Protein arginine methylation is an important modification of proteins, involved in many cellular processes. Some examples are transcription, RNA editing, cellular communication, DNA repair, viral replication and chromatin remodeling. In recent years, the significance of protein arginine methyltransferases (PRMTs) in human diseases has been increasingly studied, especially in cardiovascular disease and cancer. Although the importance of these enzymes is recognized, the understanding of how exactly PRMTs function is still limited. Very little information is available to explain how or why any of the different PRMTs interact with other proteins or, what determines where in that protein to place their methyl marks. Adding to this complexity, placing one of the three different methylation marks (products) or the other (mono methyl arginine MMA, asymmetric dimethyl ADMA, or symmetric dimethyl SDMA) on a protein can cause a cell to respond differently. Therefore, if we really want to understand how this family of proteins functions and how to control them, it’s essential that we understand how they achieve their product specificity; this means, how they decide which methyl mark to place on an interacting protein. In order to better understand the product specificity of this family of enzymes, I have been using as a model two Protein arginine methyltransferases that are responsible different methylation marks: PRMT1, which can make both ADMA and MMA and TbPRMT7, which can only make MMA. Using the information that crystal structure of these enzymes provide and what we already know about how PRMT activity is regulated, my aim is to better understand the mechanisms by which these enzymes achieve their product specificity.

PRMTs and exercise. / Protein arginine methyltransferases (PRMTs) play an important role in muscle. Using three unique but complementary approaches across human and mouse models, we examined PRMT biology during conditions of exercise-induced skeletal muscle plasticity. In response to acute and chronic cues for muscle plasticity in human muscle, an array of PRMT-specific increases and reductions in expression and activity were observed. Following this we generated coactivator-associated arginine methyltransferase 1 (CARM1) skeletal muscle-specific knockout (mKO) mice to further examine the role of this enzyme. We discovered that the rate of arginine methylation is equivalent to that of phosphorylation and ubiquitination in healthy muscle. CARM1 mKO displayed altered transcriptome and arginine methylproteomic signatures, confirming remodelled muscle contractile and neuromuscular junction characteristics, which foreshadowed the animal’s decreased acute exercise tolerance. Removal of CARM1 reduced voluntary wheel running (VWR) performance in a sex-dependent manner and eliminated the strong, positive correlation between VWR distance and mitochondrial number observed in WT mice. While CARM1 was shown to regulate AMPK-PGC-1α signaling during acute conditions of activity-induced muscle plasticity, molecular measures of PRMT biology were mostly unaffected by VWR and the removal of this enzyme. In conclusion, these results indicate that changes to expression and activity are PRMT-specific and reveal the broad impact of CARM1 in the maintenance and remodelling of skeletal muscle biology. / Thesis / Doctor of Philosophy (PhD) / Skeletal muscle is a malleable tissue that can adapt to an array of physiological demands. Past research suggests that protein arginine methyltransferases (PRMTs) regulate skeletal muscle remodelling. However, their role in exercise-induced skeletal muscle plasticity is unknown. Therefore, the purpose of this work was to investigate PRMT biology during acute and chronic exercise. Our data demonstrate that in human muscle a variety of PRMT-specific alterations in expression and activity occur in response to cues for muscle plasticity. Using mice lacking coactivator-associated methyltransferase 1 (CARM1) in skeletal muscle, we studied the impact removal of CARM1 has on the acute and chronic muscle adaptations to training. Our data demonstrate that in addition to changing molecular signals and physiological function at rest, the deletion of CARM1 decreased acute exercise ability and altered chronic training performance in a sex-dependent manner. Altogether, these findings expand our knowledge of PRMTs in skeletal muscle biology.

Protein arginine methylation is an important post-translational modification that is vital in regulating various cellular processes such as gene transcription, cell signaling, and RNA processing. Protein arginine methyltransferases (PRMTs) are responsible for performing this important modification. PRMT1 (protein arginine methyltransferase 1) and Hmt1 (hnRNP methyltransferase 1) are the predominant PRMTs in humans and yeast, respectively. Despite growing momentum in this field, relatively little is understood about PRMT regulation. Further work discovering how PRMTs are regulated will greatly advance our understanding of diseases where PRMTs have been implicated, such as heart disease, viral pathogenesis, and cancer. It has been discovered that a human protein called hCaf1 (human Ccr4-associated factor 1) is a regulator of PRMT1 with respect to certain substrates, and also colocalizes with PRMT1. We present data that suggest the yeast homolog of hCaf1, Pop2, may also perform a similar function on Hmt1. We provide data on the expression and purification of a truncation of Pop2 from S. cerevisiae, including the temperature sensitivity of one construct of Pop2 and its susceptibility to precipitation. We also demonstrated concentration-dependent inhibition of Hmt1-catalyzed methylation of histone H4 by Pop2 in vitro. Yeast cell lysates also showed altered patterns of methylation in the presence and absence of Pop2 in vivo. In an effort to understand the mechanism employed by Pop2 to accomplish this regulatory function, pull-downs were performed suggesting that Pop2 directly interacts with histone H4, a substrate of Hmt1. Mutagenic studies with Pop2 suggested a region that may be responsible for this interaction. Given these data, we hypothesized that Pop2 is able to inhibit the methylation of histone H4 via a substrate-sequestering mechanism. Further experimentation will determine the precise interaction surfaces of Pop2 and substrate, and continue to define the details of methylation inhibition by Pop2, including the scope of its influence in the cell.

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.

The synthetic methodology of tri- and pentamethine carbocyanines and their interactions with biomolecules will be discussed in two chapters. The first chapter describes the preparation of halogenated carbocyanine dyes that display multiple charges; furthermore, these particular compounds were examined for their ability to bind G-quadruplex DNA with selectivity over duplex DNA and have potential for developing novel chemotherapeutic agents. The second section discusses the synthetic methods utilized to prepare trimethine cyanine fluorophores. This chapter will show how varying the N-indolenyl substituients’ hydrophobicity from ethyl to phenylpropyl influences the binding to Human Serum Albumin (HSA); additionally, alternating the terminal heterocyclic moieties of the cyanine dye has a direct quantitative effect on the biomolecular interaction. These identical compounds were recognized to be structurally analogous to agents that commonly interact with Protein Arginine Methyl Transferase (PRMT) and these compounds display low IC50 values toward inhibition of PRMT1 with unique NIR imaging properties.

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.

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.

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.

PRMT biology during skeletal muscle disuse. / Protein arginine methyltransferase 1 (PRMT1), PRMT4 (also known as co-activator-associated arginine methyltransferase 1; CARM1), and PRMT5 are critical components of a diverse set of intracellular functions. Despite the limited number of studies in skeletal muscle, evidence strongly suggests that these enzymes are important players in the regulation of phenotypic plasticity. However, their role in disuse-induced muscle remodelling is unknown. Thus, we sought to determine whether denervation-induced muscle disuse alters PRMT expression and activity in skeletal muscle within the context of early signaling events that precede muscle atrophy. Mice were subjected to 6, 12, 24, 72, or 168 hours of unilateral hindlimb denervation. The contralateral limb served as an internal control. Muscle mass decreased by ~30% following 168 hours of disuse. Prior to atrophy, the expression of muscle RING finger 1 and muscle atrophy F-box were significantly elevated. The expression and activities of PRMT1, CARM1, and PRMT5 displayed differential responses to muscle disuse. Peroxisome proliferator-activated receptor-γ coactivator-1α, AMP-activated protein kinase (AMPK), and p38 mitogen-activated protein kinase expression and activation were altered as early as 6 hours after denervation, suggesting that adaptations in these molecules are among the earliest signals that precede atrophy. AMPK activation also predicted changes in PRMT expression and function following disuse. Our study indicates that PRMTs are important for the mechanisms that precede, and initiate muscle remodelling in response to neurogenic disuse. / Thesis / Master of Science (MSc) / Skeletal muscle is a plastic tissue that is capable of adapting to various physiological demands. Previous work suggests that protein arginine methyltransferases (PRMTs) are important players in the regulation of skeletal muscle remodelling. However, their role in disuse-induced muscle plasticity is unknown. Therefore, the purpose of this study was to investigate the role of PRMTs within the context of early, upstream signaling pathways that mediate disuse-evoked muscle remodelling. We found differential responses of the PRMTs to muscle denervation, suggesting a unique sensitivity to, or regulation by, potential upstream signaling pathways. AMP-activated protein kinase (AMPK) was among the molecules that experienced a rapid change in activity following disuse. These alterations in AMPK predicted many of the modifications in PRMT biology during inactivity, suggesting that PRMTs factor into the molecular mechanisms that precede neurogenic muscle atrophy. This study expands our understanding of the role of PRMTs in regulating skeletal muscle plasticity.

Les "protéine arginine méthyltransférases" (PRMT) sont impliquées dans de nombreux processus cellulaires : transcription, maturation et transport des ARN, traduction, transduction du signal, réplication et réparation de l'ADN, et apoptose. Différents travaux ont montré que des dérégulations de ces mécanismes impliquant les PRMT peuvent induire certains cancers, faisant de ces enzymes de nouvelles cibles potentielles en chimiothérapie. Il s’avère donc crucial de comprendre le mode d’action des PRMT à l’échelle atomique, à la fois au niveau fondamental et pour le développement de nouveaux médicaments. Les travaux décrits ici s’intéressent à la protéine PRMT4/CARM1 et s’appuient sur des études structurales par bio-cristallographie, pour comprendre les mécanismes de la réaction de méthylation catalysée par CARM1 et découvrir des inhibiteurs spécifiques, mais aussi sur des études en solution, pour caractériser l’interaction entre CARM1 et ses substrats. / Protein arginine methyltransferases (PRMTs) are involved in several cellular mechanisms: transcription, RNA maturation and transport, translation, signal transduction, DNA replication and repair, and apoptosis. Different studies showed that deregulation of those mechanisms involving PRMTs can induce some cancers, making these enzymes new potential targets for chemotherapy. It is therefore crucial to understand the mode of action of PRMTs at the atomic scale, both at the fundamental level and for the development of new drugs. The studies described here focus on PRMT4/CARM1 and rely on structural studies by bio-crystallography, in order to understand the methylation mechanisms catalyzed by CARM1 and to discover specific inhibitors, but also on in vitro studies, to characterize the interaction between CARM1 and its substrates.