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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L-arginine is one of the most versatile amino acids due to the fact that it serves as a precursor for many molecules which have important roles in bodily functions including mammalian reproduction. The current studies sought to further examine the role that L-arginine has in mammalian reproduction utilizing both in vivo and in vitro approaches. In the first study, a novel bioluminescent murine pregnancy model was developed to monitor VEGFR2 transcription activity non-invasively in the fetoplacental unit. Secondly, the effect that dietary L-arginine supplementation has during mouse gestation was examined. L-arginine supplementation increased weight gain during the latter third of gestation, total litter size, number of implantation sites, and litter birth weight. Additionally, L-arginine supplementation increased VEGFR2 transcription activity in the fetoplacental unit which may create a more favorable environment for fetal survival. Moreover, the increased number of implantation sites observed suggests an effect of L-arginine at the level of the endometrium. To this end, the effect that L-arginine has on apoptosis and cell proliferation in an established endometrial cell line was examined. The addition of L-arginine at physiological (200 micromolar) and supra-physiological (800 micromolar) concentrations increased cell proliferation , and this effect was achieved through biosynthesis of polyamines and nitric oxide. L-arginine also decreased the proportion of cells that were experiencing mitochondrial mediated apoptosis, and it was observed that this decrease in mitochondrial mediated apoptosis was concurrent with increased phosphorylation of BAD protein, which induces apoptosis when not phosphorylated. The final study examined the ability of porcine uterine epithelial (PUE) cells to synthesize L-arginine from L-citrulline. L-citrulline was able to support PUE cell proliferation in the absence of L-arginine. Additionally, ASS-1 and ASL, L-arginine synthesizing enzymes, were expressed in PUE cells and were regulated by the presence of L-arginine and L-citrulline, respectively. This data would support the hypothesis that PUE cells may be able to convert L-citrulline to L-arginine. Together, the current findings along with the plethora of relevant literature provide further evidence for the role of L-arginine in mammalian reproduction and allow for new questions to be investigated regarding this particular amino acid’s role in mammalian reproduction.

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 rations alimentaires riche en zinc (Zn) ou en arginine (Arg) sont connues pour améliorer la croissance des porcelets nouvellement sevrés mais les mécanismes d’actions ne sont pas encore bien connus. L’hypothèse à la base de ce doctorat est que le métabolisme oxydatif et la réponse anti-inflammatoire seraient impliqués dans cette réponse. Les effets de ces ajouts ont donc été évalués sur le statut antioxydant et la réponse inflammatoire des porcelets dans trois conditions différentes. Pour les trois essais, les rations étaient supplémentées ou non de Zn (2500 mg oxyde de Zn/kg) et d’Arg (1 %). Dans le premier essai, trente-deux porcelets sevrés de 20 jours ont été nourris pendant 12 jours avec l’une des quatre rations. Au jour 12, des prélèvements sanguins ont été effectués avant et 3 heures après une injection intrapéritonéale de lipopolysaccharide (LPS; 10 mg/kg) puis les porcelets ont été euthanasiés afin de prélever des échantillons de muqueuses intestinales. Les résultats ont indiqué que le Zn peut réduire l’oxydation systémique et améliorer l’état antioxydant dans les muqueuses intestinales. Par contre, les supplémentations en Zn et en Arg n’ont pas eu d’effet synergique positif sur l’état antioxydant ou l’inflammation chez les porcelets sevrés Dans le deuxième essai, quarante-huit porcelets sevrés à l’âge de 20 jours ont reçu l’une des quatre mêmes rations pendant 7 ou 14 jours. Au jour 5, les porcelets (2 par parc) ont reçu une injection intramusculaire de LPS (100 μg/kg) et des échantillons de sang ont été prélevés avant l’injection et 6h, 24h et 48h après. Au jour 7, tout de suite après avoir prélevé le dernier échantillon de sang, 1 porcelet dans chacun des parcs a été euthanasié afin de prélever des échantillons de la muqueuse de l’iléon. Au jour 12, le porcelet restant a de nouveau reçu une injection de LPS et les mêmes échantillons de sang et de muqueuse ont été prélevés. Cet essai a montré que l’ajout d’une forte dose de Zn réduisait l’oxydation des lipides et l’inflammation pendant le sevrage et que l’ajout de Zn et Arg a des effets limités sur les statuts oxydatif et inflammatoire suivant l’injection de LPS. Dans le dernier essai en conditions commerciales, des porcelets âgés de 21 jours ont été alimentés avec l’une des mêmes quatre rations alimentaires. Les échantillons sanguins ont été prélevés sur les mêmes 2 porcelets dans chacun des parcs avant la distribution des aliments le matin des jours 8 et 15. Les données ont montré que la ration avec une dose élevée en Zn améliorait les performances de croissance des porcelets (ADG et ADFI) ainsi que le status oxydatif en diminuant l’oxydation des lipides. De son côté, l’ajout de Arg a eu un effet limité sur les performances de croissance et sur le status oxydatif durant l’élevage en conditions commerciales. / High level of dietary zinc (Zn) and arginine (Arg) supplementation are known to improve growth in weanling piglets but the mechanism of action is not yet well understood. We hypothesized that antioxidant metabolism and inflammatory responses were involved. The effects of high Zn supplement and Arg supplementation on the antioxidant status and inflammatory response of piglets after lipopolysaccharide (LPS) stimulation were evaluated. In a first study, thirty-two 20-day-old weanling piglets were fed diets supplemented or not with Zn (2,500 mg Zn oxide/kg) and Arg (1%) for 12 days. On day 12, blood samples were taken before and 3h after intra-peritoneal injection of LPS (10 μg/kg) and were euthanized just after the second blood sample, and samples of mucosae were taken. These results indicated that Zn may reduce systemic oxidation and improve antioxidant status in jejunal and ileal mucosae However, Zn and Arg supplementation did not appear to act synergistically to enhance antioxidant status or reduce inflammation in weanling piglets. In a second study, forty-eight 20-days-old weanling piglets were placed for 7 or 14 days on diets supplemented or not with Zn (2,500 mg Zn oxide/kg) and Arg (1%). On day 5, piglets (two per pen) were injected with LPS injection (100 μg/kg) intramuscular and blood samples were taken just before and, 6, 24 and 48 h after. On day 7, shortly after obtaining the last post-LPS blood sample, one piglet per pen was euthanized and samples of mucosa were taken from the ileum. On day 12, remaining piglet was injected again with same LPS dose and blood and ileum samples were collected as for the piglet on day 7. This study showed that high Zn diet reduced lipid oxidation and inflammation during post-weaning period. However, Zn and Arg supplementations had limited effect on oxidative and inflammatory status following LPS challenge. In a last study, weanling piglets aged 21 d were fed for 15 days a diet supplemented or not with 2500 mg/kg of ZnO and 1% Arg under commercial breeding conditions. Blood was collected from the same two piglets in each pen before morning feeding on days 8 and 15. Under these commercial conditions, high level of Zn improved piglet growth performance (ADG and ADFI) and oxidative status by decreasing lipid oxidation however Arg supplementation had a limited effect on growth performance and oxidative status

X-ray crystallography is a popular method for resolving protein structures. Protein crystals need to be used for X-ray crystallography, but most naturally occurring proteins do not readily crystallize. The Hunt lab performed computational analyses showing that arginine is the most overrepresented amino acid in crystal-packing interfaces in the Protein Data Bank. Given the similar physicochemical characteristics of arginine and lysine, we hypothesized that multiple lysine-to-arginine (KR) substitutions should improve crystallization. To test this hypothesis, we developed software that ranks lysine sites in a target protein based on the redundancy-corrected KR substitution frequency in homologs. We demonstrate that three unrelated single-domain proteins can tolerate 5-11 KR substitutions with at most minor destabilization and that these substitutions consistently enhance crystallization propensity. This approach rapidly produced a 1.9 Å crystal structure of a human protein domain refractory to crystallization with its native sequence. Structures from bulk-KR-substituted domains show the engineered arginine residues frequently make high-quality hydrogen-bonds across crystal-packing interfaces. We thus demonstrate that bulk KR substitution represents a rational and efficient method for probabilistic engineering of protein surface properties to improve protein crystallization. This stands in direct contrast to earlier work and dogmas that posited that surface entropy reduction was the clear path forward to crystallzing proteins. Arginine is a high-entropy sidechain, yet it helps drive protein crystallization. To understand which structure and dynamical features of arginine give rise to crystal packing propensity, we performed 60 Molecular Dynamics (MD) simulations to measure the sidechain order parameter of arginine and compare it against crystal packing propensity. This work found that surface-exposed arginines with low order parameters are most likely to participate in crystal packing interactions. This is evidence against earlier thinking that high entropy surface sidechains oppose crystallization. Entropic barriers to protein crystallization can be enthalpically overcome.

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Protein arginine methyltransferase 1 (PRMT1), -4 (also known as coactivator-associated arginine methyltransferase 1; CARM1), and -5 catalyze the methylation of arginine residues on target proteins. In turn, these marked proteins mediate a variety of biological functions. By regulating molecules that are critical to the remodelling of skeletal muscle phenotype, PRMTs may influence skeletal muscle plasticity. Our study tests the hypothesis that the intracellular signals required for muscle adaptation to exercise will be associated with the induction of PRMT expression and activity. C57BL/6 mice were assigned to one of three experimental groups: sedentary (SED), acute bout of exercise (0PE), or acute exercise followed by 3 hours of recovery (3PE). The mice in the exercise groups performed a single bout of treadmill running at 15 m/min for 90 minutes. We observed that PRMT gene expression and global enzyme activity are muscle- specific, generally being higher in slow, oxidative muscle, as compared to faster, more glycolytic tissue. Despite the activation of canonical exercise-induced signalling involving AMPK and PGC-1α, PRMT expression and activity at the whole muscle level were unchanged. However, subcellular analysis revealed the exercise-evoked myonuclear translocation of PRMT1 prior to the nuclear translocation of PGC-1α, which colocalizes the proteins within the organelle after exercise. Acute physical activity also augmented the targeted methyltransferase activities of CARM1, PRMT1, and -5 in the myonuclear compartment, suggesting that PRMT-mediated histone arginine methylation is an integral part of the early signals that drive skeletal muscle plasticity. In summary, our data supports the emergence of PRMTs as important players in the regulation of skeletal muscle plasticity. / Thesis / Master of Science (MSc) / Skeletal muscle is a plastic tissue that can adapt to various physiological demands. Previous work suggests that protein arginine methyltransferases (PRMTs) are important in the regulation of skeletal muscle remodeling. However, their role in exercise-induced skeletal muscle plasticity is unknown. Therefore, the purpose of this study was to investigate the association between the intracellular signals required for muscle adaption and various metrics of PRMT biology. Our data demonstrate that PRMTs exhibit muscle-specific expression and function in mice. The movement of PRMT1 into myonuclei increased following exercise, while the specific methylation status of PRMT targets were also elevated. Overall, our data suggests that muscle-specific PRMT expression may be important for the determination and/or maintenance of different fiber type characteristics. Moreover, distinct PRMT cellular localization and methyltransferase activity may be key signals that contribute to skeletal muscle phenotypic plasticity.

Despite the emerging importance of protein arginine methyltransferases (PRMTs) in regulating skeletal muscle plasticity, the biology of these enzymes during muscle development remains poorly understood. Therefore, our purpose was to investigate PRMT1, -4, and -5 expression and function in skeletal muscle cells during the phenotypic remodeling elicited by myogenesis. C2C12 muscle cell maturation, assessed during the myoblast stage, and during days 1, 3, 5, and 7 of differentiation, was employed as an in vitro model of myogenesis. We observed PRMT-specific patterns of expression and activity during myogenesis. PRMT4 and -5 gene expression was unchanged, while PRMT1 mRNA and protein content were significantly induced. Cellular monomethylarginines and symmetric dimethylarginines, indicative of global and type II PRMT activities, respectively, remained steady during development, while type I PRMT activity indicator asymmetric dimethylarginines increased through myogenesis. Histone 4 arginine 3 (H4R3) and H3R17 contents were elevated coincident with the myonuclear accumulation of PRMT1 and -4. Collectively, this suggests that PRMTs are methyl donors throughout myogenesis and demonstrate specificity for their protein targets. Cells were then treated with TC-E 5003 (TC-E), a selective inhibitor of PRMT1 in order to specifically examine the enzymes role during myogenic differentiation. TC-E treated cells exhibited decrements in muscle differentiation, which were consistent with attenuated mitochondrial biogenesis and respiratory function. In summary, this study increases our understanding of PRMT1, -4, and -5 biology during the plasticity of skeletal muscle development. Our results provide evidence for a role of PRMT1, via a mitochondrially-mediated mechanism, in driving the muscle differentiation program. / Thesis / Master of Science (MSc) / Protein arginine methyltransferases (PRMTs) are responsible for many important functions in skeletal muscle. However, significant knowledge gaps exist with respect to PRMT expression and activity during conditions of muscle remodeling. Therefore, the purpose of this Thesis was to investigate PRMT biology throughout skeletal muscle development. Mouse muscle cells were employed to examine characteristics of PRMT1, -4, and -5 at numerous timepoints during myogenesis. PRMTs exhibited distinct patterns of gene expression and activity during muscle maturation. A PRMT1 inhibitor (TC-E) was utilized to investigate the role of this enzyme during myogenesis. Muscle differentiation was impaired in TC-E-treated cells, which coincided with reduced mitochondrial biogenesis and respiratory function. Altogether, these results suggest a PRMT-specific pattern of expression and activity during myogenesis. Furthermore, PRMT1 plays a crucial role in skeletal muscle differentiation via a mitochondrially-mediated mechanism. Our study provides a more comprehensive view on the role of PRMTs in governing skeletal muscle plasticity.