The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Steven Clarke - One of the best experts on this subject based on the ideXlab platform.
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prmt7 as a unique member of the protein arginine methyltransferase family a review
Archives of Biochemistry and Biophysics, 2019Co-Authors: Kanishk Jain, Steven ClarkeAbstract:Abstract Protein arginine methyltransferases (PRMTs) are found in a wide variety of eukaryotic organisms and can regulate gene expression, DNA repair, RNA splicing, and stem cell biology. In mammalian cells, nine genes encode a family of sequence-related enzymes; six of these PRMTs catalyze the formation of ω-asymmetric dimethyl derivatives, two catalyze ω-symmetric dimethyl derivatives, and only one (PRMT7) solely catalyzes ω-monomethylarginine formation. Purified recombinant PRMT7 displays a number of unique enzymatic properties including a substrate preference for arginine residues in R-X-R motifs with additional flanking basic amino acid residues and a temperature optimum well below 37 °C. Evidence has been presented for crosstalk between PRMT7 and PRMT5, where methylation of a histone H4 peptide at R17, a PRMT7 substrate, may activate PRMT5 for methylation of R3. Defects in muscle stem cells (satellite cells) and immune cells are found in mouse Prmt7 homozygous knockouts, while humans lacking PRMT7 are characterized by significant intellectual developmental delays, hypotonia, and facial dysmorphisms. The overexpression of the PRMT7 gene has been correlated with cancer metastasis in humans. Current research challenges include identifying cellular factors that control PRMT7 expression and activity, identifying the physiological substrates of PRMT7, and determining the effect of methylation on these substrates.
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epigenetic control via allosteric regulation of mammalian protein arginine methyltransferases
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Kanishk Jain, Cyrus Y Jin, Steven ClarkeAbstract:Arginine methylation on histones is a central player in epigenetics and in gene activation and repression. Protein arginine methyltransferase (PRMT) activity has been implicated in stem cell pluripotency, cancer metastasis, and tumorigenesis. The expression of one of the nine mammalian PRMTs, PRMT5, affects the levels of symmetric dimethylarginine (SDMA) at Arg-3 on histone H4, leading to the repression of genes which are related to disease progression in lymphoma and leukemia. Another PRMT, PRMT7, also affects SDMA levels at the same site despite its unique monomethylating activity and the lack of any evidence for PRMT7-catalyzed histone H4 Arg-3 methylation. We present evidence that PRMT7-mediated monomethylation of histone H4 Arg-17 regulates PRMT5 activity at Arg-3 in the same protein. We analyzed the kinetics of PRMT5 over a wide range of substrate concentrations. Significantly, we discovered that PRMT5 displays positive cooperativity in vitro, suggesting that this enzyme may be allosterically regulated in vivo as well. Most interestingly, monomethylation at Arg-17 in histone H4 not only raised the general activity of PRMT5 with this substrate, but also ameliorated the low activity of PRMT5 at low substrate concentrations. These kinetic studies suggest a biochemical explanation for the interplay between PRMT5- and PRMT7-mediated methylation of the same substrate at different residues and also suggest a general model for regulation of PRMTs. Elucidating the exact relationship between these two enzymes when they methylate two distinct sites of the same substrate may aid in developing therapeutics aimed at reducing PRMT5/7 activity in cancer and other diseases.
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the major protein arginine methyltransferase in trypanosoma brucei functions as an enzyme prozyme complex
Journal of Biological Chemistry, 2017Co-Authors: Lucie Kafkova, Steven Clarke, Erik W Debler, Kanishk Jain, John C Fisk, Laurie K ReadAbstract:Prozymes are catalytically inactive enzyme paralogs that dramatically stimulate the function of weakly active enzymes through complex formation. The two prozymes described to date reside in the polyamine biosynthesis pathway of the human parasite Trypanosoma brucei, an early branching eukaryote that lacks transcriptional regulation and regulates its proteome through posttranscriptional and posttranslational means. Arginine methylation is a common posttranslational modification in eukaryotes catalyzed by protein arginine methyltransferases (PRMTs) that are typically thought to function as homodimers. We demonstrate that a major T. brucei PRMT, TbPRMT1, functions as a heterotetrameric enzyme-prozyme pair. The inactive PRMT paralog, TbPRMT1PRO, is essential for catalytic activity of the TbPRMT1ENZ subunit. Mutational analysis definitively demonstrates that TbPRMT1ENZ is the cofactor-binding subunit and carries all catalytic activity of the complex. Our results are the first demonstration of an obligate heteromeric PRMT, and they suggest that enzyme-prozyme organization is expanded in trypanosomes as a posttranslational means of enzyme regulation.
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protein arginine methyltransferase product specificity is mediated by distinct active site architectures
Journal of Biological Chemistry, 2016Co-Authors: Kanishk Jain, Andrea Hadjikyriacou, Erik W Debler, Rebeccah A Warmack, Peter Stavropoulos, Steven ClarkeAbstract:In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either asymmetric or symmetric dimethylarginine (SDMA), PRMT7 is unique in producing solely monomethylarginine (MMA) products. The type of methylation on histones and other proteins dictates changes in gene expression, and numerous studies have linked altered profiles of methyl marks with disease phenotypes. Given the importance of specific inhibitor development, it is crucial to understand the mechanisms by which PRMT product specificity is conferred. We have focused our attention on active-site residues of PRMT7 from the protozoan Trypanosoma brucei. We have designed 26 single and double mutations in the active site, including residues in the Glu-Xaa8-Glu (double E) loop and the Met-Gln-Trp sequence of the canonical Thr-His-Trp (THW) loop known to interact with the methyl-accepting substrate arginine. Analysis of the reaction products by high resolution cation exchange chromatography combined with the knowledge of PRMT crystal structures suggests a model where the size of two distinct subregions in the active site determines PRMT7 product specificity. A dual mutation of Glu-181 to Asp in the double E loop and Gln-329 to Ala in the canonical THW loop enables the enzyme to produce SDMA. Consistent with our model, the mutation of Cys-431 to His in the THW loop of human PRMT9 shifts its product specificity from SDMA toward MMA. Together with previous results, these findings provide a structural basis and a general model for product specificity in PRMTs, which will be useful for the rational design of specific PRMT inhibitors.
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a glutamate aspartate switch controls product specificity in a protein arginine methyltransferase
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Erik W Debler, Steven Clarke, You Feng, Kanishk Jain, Rebeccah A Warmack, Gunter Blobel, Pete StavropoulosAbstract:Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 A resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.
Mark T Bedford - One of the best experts on this subject based on the ideXlab platform.
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the role of the prmt5 snd1 axis in hepatocellular carcinoma
Epigenomes, 2021Co-Authors: Tanner Wright, Yalong Wang, Mark T BedfordAbstract:Arginine methylation is an essential post-translational modification (PTM) deposited by protein arginine methyltransferases (PRMTs) and recognized by Tudor domain-containing proteins. Of the nine mammalian PRMTs, PRMT5 is the primary enzyme responsible for the deposition of symmetric arginine methylation marks in cells. The staphylococcal nuclease and Tudor domain-containing 1 (SND1) effector protein is a key reader of the marks deposited by PRMT5. Both PRMT5 and SND1 are broadly expressed and their deregulation is reported to be associated with a range of disease phenotypes, including cancer. Hepatocellular carcinoma (HCC) is an example of a cancer type that often displays elevated PRMT5 and SND1 levels, and there is evidence that hyperactivation of this axis is oncogenic. Importantly, this pathway can be tempered with small-molecule inhibitors that target PRMT5, offering a therapeutic node for cancer, such as HCC, that display high PRMT5–SND1 axis activity. Here we summarize the known activities of this writer–reader pair, with a focus on their biological roles in HCC. This will help establish a foundation for treating HCC with PRMT5 inhibitors and also identify potential biomarkers that could predict sensitivity to this type of therapy.
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using oriented peptide array libraries to evaluate methylarginine specific antibodies and arginine methyltransferase substrate motifs
Scientific Reports, 2016Co-Authors: Sitaram Gayatri, Martis W Cowles, Vidyasiri Vemulapalli, Donghang Cheng, Mark T BedfordAbstract:Signal transduction in response to stimuli relies on the generation of cascades of posttranslational modifications that promote protein-protein interactions and facilitate the assembly of distinct signaling complexes. Arginine methylation is one such modification, which is catalyzed by a family of nine protein arginine methyltransferases, or PRMTs. Elucidating the substrate specificity of each PRMT will promote a better understanding of which signaling networks these enzymes contribute to. Although many PRMT substrates have been identified, and their methylation sites mapped, the optimal target motif for each of the nine PRMTs has not been systematically addressed. Here we describe the use of Oriented Peptide Array Libraries (OPALs) to methodically dissect the preferred methylation motifs for three of these enzymes – PRMT1, CARM1 and PRMT9. In parallel, we show that an OPAL platform with a fixed methylarginine residue can be used to validate the methyl-specific and sequence-specific properties of antibodies that have been generated against different PRMT substrates, and can also be used to confirm the pan nature of some methylarginine-specific antibodies.
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unique features of human protein arginine methyltransferase 9 prmt9 and its substrate rna splicing factor sf3b2
Journal of Biological Chemistry, 2015Co-Authors: Andrea Hadjikyriacou, Mark T Bedford, Yanzhong Yang, Alexsandra Espejo, Steven ClarkeAbstract:Abstract Human protein arginine methyltransferase 9 (PRMT9) symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. While amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides.
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unique features of human protein arginine methyltransferase 9 prmt9 and its substrate rna splicing factor sf3b2
Journal of Biological Chemistry, 2015Co-Authors: Andrea Hadjikyriacou, Mark T Bedford, Yanzhong Yang, Alexsandra Espejo, Steven ClarkeAbstract:Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides.
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Ribosomal protein rpS2 is hypomethylated in PRMT3-deficient mice
The Journal of biological chemistry, 2007Co-Authors: Rafal Swiercz, Donghang Cheng, Daehoon Kim, Mark T BedfordAbstract:PRMT3 is a type I arginine methyltransferase that resides in the cytoplasm. A large proportion of this cystosolic PRMT3 is found associated with ribosomes. It is tethered to the ribosomes through its interaction with rpS2, which is also its substrate. Here we show that mouse embryos with a targeted disruption of PRMT3 are small in size but survive after birth and attain a normal size in adulthood, thus displaying Minute-like characteristics. The ribosome protein rpS2 is hypomethylated in the absence of PRMT3, demonstrating that it is a bona fide, in vivo PRMT3 substrate that cannot be modified by other PRMTs. Finally, the levels 40 S, 60 S, and 80 S monosomes and polyribosomes are unaffected by the loss of PRMT3, but there are additional as yet unidentified proteins that co-fractionate with ribosomes that are also dedicated PRMT3 substrates.
Kanishk Jain - One of the best experts on this subject based on the ideXlab platform.
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prmt7 as a unique member of the protein arginine methyltransferase family a review
Archives of Biochemistry and Biophysics, 2019Co-Authors: Kanishk Jain, Steven ClarkeAbstract:Abstract Protein arginine methyltransferases (PRMTs) are found in a wide variety of eukaryotic organisms and can regulate gene expression, DNA repair, RNA splicing, and stem cell biology. In mammalian cells, nine genes encode a family of sequence-related enzymes; six of these PRMTs catalyze the formation of ω-asymmetric dimethyl derivatives, two catalyze ω-symmetric dimethyl derivatives, and only one (PRMT7) solely catalyzes ω-monomethylarginine formation. Purified recombinant PRMT7 displays a number of unique enzymatic properties including a substrate preference for arginine residues in R-X-R motifs with additional flanking basic amino acid residues and a temperature optimum well below 37 °C. Evidence has been presented for crosstalk between PRMT7 and PRMT5, where methylation of a histone H4 peptide at R17, a PRMT7 substrate, may activate PRMT5 for methylation of R3. Defects in muscle stem cells (satellite cells) and immune cells are found in mouse Prmt7 homozygous knockouts, while humans lacking PRMT7 are characterized by significant intellectual developmental delays, hypotonia, and facial dysmorphisms. The overexpression of the PRMT7 gene has been correlated with cancer metastasis in humans. Current research challenges include identifying cellular factors that control PRMT7 expression and activity, identifying the physiological substrates of PRMT7, and determining the effect of methylation on these substrates.
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epigenetic control via allosteric regulation of mammalian protein arginine methyltransferases
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Kanishk Jain, Cyrus Y Jin, Steven ClarkeAbstract:Arginine methylation on histones is a central player in epigenetics and in gene activation and repression. Protein arginine methyltransferase (PRMT) activity has been implicated in stem cell pluripotency, cancer metastasis, and tumorigenesis. The expression of one of the nine mammalian PRMTs, PRMT5, affects the levels of symmetric dimethylarginine (SDMA) at Arg-3 on histone H4, leading to the repression of genes which are related to disease progression in lymphoma and leukemia. Another PRMT, PRMT7, also affects SDMA levels at the same site despite its unique monomethylating activity and the lack of any evidence for PRMT7-catalyzed histone H4 Arg-3 methylation. We present evidence that PRMT7-mediated monomethylation of histone H4 Arg-17 regulates PRMT5 activity at Arg-3 in the same protein. We analyzed the kinetics of PRMT5 over a wide range of substrate concentrations. Significantly, we discovered that PRMT5 displays positive cooperativity in vitro, suggesting that this enzyme may be allosterically regulated in vivo as well. Most interestingly, monomethylation at Arg-17 in histone H4 not only raised the general activity of PRMT5 with this substrate, but also ameliorated the low activity of PRMT5 at low substrate concentrations. These kinetic studies suggest a biochemical explanation for the interplay between PRMT5- and PRMT7-mediated methylation of the same substrate at different residues and also suggest a general model for regulation of PRMTs. Elucidating the exact relationship between these two enzymes when they methylate two distinct sites of the same substrate may aid in developing therapeutics aimed at reducing PRMT5/7 activity in cancer and other diseases.
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the major protein arginine methyltransferase in trypanosoma brucei functions as an enzyme prozyme complex
Journal of Biological Chemistry, 2017Co-Authors: Lucie Kafkova, Steven Clarke, Erik W Debler, Kanishk Jain, John C Fisk, Laurie K ReadAbstract:Prozymes are catalytically inactive enzyme paralogs that dramatically stimulate the function of weakly active enzymes through complex formation. The two prozymes described to date reside in the polyamine biosynthesis pathway of the human parasite Trypanosoma brucei, an early branching eukaryote that lacks transcriptional regulation and regulates its proteome through posttranscriptional and posttranslational means. Arginine methylation is a common posttranslational modification in eukaryotes catalyzed by protein arginine methyltransferases (PRMTs) that are typically thought to function as homodimers. We demonstrate that a major T. brucei PRMT, TbPRMT1, functions as a heterotetrameric enzyme-prozyme pair. The inactive PRMT paralog, TbPRMT1PRO, is essential for catalytic activity of the TbPRMT1ENZ subunit. Mutational analysis definitively demonstrates that TbPRMT1ENZ is the cofactor-binding subunit and carries all catalytic activity of the complex. Our results are the first demonstration of an obligate heteromeric PRMT, and they suggest that enzyme-prozyme organization is expanded in trypanosomes as a posttranslational means of enzyme regulation.
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protein arginine methyltransferase product specificity is mediated by distinct active site architectures
Journal of Biological Chemistry, 2016Co-Authors: Kanishk Jain, Andrea Hadjikyriacou, Erik W Debler, Rebeccah A Warmack, Peter Stavropoulos, Steven ClarkeAbstract:In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either asymmetric or symmetric dimethylarginine (SDMA), PRMT7 is unique in producing solely monomethylarginine (MMA) products. The type of methylation on histones and other proteins dictates changes in gene expression, and numerous studies have linked altered profiles of methyl marks with disease phenotypes. Given the importance of specific inhibitor development, it is crucial to understand the mechanisms by which PRMT product specificity is conferred. We have focused our attention on active-site residues of PRMT7 from the protozoan Trypanosoma brucei. We have designed 26 single and double mutations in the active site, including residues in the Glu-Xaa8-Glu (double E) loop and the Met-Gln-Trp sequence of the canonical Thr-His-Trp (THW) loop known to interact with the methyl-accepting substrate arginine. Analysis of the reaction products by high resolution cation exchange chromatography combined with the knowledge of PRMT crystal structures suggests a model where the size of two distinct subregions in the active site determines PRMT7 product specificity. A dual mutation of Glu-181 to Asp in the double E loop and Gln-329 to Ala in the canonical THW loop enables the enzyme to produce SDMA. Consistent with our model, the mutation of Cys-431 to His in the THW loop of human PRMT9 shifts its product specificity from SDMA toward MMA. Together with previous results, these findings provide a structural basis and a general model for product specificity in PRMTs, which will be useful for the rational design of specific PRMT inhibitors.
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a glutamate aspartate switch controls product specificity in a protein arginine methyltransferase
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Erik W Debler, Steven Clarke, You Feng, Kanishk Jain, Rebeccah A Warmack, Gunter Blobel, Pete StavropoulosAbstract:Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 A resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.
Adam Frankel - One of the best experts on this subject based on the ideXlab platform.
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the application of differential scanning fluorimetry in exploring bisubstrate binding to protein arginine n methyltransferase 1
Methods, 2020Co-Authors: Jennifer I Brown, Brent D G Page, Adam FrankelAbstract:Abstract Protein arginine N-methyltransferases (PRMTs) are a family of 9 enzymes that catalyze mono- or di-methylation of arginine residues using S-adenosyl- l -methionine (SAM). Arginine methylation is an important post-translational modification that can regulate the activity and structure of target proteins. Altered PRMT activity can lead to a variety of health issues including neurodevelopmental disease, autoimmune disorders, cancer, and cardiovascular disease. Thus, developing a robust mechanistic understanding of PRMT function may provide insight into these various disease states and enable the development of potential therapeutic agents. Although PRMTs have been studied for nearly two decades, a consensus regarding the mechanism of action for this class of enzymes has remained noticeably elusive. To address this shortcoming, differential scanning fluorimetry (DSF) was used to gain mechanistic insight into the order of PRMT substrate and cofactor binding. This methodology confirms that PRMT cofactor binding precedes target substrate binding and supports the use of DSF to study bisubstrate enzymatic reaction mechanisms.
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kinetic analysis of human protein arginine n methyltransferase 2 formation of monomethyl and asymmetric dimethyl arginine residues on histone h4
Biochemical Journal, 2009Co-Authors: Ted M Lakowski, Adam FrankelAbstract:Protein arginine N-methyltransferases (PRMTs) methylate arginine residues within proteins using S -adenosyl-L-methionine (AdoMet) to form S -adenosyl-L-homocysteine and methylarginine residues. All PRMTs produce ω- N G -monomethylarginine (MMA) residues and either asymmetric ω- N G , N G -dimethylarginine (aDMA) or symmetric ω- N G , N ′ G -dimethylarginine (sDMA) residues, referred to as Type I or Type II activity respectively. Here we report methylation activity from PRMT2 and compare it with PRMT1 activity using UPLC-MS/MS (ultra-performance liquid chromatography–tandem MS), gel electrophoresis, and thin-layer chromatography. We show that PRMT2 is a Type I enzyme and that the ratio of aDMA to MMA produced by PRMTs 1 and 2 is dependent on the substrate, regardless of rate or K m , suggesting that the reactions for both enzymes are distributive rather than processive. Using UPLC-MS/MS we find that, for PRMT2, the dissociation constant ( K A s ) and K m of AdoMet and the K m of histone H4 are similar to values for PRMT1, whereas the PRMT2 k cat is 800-fold less than the PRMT1 k cat . Although PRMT2 activity is substantially lower than PRMT1 in vitro , the fact that both enzymes selectively methylate histone H4 suggest that PRMT2, like PRMT1, may act as a transcription co-activator through this modification.
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prmt7 is a member of the protein arginine methyltransferase family with a distinct substrate specificity
Journal of Biological Chemistry, 2004Co-Authors: Tina B Miranda, Adam Frankel, Mark Miranda, Steven ClarkeAbstract:We have identified a mammalian arginine N-methyltransferase, PRMT7, that can catalyze the formation of ω-N^G-monomethylarginine in peptides. This protein is encoded by a gene on human chromosome 16q22.1 (human locus AK001502). We expressed a full-length human cDNA construct in Escherichia coli as a glutathione S-transferase (GST) fusion protein. We found that GST-tagged PRMT7 catalyzes the S-adenosyl-[methyl-^3H]-L-methionine-dependent methylation of the synthetic peptide GGPGGRGGPGG-NH_2 (R1). The radiolabeled peptide was purified by high-pressure liquid chromatography and acid hydrolyzed to free amino acids. When the hydrolyzed products were separated by high-resolution cation-exchange chromatography, we were able to detect one tritiated species which co-migrated with an ω-N^G-monomethylarginine standard. Surprisingly, GST-PRMT7 was not able to catalyze the in vitro methylation of a GST-fibrillarin (amino acids 1–148) fusion protein (GST-GAR), a methyl-accepting substrate for the previously characterized PRMT1, PRMT3, PRMT4, PRMT5, and PRMT6 enzymes. Nor was it able to methylate myelin basic protein or histone H2A, in vitro substrates of PRMT5. This specificity distinguishes PRMT7 from all of the other known arginine methyltransferases. An additional unique feature of PRMT7 is that it seems to have arisen from a gene duplication event and contains two putative AdoMet-binding motifs. To see if both motifs were necessary for activity, each putative domain was expressed as a GST-fusion and tested for activity with peptides R1 and R2 (acetyl-GGRGG-NH_2). These truncated proteins were enzymatically inactive, suggesting that both domains are required for functionality.
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the novel human protein arginine n methyltransferase prmt6 is a nuclear enzyme displaying unique substrate specificity
Journal of Biological Chemistry, 2002Co-Authors: Adam Frankel, Steven Clarke, Neelu Yadav, Jaeho Lee, Tina L Branscombe, Mark T BedfordAbstract:Protein arginine methylation is a prevalent posttranslational modification in eukaryotic cells that has been implicated in signal transduction, the metabolism of nascent pre-RNA, and the transcriptional activation processes. In searching the human genome for protein arginine N-methyltransferase (PRMT) family members, a novel gene has been found on chromosome 1 that encodes for an apparent methyltransferase, PRMT6. The polypeptide chain of PRMT6 is 41.9 kDa consisting of a catalytic core sequence common to other PRMT enzymes. Expressed as a glutathione S-transferase fusion protein, PRMT6 demonstrates type I PRMT activity, capable of forming both omega-N(G)-monomethylarginine and asymmetric omega-N(G),N(G)-dimethylarginine derivatives on the recombinant glycine- and arginine-rich substrate in a processive manner with a specific activity of 144 pmol methyl groups transferred min(-1) mg(-1) enzyme. A comparison of substrate specificity reveals that PRMT6 is functionally distinct from two previously characterized type I enzymes, PRMT1 and PRMT4. In addition, PRMT6 displays automethylation activity; it is the first PRMT to do so. This novel human PRMT, which resides solely in the nucleus when fused to the green fluorescent protein, joins a family of enzymes with diverse functions within cells.
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PRMT3 is a distinct member of the protein arginine n methyltransferase family conferral of substrate specificity by a zinc finger domain
Journal of Biological Chemistry, 2000Co-Authors: Adam Frankel, Steven ClarkeAbstract:Abstract S-Adenosyl-l-methionine-dependent protein arginine N-methyltransferases (PRMTs) catalyze the methylation of arginine residues within a variety of proteins. At least four distinct mammalian family members have now been described, including PRMT1, PRMT3, CARM1/PRMT4, and JBP1/PRMT5. To more fully define the physiological role of PRMT3, we characterized its unique putative zinc-finger domain and how it can affect its enzymatic activity. Here we show that PRMT3 does contain a single zinc-finger domain in its amino terminus. Although the zinc-liganded form of this domain is not required for methylation of an artificial substrate such as the glutathione S-transferase-fibrillarin amino-terminal fusion protein (GST-GAR), it is required for the enzyme to recognize RNA-associated substrates in RAT1 cell extracts. The recombinant form of PRMT3 is inhibited by high concentrations of ZnCl2 as well as N-ethylmaleimide, reagents that can modify cysteine sulfhydryl groups. We found that we could distinguish PRMT family members by their sensitivity to these reagents; JBP1/PRMT5 and Hsl7 methyltransferases were inhibited in a similar manner as PRMT3, whereas Rmt1, PRMT1, and CARM1/PRMT4 were not affected. We were also able to define differences in these enzymes by their sensitivity to inhibition by Tris and free arginine. Finally, we found that the treatment of RAT1 cell extracts with N-ethylmaleimide leads to a loss of the major PRMT1-associated activity that was immune to inhibition under the same conditions as a GST fusion protein. These results suggest that native forms of PRMTs can have different properties than their GST-catalytic chain fusion protein counterparts, which may lack associated noncatalytic subunits.
Andrea Hadjikyriacou - One of the best experts on this subject based on the ideXlab platform.
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protein arginine methyltransferase product specificity is mediated by distinct active site architectures
Journal of Biological Chemistry, 2016Co-Authors: Kanishk Jain, Andrea Hadjikyriacou, Erik W Debler, Rebeccah A Warmack, Peter Stavropoulos, Steven ClarkeAbstract:In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either asymmetric or symmetric dimethylarginine (SDMA), PRMT7 is unique in producing solely monomethylarginine (MMA) products. The type of methylation on histones and other proteins dictates changes in gene expression, and numerous studies have linked altered profiles of methyl marks with disease phenotypes. Given the importance of specific inhibitor development, it is crucial to understand the mechanisms by which PRMT product specificity is conferred. We have focused our attention on active-site residues of PRMT7 from the protozoan Trypanosoma brucei. We have designed 26 single and double mutations in the active site, including residues in the Glu-Xaa8-Glu (double E) loop and the Met-Gln-Trp sequence of the canonical Thr-His-Trp (THW) loop known to interact with the methyl-accepting substrate arginine. Analysis of the reaction products by high resolution cation exchange chromatography combined with the knowledge of PRMT crystal structures suggests a model where the size of two distinct subregions in the active site determines PRMT7 product specificity. A dual mutation of Glu-181 to Asp in the double E loop and Gln-329 to Ala in the canonical THW loop enables the enzyme to produce SDMA. Consistent with our model, the mutation of Cys-431 to His in the THW loop of human PRMT9 shifts its product specificity from SDMA toward MMA. Together with previous results, these findings provide a structural basis and a general model for product specificity in PRMTs, which will be useful for the rational design of specific PRMT inhibitors.
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unique features of human protein arginine methyltransferase 9 prmt9 and its substrate rna splicing factor sf3b2
Journal of Biological Chemistry, 2015Co-Authors: Andrea Hadjikyriacou, Mark T Bedford, Yanzhong Yang, Alexsandra Espejo, Steven ClarkeAbstract:Abstract Human protein arginine methyltransferase 9 (PRMT9) symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. While amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides.
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unique features of human protein arginine methyltransferase 9 prmt9 and its substrate rna splicing factor sf3b2
Journal of Biological Chemistry, 2015Co-Authors: Andrea Hadjikyriacou, Mark T Bedford, Yanzhong Yang, Alexsandra Espejo, Steven ClarkeAbstract:Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides.
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prmt9 is a type ii methyltransferase that methylates the splicing factor sap145
Nature Communications, 2015Co-Authors: Sitaram Gayatri, Andrea Hadjikyriacou, Yanzhong Yang, Daehoon Kim, Cecilia Zuritalopez, Zheng Xia, Ryan L Kelly, Ailan Guo, Steven ClarkeAbstract:The human genome encodes a family of nine protein arginine methyltransferases (PRMT1-9), whose members can catalyse three distinct types of methylation on arginine residues. Here we identify two spliceosome-associated proteins-SAP145 and SAP49-as PRMT9-binding partners, linking PRMT9 to U2 snRNP maturation. We show that SAP145 is methylated by PRMT9 at arginine 508, which takes the form of monomethylated arginine (MMA) and symmetrically dimethylated arginine (SDMA). PRMT9 thus joins PRMT5 as the only mammalian enzymes capable of depositing the SDMA mark. Methylation of SAP145 on Arg 508 generates a binding site for the Tudor domain of the Survival of Motor Neuron (SMN) protein, and RNA-seq analysis reveals gross splicing changes when PRMT9 levels are attenuated. These results identify PRMT9 as a nonhistone methyltransferase that primes the U2 snRNP for interaction with SMN.