Protein arginine methyltransferase 1: positively charged residues in substrate peptides distal to the site of methylation are important for substrate binding and catalysis
Protein arginine methyltransferases (PRMTs) are a group of eukaryotic enzymes that catalyze the methylation of Arg residues in a variety of proteins (e.g., histones H3 and H4), and their activities influence a wide range of cellular processes, including cell growth, RNA splicing, differentiation, and transcriptional regulation. Dysregulation of these enzymes has been linked to heart disease and cancer, suggesting this enzyme family as a novel therapeutic target. To aid the development of PRMT inhibitors, we characterized the substrate specificity of both the rat and human PRMT1 orthologues using histone based peptide substrates. N- and C-terminal truncations to identify a minimal peptide substrate indicate that long-range interactions between enzyme and substrate are important for high rates of substrate capture. The importance of these long-range interactions to substrate capture were confirmed by "mutagenesis" experiments on a minimal peptide substrate. Inhibition studies on S-adenosyl-homocysteine, thioadenosine, methylthioadenosine, homocysteine, and sinefungin suggest that potent and selective bisubstrate analogue inhibitor(s) for PRMT1 can be developed by linking a histone based peptide substrate to homocysteine or sinefungin. Additionally, we present evidence that PRMT1 utilizes a partially processive mechanism to dimethylate its substrates.
Protein Arginine Deiminase 4 (PAD4) has emerged as a leading target for the development of a Rheumatoid Arthritis (RA) pharmaceutical. Herein, we describe the development of a novel screen for PAD4 inhibitors that is based on a PAD4-targeted Activity-Based Protein Profiling reagent, denoted Rhodamine-conjugated F-Amidine (RFA). This screen was validated by screening 10 Disease Modifying Anti-Rheumatic Drugs (DMARDs) and identified streptomycin, minocycline, and chlortetracycline as micromolar inhibitors of PAD4 activity.
The transcriptional coactivator p300/CBP (CREBBP) is a histone acetyltransferase (HAT) that regulates gene expression by acetylating histones and other transcription factors. Dysregulation of p300/CBP HAT activity contributes to various diseases including cancer. Sequence alignments, enzymology experiments and inhibitor studies on p300/CBP have led to contradictory results about its catalytic mechanism and its structural relation to the Gcn5/PCAF and MYST HATs. Here we describe a high-resolution X-ray crystal structure of a semi-synthetic heterodimeric p300 HAT domain in complex with a bi-substrate inhibitor, Lys-CoA. This structure shows that p300/CBP is a distant cousin of other structurally characterized HATs, but reveals several novel features that explain the broad substrate specificity and preference for nearby basic residues. Based on this structure and accompanying biochemical data, we propose that p300/CBP uses an unusual 'hit-and-run' (Theorell-Chance) catalytic mechanism that is distinct from other characterized HATs. Several disease-associated mutations can also be readily accounted for by the p300 HAT structure. These studies pave the way for new epigenetic therapies involving modulation of p300/CBP HAT activity.
Protein arginine methyltransferases (PRMTs) are (S)-adenosylmethionine (SAM)-dependent methyltransferases that catalyze the post-translational methylation of Arg residues in a variety of different proteins involved in transcriptional regulation and RNA splicing (e.g., histones H2A, H3, and H4). Herein, we describe the use of an N-mustard, 5'-(diaminobutyric acid)-N-iodoethyl-5'-deoxyadenosine ammonium hydrochloride (AAI), to generate a bisubstrate analogue inhibitor of PRMT1. Using the approach outlined in this communication, it should be possible to generate bisubstrate analogue-based inhibitors of PRMT isozymes that are potent and highly selective for a particular isozyme. The fact that PRMT1 catalyzes AAI transfer is also significant because with appropriate modifications (e.g., functionalization with pendant azido or alkyne functionalities) this compound could be used for proteomic applications to identify novel PRMT substrates.
Protein arginine deiminase 4 (PAD4) is an enzyme that hydrolyzes peptidyl arginine residues to form citrulline and ammonia. This enzyme has been implicated in several disease states, e.g. rheumatoid arthritis, and therefore represents a unique target for the development of a novel therapeutic. A solution-phase synthesis of Cl-amidine, the most potent PAD4 inactivator described to date, has been developed. This synthesis proceeds in 80% yield over 4 steps at a significantly (12-fold) lower cost.
Protein Arg methyltransferases function as coactivators of the tumor suppressor p53 to regulate gene expression. Peptidylarginine deiminase 4 (PAD4/PADI4) counteracts the functions of protein Arg methyltransferases in gene regulation by deimination and demethylimination. Here we show that the expression of a tumor suppressor gene, OKL38, is activated by the inhibition of PAD4 or the activation of p53 following DNA damage. Chromatin immunoprecipitation assays showed a dynamic change of p53 and PAD4 occupancy and histone Arg modifications at the OKL38 promoter during DNA damage, suggesting a direct role of PAD4 and p53 in the expression of OKL38. Furthermore, we found that OKL38 induces apoptosis through localization to mitochondria and induction of cytochrome c release. Together, our studies identify OKL38 as a novel p53 target gene that is regulated by PAD4 and plays a role in apoptosis.
Histone Arg methylation has been correlated with transcriptional activation of p53 target genes. However, whether this modification is reversed to repress the expression of p53 target genes is unclear. Here, we report that peptidylarginine deiminase 4, a histone citrullination enzyme, is involved in the repression of p53 target genes. Inhibition or depletion of PAD4 elevated the expression of a subset of p53 target genes, including p21/CIP1/WAF1, leading to cell cycle arrest and apoptosis. Moreover, the induction of p21, cell cycle arrest, and apoptosis by PAD4 depletion is p53 dependent. Protein-protein interaction studies showed an interaction between p53 and PAD4. Chromatin immunoprecipitation assays showed that PAD4 is recruited to the p21 promoter in a p53-dependent manner. RNA polymerase II (Pol II) activities and the association of PAD4 are dynamically regulated at the p21 promoter during UV irradiation. Paused RNA Pol II and high levels of PAD4 were detected before UV treatment. At early time points after UV treatment, an increase of histone Arg methylation and a decrease of citrullination were correlated with a transient activation of p21. At later times after UV irradiation, a loss of RNA Pol II and an increase of PAD4 were detected at the p21 promoter. The dynamics of RNA Pol II activities after UV treatment were further corroborated by permanganate footprinting. Together, these results suggest a role of PAD4 in the regulation of p53 target gene expression.
Protein arginine methyltransferases (PRMTs) are SAM-dependent enzymes that catalyze the mono- and dimethylation of peptidyl arginine residues. Although all PRMTs produce monomethyl arginine (MMA), type 1 PRMTs go on to form asymmetrically dimethylated arginine (ADMA), while type 2 enzymes form symmetrically dimethylated arginine (SDMA). PRMT1 is the major type 1 PRMT in vivo, thus it is the primary producer of the competitive NOS inhibitor, ADMA. Hence, potent inhibitors, which are highly selective for this particular isozyme, could serve as excellent therapeutics for heart disease. However, the design of such inhibitors is impeded by a lack of information regarding this enzyme's kinetic and catalytic mechanisms. Herein we report an analysis of the kinetic mechanism of human PRMT1 using both an unmethylated and a monomethylated substrate peptide based on the N-terminus of histone H4. The results of initial velocity and product and dead-end inhibition experiments indicate that PRMT1 utilizes a rapid equilibrium random mechanism with the formation of dead-end EAP and EBQ complexes. This mechanism is gratifyingly consistent with previous results demonstrating that PRMT1 catalyzes substrate dimethylation in a partially processive manner.
Evasion of apoptosis and active cell proliferation are among the characteristics of cancer cells. Triggering the induction of apoptosis or reducing the proliferative rate will potentially be helpful for cancer treatment. Recently, several reports demonstrated that knockdown of the protein acetyltransferase hARD1 significantly reduced the growth rate of human cancer cell lines. Furthermore, hARD1 knockdown induced apoptosis or sensitized cells to drug induced apoptosis. hARD1 acts in complex with the NATH protein and catalyzes cotranslational acetylation of protein N-termini. Thus, it was suggested that the effects on cell proliferation and apoptosis induction are due to a reduced level of N-terminal acetylation of certain substrate proteins. NATH was originally identified as upregulated in thyroid papillary carcinomas and has lately also been found to correlate with aggressiveness and differentiation status of neuroblastic tumours. On the other hand, researchers recently reported that hARD1 acetylates Beta-catenin. Knockdown of hARD1 reduced the transcriptional activity of the Beta-Catenin/TCF4 complex, downregulating cyclin D1 and thereby promoting G1-arrest and inhibition of cell proliferation of lung cancer cells. Although the underlying molecular mechanisms need further clarification, several reports suggest that reduction of hARD1 negatively affects cell growth. Thus, hARD1 or the hARD1-NATH complex stands out as attractive drug targets in cancer treatment. One challenge will be to develop specific inhibitors that discriminate between hARD1 and the many other enzymes, including the histone acetyltransferases, using acetyl-coenzyme A as acetyl donor. This review focuses on the enzymatic and biological activities of hARD1, and potential mechanisms of functional inhibition.
The tomato brassinosteroid receptor BRI1 increases binding of systemin to tobacco plasma membranes, but is not involved in systemin signaling
The tomato wound signal systemin is perceived by a specific high-affinity, saturable, and reversible cell surface receptor. This receptor was identified as the receptor-like kinase SR160, which turned out to be identical to the brassinosteroid receptor BRI1. Recently, it has been shown that the tomato bri1 null mutant cu3 is as sensitive to systemin as wild type plants. Here we explored these contradictory findings by studying the responses of tobacco plants (Nicotiana tabacum) to systemin. A fluorescently-labeled systemin analog bound specifically to plasma membranes of tobacco suspension-cultured cells that expressed the tomato BRI1-FLAG transgene, but not to wild type tobacco cells. On the other hand, signaling responses to systemin, such as activation of mitogen-activated protein kinases and medium alkalinization, were neither increased in BRI1-FLAG-overexpressing tobacco cells nor decreased in BRI1-silenced cells as compared to levels in untransformed control cells. Furthermore, in transgenic tobacco plants BRI1-FLAG became phosphorylated on threonine residues in response to brassinolide application, but not in response to systemin. When BRI1 transcript levels were reduced by virus-induced gene silencing in tomato plants, the silenced plants displayed a phenotype characteristic of bri1 mutants. However, their response to overexpression of the Prosystemin transgene was the same as in control plants. Taken together, our data suggest that BRI1 can function as a systemin binding protein, but that binding of the ligand does not transduce the signal into the cell. This unusual behavior and the nature of the elusive systemin receptor will be discussed.
The protein arginine deiminases (PADs), and in particular PAD4, have emerged as potential therapeutic targets for the treatment of rheumatoid arthritis (RA). In this review, evidence linking dysregulated PAD activity to the onset and progression of RA is presented, and the potential role of such aberrant activity in other human diseases, such as multiple sclerosis and cancer, is discussed. The known physiological roles of the PADs, particularly PAD4, and current knowledge regarding PAD structure, catalysis and inhibition are also described.
Haloacetamidine-based inactivators of protein arginine deiminase 4 (PAD4): evidence that general acid catalysis promotes efficient inactivation
Dysregulated protein arginine deiminase (PAD) activity, particularly PAD4, has been suggested to play a role in the onset and progression of numerous human diseases, including rheumatoid arthritis (RA). Given the potential role of PAD4 in RA, we set out to develop inhibitors/inactivators that could be used to modulate PAD activity and disease progression. This effort led to the discovery of two mechanism-based inactivators, denoted F- and Cl-amidine, that inactivate PAD4 by the covalent modification of an active-site cysteine that is critical for catalysis. To gain further insights into the mechanism of inactivation by these compounds, the effect of pH on the rates of inactivation was determined. These results, combined with the results of solvent isotope effect and proton inventory studies, strongly suggest that the inactivation of PAD4 by F- and Cl-amidine proceeds by a multistep mechanism that involves the protonation and stabilization of the tetrahedral intermediate formed upon nucleophilic attack by the active-site cysteine, that is, Cys645. Stabilization of this intermediate would help to drive the halide-displacement reaction, which results in the formation of a three-membered sulfonium ring that ultimately collapses to form the inactivated enzyme. This finding-that protonation of the tetrahedral intermediate is important for enzyme inactivation-also suggests that, during catalysis, protonation of the analogous intermediate is required for efficient substrate turnover.
Helicobacter pylori encodes a potential virulence factor, agmatine deiminase (HpAgD), which catalyzes the conversion of agmatine to N-carbamoyl putrescine (NCP) and ammonia - agmatine is decarboxylated arginine. Agmatine is an endogenous human cell signaling molecule that triggers the innate immune response in humans. Unlike H. pylori, humans do not encode an AgD; it is hypothesized that inhibition of this enzyme would increase the levels of agmatine, and thereby enhance the innate immune response. Taken together, these facts suggest that HpAgD is a potential drug target. Herein we describe the optimized expression, isolation, and purification of HpAgD (10-30 mg/L media). The initial kinetic characterization of this enzyme has also been performed. Additionally, the crystal structure of wild-type HpAgD has been determined at 2.1A resolution. This structure provides a molecular basis for the preferential deimination of agmatine, and identifies Asp198 as a key residue responsible for agmatine recognition, which has been confirmed experimentally. Information gathered from these studies led to the development and characterization of a novel class of haloacetamidine-based HpAgD inactivators. These compounds are the most potent AgD inhibitors ever described.
Substrate specificity and kinetic studies of PADs 1, 3, and 4 identify potent and selective inhibitors of protein arginine deiminase 3
Protein citrullination has been shown to regulate numerous physiological pathways (e.g., the innate immune response and gene transcription) and is, when dysregulated, known to be associated with numerous human diseases, including cancer, rheumatoid arthritis, and multiple sclerosis. This modification, also termed deimination, is catalyzed by a group of enzymes called the protein arginine deiminases (PADs). In mammals, there are five PAD family members (i.e., PADs 1, 2, 3, 4, and 6) that exhibit tissue-specific expression patterns and vary in their subcellular localization. The kinetic characterization of PAD4 was recently reported, and these efforts guided the development of the two most potent PAD4 inhibitors (i.e., F- and Cl-amidine) known to date. In addition to being potent PAD4 inhibitors, we show here that Cl-amidine also exhibits a strong inhibitory effect against PADs 1 and 3, thus indicating its utility as a pan PAD inhibitor. Given the increasing number of diseases in which dysregulated PAD activity has been implicated, the development of PAD-selective inhibitors is of paramount importance. To aid that goal, we characterized the catalytic mechanism and substrate specificity of PADs 1 and 3. Herein, we report the results of these studies, which suggest that, like PAD4, PADs 1 and 3 employ a reverse protonation mechanism. Additionally, the substrate specificity studies provided critical information that aided the identification of PAD3-selective inhibitors. These compounds, denoted F4- and Cl4-amidine, are the most potent PAD3 inhibitors ever described.
Protein Arginine Deiminase (PAD) activity is dysregulated in numerous diseases, e.g., Rheumatoid Arthritis. Herein we describe the development of a fluorescence polarization-Activity Based Protein Profiling (fluopol-ABPP) based high throughput screening assay that can be used to identify PAD-selective inhibitors. Using this assay, streptonigrin was identified as a potent, selective, and irreversible PAD4 inactivator.
One subfamily of guanidino group-modifying enzymes (GMEs) consists of the agmatine deiminases (AgDs). These enzymes catalyze the conversion of agmatine (decarboxylated arginine) to N-carbamoyl putrescine and ammonia. In plants, viruses, and bacteria, these enzymes are thought to be involved in energy production, biosynthesis of polyamines, and biofilm formation. In particular, we are interested in the role that this enzyme plays in pathogenic bacteria. Previously, we reported the initial kinetic characterization of the agmatine deiminase from Helicobacter pylori and described the synthesis and characterization the two most potent AgD inactivators. Herein, we have expanded our initial efforts to characterize the catalytic mechanisms of AgD from H. pylori as well as Streptococcus mutans and Porphyromonas gingivalis. Through the use of pH rate profiles, pK(a) measurements of the active site cysteine, solvent isotope effects, and solvent viscosity effects, we have determined that the AgDs, like PADs 1 and 4, utilize a reverse protonation mechanism.
A combinatorial approach to characterize the substrate specificity of protein arginine methyltransferase 1
The dysregulation of protein arginine methyltransferases (PRMTs) is implicated in a wide variety of disease states. Here we report the design, synthesis, and screening of a combinatorial peptide library used to characterize the substrate specificity of PRMT1. The information gained from this approach was used to develop a PRMT1 inhibitor with enhanced selectivity.
The recent approvals of anticancer therapeutic agents targeting the histone deacetylases and DNA methyltransferases have highlighted the important role that epigenetics plays in human diseases, and suggested that the factors controlling gene expression are novel drug targets. Protein arginine deiminase 4 (PAD4) is one such target because its effects on gene expression parallel those observed for the histone deacetylases. We demonstrated that F- and Cl-amidine, two potent PAD4 inhibitors, display micromolar cytotoxic effects towards several cancerous cell lines (HL-60, MCF7 and HT-29); no effect was observed in noncancerous lines (NIH 3T3 and HL-60 granulocytes). These compounds also induced the differentiation of HL-60 and HT29 cells. Finally, these compounds synergistically potentiated the cell killing effects of doxorubicin. Taken together, these findings suggest PAD4 inhibition as a novel epigenetic approach for the treatment of cancer, and suggest that F- and Cl-amidine are candidate therapeutic agents for this disease.