Decoding PRMT1: Studies On the Catalytic Mechanism, Regulation, Inhibition, and Crosstalk of PRMT1-Dependent Methylation

Arginine methylation is catalyzed by the protein arginine methyltransferase (PRMT) family of enzymes, which transfer a methyl group from S-adenosylmethionine (SAM) to the guanidinium group of an arginine residue. This reaction first produces monomethylated arginine (MMA) that can then be further methylated to produce either asymmetrically dimethylated arginine (ADMA) or symmetrically dimethylated arginine (SDMA). There are nine PRMT family members described to date, with PRMT1 as the predominant member, suggested to be responsible for ~85% of asymmetric dimethylation. In addition, PRMT1-dependent methylation likely plays a significant role in a plethora of diseases (e.g., cancer, heart disease, and ALS). These observations render it imperative that the isozyme be more thoroughly characterized and suggests that potent and selective inhibitors may be useful as therapeutics. Herein we describe our efforts to decode PRMT1-dependent methylation by investigating the catalytic mechanism, the effects of post-translational modifications and protein-protein interactions on activity, the development of potent and selective inhibitors and inactivators, as well as examining crosstalk between arginine methylation and phosphorylation. Using site-directed mutagenesis and unnatural amino acid incorporation, we have identified key active site residues that are critical for catalysis and/or substrate binding, and have determined the effects of phosphorylation, if any, on enzyme activity. In vitro assays with known interacting proteins has increased our knowledge of the regulation of PRMT1 activity by protein-protein interactions. The use of MS/MS analysis aided in the identification of the site of modification for a potent inactivator of the isozyme, C21, and has led to the design of new inhibitors and inactivators that will likely be more potent and selective for not only PRMT1, but PRMT5 as well. Finally, using a peptide based model, we began to investigate crosstalk between arginine methylation and serine/threonine phosphorylation within kinase consensus sequences and hypothesize that it is an important means of regulation in regards to cell signaling. Overall, the results presented in the following chapters have enhanced our understanding of PRMT1-dependent methylation and have opened doors for future studies involving the regulation of the enzyme and inhibitor design

Using Unnatural Amino Acid Mutagenesis To Probe the Regulation of PRMT1

Protein arginine methyltransferase 1 (PRMT1)-dependent methylation contributes to the onset and progression of numerous diseases (e.g., cancer, heart disease, ALS); however, the regulatory mechanisms that control PRMT1 activity are relatively unexplored. We therefore set out to decipher how phosphorylation regulates PRMT1 activity. Curated mass spectrometry data identified Tyr291, a residue adjacent to the conserved THW loop, as being phosphorylated. Natural and unnatural amino acid mutagenesis, including the incorporation of p-carboxymethyl-L-phenylalanine (pCmF) as a phosphotyrosine mimic, were used to show that Tyr291 phosphorylation alters the substrate specificity of PRMT1. Additionally, p-benzoyl-L-phenylalanine (pBpF) was incorporated at the Tyr291 position, and cross-linking experiments with K562 cell extracts identified several proteins (e.g., hnRNPA1 and hnRNP center dot center dot H3) that bind specifically to this site. Moreover, we also demonstrate that Tyr291 phosphorylation impairs PRMT1\u27s ability to bind and methylate both proteins. In total, these studies demonstrate that Tyr291 phosphorylation alters both PRMT1 substrate specificity and protein-protein interacions

Cognitive endpoints for therapy development for neuronopathic mucopolysaccharidoses: Results of a consensus procedure

AbstractThe design and conduct of clinical studies to evaluate the effects of novel therapies on central nervous system manifestations in children with neuronopathic mucopolysaccharidoses is challenging. Owing to the rarity of these disorders, multinational studies are often needed to recruit enough patients to provide meaningful data and statistical power. This can make the consistent collection of reliable data across study sites difficult. To address these challenges, an International MPS Consensus Conference for Cognitive Endpoints was convened to discuss approaches for evaluating cognitive and adaptive function in patients with mucopolysaccharidoses. The goal was to develop a consensus on best practice for the design and conduct of clinical studies investigating novel therapies for these conditions, with particular focus on the most appropriate outcome measures for cognitive function and adaptive behavior. The outcomes from the consensus panel discussion are reported here

Cognitive endpoints for therapy development for neuronopathic mucopolysaccharidoses: Results of a consensus procedure

The design and conduct of clinical studies to evaluate the effects of novel therapies on central nervous system manifestations in children with neuronopathic mucopolysaccharidoses is challenging. Owing to the rarity of these disorders, multinational studies are often needed to recruit enough patients to provide meaningful data and statistical power. This can make the consistent collection of reliable data across study sites difficult. To address these challenges, an International MPS Consensus Conference for Cognitive Endpoints was convened to discuss approaches for evaluating cognitive and adaptive function in patients with mucopolysaccharidoses. The goal was to develop a consensus on best practice for the design and conduct of clinical studies investigating novel therapies for these conditions, with particular focus on the most appropriate outcome measures for cognitive function and adaptive behavior. The outcomes from the consensus panel discussion are reported here

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Kinase consensus sequences: a breeding ground for crosstalk

The best characterized examples of crosstalk between two or more different post-translational modifications (PTMs) occur with respect to histones. These examples demonstrate the critical roles that crosstalk plays in regulating cell signaling pathways. Recently, however, non-histone crosstalk has been observed between serine/threonine phosphorylation and the modification of arginine and lysine residues within kinase consensus sequences. Interestingly, many kinase consensus sequences contain critical arginine/lysine residues surrounding the substrate serine/threonine residue. Therefore, we hypothesize that non-histone crosstalk between serine/threonine phosphorylation and arginine/lysine modifications is a global mechanism for the modulation of cellular signaling. In this review, we discuss several recent examples of non-histone kinase consensus sequence crosstalk, as well as provide the biophysical basis for these observations. In addition, we predict likely examples of crosstalk between protein arginine methyltransferase 1 (PRMT1) and Akt and discuss the future implications of these findings

A combinatorial approach to characterize the substrate specificity of protein arginine methyltransferase 1

The dysregulation of protein arginine methyltransferases (PRMTs) is implicated in a wide variety of disease states. Here we report the design, synthesis, and screening of a combinatorial peptide library used to characterize the substrate specificity of PRMT1. The information gained from this approach was used to develop a PRMT1 inhibitor with enhanced selectivity

Automethylation of protein arginine methyltransferase 8 (PRMT8) regulates activity by impeding S-adenosylmethionine sensitivity.

Protein arginine methyltransferase (PRMT) 8 is unique among the PRMTs, as it has a highly restricted tissue expression pattern and an N terminus that contains two automethylation sites and a myristoylation site. PRMTs catalyze the transfer of a methyl group from S-adenosylmethionine (AdoMet) to a peptidylarginine on a protein substrate. Currently, the physiological roles, regulation, and cellular substrates of PRMT8 are poorly understood. However, a thorough understanding of PRMT8 kinetics should provide insights into each of these areas, thereby enhancing our understanding of this unique enzyme. In this study, we determined how automethylation regulates the enzymatic activity of PRMT8. We found that preventing automethylation with lysine mutations (preserving the positive charge of the residue) increased the turnover rate and decreased the Km of AdoMet but did not affect the Km of the protein substrate. In contrast, mimicking automethylation with phenylalanine (i.e. mimicking the increased hydrophobicity) decreased the turnover rate. The inhibitory effect of the PRMT8 N terminus could be transferred to PRMT1 by creating a chimeric protein containing the N terminus of PRMT8 fused to PRMT1. Thus, automethylation of the N terminus likely regulates PRMT8 activity by decreasing the affinity of the enzyme for AdoMet

Mechanistic studies on transcriptional coactivator protein arginine methyltransferase 1

Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to the guanidinium group of arginine residues in a number of important cell signaling proteins. PRMT1 is the founding member of this family, and its activity appears to be dysregulated in heart disease and cancer. To begin to characterize the catalytic mechanism of this isozyme, we assessed the effects of mutating a number of highly conserved active site residues (i.e., Y39, R54, E100, E144, E153, M155, and H293), which are believed to play key roles in SAM recognition, substrate binding, and catalysis. The results of these studies, as well as pH-rate studies, and the determination of solvent isotope effects (SIEs) indicate that M155 plays a critical role in both SAM binding and the processivity of the reaction but is not responsible for the regiospecific formation of asymmetrically dimethylated arginine (ADMA). Additionally, mutagenesis studies on H293, combined with pH studies and the lack of a normal SIE, do not support a role for this residue as a general base. Furthermore, the lack of a normal SIE with either the wild type or catalytically impaired mutants suggests that general acid/base catalysis is not important for promoting methyl transfer. This result, combined with the fact that the E144A/E153A double mutant retains considerably more activity then the single mutants alone, suggests that the PRMT1-catalyzed reaction is primarily driven by bringing the substrate guanidinium into the proximity of the S-methyl group of SAM and that the prior deprotonation of the substrate guanidinium is not required for methyl transfer