8 CARM1 methylation of histones is also thought to promote active transcription through the recruitment of transcriptional elongation complexes and other mechanisms.9 CARM1 methylation sites on histone substrates have been mapped to H3R17 and H3R26;10 the enzyme exhibits a preference for distinct substrate motifs compared to those preferred by other type I PRMTs.7 CARM1 overexpression has been associated with breast,11 prostate,12 and liver13 cancers; hence, CARM1 is an attractive target for cancer therapy. Although the mechanism underlying the oncogenic potential of

oactivator-associated arginine methyltransferase 1 (CARM1, also known as PRMT4) belongs to the protein arginine methyltransferase (PRMT) family.1 The human genome is believed to contain 10−50 PRMTs with at least eight members demonstrating methyltransferase activity.2 CARM1 (EC 2.1.1.125.2681) is a type I methyltransferase and catalyzes the transfer of methyl groups from S-adenosyl-L-methionine (SAM) to guanidinium nitrogens of arginine residues on protein substrates to produce both Nω-monomethyl-arginine and asymmetric Nω,Nω′-dimethyl-arginine residues. In contrast, type II arginine methyltransferases produce Nω-monomethyl-arginine and symmetric Nω,Nω-dimethyl-arginine residues. CARM1 methylates several histone and non-histone substrates and impacts many cellular processes, including transcriptional coactivation, RNA splicing and processing, control of the cell cycle, and cellular differentiation. CARM1 facilitates transcriptional coactivation by methylating nuclear receptors and nuclear receptor-associated coactivators such as SRC-3,3 © XXXX American Chemical Society

Special Issue: Epigenetics Received: October 1, 2015 Revised: January 16, 2016

A

DOI: 10.1021/acs.biochem.5b01071 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

into 20 mM Tris, 150 mM NaCl, 5% glycerol, and 5 mM β-mercaptoethanol (pH 7.8). The purity and nominal concentration of the CARM1 preparation were assessed by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) (Figure S1 of the Supporting Information) and by analysis on a Bioanalyzer (Agilent Technologies). CARM1 protein was determined to be 95% pure. LC−MS analysis of purified CARM1 revealed two peaks with molecular masses of 67934 and 68138 Da (Figure S2), equivalent to 77 and 280 Da, respectively, higher than the expected molecular mass (67858 Da). This suggests that CARM1 may contain one or more posttranslational modifications. CARM1 Methyltransferase Assay. CARM1 activity was measured as previously reported for the protein methyltransferase PRMT6.31 Buffer conditions were 20 mM bicine, 1 mM TCEP, 0.005% BSG, and 0.002% Tween 20 (pH 7.5). Assays were performed in a reaction volume of 50 μL at room temperature and quenched by the addition of 300 μM unlabeled SAM (10 uL) at various time points to measure initial rates. Biotinylated peptide was captured on streptavidincoated Flashplates (PerkinElmer), and the quantity of 3 H-labeled peptide produced was measured with a Topcount reader (PerkinElmer). Product formation was linear for 120 min. The CARM1 concentration was chosen from the range in which initial velocities were linearly dependent upon enzyme concentration [0.16−2.5 nM (Figure S3)]. Steady-state kinetic parameters, KM and kcat, were measured with 0.25 nM CARM1 by varying peptide concentrations from a top concentration of 2 or 10 μM, depending on the peptide affinity, at a constant [3H]SAM concentration equal to 30 nM. Bisubstrate analysis was performed with 0.25 nM CARM1 by varying both [3H]SAM and peptide concentrations simultaneously with top concentrations of 200 nM and 2 μM, respectively. For the inhibition studies versus varying peptide, peptide substrate concentrations were varied as described above with a fixed [3H]SAM concentration of 30 or 300 nM and 0.25 nM CARM1. When inhibitor and [3H]SAM concentrations were varied, [3H]SAM concentrations were varied from 200 or 300 nM with a fixed peptide concentration of 70 or 700 nM and 0.25 nM CARM1. SAH and SFG were dissolved in DMSO and titrated from a top concentration of 5 μM with a final DMSO concentration of 2% in the assay. Peptide inhibitors were dissolved in distilled water and titrated from a top concentration of 1 mM. All inhibitors were incubated with enzyme for 30 min at room temperature before the assay was initiated with substrates. Data Analysis. To calculate steady-state kinetic parameters kcat and KM from the initial velocity data at varying peptide concentrations, data were fit by nonlinear least-squares regression with the Michaelis−Menten equation (eq 1). Similarly, initial rates obtained when varying both concentrations of peptide and [3H]SAM simultaneously were fit by nonlinear least-squares regression with eq 2 for random substrate addition. Inhibition data were analyzed by fitting data by nonlinear leastsquares regression with competitive (eq 3), mixed noncompetitive (eq 4), and uncompetitive (eq 5) equations, and best fits were determined by Akaike’s Information Criteria (AICc).32 In contrast to the traditional hypothesis test (F test), the calculation for AICc is based on information theory and balances the goodness of fit (sum of squares) versus the degrees of freedom to determine the best fitting model (GraphPad Software, Inc.).

CARM1 is not well understood, small molecule inhibitors of CARM1 have been explored14−19 and crystal structures illustrate indole and pyrazole inhibitors binding in the arginine binding pocket of CARM1.20 The kinetics of PRMT family members, PRMT1,21−25 PRMT5,26 and PRMT6,27−29 have been previously investigated. The best studied enzyme, PRMT1, has been shown to methylate arginine residues via a rapid-equilibrium random mechanism.22 Methylation by PRMT1 was also demonstrated to be a partially processive21,25 or distributive23 process. In this case, the monomethylated intermediate can dissociate and rebind for a second methyl transfer, as opposed to a processive mechanism whereby the intermediate would remain bound until the second methyl group was added. In addition, presteady-state kinetic studies of PRMT1 reveal that a significant conformational step occurs after substrate binding and that methyl transfer is rate-limiting.24 In contrast, less has been reported on the kinetic properties of CARM1 or its kinetic mechanism. A previous crystallographic study of mouse CARM130 speculated that substrate binding follows an ordered mechanism based on the observation that binding of SAH leads to formation of the protein binding groove. Furthermore, this finding would predict a distributive mechanism to allow the exchange of SAH for a second SAM molecule. Nonetheless, further studies are required to confirm these assumptions. Providing a clear understanding of the kinetic properties of CARM1 will allow for identification of the enzyme forms (E or ES) available for inhibitor binding and therefore will be beneficial in the development of small molecule inhibitors of CARM1. In this report, the substrate specificity, processivity, and kinetic mechanism of human CARM1 were investigated. By comparing CARM1 activity on a series of histone H3 peptides, we demonstrate that CARM1 preferentially methylates H3R17. Steady-state substrate kinetics, product, and dead-end inhibitor analyses support random binding of substrate to CARM1. Finally, SAMDI mass spectrometry was used to determine that methylation proceeds by a distributive mechanism.

■

EXPERIMENTAL PROCEDURES Materials. S-Adenosyl-L-methionine (SAM), S-adenosyl-Lhomocysteine (SAH), sinefungin (SFG), bicine, Tween 20, dimethyl sulfoxide (DMSO), bovine skin gelatin (BSG), and a tris(2-carboxyethyl)phosphine hydrochloride solution (TCEP) were purchased from Sigma-Aldrich at the highest level of purity. [3H]SAM was purchased from American Radiolabeled Chemicals (St. Louis, MO) with a specific activity of 80 Ci/mmol. Streptavidin-coated Flashplates were purchased from PerkinElmer. Peptide substrates were synthesized by Biopeptide Co. Inc. (San Diego, CA) and capped with an N-terminal acetyl group and a C-terminal amide group. Biotin groups were added with an aminohexanoic acid linker to the N-terminus or to the ε-amino group of a C-terminal lysine residue. Protein Production. Full-length human FLAG-CARM1Glu-Gly-His (amino acids 2−608, accession number NP_954592) was expressed in 293F mammalian cells using standard methodologies. Cells were lysed by sonication in a buffer containing 20 mM Tris, 150 mM NaCl, 5% glycerol, and 0.1% Tween 20 (pH 7.8) (buffer A) with protease inhibitors (Roche) and then centrifuged to remove cell debris. The supernatant was loaded onto an anti-FLAG M2 affinity gel column (Sigma, St. Louis, MO) pre-equilibrated with buffer A, then washed with buffer A, and eluted with buffer A containing 400 μg/mL FLAG peptide. Protein was concentrated by ultrafiltration and dialyzed B

DOI: 10.1021/acs.biochem.5b01071 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

Table 1. Substrate Specificity of CARM1 for H3 Peptides kcat/KM (M−1 s−1)

peptide substratea

KM (μM)

kcat (s−1)

1 2 3 4 5 6 7 8 9 10 11 12 13

1.7 ± 0.6 0.15 ± 0.03 5.2 ± 2.2 0.35 ± 0.06 NDb 1.8 ± 0.5 NDb 2.5 ± 0.9 NDb NDb 0.61 ± 0.14 0.12 ± 0.04 0.095 ± 0.036

0.0057 ± 0.0010 0.0059 ± 0.0005 0.0066 ± 0.0016 0.0086 ± 0.0005 4 times the enzyme concentration in the reaction mixture (20 nM CARM1), indicating release of the monomethylated peptide from the enzyme consistent with a distributive mechanism. CARM1 Bisubstrate Kinetics Indicate a Sequential Mechanism. As a first step in understanding the kinetic mechanism of CARM1, initial rates were measured for the reactions with unmethylated R17 (peptide 11) and monomethylated R17 (peptide 13) substrates where both peptide and SAM concentrations were simultaneously varied. The data were fit with the random order bisubstrate equation (eq 2) (Figure 3A,C), and kinetic parameters were determined (Table 2). Doublereciprocal plots using the kinetic parameters obtained for peptide 11 (Figure 3B) and peptide 13 (Figure 3D) both produced a pattern of intersecting lines indicating that CARM1 forms a ternary complex with peptide and SAM in a sequential mechanism and excludes a ping-pong (i.e., double-displacement) kinetic mechanism. CARM1 methylation of peptide 11 containing unmethylated R17 produced KM values of 318 nM for peptide and 26 nM for SAM, while methylation of peptide 13, which is monomethylated at R17, produced KM values of 67 nM for peptide and 23 nM for SAM (Table 2). Furthermore, α values for both peptides were close to 1, indicating that peptide and SAM bind independently. As the steady-state kinetics are similar for both the unmethylated and monomethylated R17 peptides, the monomethylated R17 peptide (peptide 13) was chosen for further characterization by product and dead-end inhibition studies as it is capable of only a single methyl transfer, thereby simplifying interpretation of the resultant kinetics. Product and Dead-End Inhibition Studies Support Random Binding of Substrates to CARM1. Product and dead-end inhibition studies were pursued to further define the kinetic mechanism of CARM1. We used SAH and peptide 14 [acetyl-GKAP(RMe2)KQLATKAA(RMe2)KSAPK-amide] as product inhibitors (Table 3 and Figure 4) and SFG and peptide 15 [acetyl-GKAPKKQLATKAA(RMe2)KSAPK-amide] as dead-end inhibitors (Table 4 and Figure 5). Inhibition of CARM1 by SAH at varying SAM concentrations produced a competitive pattern of inhibition at both subsaturating and saturating concentrations of peptide 13, generating Ki values of 22 and 41 nM, respectively (Table 3 and Figure 4). Similarly, SFG exhibited competitive inhibition of CARM1 with respect to SAM concentrations with Ki values equal to 38 and 30 nM at subsaturating and saturating peptide 13 concentrations, respectively (Table 4 and Figure 5). When SAH and SFG were tested with varying concentrations of peptide 13, inhibition was noncompetitive or mixed noncompetitive at both subsaturating and saturating concentrations of SAM with α values ranging from 0.7 to 2.7. Ki values for SAH and SFG were 24 and 37 nM, respectively, at 30 nM SAM and increased to 160 and 54 nM,

Figure 4. Product inhibition of CARM1 by SAH (A and B) and peptide 14 (C and D). Inhibition of CARM1 was tested at varying concentrations of SAM or peptide 13, while keeping the fixed substrate at subsaturating (●) or saturating (○) concentrations (70 or 700 nM peptide 13, respectively, and 30 or 300 nM SAM, respectively). The sequence of peptide 14 was acetyl-GKAP(RMe2)KQLATKAA(RMe2)KSAPK-amide. F

DOI: 10.1021/acs.biochem.5b01071 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry Table 4. Dead-End Inhibition of CARM1 inhibitora SFG SFG SFG SFG peptide peptide peptide peptide a

15 15 15 15

variable substrate SAM SAM peptide peptide SAM SAM peptide peptide

13 13

13 13

fixed substrate 70 nM peptide 13 700 nM peptide 13 30 nM SAM 300 nM SAM 70 nM peptide 13 700 nM peptide 13 30 nM SAM 300 nM SAM

Ki (μM)

α

inhibition pattern

± ± ± ± ± ± ± ±

− − 0.7 1.7 1.7 1.9 4.7 4.7

competitive competitive noncompetitive noncompetitive noncompetitive noncompetitive mixed noncompetitive mixed noncompetitive

0.038 0.030 0.037 0.054 48 117 24 45

0.011 0.006 0.026 0.009 15 32 5 5

Same conditions as described in the footnote of Table 3.

structures with H3 peptides and SFG show that R17, and not R26, is bound in the arginine binding pocket when both residues are available.36 Our results also suggest that R26 and its four C-terminal residues (R26KSAP) provide important binding interactions with CARM1 as truncating the peptide substrate by these five residues drastically decreased the affinity for the substrate. Similar effects were presented for PRMT1 methylation of H4 N-terminal peptides, where positively charged residues (R17HRK) are important for long-range interactions.21 Like PRMT1, CARM1 may have an extended binding surface with peptide or protein substrates. Crystal structures with a truncated construct of human CARM1 and H3 peptides, however, did not reveal any electron density for these C-terminal residues, presumably because they occupied several conformational states.36 The construct used for crystallization contained only the catalytic subunit and was missing both N- and C-terminal domains. Therefore, any interactions with the C-terminal peptide residues with CARM1 residues external to the central catalytic core would not be captured in those structures. Similar results were obtained for PRMT5 and Trypanosoma brucei PRMT7 crystallized with H4 peptides, where structures revealed interactions with arginine and the adjacent residues without resolution of the structure at the C-terminus of the peptides.37,38 On the other hand, a crystal structure of rat PRMT1 with an H4 peptide suggests the presence of extended peptide interactions; however, details about the peptide−protein interactions could not be resolved, complicating any further interpretation.39 Nonetheless, a structure of a peptide complex with full-length CARM1 protein is needed to elucidate the contributions of the C-terminal peptide residues to CARM1 binding. On the other hand, the kinetic properties of the optimal peptides (peptides 4 and 11−13) are very similar to those published for full-length histone H3 with CARM1,10 indicating that these peptides contain the critical residues of H3 for interaction and reactivity with CARM1 and validating their use for the study of the kinetic mechanism. In aggregate, these results suggest that H3R17 may be the predominant methylation site for CARM1-catalyzed methylation while the predominant role of H3R26 is as an exosite binding/recognition element. While CARM1 methylation of histones is clearly involved in the regulation of gene expression,9 it is not known what differential downstream effects may be attributed to CARM1 methylation of H3R17 versus H3R26. CARM1 Methylation Is a Distributive Process. The conversion of arginine residues by CARM1 to asymmetric dimethyl-arginines occurs through the intermediate monomethyl-arginine. Here, we showed that CARM1 exhibits a similar affinity for unmethylated and monomethylated substrates consistent with a distributive mechanism. This was supported

respectively, at 300 nM SAM. Although there is a slight effect of peptide concentration on inhibitor affinity with α ∼ 2, both noncompetitive and mixed noncompetitive inhibition demonstrate that SAH and SFG can bind both free and peptide-bound enzyme forms. Together, these results indicate that SAH and SFG bind the same enzyme form as SAM and are capable of binding before and after peptide substrate. When product inhibition with peptide 14 was examined at varying peptide 13 concentrations (Table 3 and Figure 4), inhibition followed a competitive pattern with Ki values of 37 and 52 μM at subsaturating and saturating SAM concentrations, respectively. Surprisingly, product affinity (peptide 14) is 3 orders of magnitude lower than the apparent affinity of the substrate (peptide 13; KM = 67 nM). Therefore, addition of the second asymmetric methyl group has a dramatic effect on peptide affinity. The result is in good agreement with crystallographic studies, which show CARM1 with unmethylated and monomethylated H3 and PABP1 peptides but were unsuccessful in producing a ternary complex with the asymmetric dimethylated arginine residue in the binding site.36 The deadend inhibitor peptide 15 also demonstrated micromolar affinity as shown by inhibition of CARM1 at varying peptide 13 concentrations (Ki values of 24 and 45 μM at 30 and 300 nM SAM, respectively) (Table 4 and Figure 5). Inhibition by both peptides 14 and 15 was best described as mixed-type with strong competitive character as evidenced by larger α values (α > 10 and α = 5 for peptides 14 and 15, respectively). These results may arise as a result of binding of the inhibitory peptides to a CARM1−SAH complex. Finally, inhibition of CARM1 by peptides 14 and 15 was investigated with varying SAM concentrations, and both demonstrated noncompetitive inhibition with α values of 1.7−2.0. Ki values for both peptides 14 and 15 were 48 μM at 70 nM peptide 13 and increased to 108 and 117 μM, respectively, at 700 nM peptide 13. These results suggest that the peptide inhibitors bind the same enzyme form as the peptide substrate and can bind before and after SAM. Together, these inhibition data indicate that substrates bind CARM1 in a random order as depicted in Scheme 1.

■

DISCUSSION Substrate Specificity of CARM1. Previous analysis of H3 peptides radiolabeled by CARM1 demonstrated that R17 and R26 are major methylation sites.10 In this report, the interactions of human CARM1 with histone H3 were probed by testing the methylation of several peptides based on the sequence of the N-terminus of histone H3. Via examination of the kinetic properties of these peptides, methylation at R17 is preferred over that of R26 predominantly through differences in the apparent binding affinity. In agreement, recent CARM1 G

DOI: 10.1021/acs.biochem.5b01071 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

Scheme 1. Proposed Random Binding of Substrates with CARM1a

a

Kinetic parameters represent those measured for peptide 13.

by the analysis of the CARM1 reaction using SAMDI MS. In this analysis, the monomethylated product accumulates during turnover to levels exceeding the enzyme concentration, suggesting that the reaction is distributive. Likewise, previous studies of PRMT1, PRMT3, PRMT5, and PRMT6 also provided evidence of a distributive mechanism.23,26,28 Other reports support a partially processive mechanism for PRMT1,21,25 stating that SAH/SAM exchange occurs on the same time scale as the release of the monomethyl-arginine. Different substrates and protein binding partners may influence the processivity of the reaction as reported for PRMT1.25 As discussed previously, peptide 11 is a good substitute for histone H3; however, other components in the complex may influence this mechanism in vivo. It appears that all five PRMTs examined so far utilize a distributive mechanism for the production of dimethyl-arginines, indicating that this is a general mechanism for PRMTs with peptide substrates. Kinetic Mechanism of CARM1. Bisubstrate kinetics and inhibitor analysis with SAH, SFG, and peptide inhibitors produced results consistent with CARM1 operating through a random sequential mechanism (Scheme 1). This type of mechanism, in which either substrate can bind first, appears to be characteristic of the PRMT family members, as PRMT1, PRMT5, and PRMT6 also display random sequential mechanisms.22,26,29 Crystallographic data with truncated mouse CARM1, however, revealed structural rearrangement of regions of the active site upon SAH binding, which was interpreted as evidence of ordered substrate addition with SAM binding first, but kinetic experiments to support this observation have not been described.30 Similarly, isothermal calorimetry experiments have shown that select small molecule inhibitors bind to CARM1 only in the presence of SAH, suggesting an ordered binding mechanism.20 Although no structure of human apo-CARM1 has been determined to date, crystal structures of human CARM1 with nucleoside analogues show the analogues buried within the protein structure with no obvious entry path.20 Recently, crystal structures of human CARM1 with H3 and PABP1 peptides have revealed the binding interactions between CARM1 and the peptide substrates.36 These structures, however, do not provide information about the extended binding interactions that have been demonstrated in our kinetic studies. Studies with small molecule inhibitors, such as that by Sack et al.,20 would also miss the possibility for extended binding interactions that can be found with macromolecular substrates. In addition, all available structures of CARM1 were obtained using truncated proteins. Therefore, it is difficult to determine from the current structures whether peptide substrates can bind in the presence or absence of SAM or SAH. Furthermore, while crystal structures often provide important insights into structure−function studies, they are

Figure 5. Dead-end inhibition of CARM1 by SFG (A and B) and peptide 15 (C and D). Inhibition of CARM1 was tested at varying concentrations of SAM or peptide 13, while keeping the fixed substrate at subsaturating (●) or saturating (○) concentrations (70 or 700 nM peptide 13, respectively, and 30 or 300 nM SAM, respectively). The sequence of peptide 15 was acetyl-GKAPKKQLATKAA(RMe2)KSAPK-amide. H

DOI: 10.1021/acs.biochem.5b01071 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

(3) Naeem, H., Cheng, D., Zhao, Q., Underhill, C., Tini, M., Bedford, M. T., and Torchia, J. (2007) The activity and stability of the transcriptional coactivator p/CIP/SRC-3 are regulated by CARM1dependent methylation. Mol. Cell. Biol. 27, 120−134. (4) Frietze, S., Lupien, M., Silver, P. A., and Brown, M. (2008) CARM1 regulates estrogen-stimulated breast cancer growth through up-regulation of E2F1. Cancer Res. 68, 301−306. (5) Yadav, N., Cheng, D., Richard, S., Morel, M., Iyer, V. R., Aldaz, C. M., and Bedford, M. T. (2008) CARM1 promotes adipocyte differentiation by coactivating PPARgamma. EMBO Rep. 9, 193−198. (6) Cheng, D., Cote, J., Shaaban, S., and Bedford, M. T. (2007) The arginine methyltransferase CARM1 regulates the coupling of transcription and mRNA processing. Mol. Cell 25, 71−83. (7) Lee, J., and Bedford, M. T. (2002) PABP1 identified as an arginine methyltransferase substrate using high-density protein arrays. EMBO Rep. 3, 268−273. (8) Pang, L., Tian, H., Chang, N., Yi, J., Xue, L., Jiang, B., Gorospe, M., Zhang, X., and Wang, W. (2013) Loss of CARM1 is linked to reduced HuR function in replicative senescence. BMC Mol. Biol. 14, 15. (9) Wu, J., Cui, N., Wang, R., Li, J., and Wong, J. (2012) A role for CARM1-mediated histone H3 arginine methylation in protecting histone acetylation by releasing corepressors from chromatin. PLoS One 7, e34692. (10) 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) Methylation of histone H3 by coactivatorassociated arginine methyltransferase 1. Biochemistry 40, 5747−5756. (11) Cheng, H., Qin, Y., Fan, H., Su, P., Zhang, X., Zhang, H., and Zhou, G. (2013) Overexpression of CARM1 in breast cancer is correlated with poorly characterized clinicopathologic parameters and molecular subtypes. Diagn. Pathol. 8, 129. (12) 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) Aberrant expression of CARM1, a transcriptional coactivator of androgen receptor, in the development of prostate carcinoma and androgen-independent status. Cancer 101, 83−89. (13) Osada, S., Suzuki, S., Yoshimi, C., Matsumoto, M., Shirai, T., Takahashi, S., and Imagawa, M. (2013) Elevated expression of coactivator-associated arginine methyltransferase 1 is associated with early hepatocarcinogenesis. Oncol. Rep. 30, 1669−1674. (14) Allan, M., Manku, S., Therrien, E., Nguyen, N., Styhler, S., Robert, M. F., Goulet, A. C., Petschner, A. J., Rahil, G., Robert Macleod, A., Deziel, R., Besterman, J. M., Nguyen, H., and Wahhab, A. (2009) N-Benzyl-1-heteroaryl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamides as inhibitors of co-activator associated arginine methyltransferase 1 (CARM1). Bioorg. Med. Chem. Lett. 19, 1218−1223. (15) Huynh, T., Chen, Z., Pang, S., Geng, J., Bandiera, T., Bindi, S., Vianello, P., Roletto, F., Thieffine, S., Galvani, A., Vaccaro, W., Poss, M. A., Trainor, G. L., Lorenzi, M. V., Gottardis, M., Jayaraman, L., and Purandare, A. V. (2009) Optimization of pyrazole inhibitors of Coactivator Associated Arginine Methyltransferase 1 (CARM1). Bioorg. Med. Chem. Lett. 19, 2924−2927. (16) Purandare, A. V., Chen, Z., Huynh, T., Pang, S., Geng, J., Vaccaro, W., Poss, M. A., Oconnell, J., Nowak, K., and Jayaraman, L. (2008) Pyrazole inhibitors of coactivator associated arginine methyltransferase 1 (CARM1). Bioorg. Med. Chem. Lett. 18, 4438− 4441. (17) Therrien, E., Larouche, G., Manku, S., Allan, M., Nguyen, N., Styhler, S., Robert, M. F., Goulet, A. C., Besterman, J. M., Nguyen, H., and Wahhab, A. (2009) 1,2-Diamines as inhibitors of co-activator associated arginine methyltransferase 1 (CARM1). Bioorg. Med. Chem. Lett. 19, 6725−6732. (18) Wan, H., Huynh, T., Pang, S., Geng, J., Vaccaro, W., Poss, M. A., Trainor, G. L., Lorenzi, M. V., Gottardis, M., Jayaraman, L., and Purandare, A. V. (2009) Benzo[d]imidazole inhibitors of Coactivator Associated Arginine Methyltransferase 1 (CARM1)–Hit to Lead studies. Bioorg. Med. Chem. Lett. 19, 5063−5066.

limited in their ability to understand protein dynamics that occur in solution or in a cellular context. In summary, our kinetic studies indicate that human CARM1 preferentially methylates R17 over R26 of histone H3 and suggest an extended substrate binding site involving residues C-terminal to R26. Furthermore, CARM1 catalysis proceeds through a distributive, random sequential mechanism in which substrate and SAM bind independently to enzyme and monomethylated substrate is released from enzyme and must rebind to produce the asymmetrically dimethylated arginine product. From these results, the optimal peptide substrate may be chosen and the balanced assay conditions can be defined for future inhibitor screening. The random binding mechanism of CARM1 also suggests that either SAM or peptide pockets are kinetically available for small molecule targeting, thus increasing the range of potential strategies for CARM1 inhibition. These findings provide a framework for understanding CARM1 catalysis and may aid in the development of small molecule CARM1 inhibitors.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b01071. SDS−PAGE and LC−MS of full-length CARM1 protein (2−608) and initial velocities measured at increasing CARM1 concentrations (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Epizyme Inc., 400 Technology Square, 4th Floor, Cambridge, MA 02139. E-mail: [email protected]. Present Addresses †

J.G.: Alexion Pharmaceuticals, Inc., Lexington, MA 02421. M.P.S.: Genentech, Inc., South San Francisco, CA 94080.

‡

Funding

This work was funded by Epizyme, Inc. Notes

The authors declare the following competing financial interest(s): All authors were employees of Epizyme, Inc., at the time of the studies.

■ ■

ACKNOWLEDGMENTS We thank M. Scholle at SAMDI Tech for assistance with mass spectrometry and Epizyme colleagues for helpful discussions. ABBREVIATIONS BSG, bovine skin gelatin; CARM1, coactivator-associated methyltransferase 1; DMSO, dimethyl sulfoxide; LC, liquid chromatography; MS, mass spectrometry; PRMT, protein arginine methyltransferase; SAH, S-adenosyl-L-homocysteine; SAM, S-adenosyl-L-methionine; SAMDI, self-assembled monolayer and matrix-assisted laser desorption ionization; SFG, sinefungin; TCEP, tris(2-carboxyethyl)phosphine hydrochloride.

■

REFERENCES

(1) Wolf, S. S. (2009) The protein arginine methyltransferase family: an update about function, new perspectives and the physiological role in humans. Cell. Mol. Life Sci. 66, 2109−2121. (2) Copeland, R. A., Solomon, M. E., and Richon, V. M. (2009) Protein methyltransferases as a target class for drug discovery. Nat. Rev. Drug Discovery 8, 724−732. I

DOI: 10.1021/acs.biochem.5b01071 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry (19) Cheng, D., Valente, S., Castellano, S., Sbardella, G., Di Santo, R., Costi, R., Bedford, M. T., and Mai, A. (2011) Novel 3,5bis(bromohydroxybenzylidene)piperidin-4-ones as coactivator-associated arginine methyltransferase 1 inhibitors: enzyme selectivity and cellular activity. J. Med. Chem. 54, 4928−4932. (20) Sack, J. S., Thieffine, S., Bandiera, T., Fasolini, M., Duke, G. J., Jayaraman, L., Kish, K. F., Klei, H. E., Purandare, A. V., Rosettani, P., Troiani, S., Xie, D., and Bertrand, J. A. (2011) Structural basis for CARM1 inhibition by indole and pyrazole inhibitors. Biochem. J. 436, 331−339. (21) Osborne, T. C., Obianyo, O., Zhang, X., Cheng, X., and Thompson, P. R. (2007) Protein arginine methyltransferase 1: positively charged residues in substrate peptides distal to the site of methylation are important for substrate binding and catalysis. Biochemistry 46, 13370−13381. (22) Obianyo, O., Osborne, T. C., and Thompson, P. R. (2008) Kinetic mechanism of protein arginine methyltransferase 1. Biochemistry 47, 10420−10427. (23) Kolbel, K., Ihling, C., Bellmann-Sickert, K., Neundorf, I., BeckSickinger, A. G., Sinz, A., Kuhn, U., and Wahle, E. (2009) Type I Arginine Methyltransferases PRMT1 and PRMT-3 Act Distributively. J. Biol. Chem. 284, 8274−8282. (24) Feng, Y., Xie, N., Jin, M., Stahley, M. R., Stivers, J. T., and Zheng, Y. G. (2011) A transient kinetic analysis of PRMT1 catalysis. Biochemistry 50, 7033−7044. (25) Gui, S., Wooderchak-Donahue, W. L., Zang, T., Chen, D., Daly, M. P., Zhou, Z. S., and Hevel, J. M. (2013) Substrate-induced control of product formation by protein arginine methyltransferase 1. Biochemistry 52, 199−209. (26) Wang, M., Xu, R. M., and Thompson, P. R. (2013) Substrate specificity, processivity, and kinetic mechanism of protein arginine methyltransferase 5. Biochemistry 52, 5430−5440. (27) Frankel, A., Yadav, N., Lee, J., Branscombe, T. L., Clarke, S., and Bedford, M. T. (2002) The novel human protein arginine Nmethyltransferase PRMT6 is a nuclear enzyme displaying unique substrate specificity. J. Biol. Chem. 277, 3537−3543. (28) Lakowski, T. M., and Frankel, A. (2008) A kinetic study of human protein arginine N-methyltransferase 6 reveals a distributive mechanism. J. Biol. Chem. 283, 10015−10025. (29) Obianyo, O., and Thompson, P. R. (2012) Kinetic mechanism of protein arginine methyltransferase 6 (PRMT6). J. Biol. Chem. 287, 6062−6071. (30) Yue, W. W., Hassler, M., Roe, S. M., Thompson-Vale, V., and Pearl, L. H. (2007) Insights into histone code syntax from structural and biochemical studies of CARM1 methyltransferase. EMBO J. 26, 4402−4412. (31) Mitchell, L. H., Drew, A. E., Ribich, S. A., Rioux, N., Swinger, K. K., Jacques, S. L., Lingaraj, T., Boriack-Sjodin, P. A., Waters, N. J., Wigle, T. J., Moradei, O., Jin, L., Riera, T., Porter-Scott, M., Moyer, M. P., Smith, J. J., Chesworth, R., and Copeland, R. A. (2015) Aryl Pyrazoles as Potent Inhibitors of Arginine Methyltransferases: Identification of the First PRMT6 Tool Compound. ACS Med. Chem. Lett. 6, 655−659. (32) Kamikubo, K., Murase, H., Murayama, M., and Miura, K. (1986) Microcomputer-based nonlinear regression analysis of ligand-binding data: application of Akaike’s information criterion. Jpn. J. Pharmacol. 40, 342−346. (33) Copeland, R. A. (2013) Evaluation of enzyme inhibitors in drug discovery: A guide for medicinal chemists and pharmacologists, 2nd ed., John Wiley & Sons, Inc., Hoboken, NJ. (34) Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7− 18. (35) Wigle, T. J., Swinger, K. K., Campbell, J. E., Scholle, M. D., Sherrill, J., Admirand, E. A., Boriack-Sjodin, P. A., Kuntz, K. W., Chesworth, R., Moyer, M. P., Scott, M. P., and Copeland, R. A. (2015) A High-Throughput Mass Spectrometry Assay Coupled with Redox Activity Testing Reduces Artifacts and False Positives in Lysine Demethylase Screening. J. Biomol. Screening 20, 810−820.

(36) Boriack-Sjodin, P. A., Jin, L., Jacques, S. L., Drew, A., Sneeringer, C., Porter Scott, M., Moyer, M. P., Copeland, R. A., Ribich, S., and Moradei, O. (2015) Structural insights into ternary complex formation of human CARM1 with various substrates. ACS Chem. Biol., DOI: 10.1021/acschembio.5b00773. (37) Wang, C., Zhu, Y., Caceres, T. B., Liu, L., Peng, J., Wang, J., Chen, J., Chen, X., Zhang, Z., Zuo, X., Gong, Q., Teng, M., Hevel, J. M., Wu, J., and Shi, Y. (2014) Structural determinants for the strict monomethylation activity by trypanosoma brucei protein arginine methyltransferase 7. Structure 22, 756−768. (38) Antonysamy, S., Bonday, Z., Campbell, R. M., Doyle, B., Druzina, Z., Gheyi, T., Han, B., Jungheim, L. N., Qian, Y., Rauch, C., Russell, M., Sauder, J. M., Wasserman, S. R., Weichert, K., Willard, F. S., Zhang, A., and Emtage, S. (2012) Crystal structure of the human PRMT5:MEP50 complex. Proc. Natl. Acad. Sci. U. S. A. 109, 17960− 17965. (39) Zhang, X., and Cheng, X. (2003) Structure of the predominant protein arginine methyltransferase PRMT1 and analysis of its binding to substrate peptides. Structure 11, 509−520.

J

DOI: 10.1021/acs.biochem.5b01071 Biochemistry XXXX, XXX, XXX−XXX