Methylation Of Arginine Residues Research Articles

CARM1 Regulates AMPK Signaling in Skeletal Muscle.

SummaryCoactivator-associated arginine methyltransferase 1 (CARM1) is an emerging mediator of skeletal muscle plasticity. We employed genetic, physiologic, and pharmacologic approaches to determine whether CARM1 regulates the master neuromuscular phenotypic modifier AMP-activated protein kinase (AMPK). CARM1 skeletal muscle-specific knockout (mKO) mice displayed reduced muscle mass and dysregulated autophagic and atrophic processes downstream of AMPK. We observed altered interactions between CARM1 and AMPK and its network, including forkhead box protein O1, during muscle disuse. CARM1 methylated AMPK during the early stages of muscle inactivity, whereas CARM1 mKO mitigated progression of denervation-induced atrophy and was accompanied by attenuated phosphorylation of AMPK targets such as unc-51 like autophagy-activating kinase 1Ser555. Lower acetyl-coenzyme A corboxylaseSer79 phosphorylation, as well as reduced peroxisome proliferator-activated receptor-γ coactivator-1α, was also observed in mKO animals following acute administration of the direct AMPK activator MK-8722. Our study suggests that targeting CARM1-AMPK interplay may have broad impacts on neuromuscular health and disease.

HPLC and LC-MS/MS measurement methods for the quantification of asymmetric dimethylarginine (ADMA) and related metabolites

Abstract Methyl arginine derivatives such as asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), L-N-monomethyl arginine (L-NMMA) are formed by proteolytic catalysis following methylation of arginine residues in proteins. These metabolites reduce NO production. Methylated arginines are an important biomarker for various diseases such as cardiovascular and renal diseases. Therefore, many methods have been developed to reliably and accurately measure the levels of these metabolites. This review, HPLC and LC-MS/MS methods developed for the measurement of methylarginine derivatives are discussed. In HPLC methods, solid phase extraction, derivatization and subsequent separation by reverse phase chromatography were performed. Since these metabolites are polar, they are difficult to retain in conventional reverse phase columns. In addition, as serum levels of these metabolites are low, sensitivity problems have been observed in HPLC methods. Derivatization has been applied to eliminate these problems. However, there have been problems with the stability of derivatives formed. Another important problem is that the separation of stereoisomer ADMA and SDMA can only be achieved chromatographically. Tandem mass spectrometric methods are accurate, selective, sensitive and rapid since analytes are separated depending on m/z ratios rather than chromatographic separation. Therefore, tandem mass spectrometry methods might be considered as the goal standard for these analytes.

The role of protein arginine methyltransferases in kidney diseases.

The methylation of arginine residues by protein arginine methyltransferases (PRMTs) is a crucial post-translational modification for many biological processes, including DNA repair, RNA processing, and transduction of intra- and extracellular signaling. Previous studies have reported that PRMTs are extensively involved in various pathologic states, including cancer, inflammation, and oxidative stress reaction. However, the role of PRMTs has not been well described in kidney diseases. Recent studies have shown that aberrant function of PRMTs and its metabolic products-symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA)-are involved in several renal pathological processes, including renal fibrosis, acute kidney injury (AKI), diabetic nephropathy (DN), hypertension, graft rejection and renal tumors. We aim in this review to elucidate the possible roles of PRMTs in normal renal function and various kidney diseases.

Protein Arginine Methyltransferases in Cardiovascular and Neuronal Function.

The methylation of arginine residues by protein arginine methyltransferases (PRMTs) is a type of post-translational modification which is important for numerous cellular processes, including mRNA splicing, DNA repair, signal transduction, protein interaction, and transport. PRMTs have been extensively associated with various pathologies, including cancer, inflammation, and immunity response. However, the role of PRMTs has not been well described in vascular and neurological function. Aberrant expression of PRMTs can alter its metabolic products, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA). Increased ADMA levels are recognized as an independent risk factor for cardiovascular disease and mortality. Recent studies have provided considerable advances in the development of small-molecule inhibitors of PRMTs to study their function under normal and pathological states. In this review, we aim to elucidate the particular roles of PRMTs in vascular and neuronal function as a potential target for cardiovascular and neurological diseases.

Protein methylome analysis in Arabidopsis reveals regulation in RNA-related processes

Protein methylation has been proposed as an important post-translational modification, which occurs predominantly on lysine and arginine residues. Recent discoveries have revealed that protein methylation is also present on non-histones besides histones, and plays critical roles in regulating protein stability and function. However, proteome-wide identification of methylated proteins in plants remains unexplored. Here, we present the first global survey of monomethyl arginine, symmetric and asymmetric dimethyl arginine, and monomethyl, dimethyl, trimethyl lysine modifications in the proteomes of 10-day-old Arabidopsis seedlings through a combination of immunoaffinity purification and mass spectrometry analysis. In total, we identified 617 methylation sites which mapped to 412 proteins, with 263 proteins harboring 381 lysine methylation sites and 149 proteins harboring 236 arginine methylation sites. Among them, 607 methylation sites on 408 proteins were novel findings. Motif analysis revealed that glycine preferentially flanked methylated arginine residues, whereas aspartate and glutamate enriched around mono- and dimethylated lysine sites. Methylated proteins were involved in a variety of metabolic processes, showing significant enrichment in RNA-related metabolic pathways including spliceosome, RNA transport, and ribosome. Our data provide a global view of methylated non-histone proteins in Arabidopsis, laying foundations for elucidating the biological function of protein methylation in plants. SignificanceProtein methylation has emerged as a common and important modification both in eukaryotes and prokaryotes. The identification of methylated sites/peptides is fundamental for further functional analysis of protein methylation. This study was the first proteome-scale identification of lysine and arginine methylation in plants. We found that methylation occurred widely on non-histone proteins in Arabidopsis and was involved in diverse biological functions. The results provide foundations for the investigation of the protein methylome in Arabidopsis and provide powerful resources for the functional analysis of protein methylation in plants.

Separation of Isobaric Mono- and Dimethylated RGG-Repeat Peptides by Differential Ion Mobility-Mass Spectrometry.

Methylation of arginine residues in proteins, an enzyme-mediated post-translational modification (PTM), is important for mRNA processing and transport and for the regulation of many protein-protein interactions. However, proteolytic peptides resulting from alternative sites of post-translational methylation have identical masses and cannot be readily separated by standard liquid chromatography-mass spectrometry. Unlike acetylation or phosphorylation, methylation of arginine does not strongly affect the charge states of peptide ions, multiple instances of methylation can occur on a single amino acid residue, and the relative mass of the modification is <1% that of the typical proteolytic peptide. High field asymmetric waveform ion mobility spectrometry (FAIMS) is an orthogonal separation method to liquid chromatography that can rapidly separate gaseous ions prior to detection by mass spectrometry. Here, we report that FAIMS can be used to separate arginine-methylated peptides that differ by the position of a single methyl group for both mono- and dimethylated variants. Although the resolution of separation for these arginine-methylated peptides improved with increasing amounts of helium in the FAIMS carrier gas as expected, we found that the site of methylation can strongly affect the dependence of the electric field used for ion transmission on the extent of helium in the carrier gas. Thus, certain isobaric peptides can be cotransmitted at high helium concentrations whereas lower concentrations can be used for successful separations of such peptide mixtures. The capability to rapidly resolve isobaric arginine-methylated peptides should be useful in the future for the detailed analysis of protein arginine methylation in biological samples.

Peptidic transition state analogues as PRMT inhibitors.

Protein arginine N-methyltransferases (PRMTs) methylate arginine residues in target proteins using the ubiquitous methyl donor S-adenosyl-l-methionine (AdoMet) as a cofactor. PRMTs play important roles in both healthy and disease states and as such inhibition of PRMTs has gained increasing interest. A primary challenge in the development of PRMT inhibitors is achieving specificity for the PRMT of interest as the active sites are highly conserved for all nine members of the PRMT family. Notably, PRMTs show very little redundancy in vivo due to their specific sets of protein substrates. However, relatively little is known about the interactions of PRMTs with their protein substrates that drive this substrate specificity. We here describe the extended application of a methodology recently developed in our group for the production of peptide-based transition state mimicking PRMT inhibitors. Using this approach, an adenosine moiety, mimicking that of the AdoMet cofactor, is covalently linked to the guanidine side chain of a target arginine residue contained in a peptidic fragment derived from a PRMT substrate protein. Using this approach, histone H4 tail peptide-based transition state mimics were synthesized wherein the adenosine group was linked to the Arg3 residue. H4R3 is a substrate for multiple PRMTs, including PRMT1 and PRMT6. The inhibition results obtained with these new H4-based transition state mimics show low micromolar IC50 values against PRMT1 and PRMT6, indicating that the methodology is applicable to the broader family of PRMTs.

The regulation, functions and clinical relevance of arginine methylation.

Methylation of arginine residues by protein arginine methyltransferases (PRMTs) is involved in the regulation of fundamental cellular processes, including transcription, RNA processing, signal transduction cascades, the DNA damage response and liquid-liquid phase separation. Recent studies have provided considerable advances in the development of experimental tools and the identification of clinically relevant PRMT inhibitors. In this review, we discuss the regulation of PRMTs, their various cellular roles and the clinical relevance of PRMT inhibitors for the therapy of neurodegenerative diseases and cancer.

Abstract 277: Inhibition of protein arginine methylation alters RNA metabolism and DNA damage response providing a new therapeutic strategy in pancreatic ductal adenocarcinoma

Abstract With limited therapeutic options, poor overall 5-year survival rates, and increasing incidence, pancreas cancer is estimated to become the second leading cause of cancer deaths by 2030. Recognizing the need for transformative advances in pancreas cancer management, we developed an in vivo target discovery platform to uncover molecular vulnerabilities in patient-derived pancreatic ductal adenocarcinoma (PDAC) xenografts to identify and rapidly translate novel therapeutic concepts to the clinic. We identified protein arginine methyltransferase 1 (PRMT1) as a dependency in PDAC required for disease maintenance and progression. Extensive genetic and pharmacological studies support PRMT1 as a novel vulnerability, which prompted our design and synthesis of proprietary series of potent, selective PRMT Type I inhibitors (PRMTi) with compelling in vivo activity. While advancing the project in drug discovery, we deployed a comprehensive approach to elucidate the mechanism of action of PRMTi. We characterized the PRMT1 interactome via PRMT1 immunoprecipitation followed by LC/MS and observed that PRMT1 binding partners were significantly enriched in RNA-binding and -processing genes. In addition, because methylation of arginine residues is a common post-translational modification regulating protein function, we identified substrates differentially methylated upon PRMT inhibition. Integrating these results with the PRMT1 interactome confirmed a strong correlation between PRMT1 substrates and complexes that are physically associated and linked to RNA metabolism. Transcriptome assays demonstrated that PRMT inhibition globally impaired RNA metabolism, including but not limited to RNA splicing, transcription termination, and R-loop formation. In addition, PRMTi caused a profound down-regulation of multiple pathways involved in the DNA damage response (DDR) promoting genomic instability. Taken together, these data support PRMT1 as a compelling target in an area of high unmet medical need and inform a mechanism-based translational strategy for future clinical development. Citation Format: Virginia Giuliani, Alessandro Carugo, Meredith Miller, Lionel Sanz, Chiu-Yi Liu, Christopher A Bristow, Erika Suzuki, Caleb A Class, Stella R. Hartono, Guang Gao, Ningping Feng, Jason P Gay, Bhavatarini Vangamudi, Joseph R Marszalek, Jeffrey Kovacs, Maria Emilia Di Francesco, Frederic Chedin, Philip Jones, Giulio Draetta, Timothy Heffernan. Inhibition of protein arginine methylation alters RNA metabolism and DNA damage response providing a new therapeutic strategy in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 277.

Increase of arginine dimethylation correlates with the progression and prognosis of ALS.

To investigate whether arginine methylation is altered in patients with amyotrophic lateral sclerosis (ALS) and how it affects disease severity, progression, and prognosis. We compared the immunoreactivity of protein arginine methyltransferase 1 (PRMT1) and its products, asymmetric dimethylated proteins (ASYM), in postmortem spinal cord. We also measured the concentrations of total l-arginine and methylated arginine residues, including asymmetric dimethyl l-arginine (ADMA), symmetric dimethyl arginine, and monomethyl arginine, in CSF samples from 52 patients with ALS using liquid chromatography-tandem mass spectrometry, and we examined their relationship with the progression and prognosis of ALS. The immunoreactivity of both PRMT1 (p < 0.0001) and ASYM (p = 0.005) was increased in patients with ALS. The concentration of ADMA in CSF was substantially higher in patients with ALS than in disease controls. The ADMA/l-arginine ratio was correlated with the change of decline in the ALS Functional Rating Scale at 12 months after the time of measurement (r = 0.406, p = 0.010). A Cox proportional hazards model showed that the ADMA/l-arginine ratio was an independent predictor for overall survival. Moreover, a high ADMA/l-arginine ratio predicted poor prognosis, even in a group with normal percentage forced vital capacity. There was an enhancement of arginine dimethylation in patients with ALS, and the ADMA/l-arginine ratio predicted disease progression and prognosis in such patients.

Total methylated arginine load as a risk parameter in subjects with masked hypertension

ABSTRACTAsymmetric dimethylarginine, symmetric dimethylarginine, and L-monomethylarginine are originated from the subsequent proteolytic catalysis of methylated arginine residues on different proteins and inhibit the endogenous nitric oxide generation. The changes in total methylarginine load (Asymmetric dimethylarginine plus symmetric dimethylarginine plus L-monomethylarginine) may contribute to hypertension. The aim of this study was to determine serum methylarginine concentrations in patients with masked hypertension and determine the association between these biomarkers and blood pressure measurements. Control group, masked hypertension and hypertension groups consisted of 40 subjects (11 males, 28 females, mean age 48.6 ± 13.1), 28 subjects (14 males, 14 females, mean age 50.9 ± 11.0) and 36 subjects (15 males, 21 females, mean age 54.4 ± 12.3 years), respectively (P= 0.149). Serum total methylarginine load was significantly higher in hypertension group (0.63 ± 0.23) compared to masked hypertension (0.49 ± 0.16) and control groups (0.38 ± 0.13) (P= 0.008 and P< 0.001). While there was no statistically significant difference between healthy control groups [0.147 (0.03–0.29)] and masked hypertension patients [0.144 (0.05–0.42)] for serum symmetric dimethylarginine levels (P= 0.970), it was markedly elevated in hypertension group [0.25 (0.07–0.54)] compared to masked hypertension group [0.14 (0.05–0.42)] (P= 0.001). Serum total methylarginine load was positively correlated with night-time SBP (r = 0.214, P= 0.029). Serum methylarginine levels might be a useful marker for determining the courses of clinical hypertension.

Dual regulation of Arabidopsis AGO2 by arginine methylation

Argonaute (AGO) proteins are core components of RNA interference (RNAi) but the mechanisms of their regulation, especially at the post-translational level, remain unclear. Among the ten AGOs in Arabidopsis, only AGO2 is induced by bacterial infection and is known to positively regulate immunity. Here we show that the N-terminal domain of AGO2 is enriched with arginine-glycine RG/GR repeats, which are methylated by protein arginine methyltransferase5 (PRMT5). Arginine methylation has dual functions in AGO2 regulation. Methylated arginine residues can promote AGO2 protein degradation and are also bound by Tudor-domain proteins (TSNs), which can degrade AGO2-associated small RNAs (sRNAs). PRMT5 is down-regulated during infection and the prmt5 mutant is more resistant to bacteria. We speculate that reduced PRMT5 expression during infection may lead to reduced arginine methylation of AGO2, resulting in accumulation of both AGO2 and, via reduced interaction with TSNs, accumulation of AGO2-associated sRNAs, to promote plant immunity. These results reveal that both the arginine methylation writer (PRMT5) and readers (TSNs) can regulate AGO2-mediated RNAi.

Arginine methylation controls the strength of γc-family cytokine signaling in T cell maintenance.

The methylation of arginine residues in proteins is a post-translational modification that contributes to a wide range of biological processes. Many cytokines involved in T cell development and activation utilize the common cytokine receptor γ-chain (γc) and the kinase JAK3 for signal transduction, but the regulatory mechanism that underlies the expression of these factors remains unclear. Here we found that the arginine methyltransferase PRMT5 was essential for the maintenance of invariant natural killer T cells (iNKT cells), CD4+ T cells and CD8+ T cells. T cell-specific deletion of Prmt5 led to a marked reduction in signaling via γc-family cytokines and a substantial loss of thymic iNKT cells, as well as a decreased number of peripheral CD4+ T cells and CD8+ T cells. PRMT5 induced the symmetric dimethylation of Sm proteins that promoted the splicing of pre-mRNA encoding γc and JAK3, and this critically contributed to the expression of γc and JAK3. Thus, arginine methylation regulates strength of signaling via γc-family cytokines by facilitating the expression of signal-transducing components.

Further delineation of the phenotype caused by loss of function mutations in PRMT7

PRMT7 encodes for an arginine methyltransferase that methylates arginine residues on various protein substrates and has been shown to play a role in various developmental processes. Mutations in PRMT7 have been recently shown to be implicated in a phenotype with intellectual disability, short stature and brachydactyly, and considered to be a phenocopy of pseudohypoparathyroidism. We report a patient with short stature, psychomotor delay, hearing loss and brachydactyly, for whom whole exome sequencing detected two mutations in PRMT7 and parental segregation studies detected biallelic mutation inheritance. Few patients with biallelic PRMT7 mutations have been reported so far in the literature. We report a new patient and review all reported cases to date to delineate the clinical manifestations that may help in diagnosis this disorder, known as Short Stature, Brachydactyly, Intellectual Developmental Disability, and Seizures syndrome, allowing appropriate management and genetic counselling.

The ribosome: A hot spot for the identification of new types of protein methyltransferases

Cellular physiology depends on the alteration of protein structures by covalent modification reactions. Using a combination of bioinformatic, genetic, biochemical, and mass spectrometric approaches, it has been possible to probe ribosomal proteins from the yeast Saccharomyces cerevisiae for post-translationally methylated amino acid residues and for the enzymes that catalyze these modifications. These efforts have resulted in the identification and characterization of the first protein histidine methyltransferase, the first N-terminal protein methyltransferase, two unusual types of protein arginine methyltransferases, and a new type of cysteine methylation. Two of these enzymes may modify their substrates during ribosomal assembly because the final methylated histidine and arginine residues are buried deep within the ribosome with contacts only with RNA. Two of these modifications occur broadly in eukaryotes, including humans, whereas the others demonstrate a more limited phylogenetic range. Analysis of strains where the methyltransferase genes are deleted has given insight into the physiological roles of these modifications. These reactions described here add diversity to the modifications that generate the typical methylated lysine and arginine residues previously described in histones and other proteins.

JMJD1B Demethylates H4R3me2s and H3K9me2 to Facilitate Gene Expression for Development of Hematopoietic Stem and Progenitor Cells

SUMMARYThe arginine methylation status of histones dynamically changes during many cellular processes, including hematopoietic stem/progenitor cell (HSPC) development. The arginine methyltransferases and the readers that transduce the histone codes have been defined. However, whether arginine demethylation actively occurs in cells and what enzyme demethylates the methylarginine residues during various cellular processes are unknown. We report that JMJD1B, previously identified as a lysine demethylase for H3K9me2, mediates arginine demethylation of H4R3me2s and its intermediate, H4R3me1. We show that demethylation of H4R3me2s and H3K9me2s in promoter regions is correlated with active gene expression. Furthermore, knockout of JMJD1B blocks demethylation of H4R3me2s and/or H3K9me2 at distinct clusters of genes and impairs the activation of genes important for HSPC differentiation and development. Consequently, JMJD1B−/− mice show defects in hematopoiesis. Altogether, our study demonstrates that demethylase-mediated active arginine demethylation process exists in eukaryotes and that JMJD1B demethylates both H4R3me2s and H3K9me2 for epigenetic programming during hematopoiesis.

Specific Recognition of Arginine Methylated Histone Tails by JMJD5 and JMJD7

We have reported that JMJD5 and JMJD7 (JMJD5/7) are responsible for the clipping of arginine methylated histone tails to generate “tailless nucleosomes”, which could release the pausing RNA polymerase II (Pol II) into productive transcription elongation. JMJD5/7 function as endopeptidases that cleave histone tails specifically adjacent to methylated arginine residues and continue to degrade N-terminal residues of histones via their aminopeptidase activity. Here, we report structural and biochemical studies on JMJD5/7 to understand the basis of substrate recognition and catalysis mechanism by this JmjC subfamily. Recognition between these enzymes and histone substrates is specific, which is reflected by the binding data between enzymes and substrates. High structural similarity between JMJD5 and JMJD7 is reflected by the shared common substrates and high binding affinity. However, JMJD5 does not bind to arginine methylated histone tails with additional lysine acetylation while JMJD7 does not bind to arginine methylated histone tails with additional lysine methylation. Furthermore, the complex structures of JMJD5 and arginine derivatives revealed a Tudor domain-like binding pocket to accommodate the methylated sidechain of arginine, but not lysine. There also exists a glutamine close to the catalytic center, which may suggest a unique imidic acid mediated catalytic mechanism for proteolysis by JMJD5/7.

A novel splicing isoform of protein arginine methyltransferase 1 (PRMT1) that lacks the dimerization arm and correlates with cellular malignancy.

Methylation of arginine residues is an important modulator of protein function that is involved in epigenetic gene regulation, DNA damage response and RNA maturation, as well as in cellular signaling. The enzymes that catalyze this post-translational modification are called protein arginine methyltransferases (PRMTs), of which PRMT1 is the predominant enzyme. Human PRMT1 has previously been shown to occur in seven splicing isoforms, which are differentially abundant in different tissues, and have distinct substrate specificity and intracellular localization. Here we characterize a novel splicing isoform which does not affect the amino-terminus of the protein like the seven known isoforms, but rather lacks exons 8 and 9 which encode the dimerization arm of the enzyme that is essential for enzymatic activity. Consequently, the isoform does not form catalytically active oligomers with the other endogenous PRMT1 isoforms. Photobleaching experiments reveal an immobile fraction of the enzyme in the nucleus, in accordance with earlier results from our laboratory that had shown a tight association of inhibited or inactivated PRMT1 with chromatin and the nuclear scaffold. Thus, it apparently is able to bind to the same substrates as catalytically active PRMT1. This isoform is found in a variety of cell lines, but is increased in those of cancer origin or after expression of the EMT-inducing transcriptional repressor Snail1. We discuss that the novel isoform could act as a modulator of PRMT1 activity in cancer cells by acting as a competitive inhibitor that shields substrates from access to active PRMT1 oligomers.

Clipping of arginine-methylated histone tails by JMJD5 and JMJD7

Two of the unsolved, important questions about epigenetics are: do histone arginine demethylases exist, and is the removal of histone tails by proteolysis a major epigenetic modification process? Here, we report that two orphan Jumonji C domain (JmjC)-containing proteins, JMJD5 and JMJD7, have divalent cation-dependent protease activities that preferentially cleave the tails of histones 2, 3, or 4 containing methylated arginines. After the initial specific cleavage, JMJD5 and JMJD7, acting as aminopeptidases, progressively digest the C-terminal products. JMJD5-deficient fibroblasts exhibit dramatically increased levels of methylated arginines and histones. Furthermore, depletion of JMJD7 in breast cancer cells greatly decreases cell proliferation. The protease activities of JMJD5 and JMJD7 represent a mechanism for removal of histone tails bearing methylated arginine residues and define a potential mechanism of transcription regulation.

Investigating Methylated Arginine Residues in PGC‐1α

Peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α) participates in the regulation of both carbohydrate and lipid metabolism and is activated by insulin. For these reasons, it has been implicated in obesity and diabetes. PGC‐1α is subjected to many post‐translational modifications including phosphorylation of Ser570 by protein kinase B (Akt/PKB), which stops gluconeogenesis and fatty acid oxidation. In addition, PGC‐1α is methylated by protein arginine methyltransferase 1 (PRMT1) at several arginine residues, including Arg665, Arg667 and Arg669 which enhance its regulation. Evidence for additional arginine methylated sites have been found, but the exact sites remain unknown. We set out to identify methylated arginine residues on PGC‐1α. PRMT1 and truncated constructs corresponding to PGC‐1α were expressed and purified. Next, in vitro methylation reactions were carried out, followed by detection of methylated arginine residues by various methods including immunoblot and autoradiography. Identification of the methylated arginine residue(s), will enhance our understanding of PGC‐1α with the broader goal of determining its regulation in the context of obesity and diabetes.Support or Funding InformationMinority Biomedical Research Support‐Research Initiative for Scientific Enhancement (MBRS‐RISE) Fellowship. Grant: R25 GM061331