Effect of lysine side chain length on histone lysine acetyltransferase catalysis
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Abstract Histone lysine acetyltransferase (KAT)-catalyzed acetylation of lysine residues in histone tails plays a key role in regulating gene expression in eukaryotes. Here, we examined the role of lysine side chain length in the catalytic activity of human KATs by incorporating shorter and longer lysine analogs into synthetic histone H3 and H4 peptides. The enzymatic activity of MOF, PCAF and GCN5 acetyltransferases towards histone peptides bearing lysine analogs was evaluated using MALDI-TOF MS assays. Our results demonstrate that human KAT enzymes have an ability to catalyze an efficient acetylation of longer lysine analogs, whereas shorter lysine analogs are not substrates for KATs. Kinetics analyses showed that lysine is a superior KAT substrate to its analogs with altered chain length, implying that lysine has an optimal chain length for KAT-catalyzed acetylation reaction.Keywords:
PCAF
Acetyltransferases
Histone acetyltransferase
Histone H4
P300/CBP-associated factor(PCAF),an important member of histone acetyltransferase family(HATs) within eukaryotic cells,is capable of inducing the acetylation of histone,promoting the transcription of specific genes and involving in many biological effects.In the present study,full-length cDNA of PCAF was inserted into plasmid pGEX-5x-1,then the soluble protein GST-PCAF was expressed in E.coli BL21(DE3) after the optimization of inducing conditions.The recombinant protein was further purified with affinity chromatography and tested the activity by in vitro acetylation assays.High efficient PCAF protein produced by this method could serve for the study on the role of PCAF in gene regulation and the interaction between PCAF and other proteins.
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The acetylation of proteins at specific lysine residues by acetyltransferase enzymes has emerged as a posttranslational modification of high biological impact. Although lysine acetylation in histone proteins is an integral part of the histone code the acetylation of a multitude of non-histone proteins was recently recognized as a regulatory signal in many cellular processes. New substrates of acetyltransferase enzymes are continuously identified, and the analysis of acetylation sites in proteins is increasingly performed by mass spectrometry. However, the characterization of lysine acetylation in proteins using mass spectrometric techniques has some limitations and pitfalls. The non-enzymatic cysteine acetylation especially can result in false-positive identification of acetylated proteins. Here we demonstrate the application of various mass spectrometric techniques such as matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry for the analysis of protein acetylation. We describe diverse combinations of biochemical methods useful to map the acetylation sites in proteins and discuss their advantages and limitations. As an example, we present a detailed analysis of the acetylation of the HIV-1 transactivator of transcription (Tat) protein, which is known to be acetylated in vivo by the acetyltransferases p300 and p300/CBP-associated factor (PCAF).
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Histone acetyltransferase
Acetyltransferases
SAP30
P300-CBP Transcription Factors
Histone H4
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Longstanding observations suggest that acetylation and/or amino-terminal tail structure of histones H3 and H4 are critical for eukaryotic cells. For Saccharomyces cerevisiae, loss of a single H4-specific histone acetyltransferase (HAT), Esa1p, results in cell cycle defects and death. In contrast, although several yeast HAT complexes preferentially acetylate histone H3, the catalytic subunits of these complexes are not essential for viability. To resolve the apparent paradox between the significance of H3 versus H4 acetylation, we tested the hypothesis that H3 modification is essential, but is accomplished through combined activities of two enzymes. We observed that Sas3p and Gcn5p HAT complexes have overlapping patterns of acetylation. Simultaneous disruption of SAS3, the homolog of the MOZ leukemia gene, and GCN5, the hGCN5/PCAF homolog, is synthetically lethal due to loss of acetyltransferase activity. This key combination of activities is specific for these two HATs because neither is synthetically lethal with mutations of other MYST family or H3-specific acetyltransferases. Further, the combined loss of GCN5 and SAS3 functions results in an extensive, global loss of H3 acetylation and arrest in the G(2)/M phase of the cell cycle. The strikingly similar effect of loss of combined essential H3 HAT activities and the loss of a single essential H4 HAT underscores the fundamental biological significance of each of these chromatin-modifying activities.
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Acetyltransferases
Histone acetyltransferase
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Background: Many N-terminal acetyltransferases (NATs) play important role in the posttranslational modifications of histone tails. Research showed that these enzymes have been reported upregulated in many cancers. NatD is known to acetylate H4/H2A at the N-terminal. During lung cancer, this enzyme competes with the protein kinase CK2α and blocks the phosphorylation of H4 and, acetylates. It also, we observed that H4 has various mutations at the N-terminal and we considered only four mutations (S1C, R3C, G4D and G4S) to study the impacts of these mutations on H4 binding with NatD using MD simulation. Objective: Our main objective in this study was to understand the structure and dynamics of hNatD under the influence of WT and MT H4 histones bindings. The previous experimental study reported that mutations on H4 N-terminus reduce the catalytic efficiency of N-Terminal acetylation. But here, we performed a molecular- level study thus, we can understand how these mutations (S1C, R3C, G4D and G4S) cause significant depletion in catalytic efficiency of hNatD. Methods: Purely computational approaches were employed to investigate the impacts of four mutations in human histone H4 on its binding with the N-α-acetyltransferase D. Initially, molecular docking was used to dock the histone H4 peptide with the N-α-acetyltransferase. Next, all-atom molecular dynamics simulation was performed to probe the structural deviation and dynamics of N-α-acetyltransferase D under the binding of WT and MT H4 histones. Result: Our results show that R3C stabilizes the NatD whereas the remaining mutations destabilize the NatD. Thus, mutations have significant impacts on NatD structure. Our finding supports the previous analysis also. Another interesting observation is that the enzymatic activity of hNatD is altered due to the considerably large deviation of acetyl-CoA from its original position (G4D). Further, simulation and correlation data suggest which regions of the hNatD are highly flexible and rigid and, which domains or residues have the correlation and anticorrelation. As hNatD is overexpressed in lung cancer, it is an important drug target for cancer hence, our study provides structural information to target hNatD. Conclusion: In this study, we examined the impacts of WT and MTs (S1C, R3C, G4D and G4S) histone H4 decapeptides on their bindings with hNatD by using 100 ns all-atom MD simulation. Our results support the previous finding that the mutant H4 histones reduce the catalytic efficiency of hNatD. The MD posttrajectory analyses revealed that S1C, G4S and G4D mutants remarkably alter the residue network in hNatD. The intramolecular hydrogen bond analysis suggested that there is a considerable number of loss of hydrogen bonds in hNatD of hNatD-H4_G4D and hNatD-H4_G4S complexes whereas a large number of hydrogen bonds were increased in hNatD of hNatD-H4_R3C complex during the entire simulations. This implies that R3C mutant binding to hNatD brings stability in hNatD in comparison with WT and other MTs complexes. The linear mutual information (LMI) and Betweenness centrality (BC) suggest that S1C, G4D and G4S significantly disrupt the catalytic site residue network as compared to R3C mutation in H4 histone. Thus, this might be the cause of a notable reduction in the catalytic efficiency of hNatD in these three mutant complexes. Further, interaction analysis supports that E126 is the important residue for the acetyltransferase mechanisms as it is dominantly found to have interactions with numerous residues of MTs histones in MD frames. Additionally, intermolecular hydrogen bond and RMSD analyses of acetyl-CoA predict the higher stability of acetyl-CoA inside the WT complex of hNatD and R3C complex. Also, we report here the structural and dynamic aspects and residue interactions network (RIN) of hNatD to target it to control cell proliferation in lung cancer conditions.
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PCAF
P300-CBP Transcription Factors
Histone acetyltransferase
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CREB-binding protein
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Acetylation of nucleosomal histones by diverse histone acetyltransferases (HAT) plays pivotal roles in many cellular events. Discoveries of novel HATs and HAT related factors have provided new insights to understand the roles and mechanisms of histone acetylation. In this study, we identified prominent Histone H3 acetylation activity in vitro and purified its activity, showing that it is composed of the MYST acetyltransferase Chameau and Enhancer of the Acetyltransferase Chameau (EAChm) family. EAChm is a negatively charged acidic protein retaining aspartate and glutamate. Furthermore, we identified that Chameau and EAChm stimulate transcription in vitro together with purified general transcription factors. In addition, RNA-seq analysis of Chameu KD and EAChm KD S2 cells suggest that Chameau and EAChm regulate transcription of common genes in vivo. Our results suggest that EAChm regulates gene transcription in Drosophila embryos by enhancing Acetyltransferase Chameau activity.
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Here we report that PCAF and human GCN5, two related type A histone acetyltransferases, are unstable enzymes that under the commonly used assay conditions are rapidly and irreversibly inactivated. In addition, we report that free histone H1, although not acetylated in vivo, is a preferred and convenient in vitro substrate for the study of PCAF, human GCN5, and possibly other type A histone acetyltransferases. Using either histone H1 or histone H3 as substrates, we find that preincubation with either acetyl-CoA or CoA stabilizes the acetyltransferase activities of PCAF, human GCN5 and an enzymatically active PCAF deletion mutant containing the C-terminal half of the protein. The stabilization requires the continuous presence of coenzyme, suggesting that the acetyltransferase-coenzyme complexes are stable, while the isolated apoenzymes are not. Human GCN5 and the N-terminal deletion mutant of PCAF are stabilized equally well by preincubation with either CoA or acetyl-CoA, while intact PCAF is better stabilized by acetyl-CoA than by CoA. Intact PCAF, but not the N-terminal truncation mutant or human GCN5, is autoacetylated. These findings raise the possibility that the intracellular concentrations of the coenzymes affect the stability and therefore the nuclear activity of these acetyltransferases. Here we report that PCAF and human GCN5, two related type A histone acetyltransferases, are unstable enzymes that under the commonly used assay conditions are rapidly and irreversibly inactivated. In addition, we report that free histone H1, although not acetylated in vivo, is a preferred and convenient in vitro substrate for the study of PCAF, human GCN5, and possibly other type A histone acetyltransferases. Using either histone H1 or histone H3 as substrates, we find that preincubation with either acetyl-CoA or CoA stabilizes the acetyltransferase activities of PCAF, human GCN5 and an enzymatically active PCAF deletion mutant containing the C-terminal half of the protein. The stabilization requires the continuous presence of coenzyme, suggesting that the acetyltransferase-coenzyme complexes are stable, while the isolated apoenzymes are not. Human GCN5 and the N-terminal deletion mutant of PCAF are stabilized equally well by preincubation with either CoA or acetyl-CoA, while intact PCAF is better stabilized by acetyl-CoA than by CoA. Intact PCAF, but not the N-terminal truncation mutant or human GCN5, is autoacetylated. These findings raise the possibility that the intracellular concentrations of the coenzymes affect the stability and therefore the nuclear activity of these acetyltransferases. Post-translational modification of chromosomal proteins, in particular the specific acetylation of histones, is correlated with various nuclear activities such as replication, chromatin assembly, and transcription (1Allfrey V.G. Faulkner R. Mirsky A.E. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 786-794Crossref PubMed Scopus (1764) Google Scholar, 2van Holde K.E. Chromatin. Springer-Verlag, New York1988Google Scholar, 3Bradbury Bioessays. 1992; 14: 9-16Crossref PubMed Scopus (347) Google Scholar, 4Loidl P. Chromosoma (Berl.). 1994; 103: 441-449Crossref PubMed Scopus (159) Google Scholar, 5Wolffe A.P. Trends Biochem. Sci. 1994; 19: 240-244Abstract Full Text PDF PubMed Scopus (160) Google Scholar, 6Turner B.M. O'Neill L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Scopus (134) Google Scholar, 7Vettese-Dadey M. Grant P.A. Hebbes T.R. Crane-Robinson C. Allis C.D. Workman J.L. EMBO J. 1996; 15: 2508-2518Crossref PubMed Scopus (378) Google Scholar, 8Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (468) Google Scholar, 9Roth S.Y. Allis C.D. Cell. 1966; 87: 5-8Abstract Full Text Full Text PDF Scopus (223) Google Scholar, 10Tauton J. Hassing C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1534) Google Scholar). The acetylation state of specific lysine residues in the evolutionarily conserved tails of the core histones can alter the interaction between these histones and the DNA and perhaps also the interactions between the histone tail domains in adjacent nucleosomes. These interactions affect the state of chromatin compaction and therefore may be involved in regulating access to the underlying DNA sequence. Increased histone acetylation promotes chromatin decompaction (11Lee D.Y. Hayes J.J. Pruss D. Wolffe A.P. Cell. 1993; 72: 73-84Abstract Full Text PDF PubMed Scopus (966) Google Scholar) and is associated with gene activation, while loss of acetate from histone tails is associated with gene silencing (12Braunstein M. Rose A.B. Holmes S.G. Allis C.D. Broach J.R. Genes Dev. 1993; 7: 592-604Crossref PubMed Scopus (712) Google Scholar). For example, in the inactive X chromosome of female human cells the core histones are underacetylated, while in the active X chromosome the histones are acetylated (13Belyaev N.D. Keohane A.M. Turner B.M. Hum. Genet. 1996; 97: 573-578Crossref PubMed Scopus (99) Google Scholar). The recent isolation and characterization of several enzymes involved in the acetylation and deacetylation of specific histone residues provide additional insights into the possible relation between histone acetylation and gene activation (8Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (468) Google Scholar, 14Yang X.-J. Ogrryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar, 15Ogryzko V.V. Sciltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1966; 87: 953-959Abstract Full Text Full Text PDF Scopus (2402) Google Scholar). The Tetrahymenahistone acetyltransferase is homologous to the yeast protein GCN5, which is a putative transcription activator and contains intrinsic acetyltransferase activity (8Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (468) Google Scholar). We have recently demonstrated that human PCAF, a protein that competes with oncoprotein E1A for binding to the gene products of the p300/CBP and retinoblastoma gene families, has intrinsic acetyltransferase activity and is in part homologous to GCN5 protein. More recently, Ogryzko et al. demonstrated that the P300 protein has intrinsic histone acetyltransferase activity (15Ogryzko V.V. Sciltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1966; 87: 953-959Abstract Full Text Full Text PDF Scopus (2402) Google Scholar). Taken together, these results suggest that some transcription activators may be able to acetylate histone tails, thereby disrupting the nucleosomal structure and promoting transcription from chromatin templates. Although the genes coding for PCAF and GCN5 have been characterized and recombinant proteins have been produced in several systems, the enzymatic properties of these enzymes have not been extensively studied. In this article, we report that human GCN5, the catalytic subunit of a type A nuclear histone acetyltransferase (14Yang X.-J. Ogrryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar, 16Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmonston D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar), and PCAF, a histone acetyltransferase containing a domain that is highly homologous to the human GCN5 (14Yang X.-J. Ogrryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar), are highly unstable enzymes that rapidly and irreversibly lose histone acetyltransferase activity. We demonstrate that histone acetyltransferase activity is stabilized by the continuous presence of the coenzymes acetyl-CoA or CoA and that PCAF, but not hGCN5 1The abbreviations used are: hGCN5, human GCN5; PCA, perchloric acid. is autoacetylated, a modification that may further stabilize this enzyme. In addition, we demonstrate that both PCAF and hGCN5 catalyze the transfer of acetyl groups into free but not chromatin-bound histone H1. These results provide new information on the properties of these histone acetyltransferases and are pertinent to studies on the cellular function and mechanism of action of these, and perhaps other, histone acetyltransferases. Recombinant human GCN5 and PCAF were prepared as described previously (14Yang X.-J. Ogrryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar). Mutant PCAF having an internal deletion of amino acid residues 61–465 (see Ref. 14Yang X.-J. Ogrryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar) and containing the Flag tag (Kodak) was expressed and purified by the same approach as described for the intact PCAF (14Yang X.-J. Ogrryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar). Histone H1 was extracted from calf thymus with 57 trichloroacetic acid and purified by chromatography on Amberlite IRC-50 (17Bustin M. Nat. New Biol. 1973; 245: 207-209Crossref PubMed Scopus (33) Google Scholar). Histone H3 was extracted from calf thymus with 0.1 m HCl, oxidized, and purified on Sephadex G-100 (18Marzluff Jr., W.F. Sanders L.A. Miller D.M. McCarty K.S. J. Biol. Chem. 1972; 247: 2026-2033Abstract Full Text PDF PubMed Google Scholar). Chromosomal protein HMG-1 was isolated from calf thymus by extraction with 0.35 m NaCl and purified by ion exchange chromatography (19Romani M. Rodman T.C. Vidali G. Bustin M. J. Biol. Chem. 1979; 254: 2918-2922Abstract Full Text PDF PubMed Google Scholar). Lysozyme, cytochrome c, acetyl-CoA, and CoA were obtained from Sigma. [1-14C]acetyl-CoA (55 mCi/mmol) was obtained from New England Nuclear, and [3H]acetate was obtained from ICN. All assays were performed in Buffer A (50 mm Tris-HCl (pH 8.0), 107 glycerol (v/v), 1 mm dithiothreitol, 0.1 mmEDTA, 10 mm butyric acid) (20Brownell J.E. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6364-6368Crossref PubMed Scopus (240) Google Scholar). Substrate concentrations were 0.1–0.25 mg/ml and [1-14C]acetyl-CoA concentrations were at 9 ॖm (unless otherwise indicated). The assay was performed at 37 °C and initiated by addition of the protein substrate to a mixture containing the acetyltransferase and acetyl-CoA in buffer A. The radioactivity incorporated into the protein substrates was detected by either a filter, or a polyacrylamide gel assay. For the filter assay the reaction was stopped at given times by spotting onto filter papers (Whatman P81), which were then washed and counted as described previously (21Horiuchi K. Fujimoto D. Anal. Biochem. 1975; 69: 491-496Crossref PubMed Scopus (39) Google Scholar). In the polyacrylamide gel assays, the reaction was stopped by the addition of an equal volume of an SDS-gel sample buffer (100 mm Tris-HCl (pH 6.8), 200 mmdithiothreitol, 27 SDS, 0.17 bromphenol blue, 207 glycerol), boiled for 5 min, and loaded onto either a 10 or 157 polyacrylamide-SDS gel (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar) and electrophoresed at 15 V/cm until the bromphenol blue reached the bottom of the gel. The gels were stained with Coomassie Blue for estimation of protein quantities and then soaked in Enlightening Enhancer solution (Dupont) for 30 min and vacuum dried; the radioactivity incorporated into the protein bands was then visualized and quantified with a PhosphorImager (Molecular Dynamics) using Imagequant software. Acetylation of H1 in the presence of DNA was performed with DNA isolated from mononucleosomes (average length, 200 base pairs). The ratio was adjusted to 1 molecule of H1 per 200 base pairs of DNA. Estimates of the K m and V maxof PCAF for H1 were determined using Lineweaver-Burk plots. The kinetic parameters were estimated for enzyme that had been preincubated with or without acetyl-CoA (9 ॖm) for 10 min at 37 °C. The concentration of histone H1 was varied from 0.02 to 2 mg/ml. The enzymatic reactions were initiated by the addition of the protein substrate and sufficient acetyl-CoA to bring its final concentration to 18 ॖm. HeLa cells were grown in suspension in minimal Eagle's medium. Exponentially growing cells (200 ml, 5.0 × 105 cells/ml) were concentrated by centrifugation. The cells were resuspended in 8 ml of medium containing 200 ॖg/ml of cycloheximide (Sigma) and incubated at 37 °C for 10 min. After the incubation, one culture was brought to 0.5 ॖmtrichostatin A (WAKO), and 2 mCi of [3H]acetate (5 Ci/mmol, ICN) was added; the second culture was treated identically except no trichostatin A was added. The labeling was performed for 1 h at 37 °C with gentle rocking. Each culture was split into two, and the cells were harvested by centrifugation. One set of cells from each treatment was extracted with 57 perchloric acid (PCA) and precipitated with 6 volumes of acetone to measure incorporation into H1, and the second set was extracted with 0.25 m sulfuric acid and precipitated with 6 volumes of acetone to measure incorporation into the core histones. The incorporation of [3H]acetate into the given protein was measured by resolving the proteins on a 157 SDS/PAGE, staining the gel with Coomassie Blue to estimate the amount of each protein, and then excising the protein bands from the gel. The excised bands were digested at 65 °C overnight with 307 hydrogen peroxide, and the [3H]acetate incorporation into each band was determined by scintillation counting. Mouse thymus nuclei were isolated by homogenization and sedimentation in sucrose as described previously (23Garrard W.T. Hancock R. Methods Cell Biol. 1978; 17: 27-50Crossref PubMed Scopus (27) Google Scholar). The nuclei (3 mg/ml in DNA) were mildly digested with micrococcal nuclease (100 units/ml, 5 min at 37 °C; Boehringer Mannheim). The digested nuclei were harvested and extracted in 0.25 mm EDTA, 1 mm phenylmethylsulfonyl fluoride at 4 °C. The extract was centrifuged, and the supernatant was loaded onto a 12–507 sucrose gradient containing 10 mm Tris-HCl (pH 7.4), 1 mm EDTA, and 10 mm NaCl. After 20 h of centrifugation at 28,000 rpm (Beckman rotor, SW28), the gradient was fractionated, and the DNA in each fraction was analyzed using agarose gels. The fractions corresponding to oligonucleosome lengths of 4–10 nucleosomes were pooled, dialyzed to remove the sucrose, and concentrated by centrifugation dialysis (Filtron). PCAF and hGCN5, two related human histone acetyltransferases (14Yang X.-J. Ogrryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar), exhibit identical substrate preferences (Fig. 1). In addition to the previously documented acetylation of both free and nucleosomal bound histone H3 (14Yang X.-J. Ogrryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar, 24Kuo M.H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1996; 383: 269-272Crossref PubMed Scopus (507) Google Scholar), the linker histone H1 is also an excellent substrate for both enzymes. Quantitative analysis indicates that both PCAF and hGCN5 acetylate histone H1 2-fold more efficiently than histone H3 (see also Fig. 3). The acetylation of histone H1 is specific since other positively charged proteins containing numerous lysine residues (lysozyme, cytochrome c, HMG-1, nucleosomal histones H2A and H2B), similar to histone H1, were not substrates for either acetyltransferase (Fig. 1).Figure 3Loss of acetyltransferase activity in PCAF. PCAF was preincubated in either the presence (lanes 1–3) or absence (lanes 4-6) of acetyl-CoA for 10 min prior to the addition of histone H1 (lanes 1, 4,and 7), histone H3 (lanes 2, 5, and8), or nucleosome (Nuc.) cores (lanes 3, 6,and 9) as substrates. Control lacking PCAF are shown inlanes 7–9. The incorporation of acetyl groups into the substrates under various assay conditions was determined by PhosphorImager using the polyacrylamide gel assay described under 舠Materials and Methods.舡 A, protein stain; B,phosphorimage; C, bar graph indicating the amount of radioactivity in each band, normalized to the amount of protein per band, as determined by densitometric scanning of the Coomassie Blue-stained polyacrylamide gels. The amount of radioactivity was quantified with Imagequant.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Histone H1 is known to be acetylated at its N terminus; however, numerous studies on histone acetylation failed to detect post-translational acetylation of this histone (for review, see Ref.2van Holde K.E. Chromatin. Springer-Verlag, New York1988Google Scholar). Since our studies suggested that H1 is an excellent substrate for human histone acetyltransferases, we reexamined whether histone H1 is acetylated in HeLa cells. The incorporation of [3H]acetate into histones was determined in the presence or absence of a histone deacetylase inhibitor (trichostatin A) after treatment of the cells with cycloheximide to inhibit protein synthesis. Quantitation of the specific radioactivity of each histone showed that the specific activity of histone H1 was <57 of that of histone H4 (Table I). Trichostatin A increased the level of acetylation of the core histones but not that of histone H1. Thus, our results are in agreement with previous findings (2van Holde K.E. Chromatin. Springer-Verlag, New York1988Google Scholar) which indicated that in vivo, histone H1 is not acetylated post-translationally.Table IHistone H1 is not acetylated post-translationally: normalized specific activity of the radioactivity incorporated into histones in trichostatin A-treated HeLa cellsProteinSpecific activity7Histone H4100Histone H367Histone H2B51Histone H2A33Histone H1<3The specific activity of a histone was determined by dividing the number of counts obtained from scintillation counting of a histone band excised from a polyacrylamide gel by the intensity of the band as determined by densitometric analysis of the Coomassie Blue-stained gel. The intensity of the stain was within linear range and, as expected, the 4 core histones were present in equimolar rations. The specific activity of the histones was normalized to that of histone H4, the value of which was taken as 1007. Open table in a new tab The specific activity of a histone was determined by dividing the number of counts obtained from scintillation counting of a histone band excised from a polyacrylamide gel by the intensity of the band as determined by densitometric analysis of the Coomassie Blue-stained gel. The intensity of the stain was within linear range and, as expected, the 4 core histones were present in equimolar rations. The specific activity of the histones was normalized to that of histone H4, the value of which was taken as 1007. To explain the apparent discrepancy between the in vivo andin vitro acetylation of H1, we examined the ability of PCAF to acetylate H1 in the presence of DNA or in the context of purified oligonucleosomes containing endogenously bound H1. Fig.2 shows that purified histone H1 either from calf or mouse thymus is efficiently acetylated by PCAF. However, acetylation of H1 is completely abolished either by addition of DNA or in the context of oligonucleosomes. In contrast, histone H3 in oligonucleosomes was well acetylated, indicating that the enzyme was active and can acetylate its substrates in chromatin. These results indicate that the nucleosome-bound form of histone H1 is not a substrate for PCAF. During these studies, we noted that in the absence of acetyl-CoA, the acetylation activity of PCAF was rapidly lost (Fig.3). In these assays, the enzyme was preincubated in the buffer used for acetylation in either the presence or absence of acetyl-CoA prior to activity measurements using histone H1, histone H3, or nucleosome cores as substrates. After 10 min of preincubation in the absence of coenzyme, the amount of radioactivity incorporated into substrates is 5–15-fold lower than that incorporated with preincubation in the presence of coenzyme. Examination of the time course of inactivation (Fig. 4) revealed that 2 min of preincubation in the absence of acetyl-CoA resulted in a 507 loss of enzymatic activity. In contrast, no loss of enzymatic activity could be detected after 10 min of preincubation in the presence of acetyl-CoA (Fig. 4). Analysis of the initial rate of acetylation at various histone H1 concentrations indicated that after 10 min of preincubation in the absence of acetyl-CoA, theV max of PCAF was 2.38 pmol of acetyl/ॖg of enzyme/min, while the V max of the enzyme after 10 min of preincubation in the presence of the coenzyme was 27.0 pmol of acetyl/ॖg of enzyme/min, i.e. 10-fold higher. TheK m of the enzyme was not appreciably affected by preincubation with the coenzyme. A possible explanation for the rapid loss of acetyltransferase activity by PCAF is proteolytic degradation during preincubation in the absence of acetyl-CoA. Conceivably, the presence of acetyl-CoA protects PCAF from proteolytic degradation. We used polyacrylamide gels to estimate the amount of PCAF protein present after 10 min of preincubation in either the presence or absence of radioactive acetyl-CoA. The amount of PCAF protein, as determined by the intensity of the Coomassie Blue band corresponding to the M r 83,000 protein (14Yang X.-J. Ogrryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar) did not diminish under either condition (Fig.5). After a 10-min preincubation in the absence of acetyl-CoA, the self-acetylating activity of the enzyme was lost, and subsequent addition of radioactive [1-14C]acetyl-CoA to this PCAF preparation did not result in autoacetylation (Fig. 5 B). In contrast, in the presence of acetyl-CoA, a significant amount of radioactivity was incorporated into PCAF (Fig. 5 A). We conclude that in the absence of acetyl-CoA the acetylation activity of PCAF is rapidly and irreversibly lost and that this loss is not a result of degradation of the enzyme. These results and the kinetic analysis suggest that in the absence of coenzyme PCAF is irreversibly inactivated. Because the amino acid sequence of C-terminal half of PCAF is 867 homologous to the human GCN5 (14Yang X.-J. Ogrryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar) and the two proteins have identical acetyltransferase substrate specificity (Fig. 1), we tested whether preincubation with acetyl-CoA also protects hGCN5 from inactivation. In these experiments, the enzyme was preincubated in either the presence or absence of acetyl-CoA before to the addition of the histone substrate. The results indicate that in the absence of the coenzyme the ability of hGCN5 to incorporate acetyl groups into histone H1 or histone H3 is rapidly lost, suggesting that this enzyme undergoes a rapid inactivation in a manner indistinguishable from that of PCAF. As with PCAF, within 10 min of preincubation without the substrate, theV max of hGCN5 decreased 5-fold, from 12.4 pmol of acetyl/ॖg of enzyme/min for the enzyme incubated in the presence of coenzyme to 2.5 pmol of acetyl/ॖg of enzyme/min for the enzyme incubated in the absence of the coenzyme. The K m was not changed significantly. To better understand the factors associated with the stabilization, we examined the ability of both CoA and acetyl-CoA to stabilize the activity of PCAF, hGCN5, and an N-terminal truncation mutant of PCAF. The N-terminal truncation mutant of PCAF contains an internal deletion of amino acids 61–465 and retains the domain that is homologous to the human and yeast GCN5 (14Yang X.-J. Ogrryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar) and is competent in both histone H1 and histone H3 acetylation. We tested whether the continuous presence of the coenzyme is required for stabilization. In the experiments depicted in Fig.6, we incubated the intact enzyme, the N-terminal truncation mutant of PCAF, and hGCN5 for 5 min either with or without acetyl-CoA or CoA. After incubation, the unbound coenzyme was removed using a spun column (G50-Probequant column (Pharmacia)), and the enzymes were again incubated for various times in the absence of additional coenzymes prior to determination of acetyltransferase activity. The results (Fig. 6) indicate that both acetyl-CoA and CoA protect all three recombinant enzymes from inactivation albeit to differing degrees (2–10-fold). After removal of the unbound coenzymes, all three acetyltransferases exhibited a gradual decline in activity. This suggests that the stabilization is mediated by bound coenzyme and not a coenzyme-mediated modification of the structure of the enzymes. Acetyl-CoA stabilized hGCN5 and the N-terminal truncation mutant to the same extent as CoA (Fig. 6, B and C). However, with full-length PCAF, the enzymatic activity was stabilized by acetyl-CoA better than by CoA (6-fold above control and 2-fold above control, respectively), indicating that the presence of the N-terminal domain confers this preferential stabilization by acetyl-CoA. During these studies, we noted that PCAF incorporated counts during its preincubation with [1-14C]acetyl-CoA, raising the possibility that PCAF is autoacetylated. The incorporation of counts into PCAF could result from a catalytic intermediate formed at the active site of the enzyme, or alternatively, from a covalent modification. To distinguish between these possibilities, we tested whether the radioactivity incorporated into PCAF by a 10-min incubation with [1-14C]acetyl-CoA could be chased by either unlabeled acetyl-CoA, histone H1, or a mixture of both unlabeled acetyl-CoA and histone H1. As indicated by the data presented in Fig.7, neither the addition of unlabeled acetyl-CoA nor the addition of unlabeled acetyl-CoA in conjunction with substrate affected the amount of 14C incorporated into the enzyme. Throughout these experiments, the acetyltransferase activity of PCAF was not diminished. Neither hGCN5 nor the N-terminal truncated PCAF mutant incorporates counts during incubation with acetyl-CoA, suggesting that the modification occurs in the N-terminal region of PCAF. In fact, a C-terminal truncated PCAF containing only the N terminal half of PCAF is devoid of intrinsic acetyltransferase activity yet can be acetylated by full-length PCAF. 2X.-J. Yang and Y. Nakatani, unpublished data. These results suggest that the autoacetylation of PCAF occurs by an intermolecular reaction in its N-terminal domain. The N terminus is required for PCAF to distinguish, with regard to stability, between acetyl-CoA and CoA. It is conceivable that autoacetylation may play a role responding to the ratio of CoA to acetyl-CoA, providing a potential means of regulating the enzymatic activity. In summary, our experiments demonstrate that the human acetyltransferases PCAF and GCN5 are unstable enzymes that are rapidly and irreversibly inactivated. The enzymes are stable in the presence of their coenzymes. The stabilization appears to be mediated by binding of the coenzyme, suggesting that the stable species is an enzyme-coenzyme complex. Based on these findings, it is tempting to speculate that the intracellular activity of these enzymes may be regulated in part by the intracellular levels of these coenzymes.
PCAF
Acetyltransferases
Acetyltransferases
Histone acetyltransferase
P300-CBP Transcription Factors
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Lysine acetyltransferases (KATs), p300 (KAT3B), and its close homologue CREB-binding protein (KAT3A) are probably the most widely studied KATs with well documented roles in various cellular processes. Hence, the dysfunction of p300 may result in the dysregulation of gene expression leading to the manifestation of many disorders. The acetyltransferase activity of p300/CREB-binding protein is therefore considered as a target for new generation therapeutics. We describe here a natural compound, plumbagin (RTK1), isolated from Plumbago rosea root extract, that inhibits histone acetyltransferase activity potently in vivo. Interestingly, RTK1 specifically inhibits the p300-mediated acetylation of p53 but not the acetylation by another acetyltransferase, p300/CREB-binding protein -associated factor, PCAF, in vivo. RTK1 inhibits p300 histone acetyltransferase activity in a noncompetitive manner. Docking studies and site-directed mutagenesis of the p300 histone acetyltransferase domain suggest that a single hydroxyl group of RTK1 makes a hydrogen bond with the lysine 1358 residue of this domain. In agreement with this, we found that indeed the hydroxyl group-substituted plumbagin derivatives lost the acetyltransferase inhibitory activity. This study describes for the first time the chemical entity (hydroxyl group) required for the inhibition of acetyltransferase activity. Lysine acetyltransferases (KATs), p300 (KAT3B), and its close homologue CREB-binding protein (KAT3A) are probably the most widely studied KATs with well documented roles in various cellular processes. Hence, the dysfunction of p300 may result in the dysregulation of gene expression leading to the manifestation of many disorders. The acetyltransferase activity of p300/CREB-binding protein is therefore considered as a target for new generation therapeutics. We describe here a natural compound, plumbagin (RTK1), isolated from Plumbago rosea root extract, that inhibits histone acetyltransferase activity potently in vivo. Interestingly, RTK1 specifically inhibits the p300-mediated acetylation of p53 but not the acetylation by another acetyltransferase, p300/CREB-binding protein -associated factor, PCAF, in vivo. RTK1 inhibits p300 histone acetyltransferase activity in a noncompetitive manner. Docking studies and site-directed mutagenesis of the p300 histone acetyltransferase domain suggest that a single hydroxyl group of RTK1 makes a hydrogen bond with the lysine 1358 residue of this domain. In agreement with this, we found that indeed the hydroxyl group-substituted plumbagin derivatives lost the acetyltransferase inhibitory activity. This study describes for the first time the chemical entity (hydroxyl group) required for the inhibition of acetyltransferase activity. The eukaryotic genome is organized in a highly complex nucleoprotein structure, chromatin. Physiologically, chromatin is not just a DNA and histone complex, rather it is a dynamic organization of DNA associated with histone, histone-interacting non-histone proteins, and RNA (1.Batta K. Das C. Gadad S. Shandilya J. Kundu T.K. Chromatin and Disease. Springer Publications, London2007: 193-212Google Scholar). The hallmark of chromatin is its dynamic nature, which is essential for the regulation of nuclear processes that require access to genetic information. This dynamicity of chromatin is maintained by several factors, including the post-translational modifications of histone and non-histone chromatin components, especially the reversible acetylation (2.Roth S.Y. Denu J.M. Allis C.D. Annu. Rev. Biochem. 2001; 70: 81-120Crossref PubMed Scopus (1610) Google Scholar). The enzymatic activity of the acetyltransferases and deacetylases sets up a fine balance that maintains the cellular homeostasis (3.Swaminathan V. Reddy B.A. Ruthrotha Selvi B. Sukanya M.S. Kundu T.K. Chromatin and Disease. Springer Publications, London2007: 397-428Google Scholar). Any imbalance in this may result in disease manifestation. Chromatin acetylation is catalyzed by five different classes of lysine acetyltransferases (4.Grant P.A. Berger S.L. Semin. Cell Dev. Biol. 1999; 10: 169-177Crossref PubMed Scopus (104) Google Scholar). Among these, the best studied is p300 and its close homologue CBP. 4The abbreviations used are: CBPCREB-binding proteinHAThistone acetyltransferaseCCcolumn chromatographyKATlysine acetyltransferasePCAFp300/CBP-associated factorITCisothermal titration calorimetry. p300 has been shown to regulate various biological phenomena such as proliferation, cell cycle regulation, apoptosis, differentiation, and DNA damage response (5.Giles R.H. Peters D.J. Breuning M.H. Trends Genet. 1998; 14: 178-183Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 6.Giordano A. Avantaggiati M.L. J. Cell. Physiol. 1999; 181: 218-230Crossref PubMed Scopus (256) Google Scholar, 7.Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar, 8.Chan H.M. La Thangue N.B. J. Cell Sci. 2001; 114: 2363-2373Crossref PubMed Google Scholar). It is a potent transcriptional coactivator. Therefore, dysfunction of p300 may have deleterious effects on various cellular functions and thus could be the underlying cause of several diseases, especially cancer. Acetyltransferase activity has been considered as a target for new generation therapeutics (3.Swaminathan V. Reddy B.A. Ruthrotha Selvi B. Sukanya M.S. Kundu T.K. Chromatin and Disease. Springer Publications, London2007: 397-428Google Scholar, 9.Heery D.M. Fischer P.M. Drug Discov. Today. 2007; 12: 88-99Crossref PubMed Scopus (34) Google Scholar). The therapeutic potential of HAT inhibitors has been shown in cancer (10.Zheng Y. Thompson P.R. Cebrat M. Wang L. Devlin M.K. Alani R.M. Cole P.A. Methods Enzymol. 2004; 376: 188-199Crossref PubMed Scopus (39) Google Scholar, 11.Stimson L. Rowlands M.G. Newbatt Y.M. Smith N.F. Raynaud F.I. Rogers P. Bavetsias V. Gorsuch S. Jarman M. Bannister A. Kouzarides T. McDonald E. Workman P. Aherne G.W. Mol. Cancer Ther. 2005; 4: 1521-1532Crossref PubMed Scopus (188) Google Scholar, 12.Iyer N.G. Chin S.F. Ozdag H. Daigo Y. Hu D.E. Cariati M. Brindle K. Aparicio S. Caldas C. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7386-7391Crossref PubMed Scopus (123) Google Scholar), cardiac diseases (13.Davidson S.M. Townsend P.A. Carroll C. Yurek-George A. Balasubramanyam K. Kundu T.K. Stephanou A. Packham G. Ganesan A. Latchman D.S. ChemBioChem. 2005; 6: 162-170Crossref PubMed Scopus (44) Google Scholar), diabetes mellitus (14.Zhou X.Y. Shibusawa N. Naik K. Porras D. Temple K. Ou H. Kaihara K. Roe M.W. Brady M.J. Wondisford F.E. Nat. Med. 2004; 10: 633-637Crossref PubMed Scopus (118) Google Scholar), and human immunodeficiency virus (15.Varier R.A. Kundu T.K. Curr. Pharm. Des. 2006; 12: 1975-1993Crossref PubMed Scopus (24) Google Scholar). CREB-binding protein histone acetyltransferase column chromatography lysine acetyltransferase p300/CBP-associated factor isothermal titration calorimetry. Unlike the histone deacetylase inhibitors, the acetyltransferase inhibitors are relatively fewer in number. Recently, however, a few potent as well as specific KAT inhibitors have been discovered (Table 1). Among these, curcumin was found to be the only known p300-specific natural inhibitor, which is also cell-permeable (16.Balasubramanyam K. Altaf M. Varier R.A. Swaminathan V. Ravindran A. Sadhale P.P. Kundu T.K. J. Biol. Chem. 2004; 279: 33716-33726Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). Therefore, there is an active ongoing effort to identify modulators (activator/inhibitor) targeted toward p300 activity. Here we report that plumbagin, a hydroxynaphthoquinone (Fig. 1A) isolated from the roots of Plumbago rosea (known in Indian ayurvedic medicine as Chitraka), is a potent acetyltransferase inhibitor in vivo. Most of the known KAT inhibitors, possess polyhydroxy functional groups, whereas plumbagin has a single hydroxyl group. The substitution of this group with other moieties resulted in a complete loss of the p300 inhibitory activity. Therefore, these data for the first time establish the chemical entity (functional group) responsible for p300 inhibition.TABLE 1HAT inhibitorsSerial No.HAT inhibitorTargetRef.1Lysyl-CoAp30034.Lau O.D. Kundu T.K. Soccio R.E. Ait-Si-Ali S. Khalil E.M. Vassilev A. Wolffe A.P. Nakatani Y. Roeder R.G. Cole P.A. Mol. Cell. 2000; 5: 589-595Abstract Full Text Full Text PDF PubMed Google Scholar2H3-CoA-20PCAF34.Lau O.D. Kundu T.K. Soccio R.E. Ait-Si-Ali S. Khalil E.M. Vassilev A. Wolffe A.P. Nakatani Y. Roeder R.G. Cole P.A. Mol. Cell. 2000; 5: 589-595Abstract Full Text Full Text PDF PubMed Google Scholar3Anacardic acidp300/PCAF/CBP30.Balasubramanyam K. Swaminathan V. Ranganathan A. Kundu T.K. J. Biol. Chem. 2003; 278: 19134-19140Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar4Garcinolp300/PCAF/CBP16.Balasubramanyam K. Altaf M. Varier R.A. Swaminathan V. Ravindran A. Sadhale P.P. Kundu T.K. J. Biol. Chem. 2004; 279: 33716-33726Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar5Curcuminp300/CBP16.Balasubramanyam K. Altaf M. Varier R.A. Swaminathan V. Ravindran A. Sadhale P.P. Kundu T.K. J. Biol. Chem. 2004; 279: 33716-33726Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar6γ-ButyrolactonesCBP/Gcn535.Biel M. Kretsovali A. Karatzali E. Papamatheakis J. Giannis A. Angew. Chem. Int. Ed. Engl. 2004; 43: 3974-3976Crossref PubMed Scopus (131) Google Scholar7Isothiazolonesp300/PCAF11.Stimson L. Rowlands M.G. Newbatt Y.M. Smith N.F. Raynaud F.I. Rogers P. Bavetsias V. Gorsuch S. Jarman M. Bannister A. Kouzarides T. McDonald E. Workman P. Aherne G.W. Mol. Cancer Ther. 2005; 4: 1521-1532Crossref PubMed Scopus (188) Google Scholar8LTK14p30017.Mantelingu K. Reddy B.A. Swaminathan V. Kishore A.H. Siddappa N.B. Kumar G.V. Nagashankar G. Natesh N. Roy S. Sadhale P.P. Ranga U. Narayana C. Kundu T.K. Chem. Biol. 2007; 14: 645-657Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar9Epigallocatechin 3-gallateHATs29.Choi K.C. Jung M.G. Lee Y.H. Yoon J.C. Kwon S.H. Kang H.B. Kim M.J. Cha J.H. Kim Y.J. Jun W.J. Lee J.M. Yoon H.G. Cancer Res. 2009; 69: 583-592Crossref PubMed Scopus (302) Google Scholar Open table in a new tab The dried and powdered roots of P. rosea (100 g) were extracted with ethyl acetate. The ethyl acetate extract after removal of the solvent under reduced pressure gave a dark brown semisolid, which was separated into phenolic and neutral fractions by treatment with 5% NaOH and followed by acidification with 2 m HCl and extraction with diethyl ether. The phenolic fraction was subjected to flash chromatography (230–400 mesh) using increasing polarity. The fraction extracted with 4% ethyl acetate in hexane mixture gave an orange solid, which on further recrystallization yielded plumbagin/RTK1 (0.286 g). The yielded compound was compared with commercially available material. HepG2 cells (1.5 × 106 cells per 60-mm dish) were seeded overnight, and histones were extracted by acid extraction after 12 h of treatment with increasing concentrations of plumbagin. Immunoblotting analysis was performed as described elsewhere (17.Mantelingu K. Reddy B.A. Swaminathan V. Kishore A.H. Siddappa N.B. Kumar G.V. Nagashankar G. Natesh N. Roy S. Sadhale P.P. Ranga U. Narayana C. Kundu T.K. Chem. Biol. 2007; 14: 645-657Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), using polyclonal acetylated histone H3 and polyclonal histone H3 antibodies. To visualize the inhibition of histone acetylation in vivo, HeLa cells were cultured as a monolayer on the poly-l-lysine-coated coverslips in Dulbecco's modified Eagle's medium (Sigma). Immunofluorescence was carried out as described elsewhere (17.Mantelingu K. Reddy B.A. Swaminathan V. Kishore A.H. Siddappa N.B. Kumar G.V. Nagashankar G. Natesh N. Roy S. Sadhale P.P. Ranga U. Narayana C. Kundu T.K. Chem. Biol. 2007; 14: 645-657Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Fixed cells were probed with anti-acetylated histone H3 polyclonal antibodies followed by secondary antibody conjugated with Alexa 488 (Invitrogen). To stain the chromosomal DNA, Hoechst 33528 (Sigma) was used. The images were taken by using Zeiss LSM 510 laser scanning confocal microscope. Plumbagin (25 mg/kg body weight) dissolved in 50 μl of DMSO was injected intraperitoneally to 2-month-old Swiss albino mice. As controls, two mice were injected with 50 μl of water and 50 μl of DMSO intraperitoneally as negative control and solvent control, respectively. The experiment was done in triplicate. All mice were staged in animal cages for the next 6 h and anesthetized, and the liver was collected for further protein, RNA, and immunohistochemical analysis. Liver samples stored in formalin were further dehydrated in alcohol, followed by xylene treatment, and allowed to get impregnated in paraffin blocks. 3–4-μm thick paraffin-embedded sections were deparaffinized in xylene, followed by rehydration in decreasing concentrations of ethanol solutions. For routine pathological examination, deparaffinized sections from all blocks were stained with hematoxylin and eosin stains. Antigen retrieval was performed by microwaving in appropriate buffer. After washing the sections in 0.1 m phosphate buffer, blocking was done using 3% skimmed milk, following which the sections were incubated with anti-AcH3 antibody for 3 h in a humidification chamber. After 0.1 m phosphate buffer wash, the sections were incubated with link secondary antibody (DAKO LSAB+) for 3 h in a humidification chamber. After washing, the sections were developed with 3′,3′-diaminobenzidine tetrahydrochloride (Sigma). Hematoxylin was used as counterstain to identify the unstained nucleus. HEK293 cells were incubated for 3 h with 2.5, 5, and 10 μm plumbagin. After the compound treatment, the cells were treated with 500 ng/ml doxorubicin for 6 h. The cells were then harvested and lysed in TNN buffer (150 mm NaCl, 50 mm Tris, pH 7.4, 1% Nonidet P-40, 0.1% sodium deoxycholate, 1 mm EDTA, 0.5 μg/ml leupeptin, 0.5 μg/ml aprotinin, and 0.5 μg/ml pepstatin) for 3 h on ice with intermittent mixing. The whole cell lysates from the treated cells were subjected to Western blotting analysis by using mouse monoclonal anti-p53 antibody, DO1 (Calbiochem), rabbit polyclonal acetylated p53 Lys-373, and Lys-320 antibodies (Upstate) and tubulin antibody (Calbiochem). HAT assays were performed as described previously (17.Mantelingu K. Reddy B.A. Swaminathan V. Kishore A.H. Siddappa N.B. Kumar G.V. Nagashankar G. Natesh N. Roy S. Sadhale P.P. Ranga U. Narayana C. Kundu T.K. Chem. Biol. 2007; 14: 645-657Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). 2.4 μg of highly purified HeLa core histones were incubated in HAT assay buffer at 30 °C for 10 min with or without baculovirus-expressed recombinant p300 or PCAF in the presence and absence of compounds followed by addition of 1.0 μl of 3.6 Ci/mmol of [3H]acetyl-CoA (PerkinElmer Life Sciences) and further incubated for another 10 min in a 30-μl reaction. The reaction mixture was then blotted onto P81 (Whatman) filter paper, and radioactive counts were recorded on a Wallac 1409 liquid scintillation counter. 2.4 μg of highly purified HeLa core histones along with CARM1 (20 ng)/G9a (15 ng) were incubated in the buffer containing 20 mm Tris, pH 8.0, 4 mm EDTA, pH 8.0, 200 mm NaCl, and 1 μl of 15 Ci/mmol of S-[3H]adenosylmethionine (Amersham Biosciences), either in the presence or absence of compound in a final reaction volume of 30 μl at 30 °C for 30 min. The reaction was stopped on ice for 5 min before blotting onto P81 (Whatman) filter paper. The radioactive counts were recorded on Wallac 1409 Liquid Scintillation counter. The recombinant minimal HAT domain was purified as described elsewhere (18.Arif M. Kumar G.V. Narayana C. Kundu T.K. J. Phys. Chem. B. 2007; 111: 11877-11879Crossref PubMed Scopus (36) Google Scholar). The HAT reactions were carried out with the baculovirus-expressed recombinant full-length p300, in the presence of different concentrations of the inhibitor RTK1 (15, 20, 35, and 45 μm). The HAT reaction consists of two substrates, core histones and the acetyl group donor [3H]acetyl-CoA. Therefore, the kinetic analysis was done in two different sets. In the first set, concentration of core histones was kept constant at 1.6 μm, and [3H]acetyl-CoA was varied from 1.08 to 8.66 μm. In the second assay, [3H]acetyl-CoA was kept constant at 2.78 μm, and core histones were varied from 0.003 to 0.068 μm. The incorporation of the radioactivity was taken as a measure of the reaction velocity recorded as counts/min. Each experiment was performed in triplicate, and the reproducibility was found to be within 15% of the error range. Weighted averages of the values obtained were plotted as a Lineweaver-Burk plot using GraphPad Prism software. ITC experiments were carried out in a VP-ITC system (Microcal LLC) at 25 °C. Samples were centrifuged and degassed prior to titration. Titration of RTK1/RTK2 against the protein HAT domain was carried out by injecting 0.2 mm RTK1/RTK2 in 20 mm Tris, pH 7.5, 0.2 mm EDTA, 100 mm KCl buffer against 0.0034 mm HAT domain. A 2-min interval was allowed between injections for equilibration. A total of 40 injections was carried out to ensure complete titration. The protein concentration was chosen to achieve sufficiently high heat signals with a minimum enthalpy of dilution. To minimize the error associated with diffusion from the syringe during base-line equilibration, the first injection was only 1 μl, and the associated small heat change was not considered for data analysis. Blank titrations were carried out using buffer and DMSO with no ligand against the HAT domain and used for subtraction of the background heat change. The corrected heat changes were plotted against the molar ratio of the titrated products and analyzed using the manufacturer's software, which yielded the stoichiometry n (in terms of number of molecules of RTK1/protein), equilibrium constant (Ka). From the relationship ΔG0 = −RT ln Ka and the Gibbs-Helmholtz equation, the free energy of binding and the entropy of association (ΔS0) were calculated. Crystal structure of p300 HAT domain was extracted from Protein Data Bank code 3BIY. Crystal structure of the inhibitor RTK1 and its inactive derivative RTK2 was obtained and solved (Bruker X8 APEX). The HAT domain was docked with the structure of RTK1 and RTK2 to find out their interaction sites on HAT domain. RTK1 has a hydroxyl group at the 5th carbon position, and the derivative RTK2 has a methoxy group instead of the hydroxyl group. Molecular simulation and the docking of HAT domain with RTK1 and RTK2 were performed using Hex 4.5 software. The docking calculations were done using three-dimensional parametric functions of both the protein (HAT domain) and the chemical structures (RTK1/RTK2). These calculations were used to encode surface shape and electrostatic charge and potential distributions. The parametric functions are based on expansions of real orthogonal spherical polar basis functions. The docking was performed in full rotation mode; both domain and inhibitor were taken at 180 ranges for 20,000 solutions. Site-directed mutagenesis was done to obtain a HAT domain point mutant K1358A. HAT domain expression clone in pET28b was used as the template, and the mutagenesis was done by the Stratagene site-directed mutagenesis kit according to the manufacturer's instructions. The positive clones were sequenced and transformed into BL21 strain of Escherichia coli. The expression and purification of the mutant protein was done as mentioned elsewhere (18.Arif M. Kumar G.V. Narayana C. Kundu T.K. J. Phys. Chem. B. 2007; 111: 11877-11879Crossref PubMed Scopus (36) Google Scholar). NMR spectroscopy, TLC, and x-ray characterization of the compounds were done as described elsewhere (17.Mantelingu K. Reddy B.A. Swaminathan V. Kishore A.H. Siddappa N.B. Kumar G.V. Nagashankar G. Natesh N. Roy S. Sadhale P.P. Ranga U. Narayana C. Kundu T.K. Chem. Biol. 2007; 14: 645-657Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). High resolution mass spectrometry was obtained on a Bruker Daltonics APEX II (for electrospray ionization). The solution of RTK1 (100 mg, 0.53 mmol) in acetone, anhydrous potassium carbonate (146 mg, 1 mmol) was stirred for about 10 min, and then methyl iodide (43 mg, 0.3 mmol) was added dropwise and the stirring continued for about 2 h. The reaction was monitored by TLC. Then solvent was evaporated to dryness and subjected to column chromatography, which yielded the compound RTK2. Ethyl iodide (55 mg, 0.35 mmol) was added dropwise to the solution mixture of RTK1 (100 mg, 0.53 mmol) and anhydrous potassium carbonate (146 mg, 1 mmol) in acetone for about 20 min, and the reaction mixture was stirred for 2 h. The solvent was evaporated to dryness and subjected to CC to yield RTK3. The mixture of RTK1 (100 mg, 0.53 mmol) and anhydrous potassium carbonate (146 mg, 1 mmol) in acetone was stirred for about 10 min in a two-necked round bottom flask. Isopropyl iodide (63 mg, 0.37 mmol) was then added dropwise, and stirring was continued for another 2 h. The solvent was evaporated under reduced pressure and subjected to CC to yield compound RTK4. A solution of RTK1 (100 mg, 0.53 mmol) in dichloromethane was added to a cooled solution of triethylamine (148 mg, 1.4 mmol) in dichloromethane. The reaction mixture was stirred for 30 min at 0–5 °C, and then acetyl chloride (56 mg, 0.71 mmol) was slowly added into the reaction mixture for a period of 30 min. After stirring at 0–5 °C for 6 h, the temperature was slowly increased to 20–25 °C and again stirred for 4 h. The solvent was evaporated to dryness and purified by CC resulting in compound RTK5. RTK1 (100 mg, 0.53 mmol) was dissolved in dichloromethane (10 ml). Triethylamine (148 mg, 1.4 mmol) was added, and the reaction mixture was cooled to 0–5 °C. Methylsulfonyl chloride was slowly added (61 mg, 0.538 mmol) in dichloromethane. The reaction mixture was stirred for 4 h at 0–5 °C, and the temperature was raised slowly to 20–25 °C and stirred for 8–10 h. The resulting reaction mixture was isolated and subjected to CC to yield RTK6. RTK1 solution (100 mg, 0.53 mmol) in ethanol (20 ml), potassium hydroxide (59 mg, 1 mmol) was taken in a round bottom flask and stirred for about 10 min, followed by addition of 1-(2-chloroethyl)piperidine hydrochloride (117 mg, 0.63 mmol), and the reaction mixture was heated to 60 °C. After 2 h, the reaction mixture was cooled to room temperature, poured into water, acidified with dilute HCl, and extracted with ethyl acetate. The extract was evaporated to get residue, which was subjected to CC to get RTK7. The 4-(2-chloroethyl)morpholine hydrochloride (118 mg, 0.63 mmol) was added into the reaction mixture of RTK1 (100 mg, 0.53 mmol) and potassium hydroxide (59 mg, 1 mmol) in ethanol. The reaction mixture was heated up to 60 °C over a period of 90 min followed by cooling. The mixture was poured into ice-cold water and acidified with dilute HCl to pH 7.0 and extracted with ethyl acetate. The extract was evaporated to get residue and subjected to CC to yield RTK8. The solution of RTK1 in acetone (100 mg, 0.53 mmol) and potassium carbonate (146 mg, 1 mmol) was stirred at room temperature for about 10 min followed by addition of ethyl bromoacetate (76 mg, 0.46 mmol), and the temperature was slowly increased to 50 °C. The reaction mixture was heated for about 2 h and poured into ice and acidified with dilute HCl (to pH 7) and then extracted with ethyl acetate. The extract was evaporated to get residue, which was subjected to CC to get RTK9. RTK1 solution (100 mg, 0.53 mmol) in ethanol, potassium hydroxide (59 mg, 1 mmol) were taken in a two-necked round bottom flask, stirred for about 10 min, followed by addition of 2-chloro-N,N-dimethylethanamine (92 mg, 0.63 mmol), and the reaction mixture was heated to 60 °C. After 2 h, the reaction mixture was cooled to room temperature, poured in water, and extracted with ethyl acetate. The extract was evaporated to get residue, which was subjected to CC to get compound RTK10. The dysfunction of lysine acetyltransferases could be associated with several diseases, like asthma, cardiovascular disorders, diabetes, and cancer (10.Zheng Y. Thompson P.R. Cebrat M. Wang L. Devlin M.K. Alani R.M. Cole P.A. Methods Enzymol. 2004; 376: 188-199Crossref PubMed Scopus (39) Google Scholar, 11.Stimson L. Rowlands M.G. Newbatt Y.M. Smith N.F. Raynaud F.I. Rogers P. Bavetsias V. Gorsuch S. Jarman M. Bannister A. Kouzarides T. McDonald E. Workman P. Aherne G.W. Mol. Cancer Ther. 2005; 4: 1521-1532Crossref PubMed Scopus (188) Google Scholar, 12.Iyer N.G. Chin S.F. Ozdag H. Daigo Y. Hu D.E. Cariati M. Brindle K. Aparicio S. Caldas C. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7386-7391Crossref PubMed Scopus (123) Google Scholar, 13.Davidson S.M. Townsend P.A. Carroll C. Yurek-George A. Balasubramanyam K. Kundu T.K. Stephanou A. Packham G. Ganesan A. Latchman D.S. ChemBioChem. 2005; 6: 162-170Crossref PubMed Scopus (44) Google Scholar, 14.Zhou X.Y. Shibusawa N. Naik K. Porras D. Temple K. Ou H. Kaihara K. Roe M.W. Brady M.J. Wondisford F.E. Nat. Med. 2004; 10: 633-637Crossref PubMed Scopus (118) Google Scholar, 15.Varier R.A. Kundu T.K. Curr. Pharm. Des. 2006; 12: 1975-1993Crossref PubMed Scopus (24) Google Scholar). We have established a screening system of medicinal plants (described in the Indian ayurvedic literature, see Ref. 36.Dinda B. Das S.K. Hajra A.K. Indian J. Chem. 1995; 34B: 525-528Google Scholar), for acetyltransferase modulation activity. In this process, we have tested the crude extract of P. rosea and eventually isolated plumbagin (RTK1) and crystallized to characterize the compound (Fig. 1A, panel II). RTK1 is a highly cell-permeable compound with important cellular effects like anti-tumor activity (19.Kuo P.L. Hsu Y.L. Cho C.Y. Mol. Cancer Ther. 2006; 5: 3209-3221Crossref PubMed Scopus (279) Google Scholar, 20.Hsu Y.L. Cho C.Y. Kuo P.L. Huang Y.T. Lin C.C. J. Pharmacol. Exp. Ther. 2006; 318: 484-494Crossref PubMed Scopus (200) Google Scholar, 21.Gomathinayagam R. Sowmyalakshmi S. Mardhatillah F. Kumar R. Akbarsha M.A. Damodaran C. Anticancer Res. 2008; 28: 785-792PubMed Google Scholar, 22.Acharya B.R. Bhattacharyya B. Chakrabarti G. Biochemistry. 2008; 47: 7838-7845Crossref PubMed Scopus (68) Google Scholar), NF-κB activity, etc. (23.Sandur S.K. Ichikawa H. Sethi G. Ahn K.S. Aggarwal B.B. J. Biol. Chem. 2006; 281: 17023-17033Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). RTK1 is a potent apoptosis-inducing agent at higher concentrations. Histones (both H3 and H4) are known to be hyperacetylated in hepatocarcinomas (24.Bai X. Wu L. Liang T. Liu Z. Li J. Li D. Xie H. Yin S. Yu J. Lin Q. Zheng S. J. Cancer Res. Clin. Oncol. 2008; 134: 83-91Crossref PubMed Scopus (70) Google Scholar). To find out the ability of RTK1 to inhibit HAT activity in vivo, the liver cancer cell line HepG2 was treated with RTK1 at concentrations that do not induce apoptosis. In agreement with a previous report (24.Bai X. Wu L. Liang T. Liu Z. Li J. Li D. Xie H. Yin S. Yu J. Lin Q. Zheng S. J. Cancer Res. Clin. Oncol. 2008; 134: 83-91Crossref PubMed Scopus (70) Google Scholar), the histone H3 was found to be substantially acetylated in these cells (Fig. 1B, lane 1). The acetylated histone H3 level was reduced by 50% with 5 μm RTK1 treatment (Fig. 1B, lane 2). A dose-dependent inhibition of histone H3 acetylation was observed with almost 90% inhibition on 25 μm RTK1 treatment (Fig. 1B, lane 4). The overall acetylation status of the histones was also found to be significantly decreased with a prominent reduction of H3 and H4 acetylation (supplemental Fig. 1). Because the HepG2 cells grow in clumps, the immunofluorescence imaging of these cells was difficult. Therefore, the histone acetylation upon plumbagin treatment was verified by immunofluorescence analysis in HeLa cells, wherein the cells were treated with the compound and DMSO (solvent control) for 12 h. Because the acetylation level in HeLa cells is low, histone acetylation was induced by treating with histone deacetylase inhibitors (Fig. 1C, TSA/NaBu) followed by RTK1 treatment. As expected, RTK1 could inhibit the histone acetylation in HeLa cells efficiently at 5 μm concentration. There was an almost complete reduction in the acetylation levels with 25 μm concentration of RTK1, as visualized by immunofluorescence analysis (Fig. 1C, DMSO versus RTK1). Furthermore, the HAT inhibition by RTK1 was also confirmed in vivo wherein the liver tissue of RTK1-treated mice was examined for inhibition of histone acetylation. As visualized by immunohistochemistry using antibody against acetylated histone H3 (Lys-9 and Lys-14), there was a significant decrease in histone acetylation in RTK1-treated mice liver as compared with the normal and DMSO-treated controls. The acetylation status of histones in the untreated mouse liver was taken as the control for comparing the treated sections (Fig. 1D, panel I, H&E staining and AcH3 staining). There was no gross loss in morphology of cells as indicated by the hematoxylin and eosin staining. The solvent control sections show strong to moderate positive staining in hepatocytes near the central vein and portal triad regions indicating the ability of the solvent for inducing the acetylation in these cells (Fig. 1D, panel II, H&E staining and AcH3 staining). On the other hand, RTK1 dissolved in the DMSO solvent shows negative staining for Ac-H3 antibody indicating the inhibitory activity of acetylation by RTK1 in this liver sample (Fig. 1D, panel III of H&E staining and AcH3 staining). Taken together, t
Acetyltransferases
Histone acetyltransferase
CREB-binding protein
PCAF
P300-CBP Transcription Factors
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PCAF
Histone acetyltransferase
Acetyltransferases
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