Screen-identified selective inhibitor of lysine demethylase 5A blocks cancer cell growth and drug resistance.

Lysine demethylase 5A (KDM5A/RBP2/JARID1A) is a histone lysine demethylase that is overexpressed in several human cancers including lung, gastric, breast and liver cancers. It plays key roles in important cancer processes including tumorigenesis, metastasis, and drug tolerance, making it a potential cancer therapeutic target. Chemical tools to analyze KDM5A demethylase activity are extremely limited as available inhibitors are not specific for KDM5A. Here, we characterized KDM5A using a homogeneous luminescence-based assay and conducted a screen of about 9,000 small molecules for inhibitors. From this screen, we identified several 3-thio-1,2,4-triazole compounds that inhibited KDM5A with low µM in vitro IC50 values. Importantly, these compounds showed great specificity and did not inhibit its close homologue KDM5B (PLU1/JARID1B) or the related H3K27 demethylases KDM6A (UTX) and KDM6B (JMJD3). One compound, named YUKA1, was able to increase H3K4me3 levels in human cells and selectively inhibit the proliferation of cancer cells whose growth depends on KDM5A. As KDM5A was shown to mediate drug tolerance, we investigated the ability of YUKA1 to prevent drug tolerance in EGFR-mutant lung cancer cells treated with gefitinib and HER2+ breast cancer cells treated with trastuzumab. Remarkably, this compound hindered the emergence of drug-tolerant cells, highlighting the critical role of KDM5A demethylase activity in drug resistance. The small molecules presented here are excellent tool compounds for further study of KDM5A's demethylase activity and its contributions to cancer.

Identification of small molecule inhibitors of Jumonji AT-rich interactive domain 1B (JARID1B) histone demethylase by a sensitive high throughput screen.

JARID1B (also known as KDM5B or PLU1) is a member of the JARID1 family of histone lysine demethylases responsible for the demethylation of trimethylated lysine 27 in histone H3 (H3K4me3), a mark for actively transcribed genes. JARID1B is overexpressed in several cancers, including breast cancer, prostate cancer, and lung cancer. In addition, JARID1B is required for mammary tumor formation in syngeneic or xenograft mouse models. JARID1B-expressing melanoma cells are associated with increased self-renewal character. Therefore, JARID1B represents an attractive target for cancer therapy. Here we characterized JARID1B using a homogeneous luminescence-based demethylase assay. We then conducted a high throughput screen of over 15,000 small molecules to identify inhibitors of JARID1B. From this screen, we identified several known JmjC histone demethylase inhibitors, including 2,4-pyridinedicarboxylic acid and catechols. More importantly, we identified several novel inhibitors, including 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), which inhibits JARID1B with an IC50 of about 3 µm in vitro. Consistent with this, PBIT treatment inhibited removal of H3K4me3 by JARID1B in cells. Furthermore, this compound inhibited proliferation of cells expressing higher levels of JARID1B. These results suggest that this novel small molecule inhibitor is a lead compound that can be further optimized for cancer therapy.

Epigenetic Regulation by Lysine Demethylase 5 (KDM5) Enzymes in Cancer.

Similar to genetic alterations, epigenetic aberrations contribute significantly to tumor initiation and progression. In many cases, these changes are caused by activation or inactivation of the regulators that maintain epigenetic states. Here we review our current knowledge on the KDM5/JARID1 family of histone demethylases. This family of enzymes contains a JmjC domain and is capable of removing tri- and di- methyl marks from lysine 4 on histone H3. Among these proteins, RBP2 mediates drug resistance while JARID1B is required for melanoma maintenance. Preclinical studies suggest inhibition of these enzymes can suppress tumorigenesis and provide strong rationale for development of their inhibitors for use in cancer therapy.

TbPRMT6 is a type I protein arginine methyltransferase that contributes to cytokinesis in Trypanosoma brucei.

Arginine methylation is a widespread posttranslational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). In Saccharomyces cerevisiae and mammals, this modification affects multiple cellular processes, such as chromatin remodeling leading to transcriptional regulation, RNA processing, DNA repair, and cell signaling. The protozoan parasite Trypanosoma brucei possesses five putative PRMTs in its genome. This is a large number of PRMTs relative to other unicellular eukaryotes, suggesting an important role for arginine methylation in trypanosomes. Here, we present the in vitro and in vivo characterization of a T. brucei enzyme homologous to human PRMT6, which we term TbPRMT6. Like human PRMT6, TbPRMT6 is a type I PRMT, catalyzing the production of monomethylarginine and asymmetric dimethylarginine residues. In in vitro methylation assays, TbPRMT6 utilizes bovine histones as a substrate, but it does not methylate several T. brucei glycine/arginine-rich proteins. As such, it exhibits a relatively narrow substrate specificity compared to other T. brucei PRMTs. Knockdown of TbPRMT6 in both procyclic form and bloodstream form T. brucei leads to a modest but reproducible effect on parasite growth in culture. Moreover, upon TbPRMT6 depletion, both PF and BF exhibit aberrant morphologies indicating defects in cell division, and these defects differ in the two life cycle stages. Mass spectrometry of TbPRMT6-associated proteins reveals histones, components of the nuclear pore complex, and flagellar proteins that may represent TbPRMT6 substrates contributing to the observed growth and morphological defects.

A type III protein arginine methyltransferase from the protozoan parasite Trypanosoma brucei.

Arginine methylation is a widespread post-translational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). The ancient protozoan parasite, Trypanosoma brucei, possesses five putative PRMTs, a relatively large number for a single-celled eukaryote. Trypanosomatids lack gene regulation at the level of transcription, instead relying on post-transcriptional control mechanisms that act at the levels of RNA turnover, translation, and editing, all processes that likely involve multiple RNA-binding proteins, which are common targets of arginine methylation. Here, we report the characterization of a trypanosome PRMT, TbPRMT7, which is homologous to human PRMT7. Interestingly, trypanosomatids are the only single-celled eukaryotes known to harbor a PRMT7 homologue. TbPRMT7 differs dramatically from all known metazoan PRMT7 homologues in lacking the second AdoMet binding-like domain that is required for activity of the human enzyme. Nevertheless, bacterially expressed TbPRMT7 exhibits robust methyltransferase activity toward multiple targets in vitro. High resolution ion exchange chromatography analysis of methylated substrates reveals that TbPRMT7 is a type III PRMT, catalyzing the formation of only monomethylarginine, thereby representing the only exclusively type III PRMT identified to date. TbPRMT7 is expressed in both mammalian and insect stage T. brucei and is apparently dispensable for growth in both life cycle stages. The enzyme is cytoplasmically localized and is a component of several higher order complexes in vivo. Together, our studies indicate that TbPRMT7 is a Type III PRMT, and its robust activity and presence in numerous complexes suggest it plays multiple roles during the complex T. brucei life cycle.

Hsl7 is a substrate-specific type II protein arginine methyltransferase in yeast.

The Saccharomyces cerevisiae protein Hsl7 is a regulator of the Swe1 protein kinase in cell cycle checkpoint control. Hsl7 has been previously described as a type III protein arginine methyltransferase, catalyzing the formation of omega-monomethylarginine residues on non-physiological substrates. However, we show here that Hsl7 can also display type II activity, generating symmetric dimethylarginine residues on calf thymus histone H2A. Symmetric dimethylation is only observed when enzyme and the methyl-accepting substrate were incubated for extended times. We confirmed the Hsl7-dependent formation of symmetric dimethylarginine by amino acid analysis and thin layer chromatography with wild-type and mutant recombinant enzymes expressed from both bacteria and yeast. This result is significant because no type II activity has been previously demonstrated in S. cerevisiae. We also show that Hsl7 has little or no activity on GST-GAR, a commonly used substrate for protein arginine methyltransferases, and only minimal activity on myelin basic protein. This enzyme thus may only recognize only a small subset of potential substrate proteins in yeast, in contrast to the situation with Rmt1, the major type I methyltransferase.

Regulation of protein arginine methyltransferase 8 (PRMT8) activity by its N-terminal domain.

Human protein arginine methyltransferase PRMT8 has been recently described as a type I enzyme in brain that is localized to the plasma membrane by N-terminal myristoylation. The amino acid sequence of human PRMT8 is almost 80% identical to human PRMT1, the major protein arginine methyltransferase activity in mammalian cells. However, the activity of a recombinant PRMT8 GST fusion protein toward methyl-accepting substrates is much lower than that of a GST fusion of PRMT1. We show here that both His-tagged and GST fusion species lacking the initial 60 amino acid residues of PRMT8 have enhanced enzymatic activity, suggesting that the N-terminal domain may regulate PRMT8 activity. This conclusion is supported by limited proteolysis experiments showing an increase in the activity of the digested full-length protein, consistent with the loss of the N-terminal domain. In contrast, the activity of the N-terminal truncated protein was slightly diminished by limited proteolysis. Significantly, we detect automethylation at two sites in the N-terminal domain, as well as binding sites for SH3 domain-containing proteins. We suggest that the N-terminal domain may function as an autoregulator that may be displaced by interaction with one or more physiological inducers.

Evolutionarily divergent type II protein arginine methyltransferase in Trypanosoma brucei.

Protein arginine methylation is a posttranslational modification that impacts cellular functions, such as RNA processing, transcription, DNA repair, and signal transduction. The majority of our knowledge regarding arginine methylation derives from studies of yeast and mammals. Here, we describe a protein arginine N-methyltransferase (PRMT), TbPRMT5, from the early-branching eukaryote Trypanosoma brucei. TbPRMT5 shares the greatest sequence similarity with PRMT5 and Skb1 type II enzymes from humans and Schizosaccharomyces pombe, respectively, although it is significantly divergent at the amino acid level from its mammalian and yeast counterparts. Recombinant TbPRMT5 displays broad substrate specificity in vitro, including methylation of a mitochondrial-gene-regulatory protein, RBP16. TbPRMT5 catalyzes the formation of omega-N(G)-monomethylarginine and symmetric omega-N(G),N(G')-dimethylarginine and does not require trypanosome cofactors for this activity. These data establish that type II PRMTs evolved early in the eukaryotic lineage. In vivo, TbPRMT5 is constitutively expressed in the bloodstream form and procyclic-form (insect host) life stages of the parasite and localizes to the cytoplasm. Genetic disruption via RNA interference in procyclic-form trypanosomes indicates that TbPRMT5 is not essential for growth in this life cycle stage. TbPRMT5-TAP ectopically expressed in procyclic-form trypanosomes is present in high-molecular-weight complexes and associates with an RG domain-containing DEAD box protein related to yeast Ded1 and two kinetoplastid-specific proteins. Thus, TbPRMT5 is likely to be involved in novel methylation-regulated functions in trypanosomes, some of which may include RNA processing and/or translation.

The regulatory role of residues 226-232 in phosphoenolpyruvate carboxylase from maize.

The regulatory properties of maize phosphoenolpyruvate carboxylase were significantly altered by site-directed mutagenesis of residues 226 through 232. This conserved sequence element, RTDEIRR, is part of a surface loop at the dimer interface. Mutation of individual residues in this sequence caused various kinetic changes, including desensitization of the enzyme to key allosteric effectors or alteration of the K(0.5 PEP) for the substrate phosphoenolpyruvate. R231A, and especially R232Q, displayed decreased apparent affinity for the activator glucose-6-phosphate. Apparent affinity for the activator glycine was reduced in D228N and R232Q, while the maximum activation caused by glycine was greatly reduced in R226Q and E229A. R226Q and E229A also showed significantly lower sensitivity to the inhibitors malate and aspartate. E229A exhibited a low K(0.5 PEP), while the K(0.5 PEP )of R232Q was significantly higher than that of wild type. Thus these seven residues are critical determinants of the enzyme's kinetic responses to activators, inhibitors and substrate. The present results support an earlier suggestion that Arg 231 contributes to the binding site of the allosteric activator glucose-6-phosphate, and are consistent with other proposals that the substrate phosphoenolpyruvate allosterically activates the enzyme by binding at or near the glucose-6-phosphate site. The results also suggest that the glycine binding site may be contiguous with the glucose-6-phosphate binding site. Glu 229, which extends from this interface region through the interior of the protein and emerges near the aspartate binding site, may provide a physical link for propagating conformational changes between the allosteric activator and inhibitor binding regions.

Yeast Hsl7 (histone synthetic lethal 7) catalyses the in vitro formation of omega-N(G)-monomethylarginine in calf thymus histone H2A.

The HSL7 (histone synthetic lethal 7) gene in the yeast Saccharomyces cerevisiae encodes a protein with close sequence similarity to the mammalian PRMT5 protein, a member of the class of protein arginine methyltransferases that catalyses the formation of omega-N(G)-monomethylarginine and symmetric omega-N(G),N'(G)-dimethylarginine residues in a number of methyl-accepting species. A full-length HSL7 construct was expressed as a FLAG-tagged protein in Saccharomyces cerevisiae. We found that FLAG-tagged Hsl7 effectively catalyses the transfer of methyl groups from S-adenosyl-[methyl-3H]-L-methionine to calf thymus histone H2A. When the acid-hydrolysed radiolabelled protein products were separated by high-resolution cation-exchange chromatography, we were able to detect one tritiated species that co-migrated with an omega-N(G)-monomethylarginine standard. No radioactivity was observed that co-migrated with either the asymmetric or symmetric dimethylated derivatives. In control experiments, no methylation of histone H2A was found with two mutant constructs of Hsl7. Surprisingly, FLAG-Hsl7 does not appear to effectively catalyse the in vitro methylation of a GST (glutathione S-transferase)-GAR [glycine- and arginine-rich human fibrillarin-(1-148) peptide] fusion protein or bovine brain myelin basic protein, both good methyl-accepting substrates for the human homologue PRMT5. Additionally, FLAG-Hsl7 demonstrates no activity on purified calf thymus histones H1, H2B, H3 or H4. GST-Rmt1, the GST-fusion protein of the major yeast protein arginine methyltransferase, was also found to methylate calf thymus histone H2A. Although we detected Rmt1-dependent arginine methylation in vivo in purified yeast histones H2A, H2B, H3 and H4, we found no evidence for Hsl7-dependent methylation of endogenous yeast histones. The physiological substrates of the Hsl7 enzyme remain to be identified.

Identification and characterization of the methyl arginines in the fragile X mental retardation protein Fmrp.

Fragile X syndrome is the most common form of inherited mental retardation and is caused by the absence of expression of the FMR1 gene. The protein encoded by this gene, Fmrp, is an RNA-binding protein that binds a subset of mRNAs and regulates their translation, leading to normal cognitive function. Although the association with RNAs is well established, it is still unknown how Fmrp finds and assembles with its RNA cargoes and how these activities are regulated. We show here that Fmrp is post-translationally methylated, primarily on its arginine-glycine-glycine box. We identify the four arginines that are methylated and show that cellular Fmrp is monomethylated and asymmetrically dimethylated. We also show that the autosomal paralog Fxr1 and the Drosophila ortholog dFmr1 are methylated post-translationally. Recombinant protein arginine methyl transferase 1 (PRMT1) methylates Fmrp on the same arginines in vitro as in cells. In vitro methylation of Fmrp results in reduced binding to the minimal RNA sequence sc1, which encodes a stem loop G-quartet structure. Our data identify an additional mechanism, arginine methylation, for modifying Fmrp function and suggest that methylation occurs to limit or modulate RNA binding by Fmrp.

PRMT8, a new membrane-bound tissue-specific member of the protein arginine methyltransferase family.

Protein arginine methylation is a common post-translational modification that has been implicated in signal transduction, RNA processing, transcriptional regulation, and DNA repair. A search of the human genome for additional members of the protein arginine N-methyltransferase (PRMT) family of enzymes has identified a gene on chromosome 12 that we have termed PRMT8. This novel enzyme is most closely related to PRMT1, although it has a distinctive N-terminal region. The unique N-terminal end harbors a myristoylation motif, and we have shown here that PRMT8 is indeed modified by the attachment of a myristate to the glycine residue after the initiator methionine. The myristoylation of PRMT8 results in its association with the plasma membrane. The second singular property of PRMT8 is its tissue-specific expression pattern; it is largely expressed in the brain. A glutathione S-transferase fusion protein of PRMT8 has type I PRMT activity, catalyzing the formation of omega-NG-monomethylated and asymmetrically omega-NG,NG-dimethylated arginine residues on a recombinant glycine- and arginine-rich substrate. PRMT8 is thus an active arginine methyltransferase that is membrane-associated and tissue-specific, two firsts for this family of enzymes.

Human PAD4 regulates histone arginine methylation levels via demethylimination.

Methylation of arginine (Arg) and lysine residues in histones has been correlated with epigenetic forms of gene regulation. Although histone methyltransferases are known, enzymes that demethylate histones have not been identified. Here, we demonstrate that human peptidylarginine deiminase 4 (PAD4) regulates histone Arg methylation by converting methyl-Arg to citrulline and releasing methylamine. PAD4 targets multiple sites in histones H3 and H4, including those sites methylated by coactivators CARM1 (H3 Arg17) and PRMT1 (H4 Arg3). A decrease of histone Arg methylation, with a concomitant increase of citrullination, requires PAD4 activity in human HL-60 granulocytes. Moreover, PAD4 activity is linked with the transcriptional regulation of estrogen-responsive genes in MCF-7 cells. These data suggest that PAD4 mediates gene expression by regulating Arg methylation and citrullination in histones.