Adam Frankel

Protein arginine N-methyltransferases (PRMTs) catalyze the transfer of methyl groups from the methyl donor S-adenosyl-L-methionine (SAM) to polypeptide substrates. PRMTs are conserved among eukaryotes and many of their biological roles, including transcriptional regulation, DNA repair, and RNA processing, have been well elucidated. Another emerging research area is their role in stress pathways. Because of their significance in biological processes, PRMTs are promising drug targets for many human diseases. Knowledge of an enzyme’s kinetic mechanism and roles in cellular pathways can contribute to effective design of PRMT inhibitors. The purpose of this project was to examine how PRMTs bind to and methylate their substrates, and explore their roles in the stress response using Saccharomyces cerevisiae as a model organism. Using a combination of steady-state enzyme kinetics and biophysical assays, including differential scanning fluorimetry (DSF), we show that the predominant mammalian PRMT binds its substrates in a sequential manner where target substrate binding follows cofactor binding. By using knockout strains, we demonstrate that methylarginines are expelled from cells undergoing stress and that methylarginine expulsion is driven by autophagy. Further, we demonstrate that yeast do not take up methylarginines from their environment because methylarginines can inhibit nitric oxide production which is important for long-term cell survival under stressful growth conditions. Last, through studying in vitro methylation of yeast lysates using recombinantly expressed enzymes, we identify putative yeast methyltransferase Ynl092wp as a protein histidine N-methyltransferase that binds its substrates using a sequential mechanism and predict that Ykl162cp is an RNA methyltransferase. Here, we use a novel approach to examine the PRMT binding mechanism and we demonstrate that this novel technique is applicable to other enzyme families. Further, we demonstrate how yeast cells can evade cell toxicity by controlling methylarginine flux. Therefore, the research described herein encompasses the full importance and impact of PRMTs and their substrates in eukaryotic biological pathways.

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Within the last two decades research in the field of epigenetics has increased significantly as targeting the epigenetic enzymes has the potential to alter the transcription of genes. Aberrant regulation of transcription is seen in several disease states, and drugs targeting the epigenetic histone deacetylases and DNA methylases are already marketed for cancer treatment. The Protein Arginine Methyl Transferases (PRMTs) belong to an epigenetic enzyme family that is upregulated in several cancers. However, currently no inhibitors of the PRMTs have been marketed. In this thesis several peptidomimetic strategies were utilised to modify the tryptophan residues in two peptide leads in order to discover new inhibitors of the PRMTs. One of these strategies involved constraining the side chain indole of tryptophan to the peptide backbone, thus producing a seven membered azepinone mimetic, Aia. The peptidomimetic efforts resulted in a structure-activity relationship study from which a constrained peptidomimetic containing two Aias was discovered to be a low micromolar inhibitor of several PRMTs. To characterise the inhibitor the conformation of the inhibitor was examined using solution-phase NMR and was shown to display an interesting turn-structure. The original peptide lead was fluorescently tagged and investigated in a cellular setting, but did not reveal any PRMT-specific localisation.In an effort to study the binding of the discovered inhibitor with the PRMTs, protein expression in E. coli and purification was performed. This resulted in the optimisation of PRMT6 purification in order to obtain highly pure PRMT6 for isothermal titration calorimetry (ITC) studies. Unfortunately these ITC studies were unsuccessful.Furthermore, as the constrained tryptophan mimetic had proven very useful in the peptidomimetic inhibitors of the PRMTs, we attempted to synthesise a lysine/arginine dipeptide mimetic using aziridine chemistry.

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Protein arginine N-methyltransferases (PRMTs) are a family of enzymes involved in signaling pathways and gene expression by methylating arginine residues of substrate proteins. PRMT2 has been demonstrated to play a role in the NF-κB signaling pathway. Moreover, our lab recently revealed association using proteomic techniques between PRMT2 and splicing factors including Src-associated in mitosis 68 kDa protein (SAM68) that mediates the alternative splicing of BCL-X involved in the NF-κB mediated inflammatory pathway. I wanted to investigate if the PRMT activity plays a role in response to inflammation under the treatment of inflammatory cytokine tumor necrosis factor-α (TNF-α) or pro-inflammatory bacterial lipopolysaccharide (LPS) in A549 cells. My proteomic experiments revealed that TNF-α and LPS cause similar changes to arginine methylation for proteins primarily involved in mRNA processing, RNA splicing, and nuclear transport, indicating that these two inflammatory stimuli share mutual downstream pathways involving methyltransferase activity. Among the proteins that showed hypermethylation upon treatment relative to control in mass spectrometry analysis, GAP SH3 domain-binding protein 2 (G3BP2) showed consistently in all three replicates on average a 1.5-fold increase in methylation at the R468 site in both TNF-α and LPS-treated cells. G3BP2 binds to IκB-α and prevents NF-κB translocation into the nucleus for subsequent signaling. G3BP2 methylation was necessary for signaling to occur in the Wnt/β-catenin signaling pathway. Methylation of G3BP2 might also have similar role in the inflammatory pathway that demands further study. Moreover, consistent with an inflammatory response, proteins particularly involved in innate immunity and viral response increased upon TNF-α treatment that could be related to the observed change in methylation of proteins involved in RNA processing. Our finding that PRMT2 interacts with SAM68, prompted me to investigate the potential role of PRMT2 in BCL-X alternative splicing. I found that reduced expression of PRMT2 by siRNA caused a decrease in the BCL-X(L)/BCL-X(s) ratio, suggesting that PRMT2 may contribute to BCL-X alternative splicing. This effect was replicated in TNF-α or LPS stimulated cells when PRMT2 expression was reduced by shRNA, and reversed when PRMT2 expression was increased. These results indicate that PRMT2 may play a role during inflammation in alternative splicing regulation.

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Protein arginine N-methyltransferases (PRMTs) constitute a family of post-translationalmodifying enzymes that modulate protein-protein interactions via the addition of methyl groupsto arginine residues in protein substrates (1). PRMTs have been demonstrated to homooligomerizevia a dimerization arm that binds with the outer surface of the S-adenosyl-Lmethionine(AdoMet) binding domain (2-5). In this body of work, I have demonstrated andquantified in vitro the strength of homodimerization for PRMT1 and PRMT6 and demonstratedthat saturating concentrations of S-adenosyl-L-methionine (AdoMet) or S-adenosyl-Lhomocysteine(AdoHcy) respectively strengthen or weaken this interaction. This findingsupports an ordered bisubstrate mechanism in which AdoMet binding promotes formation of thecomplete peptide-substrate binding groove through dimerization, and AdoHcy generationpromotes dissociation of the dimeric complex and turnover of substrate.A kinetic study using HIV Tat peptides revealed oligomerization-dependent kineticpatterns with these substrates. Kinetic experiments were initially performed on HIV Tat peptidewith novel ωN-substitutions to probe their ability to inhibit PRMT1, 4 and 6. It was found thatthese Tat-peptides act as substrate inhibitors for both PRMT1 and PRMT6 and that this substrateinhibition was mitigated as the enzyme concentration increased. A model was proposed thatrepresents activity as the sum of each ordered oligomer in solution, with the monomer beinguniquely susceptible to substrate inhibition.Diverging from strictly oligomerization effects, R1 fibrillarin-like peptide containing asingle arginine was substituted to alter the pKa of the terminal guanidino group to better probethe physicochemical properties that control methyltransfer. Surprisingly, hydroxyl substitutedR1 peptide demonstrated an enhanced catalytic constant with PRMT1. MS and MS² experiments demonstrate that only monomethylation occurs on substituted arginines with PRMT1, and that this addition is asymmetric. PRMT1 D51N, a catalytically compromised mutant, revealed the kcat as rate limiting in the presence of D₂O, and electrostatic potential maps indicate that deprotonation of hydroxyl substituted arginine produces a strong nucleophile capable of enhanced methyltransfer.Altogether, these studies support water mediated, ordered bisubstrate mechanism in which oligomerization modulates activity. Substrate inhibition and active site chemistry were investigated using novel chemically substituted peptide probes that highlight trends beyond what site-directed mutagenesis can reveal alone.

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Protein arginine N-methyltransferases (PRMTs) act in signaling pathways and geneexpression by methylating arginine residues within target proteins. PRMT1 is responsible formost cellular arginine methylation activity and can work independently or in collaboration withother PRMTs. In this Ph.D. thesis I demonstrated an interaction between PRMT1 and -2 usingco-immunoprecipitation and bimolecular fluorescence complementation (BiFC). As a result ofthis interaction, PRMT2 stimulated PRMT1 methyltransferase activity, affecting its apparentVmax and Km values in vitro, and increasing the production of methylarginines in cells. Activesite mutations and regional deletions on PRMT1 and -2 were also investigated, whichdemonstrated that complex formation required full-length, active PRMT1. However, theinteraction between PRMT1 and -2 proved insensitive to methylation inhibition in the absence ofthe PRMT2 Src homology 3 (SH3) domain, which suggests that the PRMT2 SH3 domain maymediate this interaction between PRMT1 and -2 in a methylation-dependent fashion.The role of the PRMT2 SH3 domain was investigated through screening for its associatedproteins using GST-pull down assays followed by LC-MS/MS proteomic analysis. The result ofthis study revealed associations of the PRMT2 SH3 domain with at least 29 splicing-relatedproteins, 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 domaincould affect Sam68 sub-cellular localization in hypomethylated HeLa cells. The biological functions of PRMT2 and the PRMT1/2 heteromeric complex were exploredby pursuing the identity of associated proteins common to both PRMT1 and -2 using massspectrometry proteomics. Approximately 50% of the identified protein hits have reported rolesin controlling gene expression, while other hits are involved in diverse cellular processes such asprotein 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 throughPRMT2-protein interactions.

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