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1
Zamiri, Maryam. "Synthesis of protein arginine N-methyltransferase 6 inhibitors." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/43808.
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Protein arginine N-methyltransferases (PRMTs) are pertinent targets for drug discovery as their dysfunction is associated with a number of diseases such as cancers, cardiovascular diseases and viral pathogenesis. The precise role of PRMTs in the initiation, development, or progression of diseases is not known yet.
Due to association of PRMT1 and 4 with transcriptional activation, the main focus of inhibitor discovery has been on these two enzymes. On the other hand, the goal of this study is to find a PRMT6 specific inhibitor. PRMT6 methylates DNA polymerase β, histones H3 and H4 and HIV proteins: Rev and Tat.
PRMT6 uses S-adenosyl-L-methionine (AdoMet) as the “methyl group” source. AdoMet fits into a distinct conserved binding site in the enzyme, which is located adjacent to the protein substrate/catalytic site such that its S⁺-Me motif is correctly positioned with respect to the substrate arginine nitrogen atom that undergoes methylation.
Based on crystallography data for PRMT1, the purine C8 center in AdoMet is in close proximity to the methionine sulfur atom (M166 in PRMT6). As shown by Frankel et al. (Faculty of Pharmaceutical Sciences, UBC), the M166C PRMT6 mutant displays activity. Based upon this observation, we hypothesize that Ado-Met analogues with reactive substituents (e.g., CHO) at C8 position of adenine ring will form a covalent bond with the proximal Cys SH group in M166C PRMT6. This validates our further hypothesize that in appropriately designed analogues, it will be possible to subsequently detach the sugar and amino acid components of Ado-Met to leave the adenine ring component alone bound to the enzyme. This provides a unique opportunity to explore the “fragment based approach in drug discovery” to design PRMT6 specific inhibitors.
2
Hong, Wei. "Design and synthesis of protein arginine methyltransferase inhibitors." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/12835/.
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Biological methylation is defined as the transfer of a methyl group from S-adenosyl-L-methionine(SAM) to one of a wide range of potential acceptors such as DNA, RNA, protein, hormones and neurotransmitters. Protein arginine methylation is a common post-translational modification facilitated by protein arginine methyltransferases(e.g. PRMTI). The roles of these enzymes in vivo are currently poorly understood. The focus of the project is design and synthesis of PRMT inhibitors with the ultimate goal of evaluating their activities in cells. Preliminary work toward the synthesis of S-adenosyl-trifluoromethyl-L-homocystein and adenosyl 5'-[2-(tert-butoxycarbonylamino)ethyl-trifluoro methyl] thiophenium is described. The ternary crystal structure of PRMTI in complex with S-adenoSyl-L-homocystein(eSAH) and an arginine containing peptide (PBD IOR8) was used to design a series of potential bisubstrate inhibitors of PRMTI. The prototypical SAM analogues bearing guanidine group were sought to replace the reactive sulfonium centre with nitrogen. Analogue synthesis proceeded via successive reductive arnination of Y-arnino-Y-deoxyadenosine and deprotection in good overall yields. An alkyne SAM analogue, 5'-[(S-3-amino-3-carboxypropyl)-propargylaminol-5'-deoxyadenosine was prepared, which underwent efficient Cu(1) catalysed Huisgen reaction to yield a triazole derived SAM analogue 5'-[(S-3-amino-3-carboxypropyl)-[I-(2-guanidinoethyl)-IH-1,2,3-triazol-4-yl]methyl-amino]-5'-deoxyadenosine. Preliminary biological evaluation of the compounds by collaborators Professor Steve Ward and Dr Richard Parry at the University of Bath, confirmed that 5'-[(S-3-amino-3-carboxypropyl)- 3-guanidinopropyl-amino]-5'-deoxyadenosine and 5-[(S-3-amino-3-carboxypropyl)-5-guanidinopentyl-amino]-5'-deoxyadenosine are potent inhibitors of PRMTI but not the lysine methyltransferase SET7. A related N-6 modified SAM analogue 5'-[(S-3-amino-3-carboxypropyl)-3-guanidinopropylamino]-5'-deoxy-N6-(lI-azido-3,6,9-trioxaundecane)-amino adenosine bearing an azide tether was developed with the aim of allowing facile introduction of biotin or fluorescent dyes, using either Staudinger ligation, or Cu(1) catalysed Huisgen reaction to provide compounds that can be used for affinity purification of the target protein or study of its localisation in cells respectively. Finally, progress toward a novel, rapid and enantioselective synthesis of the natural product (+)-sinefungin is reported. Key dihydropyridazine intermediates were generated from adenosyl 5'-propaldehyde, commercially available azodicarboxylate derivatives and ester substituted vinyltriphenylphosphonium salt by successful extension of methodology first reported by Ley and co-workers. Deprotection and ring opening of clihydropyridazine compounds was attempted, and unfortunately we were not able to generate (+)-sinefungin, although it is hoped that this route can be developed to achieve this in the future.
3
Pak, Laam. "Insights into a heteromeric protein arginine N-methyltransferase complex." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/42123.
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Protein arginine N-methyltransferases (PRMTs) act in signaling pathways and gene
expression by methylating arginine residues within target proteins. PRMT1 is responsible for
most cellular arginine methylation activity and can work independently or in collaboration with
other PRMTs. In this Ph.D. thesis I demonstrated an interaction between PRMT1 and -2 using
co-immunoprecipitation and bimolecular fluorescence complementation (BiFC). As a result of
this interaction, PRMT2 stimulated PRMT1 methyltransferase activity, affecting its apparent
Vmax and Km values in vitro, and increasing the production of methylarginines in cells. Active
site mutations and regional deletions on PRMT1 and -2 were also investigated, which
demonstrated that complex formation required full-length, active PRMT1. However, the
interaction between PRMT1 and -2 proved insensitive to methylation inhibition in the absence of
the PRMT2 Src homology 3 (SH3) domain, which suggests that the PRMT2 SH3 domain may
mediate this interaction between PRMT1 and -2 in a methylation-dependent fashion.
The role of the PRMT2 SH3 domain was investigated through screening for its associated
proteins using GST-pull down assays followed by LC-MS/MS proteomic analysis. The result of
this study revealed associations of the PRMT2 SH3 domain with at least 29 splicing-related
proteins, suggesting a potential role of PRMT2 in regulating pre-mRNA processing and splicing.
The interaction between PRMT2 and the Src substrate associated in mitosis of 68 kDa (Sam68)
possibly through the PRMT2 SH3 domain was demonstrated using co-immunoprecipitation.
Additionally, immunofluorescence results present herein imply that the PRMT2 SH3 domain
could affect Sam68 sub-cellular localization in hypomethylated HeLa cells. The biological functions of PRMT2 and the PRMT1/2 heteromeric complex were explored
by pursuing the identity of associated proteins common to both PRMT1 and -2 using mass
spectrometry proteomics. Approximately 50% of the identified protein hits have reported roles
in controlling gene expression, while other hits are involved in diverse cellular processes such as
protein folding, degradation, and metabolism. Importantly, three novel PRMT2 binders, p53,
promyelocytic leukemia protein (PML), and extra eleven nineteen (EEN) were uncovered,
suggesting that PRMT2 could participate in regulation of transcription and apoptosis through
PRMT2-protein interactions.
4
Smith, Porsha L. "Protein Arginine Methyltransferase 5 as a Driver of Lymphomagenesis." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1468975682.
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5
May, Kyle M. "Investigation of Protein Dynamics and Communication in Adomet-Dependent Methyltransferases: Non-Ribosomal Peptide Synthetase and Protein Arginine Methyltransferase." DigitalCommons@USU, 2019. https://digitalcommons.usu.edu/etd/7550.
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For many enzymes to function correctly they must have the freedom to display a level of dynamics or communication during their catalytic cycle. The effects that protein dynamics and communication can have are wide ranging, from changes in substrate specificity or product profiles, to speed of reaction or switching activity on or off. This project investigates the protein dynamics and communication in two separate systems, a non-ribosomal peptide synthetase (NRPS), and a protein arginine methyltransferase (PRMT).
PRMT1, the enzyme responsible for 80% of arginine methylation in humans, has been implicated in a variety of disease states when functioning incorrectly. For this reason, much focus has been placed on better understanding how PRMT1 determines which products it creates and at what times. This project aims to shed light on how dynamics and communication within PRMT1 dictate its activity. We have to this point developed a protocol for creating and purifying a linked PRMT1 construct which will enable us to conduct the necessary experiments capable of answering our larger questions about the PRMT1 catalytic mechanism.
Our collaborators in the Zhan lab discovered the presence of a methyltransferase (Mt) in the two NRPS systems they study, which produce two different and medically relevant compounds, bassianolide and beauvericin. The Hevel lab is well suited to study methyltransferases and so were asked to help evaluate the role of these Mt domains and how they affect the production of the relevant natural products. Achieving a more complete understanding of these systems will move us closer toward the “holy grail” of being able to manipulate and harness NRPS systems for the engineering of novel medically relevant compounds. This project has found that the Mt domain substrate specificity is affected by the surrounding protein domains, or even small portions of them.
6
Feng, You. "Kinetic Mechanism and Inhibitory Study of Protein Arginine Methyltransferase 1." Digital Archive @ GSU, 2012. http://digitalarchive.gsu.edu/chemistry_diss/68.
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Protein arginine methyltransferase 1 (PRMT1) is a key posttranslational modification enzyme that catalyzes the methylation of specific arginine residues in histone and nonhistone protein substrates, regulating diverse cellular processes such as transcriptional initiation, RNA splicing, DNA repair, and signal transduction. Recently the essential roles of PRMT1 in cancer and cardiovascular complications have intrigued much attention. Developing effective PRMT inhibitors therefore is of significant therapeutic value. The research on PRMT inhibitor development however is greatly hindered by poor understanding of the biochemical basis of protein arginine methylation and lack of effective assays for PRMT1 inhibitor screening.
Herein, we report our effort in the kinetic mechanism study as well as the fluorescent probe and inhibitor development for PRMT1. New fluorescent reporters were designed and applied to perform single-step analysis of substrate binding and methylation of PRMT1. Using these reporters, we performed transient-state fluorescence measurements to dissect the rate constants along the PRMT1 catalytic coordinate. The data give evidence that the chemistry of methyl transfer is the major rate-limiting step, and that binding of the cofactor SAM or SAH affects the association and dissociation of H4 with PRMT1. Importantly, we identified a critical kinetic step suggesting a precatalytic conformational transition induced by substrate binding. On the other hand, we discovered a type of naphthyl-sulfo (NS) compounds that block PRMT1- mediated arginine methylation at micromolar potency through a unique mechanism: they directly target the substrates but not PRMT enzymes for the observed inhibition. We also found that suramin, an anti-parasite and anti-cancer drug bearing similar functional groups, effectively inhibited PRMT1 mediated methylation. These findings about novel PRMT inhibitors and their unique inhibition mechanism provide a new way for chemical regulation of protein arginine methylation. Addionally, to dissect the interplaying relationship between different histone modification marks, we investigated how individual lysine acetylations and their different combinations at the H4 tail affect Arg-3 methylation in cis. Our data reveal that the effect of lysine acetylation on arginine methylation depends on the site of acetylation and the type of methylation. While certain acetylations present a repressive impact on PRMT-1 mediated methylation (type I methylation), lysine acetylation generally is correlated with enhanced methylation by PRMT5 (type II dimethylation). In particular, Lys-5 acetylation decreases activity of PRMT1 but increases that of PRMT5. Furthermore, hyperacetylation increases the content of ordered secondary structures of H4 tail. These findings provide new insights into the regulatory mechanism of Arg-3 methylation by H4 acetylation, and unravel that complex intercommunications exist between different posttranslational marks in cis.
7
Pelletier, Marie-Eve. "PRMT8: Characterization of a novel neuron-specific protein arginine methyltransferase." Thesis, University of Ottawa (Canada), 2010. http://hdl.handle.net/10393/28467.
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Methylation of arginine residues is a post-translational modification mediated by specific enzymes known as the Protein Arginine Methyltransferase (PRMT) family. In this thesis, we present our research on PRMT8, an enzyme catalyzing the formation of asymmetric dimethylarginine (aDMA), which displays a unique distribution at the plasma membrane of neuronal cells of the central nervous system (CNS). An ontogenic analysis of PRMT8 in mouse tissues revealed that its expression in the brain is induced during the perinatal stage and is maintained in adulthood. The P19 cell line was identified as a valid model for endogenous PRMT8 and showed the induction and requirement of PRMT8 to achieve neuronal processes. A P19 PRMT8 knock-down cell line does not survive neuronal differentiation while, in contrast, no cell death is observed when the muscle lineage is induced. Taken together, these findings suggest an important role for PRMT8 in neuronal differentiation and/or function in the CNS.
8
Morales, Yalemi. "Characterization of the Substrate Interactions and Regulation of Protein Arginine Methyltransferase." DigitalCommons@USU, 2016. https://digitalcommons.usu.edu/etd/5074.
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Protein arginine methylation is a posttranslational modification catalyzed by the family of proteins known as the protein arginine methyltransferases (PRMTs). Thousands of methylated arginines have been found in mammalian cells. Many targets of arginine regulation are involved in important cellular processes like transcription, RNA transport and processing, translation, cellular signaling, and DNA repair. Since PRMT dysregulation has been linked to a variety of disease states, understanding how the activity of the PRMTs is regulated is of paramount importance. PRMT1 is the predominant PRMT, responsible for about 85% of all arginine methylation in cells, but very little is known about how PRMT1 is regulated. Although a few methods to regulate PRMT1 activity have been reported, the details of interaction and regulatory mechanisms remain largely unknown.
To better understand how PRMT1 is able to bind its substrates and how PRMT1 activity is regulated, we followed a mechanistic and structural biology approach to better understand how PRMT1 interacts with its substrates and protein regulators. In this study the regulation of Hmt1 methyltransferase activity by the Air1 and Air2 proteins was analyzed and only one was determined to affect Hmt1 activity. The posttranslational phosphorylation of Hmt1 had also been reported to affect Hmt1 activity in vivo and our preliminary studies suggest that additional factors may help influence the regulatory effect of phosphorylation. Lastly, we report a new method of PRMT regulation through the reversible oxidation of key PRMT1 cysteine residues. We are also able to show that this regulation occurs in cells and affects several PRMT isoforms.
9
Dacwag, Caroline S. "Analysis of Protein Arginine Methyltransferase Function during Myogenic Gene Transcription: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/402.
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Skeletal muscle differentiation requires synergy between tissue-specific transcription factors, chromatin remodeling enzymes and the general transcription machinery. Here we demonstrate that two distinct protein arginine methyltransferases are required to complete the differentiation program. Prmt5 is a type II methyltransferase, symmetrically dimethylates histones H3 and H4 and has been shown to play a role in transcriptional repression. An additional member of the Prmt family, Carm1 is a type I methyltransferase, and asymmetrically methylates histone H3 and its substrate proteins. MyoD regulates the activation of the early class of skeletal muscle genes, which includes myogenin. Prmt5 was bound to and dimethylates H3R8 at the myogenin promoter in a differentiation-dependent fashion. When proteins levels of Prmt5 were reduced by antisense, disappearance of H3R8 dimethylation and Prmt5 binding was observed. Furthermore, binding of Brg1 to regulatory sequences of the myogenin promoter was abolished. All subsequent events relying on Brg1 function, such as chromatin remodeling and stable binding by muscle specific transcription factors such as MyoD, were eliminated. Robust association of Prmt5 and dimethylation of H3R8 at myogenin promoter sequences was observed in mouse satellite cells, the precursors of mature myofibers. Prmt5 binding and histone modification were observed to a lesser degree in mature myofibers. Therefore, these results indicate that Prmt5 is required for dimethylating histone at the myogenin locus during skeletal muscle differentiation in order to facilitate the binding of Brg1, the ATPase subunit of the chromatin remodeling complex SWI/SNF.
Further exploration of the role of Prmt5 during the activation of the late class of muscle genes revealed that though Prmt5 is associated with and dimethylates histones at the regulatory elements of late muscle genes in tissue and in culture, it was dispensable for late gene activation. Previous reports had indicated that Carm1 was involved during late gene activation. We observed that Carm1 was bound to and responsible for dimethylating histones at late muscle gene promoters in tissue and in culture. In contrast to Prmt5, a complete knockout of Carm1 resulted in abrogation of late muscle gene activation. Furthermore, loss of Carm1 binding and dimethylated histones resulted in a disappearance of Brg1 binding and chromatin remodeling at late muscle gene loci. Time course chromatin immunoprecipitations revealed that Carm1 binding and histone dimethylation occurred concurrently with the onset of late gene activation. In vitro binding assays revealed that an interaction between Carm1, myogenin and Mef2D exists. These results demonstrate that Carm1 is recruited to the regulatory sequences of late muscle genes via its interaction with either myogenin or Mef2D and is responsible for dimethylates histones in order to facilitate the binding of Brg1. Therefore, these results indicate that during skeletal muscle differentiation, distinct roles exist for these Prmts such that Prmt5 is required for activation of early genes while Carm1 is essential for late gene induction.
10
Haghandish, Nasim. "Characterizing the Role of Protein Arginine Methyltransferase 7 (PRMT7) in Breast Cancer." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/38672.
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The development of more efficient therapeutic strategies in the treatment of breast cancer relies on understanding the biological events that promote its progression. Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of arginine residues within proteins resulting in changes in several biological processes. PRMTs have been shown to be aberrantly expressed in many cancers and promote tumourigenesis and cancer progression. Specifically, PRMT7 mRNA expression correlates with breast cancer aggressiveness and invasiveness. Thus, we sought to determine whether PRMT7 promotes breast cancer progression/tumourigenesis and to further identify the functional mechanisms through which this is possible. We have shown that PRMT7 is upregulated in both breast cancer tissues and cell lines. Moreover, we have shown both in vitro and in vivo that PRMT7 enhances breast cancer cell invasion and metastasis. Using biochemical experimentation, we demonstrated that PRMT7 induces the expression of matrix metalloproteinase 9 to promote invasion and subsequent metastasis. Furthermore, using proteomic experiments, we discovered many novel PRMT7-interacting proteins. Further biochemical experimentation identified eukaryotic translation initiation factor eIF2α as an interacting protein and substrate of PRMT7. We demonstrated a regulatory interplay between eIF2α methylation and phosphorylation upon cellular stress: methylation is required for S51 phosphorylation. Accordingly, we have shown that stress granule formation, in the face of cellular stresses, was significantly diminished in PRMT7-knockdown cells. We additionally found that PRMT7 plays a regulatory role in protein translation. Overall, these findings suggest that PRMT7 plays a critical role in promoting breast cancer cell invasion, metastasis, stress regulation, and protein translation.
11
Wooderchak, Whitney Lyn. "Characterization of the Substrate Specificity and Mechanism of Protein Arginine Methyltransferase 1." DigitalCommons@USU, 2009. https://digitalcommons.usu.edu/etd/310.
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Protein arginine methyltransferases (PRMTs) posttranslationally modify protein arginine residues. Type I PRMTs catalyze the formation of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA) via methyl group transfer from S-adenosyl methionine onto protein arginine residues. Type II PRMTs generate MMA and symmetric dimethylarginine. PRMT-methylation affects many biological processes. Although PRMTs are vital to normal development and function, PRMT-methylation is also linked to cardiovascular disease, stroke, multiple sclerosis, and cancer.
Thus far, nine human PRMT isoforms have been identified with orthologues present in yeast, plants, and fish. PRMT1 predominates, performing an estimated 85% of all protein arginine methylation in vivo. Yet, the substrate specificity and catalytic mechanism of PRMT1 remain poorly understood.
Most PRMT1 substrates are methylated within repeating `RGG' and glycine-arginine rich motifs. However, PRMT1 also methylates a single arginine on histone-H4 that is not embedded in a glycine-arginine motif, indicating that PRMT1 protein substrates are not limited to proteins with `RGG' sequences. In order to determine if PRMT1 displays broader substrate selectivity, I first developed a continuous spectrophotometric assay to measure AdoMet-dependent methyltransferase activity. Using this assay and a focused peptide library based on a sequence derived from the in vivo PRMT1 substrate fibrillarin, we observed that PRMT1 demonstrates amino acid sequence selectivity in peptide and protein substrates. PRMT1 methylated eleven substrate motifs that went beyond the `RGG' and glycine-arginine rich paradigm, suggesting that the methyl arginine proteome may be larger and more diverse than previously thought.
PRMT1 methylates multiple arginine residues within the same protein to form protein-associated MMA and ADMA. Interestingly, ADMA is the dominant biological product formed and is a predictor of mortality and cardiovascular disease. To understand why PRMT1 preferentially forms ADMA in vivo, we began to 1) probe the mechanism of ADMA formation and 2) examine the catalytic role of certain active site residues and their involvement in ADMA formation. We found that PRMT1 dissociatively methylated several peptide substrates and preferred to methylate mono-methylated substrates over their non-methylated counterparts. Methylation of a multiple arginine-containing substrate was systematic (not random), a phenomenon that may be important biologically. All in all, our data help explain how PRMT1 generates ADMA in vivo.
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Webb, Lindsay M. Webb. "Protein Arginine MethylTransferase 5 (PRMT5) Drives Inflammatory T cell Responses and Autoimmunity." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1540137110161319.
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Gui, Shanying. "Characterization of the Product Specificity and Kinetic Mechanism of Protein Arginine Methyltransferase 1." DigitalCommons@USU, 2013. https://digitalcommons.usu.edu/etd/1980.
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Protein arginine methylation is an essential post-translational modification catalyzed by protein arginine methyltransferases (PRMTs). Type I PRMTs transfer the methyl group from S-adenosyl-L-methionine (AdoMet) to the arginine residues and catalyze the formation of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA). Type II PRMTs generate MMA and symmetric dimethylarginine (SDMA). PRMT-catalyzed methylation is involved in many biological processes and human diseases when dysregulated. As the predominant PRMT, PRMT1 catalyzes an estimated 85% of all protein arginine methylation in vivo. Nevertheless, the product specificity of PRMT1 remains poorly understood. A few articles have been published regarding the kinetic mechanism of PRMT1, yet with controversial conclusions.
To gain more insights into the product specificity of PRMT1, we dissected the active site of PRMT1 and identified two conserved methionines (Met-48 and Met-155) significant for the enzymatic activity and the product specificity. These two methionines regulate the final product distribution between MMA and ADMA by differentially affecting the first and second methyl transfer step. Current data show that Met-48 also specifies ADMA formation from SDMA. To further understand the kinetic mechanism of PRMT1, we developed a double turnover experiments to conveniently assay the processivity of the two-step methyl transfer. Using the double turnover experiments, we observed that PRMT1-catalyzed dimethylation is semi-processive. The degree of processivity depends on the substrate sequences, which satisfies the controversy between the distributive or partially processive mechanisms previously reported. We are using transient kinetics and single turnover experiments to further investigate the mechanism of PRMT1. Interestingly, during these studies, we found that PRMT1 may incur oxidative damage and the histidine affinity tag influences the protein characteristics of PRMT1. These studies have given important insights into the product specificity and kinetic mechanism of PRMT1, and provided a strong foundation for future studies on PRMT1.
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Goulet, Isabelle. "New Roles for Arginine Methylation in RNA Metabolism and Cancer." Thèse, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/20293.
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Because it can expand the range of a protein’s interactions or modulate its activity, post-translational methylation of arginine residues in proteins must be duly coordinated and ‘decoded’ to ensure appropriate cellular interpretation of this biological cue. This can be achieved through modulation of the enzymatic activity/specificity of the protein arginine methyltransferases (PRMTs) and proper recognition of the methylation ‘mark’ by a subset of proteins containing ‘methyl-sensing’ protein modules known as ‘Tudor’ domains. In order to gain a better understanding of these regulatory mechanisms, we undertook a detailed biochemical characterization of the predominant member of the PRMT family, PRMT1, and of the novel Tudor domain-containing protein 3 (TDRD3). First, we found that PRMT1 function can be modulated by 1) the expression of up to seven PRMT1 isoforms (v1-7), each with a unique N-terminal region that confers distinct substrate specificity, and by 2) differential subcellular localization, as revealed by the presence of a nuclear export sequence unique to PRMT1v2. Second, our findings suggest that TDRD3 is recruited to cytoplasmic stress granules (SGs) in response to environmental stress potentially by engaging in methyl-dependent protein-protein interactions with proteins involved in the control of gene expression. We also found that arginine methylation may serve as a general regulator of overall SG dynamics. Finally, we uncovered that alteration of PRMT1, TDRD3, and global arginine methylation levels in breast cancer cells may be closely associated with disease progression and poor prognosis. Therefore, further studies into the pathophysiological consequences ensuing from misregulation of arginine methylation will likely lead to the development of novel strategies for the prevention and treatment of breast cancer.
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Hu, Yu-Jie. "Roles of Protein Arginine Methyltransferase 7 and Jumonji Domain-Containing Protein 6 in Adipocyte Differentiation: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/797.
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Regulation of gene expression comprises a wide range of mechanisms that control the abundance of gene products in response to environmental and developmental changes. These biological processes can be modulated by posttranslational modifications including arginine methylation. Among the enzymes that catalyze the methylation, protein arginine methyltransferase 7 (PRMT7) is known to modify histones to repress gene expression. Jumonji domain-containing protein 6 (JMJD6) is a putative arginine demethylase that potentially antagonize PRMT7. However, the biological significance of these enzymes is not well understood. This thesis summarizes the investigation of both PRMT7 and JMJD6 in cell culture models for adipocyte differentiation. The results suggest that PRMT7 is not required for the differentiation, whereas JMJD6 is necessary for the differentiation by promoting the expression of the lineage determining transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancerbinding proteins (C/EBPs). The underlying mechanisms by which JMJD6 regulate differentiation involve transcriptional and post-transcriptional control of gene expression. Unexpectedly, the adipogenic function of JMJD6 is independent of its enzymatic activity. Collectively, the present research reveals a novel role of JMJD6 in gene regulation during the differentiation of adipocytes.
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Hu, Yu-Jie. "Roles of Protein Arginine Methyltransferase 7 and Jumonji Domain-Containing Protein 6 in Adipocyte Differentiation: A Dissertation." eScholarship@UMMS, 2010. http://escholarship.umassmed.edu/gsbs_diss/797.
Full textAbstract:
Regulation of gene expression comprises a wide range of mechanisms that control the abundance of gene products in response to environmental and developmental changes. These biological processes can be modulated by posttranslational modifications including arginine methylation. Among the enzymes that catalyze the methylation, protein arginine methyltransferase 7 (PRMT7) is known to modify histones to repress gene expression. Jumonji domain-containing protein 6 (JMJD6) is a putative arginine demethylase that potentially antagonize PRMT7. However, the biological significance of these enzymes is not well understood. This thesis summarizes the investigation of both PRMT7 and JMJD6 in cell culture models for adipocyte differentiation. The results suggest that PRMT7 is not required for the differentiation, whereas JMJD6 is necessary for the differentiation by promoting the expression of the lineage determining transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancerbinding proteins (C/EBPs). The underlying mechanisms by which JMJD6 regulate differentiation involve transcriptional and post-transcriptional control of gene expression. Unexpectedly, the adipogenic function of JMJD6 is independent of its enzymatic activity. Collectively, the present research reveals a novel role of JMJD6 in gene regulation during the differentiation of adipocytes.
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Huang, Jinghan. "Defining the role of Quaking RNA binding protein and arginine methyltransferase PRMT5 in myelination." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=96921.
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Oligodendrocyte differentiation is controlled by a complex network of epigenetic regulators, transcription factors, RNA binding proteins, and cell cycle regulators. The involvement of QKI, an RNA binding protein that regulates mRNA stability, splicing and transport, is only well characterized in rodent myelin development. QKI regulates oligodendrocyte differentiation by controlling in part the expression of cyclin-dependent kinase inhibitor p27 (p27Kip1), a cell cycle inhibitor, and myelin basic protein (MBP), a major myelin component. In this thesis, we first show that human glial progenitor cells readily expressed QKI-6 and QKI-7 but not QKI-5. These cells increased the expression of all three major QKI isoforms as they commit to the OPC lineage and become mature oligodendrocytes. The ectopic QKI-6 and QKI-7 expression promoted human oligodendrocyte differentiation, while QKI-5 played a negative role in this process. Second, we show that protein arginine methyl transferase 5 (PRMT5), an enzyme known to symmetrically dimethylate both QKI and MBP in vitro, was up-regulated during myelin development and indeed methylated MBP in vivo. PRMT5 deficient oligodendrocytes demonstrated impaired differentiation, which was reflected by both the morphology and the transcription factor expression profile. Our findings implicate that QKI-6, QKI-7 and PRMT5 are promoters of oligodendrocyte differentiation, while QKI-5 is an inhibitor of the maturation process.La différenciation d'oligodendrocyte est contrôlée par un réseau complexe de régulateurs épigéniques, de facteurs de transcription, de protéines de liaison à l'ARN ainsi que de régulateurs du cycle cellulaire. L'implication de QKI, une protéine de liaison à l'ARN qui régule la stabilité, l'épissage et le transport de l'ARNm, n'est que bien caractérisée dans le développement de la myéline chez les rongeurs. En régulant l'expression de p27Kip1, un inhibiteur du cycle cellulaire, ainsi que MBP, un composant majeur de la myéline, QKI-6 et QKI-7 stimulent tandis que QKI-5 réprime la différenciation d'oligodendrocytes. Nous avons démontré que les effets d'une expression exogène de QKI chez des oligodendrocytes humains sont consistants avec ceux observés chez les cellules des rongeurs. On a d'ailleurs établi que PRMT5, une enzyme capable de méthyler QKI ainsi que MBP in vitro, est régulée positivement pendant le développement de la myéline du SNC. De plus, nous avons confirmé que MBP est méthylé par PRMT5 in vivo. L'ablation de PRMT5 dans les oligodendrocytes résulte en une différenciation anormale, ainsi caractérisée morphologiquement et en étudiant le profil des facteurs de transcription. Prise ensemble, ces résultats suggèrent que PRMT5 est un promoteur de la différenciation d'oligodendrocytes.
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Canup, Brandon S. "Discovery of Novel Cross-Talk between Protein Arginine Methyltransferase Isoforms and Design of Dimerization Inhibitors." Digital Archive @ GSU, 2013. http://digitalarchive.gsu.edu/chemistry_theses/58.
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Protein arginine methyltransferase, PRMT, is a family of epigenetic enzymes that methylate arginine residues on histone and nonhistone substrates which result in a monomethylation, symmetric dimethylation or asymmetric dimethylation via the transfer of a methyl group from S-adenosyl-L-methionine (SAM). We discovered a novel interaction between two PRMT isoforms: PRMT1 interacts and methylates PRMT6. In this study site-directed mutagenesis was performed on selected arginines identified from tandem mass spectrometric analysis to investigate major methylation sites of PRMT6 by PRMT1. In combination with radiometric methyltransferase assays, we determined two major methylation sites. Methylations at these sites have significant effects on the nascent enzymatic activity of PRMT6 in H4 methylation. PRMTs have the ability to homodimerize which have been linked to methyltransferase activity. We designed dimerization inhibitors (DMIs) to further investigate the need for dimerization for enzyme activity. Preliminary results suggest that the monomeric form of PRMT1 retains methyltransferase activity comparable to that of the uninhibited PRMT1.
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Weimann, Mareike. "A proteome-wide screen utilizing second generation sequencing for the identification of lysine and arginine methyltransferase protein interactions." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16581.
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Proteinmethylierung spielt eine immer größere Rolle in der Regulierung zellulärer Prozesse. Die Entwicklung effizienter proteomweiter Methoden zur Detektion von Methylierung auf Proteinen ist limitiert und technisch schwierig. In dieser Arbeit haben wir einen neuen Hefe-Zwei-Hybrid-Ansatz (Y2H) entwickelt, der Proteine, die miteinander wechselwirken, mit Hilfe von Sequenzierungen der zweiten Generation identifiziert (Y2H-Seq). Der neue Y2H-Seq-Ansatz wurde systematisch mit dem Y2H-Seq-Ansatz verglichen. Dafür wurde ein Bait-Set von 8 Protein-Arginin-Methyltransferasen, 17 Protein-Lysin-Methyltransferasen und 10 Demethylasen gegen 14,268 Prey-Proteine getestet. Der Y2H-Seq-Ansatz ist weniger arbeitsintensiv, hat eine höhere Sensitivität als der Standard Y2H-Matrix-Ansatz und ist deshalb besonders geeignet, um schwache Interaktionen zwischen Substraten und Protein-Methyltransferasen zu detektieren. Insgesamt wurden 523 Wechselwirkungen zwischen 22 Bait-Proteinen und 324 Prey-Pr oteinen etabliert, darunter 11 bekannte Methyltransferasen-Substrate. Netzwerkanalysen zeigen, dass Methyltransferasen bevorzugt mit Transkriptionsregulatoren, DNA- und RNA-Bindeproteinen wechselwirken. Diese Daten repräsentieren das erste proteomweite Wechselwirkungsnetzwerk über Protein-Methyltransferasen und dienen als Ressource für neue potentielle Methylierungssubstrate. In einem in vitro Methylierungsassay wurden exemplarisch mit Hilfe massenspektrometrischer Analysen die methylierten Aminosäurereste einiger Kandidatenproteine bestimmt. Von neun getesteten Proteinen waren sieben methyliert, zu denen gehören SPIN2B, DNAJA3, QKI, SAMD3, OFCC1, SYNCRIP und WDR42A. Wahrscheinlich sind viele Methylierungssubstrate im Netzwerk vorhanden. Das vorgestellte Protein-Protein-Wechselwirkungsnetzwerk zeigt, dass Proteinmethylierung sehr unterschiedliche zelluläre Prozesse beeinflusst und ermöglicht die Aufstellung neuer Hypothesen über die Regulierung Molekularer Mechanismen durch Methylierung.Protein methylation on arginine and lysine residues is a largely unexplored posttranslational modification which regulates diverse cellular processes. The development of efficient proteome-wide approaches for detecting protein methylation is limited and technically challenging. We developed a novel workload reduced yeast-two hybrid (Y2H) approach to detect protein-protein interactions utilizing second generation sequencing. The novel Y2H-seq approach was systematically evaluated against our state of the art Y2H-matrix screening approach and used to screen 8 protein arginine methyltransferases, 17 protein lysine methyltransferases and 10 demethylases against a set of 14,268 proteins. Comparison of the two approaches revealed a higher sensitivity of the new Y2H-seq approach. The increased sampling rate of the Y2H-seq approach is advantageous when assaying transient interactions between substrates and methyltransferases. Overall 523 interactions between 22 bait proteins and 324 prey proteins were identified including 11 proteins known to be methylated. Network analysis revealed enrichment of transcription regulator activity, DNA- and RNA-binding function of proteins interacting with protein methyltransferases. The dataset represents the first proteome-wide interaction network of enzymes involved in methylation and provides a comprehensively annotated resource of potential new methylation substrates. An in vitro methylation assay coupled to mass spectrometry revealed amino acid methylation of candidate proteins. Seven of nine proteins tested were methylated including SPIN2B, DNAJA3, QKI, SAMD3, OFCC1, SYNCRIP and WDR42A indicating that the interaction network is likely to contain many putative methyltransferase substrate pairs. The presented protein-protein interaction network demonstrates that protein methylation is involved in diverse cellular processes and can inform hypothesis driven investigation into molecular mechanisms regulated through methylation.
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Samadzadeh, Tarighat Somayeh. "Novel epigenetic role and therapeutic targeting of protein arginine methyltransferase 5 (PRMT5) in Acute Myeloid Leukemia." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1406025487.
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Nicholas, Courtney. "The Protein Arginine Methyltransferase PRMT5 Regulates Proliferation and the Expression of MITF and p27Kip1 in Human Melanoma." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1343078125.
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Lattouf, Hanine. "Crosstalk entre la kinase LKB1 et l'arginine methyltransferase PRMT5 dans le cancer du sein." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1252/document.
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La protéine arginine méthyltransférase 5 est la majeure arginine méthyltransférase de type II chez les mammifères, responsable de la génération de la majorité des arginines protéiques symétriquement diméthylées. Elle est impliquée dans divers processus oncogéniques tel que la progression tumorale et la croissance indépendante de l'ancrage. PRMT5 est surexprimée dans plusieurs cancers comme le cancer de l'ovaire, des poumons et du colon. Cependant, son expression dans le cancer du sein n'est pas assez étudiée. Dans ce projet de thèse, nous avons analysé l'expression de PRMT5 dans une cohorte de 440 tumeurs mammaires. Nos résultats montrent que son expression nucléaire est un facteur de bon pronostic, notamment dans les tumeurs ERa-positives. Nous avons aussi mis en évidence une corrélation entre PRMT5 et la sérine/thréonine kinase LKB1, suggérant un lien entre ces deux protéines. Plusieurs approches in vitro et in cellulo nous ont permis de démontrer que PRMT5 et LKB1 interagissent dans le cytoplasme des cellules mammaires épithéliales. Bien que PRMT5 soit incapable de méthyler LKB1, nous avons montré pour la première fois que PRMT5 est un substrat de cette kinase. Nous avons par la suite identifié les Thr132, 139 et 144 comme cibles de la phosphorylation, au niveau du tonneau TIM en N-terminal de PRMT5. La mutation des thréonines T139/144 en alanine diminue significativement l'activité de PRMT5, probablement suite à une perte de son interaction avec des protéines régulatrices comme MEP50, pICLn et RiOK1. De plus, la modulation de l'expression de LKB1 altère l'activité de PRMT5, témoignant d'un nouveau mécanisme de régulation médié par la phosphorylation identifiéeProtein arginine methyltrasferase 5 is the major type II arginine methyltransferase in humans. It symmetrically dimethylates arginine residues on target proteins in both the cytoplasm and the nucleus. PRMT5 was reported to be an oncoprotein implicated in anchorage independent growth and tumor progression. So far, it has been involved in various cancers such as ovarian cancer, lung cancer and colon cancer, but its expression pattern in breast cancer has not been deeply studied. In this thesis project, we analyzed PRMT5 expression in a cohort of 440 breast tumor samples and we found that its nuclear expression is a good prognosis factor, mainly in ERa-positive tumors. Interestingly, our clinical results analysis showed that PRMT5 expression is correlated with the serine/threonine kinase LKB1, suggesting a relationship between both proteins. Several in vitro and in cellulo approaches gave evidence that PRMT5 and LKB1 interact directly in the cytoplasm of mammary epithelial cells. Moreover, although PRMT5 is not able to methylate LKB1, we found that PRMT5 is a bona fade substrate for LKB1. We next identified Thr132, 139 and 144 residues as target sites for phosphorylation, located in the TIM barrel domain of PRMT5. Interestingly, the Thr139/144 mutation to alanine decreased drastically PRMT5 methyltransferase activity, probably due to the loss of PRMT5 interaction with regulatory proteins such as MEP50, pICLn and RiOK1. In addition, the modulation of LKB1 expression modifies PRMT5 enzymatic activity, highlighting a new regulatory mechanism mediated by the discovered posttranslational modification of this arginine methyltransferase
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Suh-Lailam, Brenda Bienka. "Development of Novel Methods and their Utilization in the Analysis of the Effect of the N-terminus of Human Protein Arginine Methyltransferase 1 Variant 1 on Enzymatic Activity, Protein-protein Interactions, and Substrate Specificity." DigitalCommons@USU, 2010. https://digitalcommons.usu.edu/etd/863.
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Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of protein arginine residues, resulting in the formation of monomethylarginine, and/or asymmetric or symmetric dimethylarginines. Although understanding of the PRMTs has grown rapidly over the last few years, several challenges still remain in the PRMT field. Here, we describe the development of two techniques that will be very useful in investigating PRMT regulation, small molecule inhibition, oligomerization, protein-protein interaction, and substrate specificity, which will ultimately lead to the advancement of the PRMT field. Studies have shown that having an N-terminal tag can influence enzyme activity and substrate specificity. The first protocol tackles this problem by developing a way to obtain active untagged recombinant PRMT proteins. The second protocol describes a fast and efficient method for quantitative measurement of AdoMet-dependent methyltranseferase activity with protein substrates. In addition to being very sensitive, this method decreases the processing time for the analysis of PRMT activity to a few minutes compared to weeks by traditional methods, and generates 3000-fold less radioactive waste. We then used these methods to investigate the effect of truncating the NT of human PRMT1 variant 1 (hPRMT1-V1) on enzyme activity, protein-protein interactions, and substrate specificity. Our studies show that the NT of hPRMT1-V1 influences enzymatic activity and protein-protein interactions. In particular, methylation of a variety of protein substrates was more efficient when the first 10 amino acids of hPRMT1v1 were removed, suggesting an autoinhibitory role for this small section of the N-terminus. Likewise, as portions of the NT were removed, the altered hPRMT1v1 constructs were able to interact with more proteins. Overall, my studies suggest the the sequence and length of the NT of hPRMT1v1 is capable of enforcing specific protein interactions.
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Ajebbar, Samira. "Synthèse de ligands à la proteine CARM1 pour l'étude de son activité enzymatique et la synthèse d'inhibiteurs sélectifs." Phd thesis, Université de Strasbourg, 2012. http://tel.archives-ouvertes.fr/tel-00769956.
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Les protéines arginine méthyl transférases ("PRMTs") sont impliquées dans de nombreux processus cellulaires essentiels. La protéine CARMI ("Coactivator-associated arginine methyltransferase 1", appelée aussi "PRMT4") a été initialement identifiée par sa fonction co-activatrice de la transcription impliquantplusieurs récepteurs nucléaires des hormones. CARMI est une enzyme qui catalyse la réaction de méthylation sur les histones via un donneur de méthyl naturel, la S-adénosY-L -méthionine (SAM). De nombreux travaux ont montré que CARMI est surexprimée dans les cancers du sein et de la prostate. L' objectif de ce travail est la compréhension à l'échelle moléculaire du mode d'action de CARMI et l'étude du mécanisme de reconnaissances moléculaires et de transferts d' informations gouvernés par la protéine CARMI. La structure cristallographique obtenue de cette enzyme en présence de cofacteur, la S-AdénosyhHomocystéine ou la Sinefungine a eu un effet stabilisant. Ainsi, notre stratégie a été de créer des molécules hameçons basées sur le motif de la SAM capables d' ancrer un peptide mimant la séquence de l' histone H3, pour ensuite les tester en co-cristallisation avec CARMI. Ainsi, grâce à la diffraction aux rayons X, les interactions mises en jeu dans le complexe CARMlImolécules hameçons/peptide pourront être déterminées. Cette stratégie s'est effectuée en trois étapes : la première étape, décrite dans le chapitre 2, a consisté en la synthèse d'analogues de la SAM obtenus grâce à des modifications réalisées autour de l'atome de soufre. Ces composés nous ont permis d' explorer la " poche du sulfonium ". Puis la seconde étape, décrite dans le chapitre 3, a été la synthèse * d'analogues de bisubstrats nécessaires pour l'exploration de la " poche de l'arginine ". Dans une dernière étape, décrite dans les chapitres 4 et 5, nous avons abordé la synthèse d'adduits SAM-peptide pouf pouvoir étudier le " domaine de fixation du peptide ". Dans le quatrième chapitre, la méthode de choix est la création d'un lien covalent entre une molécule hameçon électrophile etun peptide par chimie de click in-situ : par réaction de cycloaddition de Huisgen; par réaction entre des molécules hameçons électrophiles capables de piéger un peptide cystéine ou un peptide arginine. Ces essais se sont révélés infructueux et une nouvelle stratégie a été employée en utilisant des molécules ancres. Dansle cinquième chapitre des molécules ancres ont donc été préformés pour ensuite être testés en cocristallisation dans CARMl.
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Stopa, Nicole. "Chromatin modifiers in Xenopus laevis| Protein arginine methyltransferase 5 function and Williams syndrome transcription factor complexes in development." Thesis, University of Alaska Anchorage, 2014. http://pqdtopen.proquest.com/#viewpdf?dispub=1556018.
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Cellular DNA is condensed and stored with the help of proteins, especially histones. This mix of DNA and proteins is termed chromatin. Manipulations of chromatin include post-translational modifications (PTMs) of histone proteins, such as the addition of methyl or acetyl groups, and the movement of histones by chromatin remodeling complexes to control how tightly or which regions of DNA are condensed or exposed. These actions can impact gene expression and thus influence the differentiation of tissues during development. I investigated if the enzyme protein arginine methyltransferase 5 (PRMT5), which mono- or symmetrically dimethylates arginine, is acting on histones during early development in Xenopus laevis. I also investigated if Williams syndrome transcription factor (WSTF) occurs within chromatin remodeling complexes during early development in X. laevis. WSTF interacts with the protein ISWI in early embryos, indicating it is part of a chromatin remodeling complex during this period of development.
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Chénard, Carol Anne. "Ribonucleoprotein complexes and protein arginine methylation : a role in diseases of the central nervous sytem." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=115894.
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For the past 45 years, QKI has been studied for its role in the processes of development and central nervous system myelination using the qkv mouse. The presence of a single KH domain and the recent identification of a high-affinity binding site in mRNAs, suggests that it can bind to and regulate mRNAs through processes such as stability, splicing and transport. As a member of the STAR RNA binding family of proteins the QKI isoforms may also be involved in cell signaling pathways.QKI's involvement in all of these processes, lead us to examine both the protein partners and the mRNA targets of the QKI complex in order to identify potentially new pathways regulated by QKI. In doing so, we identified a novel direct protein-protein interaction with PABP and for the first time described the relocalization of QKI to cytoplasmic granules following oxidative stress. In addition, in vivo mRNA interaction studies were performed and allowed the identification of approximately 100 new mRNA targets in human glioblastoma cells. One of the targets identified was VEGF mRNA.
Another QKI target mRNA is MBP, a major protein component of the myelin sheath and the candidate auto-antigen in multiple sclerosis (MS). In vivo MBP is symmetrically dimethylated on a single arginine residue. To further establish the role of the methylation of MBP in myelination, a methyl-specific antibody and an adenovirus expressing a recombinant protein arginine methyltransferase 5 (PRMT5) was generated. We show that methylated MBP is found in areas of mature myelin and that overexpression of the PRTM5 blocked the differentiation of oligodendrocytes.
Taken together these datas implicate QKI for the first time in the process of human cancer angiogenesis and could explain the vascularization defects observed in some of the qkI mutant mice. In addition, arginine methylation of MBP may prove to have an important role in the process of myelination and in the pathogenesis of demyelination and the autoimmune reaction in diseases such as MS.
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Vinet, Mathilde. "Analyse de la protéine arginine méthyltransférase 5 comme cible thérapeutique dans les cancers du sein triple-négatifs." Thesis, Paris Sciences et Lettres (ComUE), 2019. https://tel.archives-ouvertes.fr/tel-02631827.
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Les cancers du sein triple-négatifs (TNBC) représentent un sous-type de cancer du sein très hétérogène et agressif, pour lequel aucune thérapie ciblée n’a été montrée efficace à ce jour. La protéine arginine méthyltransférase 5 (PRMT5), une enzyme catalysant la méthylation des arginines protéiques, et son cofacteur, la protéine méthylosome 50 (MEP50), ont récemment été associés à des processus oncogéniques. Dans ce projet de thèse, nous analysons l’expression et la localisation de PRMT5 et de MEP50 dans une cohorte de 150 tumeurs mammaires, et étudions le potentiel thérapeutique de l’inhibition de PRMT5 dans les TNBC, à l’aide de siRNAs et d’un inhibiteur spécifique de PRMT5 : EPZ015666. Nous montrons que les protéines PRMT5 et MEP50 sont exprimées à un niveau similaire dans les TNBC que dans les tissus mammaires sains et que dans les autres sous-types de cancer du sein, mais avec une plus faible localisation nucléaire, suggestive d’une valeur pronostique de la localisation subcellulaire de PRMT5/MEP50. Nous mettons en évidence le potentiel thérapeutique de l’inhibition de PRMT5, seule ou en combinaison, pour un sous-ensemble de TNBC. Finalement, nous identifions de nouveaux partenaires protéiques potentiels de PRMT5/MEP50, dont la fonction mérite d’être plus précisément explorée dans le contexte des TNBCTriple-negative breast cancers (TNBC) are highly heterogeneous and aggressive breast cancers for which no targeted therapy yet exists. Protein arginine methyltransferase 5 (PRMT5), an enzyme which catalyzes the methylation of arginines on histone and non‐histone proteins, and its cofactor methylosome protein 50 (MEP50), have recently been attributed oncogenic functions. This thesis analyzes PRMT5/MEP50 expression and localization in a cohort of 150 breast tumors, and explores the therapeutic potential of PRMT5 targeting in TNBC, using siRNA and the specific, small-molecule inhibitor EPZ015666. We show that TNBC express similar levels of PRMT5 and MEP50 proteins compared to healthy breast tissues and to other breast cancer subtypes, but with a distinctively low nuclear component, suggesting a prognostic value of PRMT5/MEP50 localization in breast cancers. We find PRMT5 to be a relevant therapeutic target, alone or in combination, for a subset of TNBC. Finally, we identify putative novel PRMT5/MEP50 partners, whose function merit further investigation in the context of TNBC
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Weimann, Mareike Verfasser], Christian [Akademischer Betreuer] Spahn, Erich [Akademischer Betreuer] [Wanker, and Hans [Akademischer Betreuer] Lehrach. "A proteome-wide screen utilizing second generation sequencing for the identification of lysine and arginine methyltransferase protein interactions / Mareike Weimann. Gutachter: Christian Spahn ; Erich Wanker ; Hans Lehrach." Berlin : Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://d-nb.info/1026081971/34.
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Weimann, Mareike [Verfasser], Christian Akademischer Betreuer] Spahn, Erich [Akademischer Betreuer] [Wanker, and Hans [Akademischer Betreuer] Lehrach. "A proteome-wide screen utilizing second generation sequencing for the identification of lysine and arginine methyltransferase protein interactions / Mareike Weimann. Gutachter: Christian Spahn ; Erich Wanker ; Hans Lehrach." Berlin : Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://nbn-resolving.de/urn:nbn:de:kobv:11-100204062.
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Sayegh, Joyce Ellen. "Identification and characterization of eukaryotic protein arginine methyltransferases." Diss., Restricted to subscribing institutions, 2007. http://proquest.umi.com/pqdweb?did=1495958991&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Anthony, Shelagh. "Analysis of mammalian protein arginine N-methyltransferases in the vasculature." Thesis, University College London (University of London), 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424909.
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Varney, Amy. "Small molecule inhibitors and substrate analyses of protein arginine methyltransferases." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:58350487-397d-4fa7-a545-429b16d23540.
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Chapter 1 introduces the Protein Arginine Methyltransferases (PRMTs) as epigenetic regulators that decorate peptidic arginine with methyl groups. Evidence for PRMT involvement in cancer pathogenesis is reviewed and their plausibility as therapeutic targets is introduced. The methylation patterns conferred by the PRMTs is described, and the existence of novel patterns is considered. Techniques for assaying PRMT activity are compared and contrasted and a discussion of current PRMT inhibitors is presented. The chapter concludes by outlining the aims of the thesis. Chapter 2 describes synthesis towards novel methylated arginine molecules that are fully protected for inclusion in peptides via solid phase peptide synthesis. Chapter 3 outlines the total synthesis of protected d-monomethylated arginine for peptide synthesis. This methylation pattern is known in yeast but has not yet been identified in humans. Chapter 4 details a new mass spectrometry-based assay that can be applied for inhibitor and substrate analyses. Synthesis of a novel histone peptide containing the d-monomethylated arginine, produced in Chapter 3, is also described and this is tested for relevance as a human epigenetic marker. Novel polymethylation patterns are also explored in a total of five histone peptides. This chapter concludes with discussion of possible methylation pattern rearrangements. Chapter 5 describes the synthesis and testing of two series of putative PRMT inhibitors based on previously identified scaffolds within this research group. Data obtained from three different assays, including that outlined in Chapter 4, are analysed and suggestions as to the direction of PRMT assay design are offered. Chapter 6 provides the experimental data to support Chapters 2-5, including all organic synthesis procedures, protein & peptide syntheses and assay methodology.
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Morettin, Alan James. "Investigating the Role of Protein Arginine Methyltransferases in Breast Cancer Etiology." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/31920.
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Breast cancer is the most commonly diagnosed cancer amongst Canadian women. Though numerous treatments are available, in many instances tumours become refractory or recur. Therefore, understanding the biological events that lead to the progression and therapeutic resistance of breast cancer is essential for the development of novel treatment options for this disease. Numerous members of the protein arginine methyltransferase (PRMT) family, which are the enzymes responsible for catalyzing methylation on arginine residues are aberrantly regulated in breast cancer. Hence, understanding the precise contribution of PRMTs to the development and progression of breast cancer is important. This Thesis will present my findings on the alternatively spliced PRMT1 isoform, PRMT1v2, previously identified to be overexpressed in breast cancer cell lines and here shown to promote breast cancer cell survival and invasion. Second, a novel role is ascribed to PRMT6, another PRMT aberrantly expressed in breast cancer. PRMT6 promotes chemoresistance to the drug bortezomib by mediating stress granule formation through down-regulation of eIF4E. Increased stress granule formation in bortezomib-resistant cancer cells promotes cell survival. Third, DDX3, a prototypical PRMT substrate which is overexpressed in breast cancer cell lines and stimulates transformation of mammary epithelial cells is a novel substrate of PRMT1, CARM1, and PRMT6. Lastly, TDRD3, a reader/effector of arginine methylation also overexpressed in breast tumours regulates breast cancer cell proliferation, anchorage-independent growth and cell motility and invasion.
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Thomas, Dylan. "Oligomerization dependent enzyme kinetics and mechanistic characterization of type I protein arginine N-methyltransferases." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44623.
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Herglotz, Julia [Verfasser], and Rolf [Akademischer Betreuer] Marschalek. "Regulation des hämatopoietischen Transkriptionsfaktors RUNX1 durch die Protein-Arginin-Methyltransferase 6 / Julia Herglotz. Gutachter: Rolf Marschalek." Frankfurt am Main : Univ.-Bibliothek Frankfurt am Main, 2011. http://d-nb.info/104394754X/34.
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Marechal, Nils. "Étude structurale des protéine arginine méthyltransférases : reconnaissance des substrats et conception rationnelle de modulateurs." Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ048.
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Les protéine arginine méthyltransférases (PRMT) sont impliquées dans de nombreux processus cellulaires, incluant la régulation de l’expression des gènes, le contrôle de l’épissage, le maintien de l’intégrité du génome et la transduction du signal. De nombreuses études montrent que la dérégulation de l’activité des PRMT est associée au développement de pathologies, et en particulier de cancers. Les PRMT constituent ainsi une des nouvelles cibles potentielles en chimiothérapie. Les travaux présentés dans ce manuscrit portent sur trois cibles : PRMT2, PRMT3 et PRMT4/CARM1. Combinant des approches biochimiques, biophysiques et structurales (cristallographie et cryo- microscopie électronique), ces travaux comportent deux aspects : (I) comprendre au niveau atomique la régulation de la réaction de méthylation des protéines (reconnaissance protéines-protéines et interactions entre modifications post-traductionnelles) ; (II) découvrir des inhibiteurs spécifiques et puissants de plusieurs PRMT ciblesProtein arginine methyltransferases (PRMT) are involved in many cellular processes, including gene expression regulation, splicing control, maintenance of genome integrity and signal transduction.Since deregulation of those biological processes appears to be implicated in the pathogenesis of different diseases, PRMTs have emerged as potential new targets for the development of novel therapeutic modulators. Despite the large amount of biological and structural data on PRMTs, two challenges remain to be solved by structural biology ; (I) understanding how PRMTs recognize and bind their full-length substrates ; (II) revealing how PRMTs achieve specific arginine methylation on different target sites. The works presented here focused on 3 targets: PRMT2, PRMT3 and PRMT4/ CARM1. We used biochemical, biophysical and structural methods (bio-crystallography and cryo- electron microscopy) to decipher structural clues that drive PRMT-substrate recognition. We developed new chemical probes that can be used in early drug discovery for the conception of PRMT inhibitors
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Chen, Ta-Huang, and 陳大晃. "Characterization of Protein Arginine Methyltransferase and Studies of the Subcellular Localization of Arginine Methyl-accepting Proteins." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/48821237174141385921.
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碩士中山醫學大學
醫學研究所
90
Abstract Protein arginine methylation is a posttranslation modification catalyzed by protein arginine methyltransferase (PRMT). The type I methyltransferase catalyzes the formation of mono- and asymmetric di-methylarginine and another type II activity can catalyze the formation of mono- and symmetric di-methylarginine on other methylaccepting proteins. Before the identification of five different PRMT genes recently, the type I and II PRMT from various biological resources were biochemically characterized to be of molecular weight from 275 kDa to 500 kDa. All these PRMT monomers are of molecular weight from 40 kDa to 70 kDa, thus are likely to be the catalytic subunit in a methyltransferase complex. In this study, we used the biochemical approach to isolate and analyze the arginine methyltransferase from a natural source porcine brain. We fractionated the lysates by differential centrifugation to obtain the supernatant S1, S2 and S3, as well as the pellet fractions P1, P2 and P3. Part of the P3 was resuspended in lysis buffer with 0.5M KCl and subjected to ultracentrifuge again to obtain S4 and P4. The majority of the PRMT activity (assayed by a type I substrate fibrillarin) was in the S3 (cytosolic) fraction while the highest specific activity was in the S4 fraction. Addition of KCl increase the arginine methyltransferase activity in the P3 fraction as well as that in the lymphoblastoid cell. Removal of the KCl in the S4 fraction decrease the PRMT activity. It is possible that high KCl concentrate can help to release the PRMT in P4 from associated inhibiting factors, thus increase the methyltransferase activity. However, PRMT1, the predominant type I PRMT in the mammalian system is much more abundant in S3 than S4, and the PRMT activities in S3 and recombinant PRMT1 were not affect by 0.5M KCl. Whether the methyltransferase in S3 and S4 were the same or different enzyme is under investigation. The type I activity in both fractions almost eluted at the same position by anion-exchange (Mono Q) column. We are now trying to purify the enzyme in both fractions to understand their identities. Arginine methylation in RNA-binding proteins containing arginine- and glycine-rich RGG motifs is catalyzed by specific protein arginine N-methyltransferase in cells. We previously showed that lymphoblastoid cells grown in the presence of an indirect methyltransferase inhibitor, adenosine dialdehyde (AdOx), accumulated high level of hypomethylated protein substrates. We also demonstrated that protein-arginine methyltransferases as well as their methyl-accepting substrates were widely distributed in different subcellular fractions of lymphoblastoid cells. Recently arginine methylation has been suggested to be related with protein-protein interaction and subcellular localization of some methyl-accepting proteins. Therefore, in this study we fractioned lymphoblastoid cells into different subcellular fractions to study if localization of certain RGG containing proteins such as FMRP, hnRNP-A1, hnRNP-A2/B1, and nucleolin would be interfered upon Adox treatment. By western blot analyses using antibodies against these proteins, we demonstrated that subcellular localization of FMRP and hnRNP-A2/B1 appeared to be different in AdOx-treated and normal cells. Further studies utilizing immunofluorescent staining and 2-dimensional gel electrophoresis will provide more information of the relationship between the methylation status of arginine methyl-accepting proteins and their subcellular localization.
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Hua, Wei-Kai, and 華尉愷. "Roles of Protein Arginine Methyltransferase 1 in Erythroid Differentiation." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/74225459761417668532.
Full textAbstract:
博士國立陽明大學
生物藥學研究所
101
Protein arginine methylation is emerging as a pivotal posttranslational modification involved in regulating various cellular processes including cell growth, gene expression and signal transduction. PRMT1 (protein arginine methyltransferase 1) is a predominant PRMT in mammalian cells. Erythropoiesis generates circulating red blood cells. Despite extensive studies, the molecular events regulating erythropoiesis are not fully understood. This study showed that the increase in PRMT1 levels significantly promoted erythroid differentiation in the bipotent human leukemic K562 cell line. PRMT1 expression enhanced the production of hemoglobin and the erythroid surface marker glycophorin A, and also up-regulated key transcription factors, GATA1, NF-E2 and EKLF, which are critical for lineage-specific differentiation. The shRNA-mediated knockdown of PRMT1 suppressed erythroid differentiation. The methyltransferase activity-deficient mutant failed to stimulate differentiation, indicating the requirement of arginine methylation of target proteins. PRMT1 also enhanced EPO (erythropoietin)-induced erythroid differentiation in human primary hematopoietic progenitor CD34+ cells, confirming the crucial role of PRMT1-mediated arginine methylation in erythroid differentiation. The p38 MAPK signaling pathway is known to promote erythropoiesis. This study further identified that a specific isoform, p38a, promoted erythroid differentiation, whereas p38b did not play a role. PRMT1 enhanced the activation of p38 during erythroid differentiation. The stimulation of erythroid differentiation by PRMT1 was diminished in p38a- but not p38b-knockdown cells. Importantly, this study showed for the first time that PRMT1 was associated with p38a in cells by co-immunoprecipitation and that PRMT1 directly methylated p38a in in vitro methylation assays indicating the direct arginine methylation by PRMT1 may mediate the stimulatory role of p38a in erythroid differentiation. Further studies revealed that the calcium concentrations could regulate the PRMT1-mediated promotion of erythroid differentiation suggesting, for the first time, that calcium may be an important intracellular regulator of PRMT1 activity. This study provides evidence suggesting a novel regulatory mechanism for erythropoiesis.
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Tsai, Cheng-Kang, and 蔡政剛. "Specific interacting proteins of the N-terminus of protein arginine methyltransferase PRMT8." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/68640092608876629178.
Full textAbstract:
碩士中山醫學大學
生物醫學科學學系碩士班
104
Protein arginine methyltransferase (PRMT) 8 is a vertebrate-restricted paralogue of PRMT1 with an extra N-terminal sequence. Its expression is limited in brain and is the only PRMT with tissue-restricted expression pattern. The N-terminus of PRMT8 contains a unique proline-rich sequence in human and a unique glutamine-rich (Q-rich) sequence before a proline- and histidine-rich region in zebrafish. Therefore, we are interested in the function of the N-terminus of PRMT8 and would like to analyze the interacting partners of this segment cross species. We performed the yeast two-hybrid (Y2H) screen for specific interacting proteins of the N-terminus of human PRMT8 (H8N) against a human brain cDNA library. We identified ten proteins as potential PRMT8 N-terminal interactors that had not previously been reported to interact with PRMT8. These diverse binding partners of H8N are associated with cell adhesion, cellular metabolic process, RNA binding, transcriptional regulation, actin filament binding, mitotic spindle organization and aging. Based on the Y2H system, we performed 3-AT spot dilute assay to examine the binding strength between PRMT8 and PRMT8 interactors. We also conducted a Y2H-based analysis in cross species and showed that two H8N interactors, SDHB and TMEFF2, can interact with the N-terminal 96 amino acid of zebrafish Prmt8 (ZF8N). Besides, two more H8N interactors DCTN6 and CACYBP interacted with ZF8N when a unique glutamine-rich (Q-rich) sequence before the proline and histidine-rich region in zebrafish PRMT8 was deleted (∆ polyQ). In addition, we identified that the N-terminus of human PRMT8 interact with SDHB at the C-terminal 134 amino acids. Moreover, we used co-immunoprecipitation assay to validate specific interaction between H8N and the putative interactors such as SYNE1, CACYBP and CNRIP1a. In this study, we finally validated the interaction of a few putative ZF8N interacting proteins identified by my previous pulldown experiments that are non-overlapping with H8N interactors. We used the pull-down assay and the Y2H analysis to study the interactions between ZF8N and two putative interactors Epd and ZFYwhaba. Co-immunoprecipitation assay was also used to examine the interaction of other specific interactors including INA, actin, tubulin, and GAPDH. Results of this study should extend the interactome of PRMT8 and provide more specific understanding of the function of PRMT8.
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Xie, Ying. "Transcriptional Regulation of Pregnane X Receptor by Protein Arginine Methyltransferase." Thesis, 2010. http://hdl.handle.net/1969.1/ETD-TAMU-2010-05-7978.
Full textAbstract:
Pregnane X receptor (PXR) is a ligand-dependent transcription factor that plays an important role in xenobiotic/drug metabolism. The ligand-receptor interaction transcriptionally activates phase I and phase II enzymes, and membrane-bound transporters in a coordinated manner and ultimately leads to detoxification and excretion of the ligands. One of the direct target genes is cytochrome P450 3A4 (CYP3A4) which is responsible for metabolism of over 50% of clinically used drugs. Understanding the regulation of PXR is important for treatment of disease and avoidance of untoward drug-drug interactions.
In this research, we have used various biochemical and molecular approaches to investigate factors that regulate the transcriptional activity of PXR. We have stably transfected PXR into HepG2 human liver hepatoma cells. Using these PXR-HepG2 cells, we surveyed the histone methyltransferases that interact with PXR. Based on results from co-immunoprecipitation/methyltransferase, N-terminal peptide sequencing, GST-pulldown assays, we found that protein arginine methyltransferase 1 (PRMT1) is a predominant histone methyltransferase in HepG2 cells.
Evidence from other laboratories suggests that histone methylation by PRMT1 sets the stage for subsequent histone modifications such as the acetylation of histone H4. These modifications are believed to be important for transcriptional and epigenetic regulation of gene expression. We hypothesize that PRMT1 plays a role in the epigenetic changes regulated by PXR. PRMT1-dependent histone methylation changes may be involved in epigenetic cell memory where prior exposure to certain agents may alter the chromatin (or priming the chromatin) with a "primed" state which alters the subsequent magnitude or duration of gene expression.
In our study, we have found that pretreatment of PXR-HepG2 cells with DMSO greatly enhanced PXR-mediated activation of CYP3A4 upon rifampicin treatment. DMSO pretreatment altered histone modifications association with the promoter of the PXR-regulated gene (CYP3A4). Inhibition of histone methylation by PRMT1 either through RNAi or the methyltransferase inhibitor (Adox) abolished the priming effects.
My research results strongly indicate that PRMT1 is involved in transcriptional regulation of PXR and may be involved in epigenetic memory of liver cells where prior exposure to agents changes the subsequent detoxification responses.
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Chang, Yuan-I., and 張原翊. "The Role of Protein Arginine Methyltransferase 1 (PRMT1) in Megakaryocytic Differentiation." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/56450062880750444889.
Full textAbstract:
博士國立陽明大學
生物藥學研究所
98
Protein arginine methyltransferase 1 (PRMT1) plays pivotal roles in various cellular processes. However its role in megakaryocytic differentiation is yet to be investigated. This study showed that ectopic expression of HA-PRMT1 in human leukemia K562 cells suppressed phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation as demonstrated by changes in cytological characteristics, adhesive properties and CD41 expression, while knockdown of PRMT1 by small interference RNA promoted differentiation. Impairment of the methyltransferase activity of PRMT1 diminished the suppressive effect. In addition, enforced expression of PRMT1 in human CD34+ hematopoietic cells also suppressed thrombopoietin (TPO)-induced megakaryocytic differentiation. These results provide evidence for a novel role of PRMT1 in negative regulation of megakaryocytic differentiation. Activation of Erk MAPK has been shown to be essential for megakaryocytic differentiation, however, the role of p38 MAPK is still poorly understood. Here, knockdown of p38α MAPK or treatment with the p38 inhibitor SB203580 significantly enhanced PMA-induced megakaryocytic differentiation. Further investigation revealed that PRMT1 promotes the activation of p38 MAPK without inhibiting the activation of Erk. In p38α-knockdown cells, PRMT1 could no longer suppress differentiation. In contrast, enforced expression of p38α MAPK suppressed PMA-induced megakaryocytic differentiation of parental K562 as well as PRMT1-knockdown cells. Together, I propose modulation of the p38 MAPK pathway by PRMT1 as a novel mechanism regulating megakaryocytic differentiation. This study thus provides a new perspective on the regulation of megakaryopoiesis. In mammalian cells, PRMT1 proteins exist in a large protein complex, which has been implied in modulating the regulatory and catalytic properties of this enzyme. Here, I showed that ectopically expressed PRMT1 in mammalian HEK293 cells not only exhibited catalytic properties comparable to the endogenous enzyme but also existed in a functional complex together with the endogenous PRMT1. Thus, this cell model was used to systemically identify putative PRMT1 substrates in mammalian cell contexts by a proteomic approach. Two proteins identified by this approach, the eukaryotic translation initiation factor 4A-I (eIF4A-I) and the intermediate filament vimentin, were demonstrated indeed as novel PRMT1 substrates by an in vitro methylation assay. Interestingly, these two proteins do not contain the previously known PRMT1 recognition motif glycine- and arginine-rich (GAR) sequence. Thus, I have demonstrated the functional equivalence of an ectopically expressed HA-PRMT1 in HEK293 cells and the application of this cell model in combination with a proteomic analysis to systematically identify the putative substrate proteins.
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Hsu, Min, and 徐鳴. "Ectopic Expression of Protein Arginine Methyltransferase 1 (PRMT1) in Mammalian Cells." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/53106441423199942693.
Full textAbstract:
碩士國立陽明大學
生物藥學研究所
89
PRMT1 (protein-arginine N-methyltransferase 1) is the predominant arginine methyltransferase in mammalian cells that contributes > 90% of protein-arginine methylated activity. PRMT1 was first cloned because of its ability to interact with mammalian TIS21 immediate-early protein. To understand the enzymatic activity of PRMT1, we used hnRNP A2 as a substrate for GST-PRMT1. We showed that bivalent cations affected the methyltransferase activity of PRMT1. That display distinct methylated degrees in the presences of different cations and concentrations and we found that Ca2+ has conspicuous influence. Relative, univalent cation has slightly effect. PRMT1 activity appears to be positively regulated through its association with two proteins, the TIS21/BTG1 gene product and the interferon receptor, each of which plays an important role in cell growth regulation. We characterized the growth property of RAT1 cells overexpressing HA-PRMT1 by growth curve counting and BrdU incorporation analysis, and demonstrated that there is no difference between RAT1 and RAT1 overexpressing HA-PRMT1 cells in growth patterns. In addition, in vitro methylation of Trx-hnRNP A2, using cell lysates from RAT1 or RAT1 overexpressing HA-PRMT1, showed that there was no apparent difference in methyltransferases activity.
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(9732323), Elena Wild. "Protein Arginine Methyltransferase 5 in Castration-Resistant and Neuroendocrine Prostate Cancer." Thesis, 2020.
Find full textAbstract:
Prostate cancer is one of the most frequently diagnosed cancers and the second leading
cause of cancer-related deaths in male population. While localized prostate cancer can be
successfully treated with surgery or radiation therapy, the metastatic disease has no curable options.
Metastasis can be developed as a result of failed therapy of localized cancer or present at initial
diagnosis. As metastasis is the most common cause of prostate cancer-related death, developing
novel approaches and improving the efficiency of existing therapies for the metastatic prostate
cancer treatment will significantly improve patients’ survival. The first-line treatment option for metastatic prostate cancer and localized prostate cancer
with high risk of recurrence is androgen deprivation therapy (ADT) that decreases androgen
receptor (AR) signaling. However, targeting AR signaling inevitably leads to AR reactivation and
cancer progression to the castration-resistant prostate cancer (CRPC) that has no curable treatment
options. Moreover, about 30% of CRPC cases progress to neuroendocrine prostate cancer (NEPC),
highly aggressive and lethal type of prostate cancer.
Recently my group has shown that protein arginine methyltransferase 5 (PRMT5) functions
as an activator of AR expression in hormone-naïve prostate cancer (HNPC). In this dissertation, I
demonstrate that PRMT5 also functions as an epigenetic activator of AR transcription in CRPC via
symmetric dimethylation of H4R3 at the AR promoter. This epigenetic activation is dependent on
pICln, a PRMT5 interaction partner involved in spliceosome assembly, and independent of MEP50,
the canonical cofactor of PRMT5. PRMT5 and pICln, but not MEP50, were required for the
expression of AR signaling pathway genes. In clinical samples of both HNPC and CRPC, nuclear
PRMT5 and pICln protein expressions were highly positively correlated with nuclear AR protein
expression. In xenograft tumors, targeting PRMT5 or pICln significantly decreased tumor growth
and AR expression.
Overall, this work identifies PRMT5/pICln as a therapeutic target for HNPC and CRPC
treatment that needs to be further evaluated in clinical setting.
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Lu, Yu-Ling, and 呂玉琳. "Biochemical analysis of the catalytic activity of protein arginine methyltransferase 1 (PRMT1)." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/85887402131896497397.
Full textAbstract:
碩士國立陽明大學
生物藥學研究所
90
Protein arginine methyltransferase 1 (PRMT1) is a type I protein arginine methyltransferase, which can catalyze the formation of asymmetric ω-NG,N’G-dimethylarginine residues by transferring methyl groups from S-adenosyl-L-methionine to guanidine groups of arginine residues in a variety of eukaryotic proteins. To study the catalytic properties of PRMT1, we have successfully purified recombinant GST-PRMT1 and showed that GST-PRMT1 was enzymatically active using Trx-hnRNP A2 as a substrate. The kinetic properties of GST-PRMT1 with either Trx-hnRNP A2 or a peptide substrate containing RGG motif have been examined. Divalent cations showed differential effects on methylation of Trx-hnRNP A2 and the peptide substrate by GST-PRMT1. These suggested a potential regulation of PRMT1 activity through metal ions in cells.
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Lin, Sheng-Wei, and 林盛偉. "Identification and characterization of protein substrates of protein arginine methyltransferase 1 (PRMT1) in HEK293 cells." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/91216993202343580209.
Full textAbstract:
碩士國立陽明大學
生物藥學研究所
92
Abstract In the complicated cellular processes, post-translational modifications such as phosphorylation, glycosylation, and methylation play significant roles. Accumulating evidence shows that protein arginine methylation participates in various mechanisms controling cell growth, gene expression and signal transduction. PRMT1 (protein arginine methyltransfrease 1) is the predominant type I protein arginine methyltransferase in mammalian cells. Arginine methylation has been shown to affect protein-protein interaction, signal transduction and RNA transport. This study is aimed to identify potential substrates of PRMT1 by proteomic approach. PRMT1 was overexpressed, either transiently or stably in HEK293 cells. The two-dimensional gel electrophoresis (2D) analysis followed by fluorography clearly displayed proteins preferentially methylated in PRMT1-expressing cell lysates suggesting that they were potential PRMT1 substrates. Several proteins including heterogeneous nuclear ribonucleoprotein K (hnRNP K), a RNA-binding protein, was identified by ESI-Q-TOF mass spectrometry. hnRNP K was methylated readily by GST-PRMT1 in the in vitro methylation assay. Methylation of hnRNP K was also enhanced by HEK293 cells transfected with PRMT1, both suggesting that hnRNP K was a substrate of PRMT1. Methylation of hnRNP K by PRMT1 was modulated by small molecules. This study also showed the different regulation between hnRNP K and hnRNP A1, a known protein substrate of PRMT1 by metal ions.
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Lin, Yi-Chin, and 林苡勤. "Role of protein arginine methyltransferase 3 in colorectal cancer cell invasion and progression." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/c8snxt.
Full textAbstract:
碩士國立臺灣大學
生物化學暨分子生物學研究所
107
Colorectal cancer is one of the most common cancers and the third leading cause of cancer-related mortality in Taiwan and worldwide. Although cancer therapies have been improved, the mortality of metastatic colorectal cancer patients still remains high. The epigenetic modification of methylation has been proposed to be involved in colorectal cancer development and progression and arginine methylation is a newly identified mechanism for post translation modification. In this study, I analyzed if there was an alteration of a protein arginine methyltransferase (PRMT) in human colorectal cancer. According to a public database, the bioinformatics analysis showed that the alterations of the PRMT2, PRMT3 and PRMT5 gene expression levels were correlated with the survival rates of colorectal cancer patients. Among these three genes, I further found that the expression levels of PRMT3 were decreased in the colorectal cancer cells isolated from Dukes’ D, compared to those cells from Dukes’ B and C. Moreover, PRMT3 silencing increased colorectal DLD-1 and HCT116 cancer cell invasion. The PRMT3 knockdown-induced colorectal cancer cell invasion was able to be inhibited by a broad serine protease inhibitor, suggesting a serine protease is involved in PRMT3 signaling of colorectal cancer cells. Moreover, I used a protease array analysis and found that the expression levels of kallikrein 5 were dramatically increased in PRMT3 silencing colorectal cancer cells. The results together indicate that PRMT3 plays a suppression role in colorectal cancer cell invasion and kallikerin 5 may be involved in PRMT3-mediated colorectal cancer progression.
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Chen, Jo-Hsin, and 陳若昕. "The Role of Protein Arginine Methyltransferase 6 in PMA-induced Megakaryocytic Differentiation of K562 Cells." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/51229105068385324130.
Full textAbstract:
碩士國立陽明大學
生物藥學研究所
100
Protein arginine methylation adds methyl groups onto arginine residues in protein and has been emerging as a pivotal posttranslational modification regulating a variety of cellular processes. There are at least 11 PRMTs (protein arginine methyltransferases) identified. PRMT6 (Protein arginine methyltransferase 6) has been implicated in gene transcription, viral pathogenesis, and DNA repair, however its role in MK (megakaryocytic) differentiation has not been investigated. In this study, we used human leukemia cell line K562 as a model and showed that overexpression of PRMT6 promoted PMA (phorbol myristate acetate) -induced MK differentiation of K562 as examined by formation of lobed nuclei, increased vacuoles, and surface expression of CD41. This stimulatory effect was particularly remarkable at low concentrations of PMA. Knockdown of PRMT6 suppressed MK differentiation. Activation of the ERK1/2 (extracellular signal-related kinase 1/2) pathway is known to be essential for MK differentiation. This study showed that PRMT6 enhanced activation of ERK1/2 particularly at low concentrations of PMA, suggesting PRMT6 may promote MK differentiation by promoting activation of ERK1/2. Inhibition of MEK-ERK1/2 pathway by using a MEK inhibitor (PD98059) only partly suppressed PRMT6-meditated effects, suggesting that PRMT6 regulates, in addition to the MEK-ERK1/2 pathway, other molecular events to promote MK differentiation. Our previous studies revealed that PRMT1 negatively regulated MK differentiation. This study showed that overexpression of PRMT6 could override the suppressive effect of PRMT1 on MK differentiation despite that PRMT1 is the predominant member of mammalian PRMTs. PRMT6 modestly but significantly affected PRMT1 activity as detected by in vitro methylation assays. Our results showed for the first time that PRMT6 regulated MK differentiation and suggest that members of the PRMT family can functionally interact to regulate MK differentiation.
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Hung, Chuan Mao, and 洪銓錨. "Identification and phylogenetic analyses of the protein arginine methyltransferase gene family in fish and ascidians." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/16995426980737446230.
Full textAbstract:
碩士中山醫學大學
醫學研究所
92
Protein arginine methyltransferases (PRMT) involved in the regulations of signal transduction, protein subcellular localization, and transcription have been mostly studied in mammals and yeast. In this study orthologues of eight human PRMT genes (PRMT1-7 and HRMT1L3) were identified in both puffer fish Fugu rubripes and zebrafish Danio rerio. The fish PRMT genes appear to be conserved with their mammalian orthologues at the levels of amino acid sequences as well as genomic structures. All vertebrate PRMT genes contain 10-16 coding exons except PRMT6 that contains only one coding exon. Western blot analyses of zebrafish tissue extracts confirmed the expression of some PRMT proteins in zebra fish. We further identified six PRMT members (PRMT1, 3-7) in an invertebrate chordate Ciona intestinalis. Genomic structures of the PRMT orthologues are no more conserved in the ascidians, as PRMT3 and PRMT5 contain only one coding exon while PRMT6 contains 6 exons. PRMT2 and HRMT1L3 that are missing in Ciona appear to be vertebrate-specific. HRMT1L3 is a PRMT1 paralogue with highly conserved sequences and exact exon junctions, whereas the PRMT2 orthologues are very diverged. Different PRMT orthologues are likely to evolve at different rates and the PRMT1 orthologues appear to be most conserved through evolution. Furthermore, phylogenetic analyses using the core regions of various PRMT genes show that PRMT5 with the type II PRMT activity is separated in one branch. All other PRMT genes including PRMT1, 2, 3, 4, 6, 7 and HRMT1L3 clustered in the other branch, probably represent the genes for the type I activity.
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Prabhu, Lakshmi Milind. "Targeting Protein Arginine Methyltransferase 5 as a Novel Therapeutic Approach in Pancreatic & Colorectal Cancer." Diss., 2018. http://hdl.handle.net/1805/18096.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)Pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) are among the most commonly diagnosed forms of cancer in the United States. Due to their widespread prevalence and high mortality rate, it is vital to develop effective therapeutic drugs to combat these deadly diseases. In both PDAC and CRC, the multifunctional factor nuclear factor kappa B (NF-kB), a central coordinator of cellular immune responses, is activated abnormally, leading to tumorigenesis and cancer progression. Therefore, controlling NF-kB activity is critical in the treatment of these cancers. In a previous study, we identified a new mechanism by which NF-kB activity is regulated by an epigenetic enzyme known as protein arginine methyltransferase 5 (PRMT5). We showed that overexpression of PRMT5 not only activated NF-kB, but also significantly promoted several characteristics associated with cancer, including increased cell proliferation, migration, and anchorage-independent growth in both PDAC and CRC cells. Moreover, in order to examine the therapeutic potential of PRMT5 in these cancers, we adapted the state-of-the-art AlphaLISA technique into a high throughput screen (HTS) platform to screen for PRMT5 inhibitors. As a result, we successfully identified the small molecule PR5-LL-CM01 as our lead hit. Further validation experiments confirmed that PR5-LL-CM01 is a potent and specific PRMT5 inhibitor that exhibits significant anti-tumor efficacy in both in vitro and in vivo models of PDAC and CRC. Additionally, in a second screen, we discovered two natural compounds, P1608K04 and P1618J22, that can also function as the PRMT5 inhibitors. These findings further highlight the robustness of the PRMT5- specific AlphaLISA HTS technique. To conclude, we describe here for the first time a novel role of PRMT5 as a tumor-promoting factor in PDAC and CRC through NF-kB activation. By successfully developing and applying an innovative AlphaLISA HTS technique, we discovered PR5-LL-CM01, P1608K04, and P1618J22 as novel PRMT5 inhibitors, with PR5-LL-CM01 showing the strongest potency in both PDAC and CRC models. Therefore, we demonstrated that PRMT5 is a promising therapeutic target in PDAC and CRC, and the novel PRMT5 inhibitor PR5-LL-CM01 could serve as a promising basis for new drug development in PDAC and CRC.
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Wang, Chien-Wen, and 王千文. "Analyses of the deletion of the most evolutionarily conserved protein arginine methyltransferase PRMT1 in avian." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/56960787617767661280.
Full textAbstract:
碩士中山醫學大學
生物醫學科學學系碩士班
104
Protein arginine methyltransferases (PRMTs) preform posttranslational modification involved in transcriptional regulation, RNA processing and many different cellular processes. Nine prmt genes are conserved in vertebrates, while prmt1, 2, 4 and 6 are not present in avian. Among these prmt1 is the most conserved and widely distributed prmt gene in eukaryotes, and its deletion is embryonic lethal in mouse. No prmt1 but only its vertebrate paralogue prmt8 could be identified in birds. As avian underwent large segmental chromosomal deletions and gene loss during their evolution, we first determined whether in birds the prmt1 gene was specifically eliminated alone or with neighboring chromosomal segments. We obtained 11 downstream and upstream genes of prmt1 and prmt8 in typical vertebrate species as shown in the UCSC genome browser, collected their homologous genes in other species by BLAST, and then recorded and compared their chromosome localizations. In pair-wise species comparison, orthologous genes near prmt8 have higher conserved level and more gene clusters than prmt1. As most vertebrate type I PRMTs (PRMT1, 2, 3, 4, 6, and 8) that catalyze the formation of asymmetric dimethylarginine (ADMA) are missing in avian, we are interested in whether the ADMA level in birds may be lower than that in other vertebrates. We prepared brain, liver and muscle extracts from chicken and mouse. The ADMA levels were not reduced in different chicken tissues as shown by western blot analyses with an ADMA-specific antibody. We then suspected that loss of prmt1 may be compensated by its paralogue prmt8. However, as shown by RT-PCR analyses, prmt8 mRNA expression was still brain-specific in chicken. It is thus unlikely that prmt8 can complement the loss other type I PRMTs. We also fractionated mouse and chicken brain tissue to examine the PRMT distribution pattern. The result reveals subcellular distribution of type I PRMTs in chicken and mouse have high similarity. Furthermore, in vitro methylation revealed that chicken brain still has similar capability to catalyze arginine methylation as mouse brain extract. Our study provides information about chromosome rearrangement and syntenic blocks deletion near prmt1 in avian evolution. Chicken can be considered as a natural multiple prmt-deleted biological system to further elucidate the critical function of protein arginine methylation conserved and not conserved in vertebrates.