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1.  Scintillation Proximity Assay of Arginine Methylation 
Journal of Biomolecular Screening  2011;17(2):237-244.
Methylation of arginine residues, catalyzed by protein arginine methyltransferases (PRMTs), is one important protein post-translational modification involved in epigenetic regulation of gene expression. A fast and effective assay for PRMT can provide valuable information for dissecting the biological functions of PRMTs, as well as for screening small-molecule inhibitors of arginine methylation. Currently, among the methods used for PRMT activity measurement, many contain laborious separation procedures, which restrict the applications of these assays for high-throughput screening (HTS) in drug discovery. The authors report here a mix-and-measure method to measure PRMT activity based on the principle of scintillation proximity assay (SPA). In this assay, 3H-AdoMet was used as methyl donor, and biotin-modified histone H4 peptide served as a methylation substrate. Following the methylation reaction catalyzed by PRMTs, streptavidin-coated SPA beads were added to the reaction solution, and SPA signals were detected by a MicroBeta scintillation counter. No separation step is needed, which simplifies the assay procedure and greatly enhances the assay speed. Particularly, the miniaturization and robustness suggest that this method is suited for HTS of PRMT inhibitors.
PMCID: PMC3236808  PMID: 21821785
protein arginine methyltransferases; PRMT; scintillation proximity assay; SPA; high-throughput screening; HTS
2.  Rational Design of Substrate-Based Multivalent Inhibitors of the Histone Acetyltransferase 
ChemMedChem  2014;9(3):537-541.
Tip60 (the 60-kDa HIV-1 Tat-interactive protein) is a key member of the MYST family of histone acetyltransferases (HATs) and plays critical roles in apoptosis and DNA repair. Potent and selective inhibitors for Tip60 are valuable tools for studying the functions of the enzyme. In this work, we designed, synthesized and evaluated a new set of substrate-based inhibitors containing multiple binding modalities. In addition to the CoA moiety conjugated to H3 peptide, mono- and trimethylated lysine residues were incorporated at Lys-4 and/or Lys-9 sites in the H3 peptide substrate. The biochemical assay results showed that the presence of methyl group(s) on the substrate resulted in more potent inhibitors to Tip60, relative to the parent H3-CoA bisubstrate inhibitor. Importantly, by comparing the inhibitory properties of the ligands against full-length Tip60 versus the HAT domain, we determined that the K4me1 and K9me3 marks contributed to the potency augmentation by interacting with the catalytic region of the enzyme.
PMCID: PMC4150253  PMID: 24446345
Tip60; bisubstrate inhibitor; multivalency; HAT; methyllysine
3.  Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells 
Molecular Oncology  2015;9(3):617-627.
Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over-expression. Here we present data showing that short-interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell-lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN-negative SH-SY5Y NB cell-line, or in two immortalized human fibroblast cell-lines. Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, Mann–Whitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post-transcriptional level. Reciprocal co-immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK-N-BE(2)C and NGP cell lines. By using liquid chromatography – tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small-molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene.
Graphical abstract
•PRMT5 targeting siRNAs induce neuroblastoma cell apoptosis.•PRMT5 expression correlates with poor prognosis neuroblastoma and MYCN amplification.•MYCN protein stability is regulated by PRMT5 depletion.•PRMT5 and MYCN proteins physically interact.•MYCN protein is post-translationally modified by arginine methylation.
PMCID: PMC4359099  PMID: 25475372
Neuroblastoma; MYCN; PRMT5; Arginine methylation
4.  Chemical and Biochemical Approaches in the Study of Histone Methylation and Demethylation 
Medicinal research reviews  2012;32(4):815-867.
Histone methylation represents one of the most critical epigenetic events in DNA function regulation in eukaryotic organisms. Classic molecular biology and genetics tools provide significant knowledge about mechanisms and physiological roles of histone methyltransferases and demethylases in various cellular processes. In addition to this stream line, development and application of chemistry and chemistry-related techniques are increasingly involved in biological study, and provide information otherwise difficulty to obtain by standard molecular biology methods. Herein, we review recent achievements and progress in developing and applying chemical and biochemical approaches in the study of histone methylation, including chromatin immunoprecipitation (ChIP), chemical ligation, mass spectrometry (MS), biochemical assays, and inhibitor development. These technological advances allow histone methylation to be studied from genome-wide level to molecular and atomic levels. With ChIP technology, information can be obtained about precise mapping of histone methylation patterns at specific promoters, genes or other genomic regions. MS is particularly useful in detecting and analyzing methylation marks in histone and nonhistone protein substrates. Chemical approaches that permit site-specific incorporation of methyl groups into histone proteins greatly facilitate the investigation of the biological impacts of methylation at individual modification sites. Discovery and design of selective organic inhibitors of histone methyltransferases and demethylases provide chemical probes to interrogate methylation-mediated cellular pathways. Overall, these chemistry-related technological advances have greatly improved our understanding of the biological functions of histone methylation in normal physiology and diseased states, and also are of great potential to translate basic epigenetics research into diagnostic and therapeutic application in the clinic.
PMCID: PMC4129395  PMID: 22777714
histone methylation; demethylation; ChIP; mass spectrometry; chemical ligation; inhibitor
5.  Arginine methylation initiates BMP-induced Smad signaling 
Molecular cell  2013;51(1):5-19.
Kinase activation and substrate phosphorylation commonly form the backbone of signaling cascades. Bone morphogenetic proteins (BMPs), a subclass of TGF-β family ligands, induce activation of their signaling effectors, the Smads, through C-terminal phosphorylation by transmembrane receptor kinases. However, the slow kinetics of Smad activation in response to BMP suggests a preceding step in the initiation of BMP signaling. We now show that arginine methylation, which is known to regulate gene expression, yet also modifies some signaling mediators, initiates BMP-induced Smad signaling. BMP-induced receptor complex formation promotes interaction of the methyltransferase PRMT1 with the inhibitory Smad6, resulting in Smad6 methylation and relocalization at the receptor, leading to activation of effector Smads through phosphorylation. PRMT1 is required for BMP-induced biological responses across species, as evidenced by the role of its ortholog Dart1 in BMP signaling during Drosophila wing development. Activation of signaling by arginine methylation may also apply to other signaling pathways.
PMCID: PMC3951972  PMID: 23747011
6.  6-alkylsalicylates are selective Tip60 inhibitors and target the acetyl-CoA binding site 
Histone acetyltransferases are important enzymes that regulate various cellular functions, such as epigenetic control of DNA transcription. Development of HAT inhibitors with high selectivity and potency will provide powerful mechanistic tools for the elucidation of the biological functions of HATs and may also have pharmacological value for potential new therapies. In this work, analogs of the known HAT inhibitor anacardic acid were synthesized and evaluated for inhibition of HAT activity. Biochemical assays revealed novel anacardic acid analogs that inhibited the human recombinant enzyme Tip60 selectively compared to PCAF and p300. Enzyme kinetics studies demonstrated that inhibition of Tip60 by one such novel anacardic acid derive, 20, was essentially competitive with Ac-CoA and noncompetitive with the histone substrate. In addition, these HAT inhibitors effectively inhibited acetyltransferase activity of nuclear extracts on the histone H3 and H4 at micromolar concentrations.
PMCID: PMC3399519  PMID: 22100137
histone acetylation; epigenetics; anacardic acid; histone acetyltransferase (HAT); inhibitors
7.  Function of the Active Site Lysine Autoacetylation in Tip60 Catalysis 
PLoS ONE  2012;7(3):e32886.
The 60-kDa HIV-Tat interactive protein (Tip60) is a key member of the MYST family of histone acetyltransferases (HATs) that plays critical roles in multiple cellular processes. We report here that Tip60 undergoes autoacetylation at several lysine residues, including a key lysine residue (i.e. Lys-327) in the active site of the MYST domain. The mutation of K327 to arginine led to loss of both the autoacetylation activity and the cognate HAT activity. Interestingly, deacetylated Tip60 still kept a substantial degree of HAT activity. We also investigated the effect of cysteine 369 and glutamate 403 in Tip60 autoacetylation in order to understand the molecular pathway of the autoacetylation at K327. Together, we conclude that the acetylation of K327 which is located in the active site of Tip60 regulates but is not obligatory for the catalytic activity of Tip60. Since acetylation at this key residue appears to be evolutionarily conserved amongst all MYST proteins, our findings provide an interesting insight into the regulatory mechanism of MYST activities.
PMCID: PMC3314657  PMID: 22470428
8.  Small molecule inhibitors of histone acetyltransferase Tip60 
Bioorganic chemistry  2010;39(1):53-58.
Tip60 is a key member of the MYST family of histone acetyltransferases and involved in a broad spectrum of cellular pathways and disease conditions. So far, small molecule inhibitors of Tip60 and other members of MYST HATs are rarely reported. To discover new small molecule inhibitors of Tip60 as mechanistic tools for functional study and as chemical leads for therapeutic development, we performed virtual screening using the crystal structure of Esa1 (the yeast homolog of Tip60) on a small molecule library database. Radioactive acetylation assays were carried out to further evaluate the virtual screen hits. Several compounds with new structural scaffolds were identified with micromolar inhibition potency for Tip60 from the biochemical studies. Further, computer modeling and kinetic assays suggest that these molecules target the acetyl-CoA binding site in Tip60. These new inhibitors provide valuable chemical hits to develop further potent inhibitors for the MYST HATs.
PMCID: PMC3144758  PMID: 21186043
Tip60; MYST; HAT; Histone; Inhibitor

Results 1-8 (8)