scholarly journals Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

2020 ◽  
Author(s):  
Stephanie M. Lehman ◽  
Hongshan Chen ◽  
Emmanuel S. Burgos ◽  
Maxim Maron ◽  
Sitaram Gayatri ◽  
...  
Keyword(s):  
Electron Transfer Dissociation ◽  
Rna Binding ◽  
A549 Cells ◽  
Arginine Methylation ◽  
Type I ◽  
Type Ii ◽  
Intrinsically Disordered ◽  
Substrate Preferences ◽  
Total Proteome ◽  
A Minor

AbstractArginine methylation is essential for both cellular viability and development and is also dysregulated in cancer. PRMTs catalyze the post translational monomethylation (Rme1/MMA, catalyzed by Type I-III), asymmetric (Rme2a/ADMA, Type I enzymes)-, or symmetric (Rme2s/SDMA, Type II enzymes) dimethylation of arginine. Despite many studies, a thorough integration of PRMT enzyme substrate determination and proteomic and transcriptomic consequences of inhibiting Type I and II PRMTs is lacking. To characterize cellular substrates for Type I (Rme2a) and Type II (Rme2s) PRMTs, human A549 lung adenocarcinoma cells were treated with either Type I (MS023) or Type II (GSK591) inhibitors. Using total proteome hydrolysis, we developed a new mass spectrometry approach to analyze total arginine and lysine content. We showed that Rme1 was a minor population (∼0.1% of total arginine), Rme2a was highly abundant (∼1.1%), and Rme2s was intermediate (∼0.4%). While Rme2s was mostly eliminated by GSK591 treatment, total Rme1 and Rme2a were more resistant to perturbation. To quantitatively characterize substrate preferences of the major enzymes PRMT1, PRMT4(CARM1), and PRMT5, we used oriented peptide array libraries (OPAL) in methyltransferase assays. We demonstrated that while PRMT5 tolerates aspartic acid residues in the substrate, PRMT1 does not. Importantly, PRMT4 methylated previously uncharacterized hydrophobic motifs. To integrate our studies, we performed PTMScan on PRMT-inhibited A549 cells and enriched for methylated arginine containing tryptic peptides. For detection of highly charged peptides, a method to analyze the samples using electron transfer dissociation was developed. Proteomic analysis revealed distinct methylated species enriched in nuclear function, RNA-binding, intrinsically disordered domains, and liquid-liquid phase separation. Parallel studies with proteomics and RNA-Seq revealed distinct, but ontologically overlapping, consequences to PRMT inhibition. Overall, we demonstrate a wider PRMT substrate diversity and methylarginine functional consequence than previously shown.

scholarly journals Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4

2004 ◽  
Vol 379 (2) ◽  
pp. 283-289 ◽  
Author(s):  
Marie-Chloé BOULANGER ◽  
Tina Branscombe MIRANDA ◽  
Steven CLARKE ◽  
Marco di FRUSCIO ◽  
Beat SUTER ◽  
...  
Keyword(s):  
Rna Binding ◽  
Developmental Stages ◽  
Rna Binding Proteins ◽  
Genetic System ◽  
Arginine Methylation ◽  
Type I ◽  
Protein Arginine Methyltransferases ◽  
Arginine Residues ◽  
Drosophila Protein

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 (Drosophilaarginine methyltransferases 1–9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation.

scholarly journals Heterogeneity in Biodistribution and Cytotoxicity of Silver Nanoparticles in Pulmonary Adenocarcinoma Human Cells

Nanomaterials ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 36 ◽  
Author(s):  
My Kieu Ha ◽  
Kyung Hwun Chung ◽  
Tae Hyun Yoon
Keyword(s):  
Silver Nanoparticles ◽  
Single Cell ◽  
Dose Response ◽  
A549 Cells ◽  
In Vitro Assays ◽  
Response Analysis ◽  
Complex Nature ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii

Cellular association of nanoparticles (NPs) and their resultant cytotoxicity are heterogeneous in nature and can be influenced by the variances in NPs’ properties, cell types, and status. However, conventional in vitro assays typically consider the administered NP dose and the averaged cellular responses based on the assumption of a uniform distribution of monodisperse NPs in homogeneous cells, which might be insufficient to describe the complex nature of cell–NP interactions. Here, using flow cytometry, we report observations of the heterogeneity in the cellular association of silver nanoparticles (AgNPs) in A549 cells, which resulted in distinct dose-response relationships and cytotoxicity. Type I and Type II cells were moderately associated with AgNPs but as the cellular AgNP dose increased, Type I cells remained viable while Type II cells became less viable. Type III cells did not have high affinity with AgNPs but were, however, the least viable. Transmission electron microscopic images revealed that the biodistribution and the released Ag+ ions contributed to the distinct toxic effects of AgNPs in different populations. This single-cell dose-response analysis approach enabled the examination of how differently individual cells responded to different cellular NP doses and provided insights into nanotoxicity pathways at a single-cell level.

Odour measurement - factors affecting olfactometry panel performance

1996 ◽  
Vol 34 (3-4) ◽  
pp. 549-556 ◽  
Author(s):  
P. J. Bliss ◽  
T. J. Schulz ◽  
T. Senger ◽  
R. B. Kaye
Keyword(s):  
Data Bank ◽  
Type I ◽  
Type Ii ◽  
Factors Affecting ◽  
Natural Logarithm ◽  
Odour Threshold ◽  
One Step ◽  
The Mean ◽  
A Minor ◽  
Over 40 Years

To identify factors affecting olfactometry panel performance in the measurement of environmental odours, a data bank of odour threshold measurements including 923 individual panel tests on environmental odours and 145 tests on standards were analysed statistically. There is an evident decrease in olfactory sensitivity to environmental odours with age. The group threshold tends to be one step lower for a 25 year increase in average age of panel members for Type I odours (piggery, feedlot, landfill and mushroom composting) and for 36 years increase for Type II odours (sewage and industrial coke works). The threshold for N-butanol tends to be 1 step lower for an increase of 15 years in age. People who are over 40 years old exhibited a greater variation than younger people. Although there was a minor gender difference in the sensitivity to butanol standard, it was not statistically significant (mean natural logarithm butanol threshold was 3.65 for males and 3.84 for females). Similar minor differences were exhibited in Confidence Index (CI), 1.72 for females (std. dev. 0.73) and 1.81 for males (std. dev. 0.77). Using “guess and correct” as criterion to determine individual thresholds in the forced choice olfactometry, the mean natural logarithm of ppb butanol is 1.365 lower than that for “certain and correct”. The standard deviation for “guess” and “certain” criteria were 1.093 and 0.911 respectively. The “certainty” criterion gave a better repeatability than the “guess” criterion.

scholarly journals Type I and II PRMTs inversely regulate post-transcriptional intron detention through Sm and CHTOP methylation

eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Maxim I Maron ◽  
Alyssa D Casill ◽  
Varun Gupta ◽  
Jacob S Roth ◽  
Simone Sidoli ◽  
...  
Keyword(s):  
Arginine Methylation ◽  
Targeted Mutagenesis ◽  
Type I ◽  
Type Ii ◽  
Protein Arginine Methyltransferases ◽  
Polyadenylated Rna ◽  
Transcript Elongation ◽  
Retained Introns ◽  
Method Analysis ◽  
Processing Factors

Protein arginine methyltransferases (PRMTs) are required for the regulation of RNA processing factors. Type I PRMT enzymes catalyze mono- and asymmetric dimethylation; Type II enzymes catalyze mono- and symmetric dimethylation. To understand the specific mechanisms of PRMT activity in splicing regulation, we inhibited Type I and II PRMTs and probed their transcriptomic consequences. Using the newly developed Splicing Kinetics and Transcript Elongation Rates by Sequencing (SKaTER-seq) method, analysis of co-transcriptional splicing demonstrated that PRMT inhibition resulted in altered splicing rates. Surprisingly, co-transcriptional splicing kinetics did not correlate with final changes in splicing of polyadenylated RNA. This was particularly true for retained introns (RI). By using actinomycin D to inhibit ongoing transcription, we determined that PRMTs post-transcriptionally regulate RI. Subsequent proteomic analysis of both PRMT-inhibited chromatin and chromatin-associated polyadenylated RNA identified altered binding of many proteins, including the Type I substrate, CHTOP, and the Type II substrate, SmB. Targeted mutagenesis of all methylarginine sites in SmD3, SmB, and SmD1 recapitulated splicing changes seen with Type II PRMT inhibition, without disrupting snRNP assembly. Similarly, mutagenesis of all methylarginine sites in CHTOP recapitulated the splicing changes seen with Type I PRMT inhibition. Examination of subcellular fractions further revealed that RI were enriched in the nucleoplasm and chromatin. Together, these data demonstrate that, through Sm and CHTOP arginine methylation, PRMTs regulate the post-transcriptional processing of nuclear, detained introns.

scholarly journals Arginine Methylation Regulates SARS-CoV-2 Nucleocapsid Protein Function and Viral Replication

2021 ◽  
Author(s):  
Ting Cai ◽  
Zhenbao Yu ◽  
Zhen Wang ◽  
Chen Liang ◽  
Stephane Richard
Keyword(s):  
Protein Function ◽  
Rna Binding ◽  
Binding Capacity ◽  
Viral Proteins ◽  
Arginine Methylation ◽  
Host Protein ◽  
Hek293 Cells ◽  
Type I ◽  
Dependent Manner ◽  
N Protein

Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) playing critical roles during viral infections. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG sequences. Arginine methylation of N protein was confirmed by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated by cell culture. We demonstrate that arginine methylation of N protein is required for its RNA binding capacity, since treatment with a type I PRMT inhibitor (MS023) or substitution of R95K or R177K inhibited interaction with the 5'-UTR of the SARS-CoV-2 genomic RNA. We defined the N interactome in HEK293 cells with or without MS023 treatment and identified PRMT1 and many of its RGG/RG substrates including the known interactor, G3BP1, and other components of stress granules (SG). Methylation of N protein at R95 regulates another function namely its property to suppress the formation of SGs. MS023 treatment or R95K substitution blocked N-mediated suppression of SGs. Also, the co-expression of methylarginine reader TDRD3 quenched N-mediated suppression of SGs in a dose-dependent manner. Finally, pre-treatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. With type I PRMT inhibitors being in clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may be an additional therapeutic application of these drugs.

Abstract 119: PRMT5 Is A New Epigenetic Regulator In Cardiovascular Disease

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Tobias S Merkel ◽  
David Hassel ◽  
Marcus Krüger ◽  
Dieter Weichenhan ◽  
Christoph Plass ◽  
...  
Keyword(s):  
Arginine Methylation ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Cardiomyocyte Hypertrophy ◽  
Type I ◽  
Type Ii ◽  
Cardiac Phenotype ◽  
Epigenetic Regulator

Protein arginine methylation is an abundant posttranslational modification involved in various cellular events such as DNA repair, splicing, and transcription. Protein arginine methyltransferases (PRMTs) catalyze the symmetric and asymmetric dimethylation of arginines and can be subdivided into two distinct families: type I enzymes catalyze the formation of asymmetric dimethylation whereas type II enzymes enhance the addition of symmetric methyl groups. Here, we report evidence that PRMT5 a type II enzyme serves as an important epigenetic regulator in myocardial disease. We identified PRMT5 via mass spectrometry as an interactant of class IIa histone deacetylases (HDACs). Additional cardiac interaction partners of PRMT5 were identified by a human cDNA library based yeast-two-hybrid screen. Thereby we elucidated Interleukin enhancer binding factor 3 (ILF3) as a putative interacting protein. ILF3 is a member of the NFAT family. NFAT signalling is known to be a potent driver of cardiomyocyte hypertrophy. Accordingly, we detected a 40% decrease in the size of neonatal rat ventricular myocytes in response to siRNA mediated knockdown of prmt5 . A dominant-negative version of PRMT5 is capable of interfering with the Calcineurin-NFAT axis and blocks NFAT activation. In vivo knockdown of endogenous prmt5 in the zebrafish led to a distinct cardiac phenotype with altered morphology and impaired function. To further elucidate prmt5 function in vivo we use mice with floxed prmt5 alleles. As a loss of prmt5 is lethal in the ES cell stadium we use an inducible α myosin heavy chain Mer-Cre-Mer system. Knockout mice display a complete loss of PRMT5 in cardiomyocytes. In line with our in vitro studies these mice are viable and show no phenotype under baseline conditions. Our work in progress investigates the hypothesis that class IIa HDACs serve as a scaffold to recruit PRMT5 to the DNA, forming a complex with ILF3 to block NFAT activity and the pro-hypertrophic gene program.

Erythrocyte uroporphyrinogen decarboxylase activity and therapeutic phlebotomy in porphyria cutanea tarda

2000 ◽  
Vol 04 (08) ◽  
pp. 736-738
Author(s):  
FERENC KÓSZÓ ◽  
MÁRTA MORVAY ◽  
ATTILA DOBOZY ◽  
KRISZTINA BODA
Keyword(s):  
Porphyria Cutanea Tarda ◽  
Decarboxylase Activity ◽  
Type I ◽  
Type Ii ◽  
Uroporphyrinogen Decarboxylase ◽  
The Family ◽  
Different Types ◽  
History Of ◽  
A Minor ◽  
Genetically Determined

On the basis of the uroporphyrinogen decarboxylase ( UD ) activity in the erythrocytes, and the family history of the disease, different types of porphyria cutanea tarda ( PCT ) can be distinguished. In some cases, however, the distinction may involve some uncertainty (overlapping of subgroups). The question arises of whether the current erythrocyte UD activities in the different types of PCT are determined merely genetically. The erythrocyte UD activities in 72 unrelated patients with different forms of PCT (62 with type I PCT and 10 with type II PCT ), in different stages of the disease, were measured in order to test whether the activity exhibits any change during the long period of recovery. In both types the activities were faintly but significantly increased, from 94.9% (in PCT I) or 54.3% (in PCT II) up to 98.4% or 56.1% respectively. In both types the lower activity in the untreated condition can be attributed to a combination of several factors, including oxidative damage to UD , which results in a minor additional inhibition of the genetically determined enzyme activities.

Nuclear localization of leukotriene A4 hydrolase in type II alveolar epithelial cells in normal and fibrotic lung

2005 ◽  
Vol 289 (2) ◽  
pp. L224-L232 ◽  
Author(s):  
Thomas G. Brock ◽  
Young-Jik Lee ◽  
Elana Maydanski ◽  
Tessa L. Marburger ◽  
Ming Luo ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Nuclear Localization ◽  
A549 Cells ◽  
Alveolar Epithelial Cells ◽  
Nuclear Accumulation ◽  
Aminopeptidase Activity ◽  
Type I ◽  
Type Ii ◽  
Alveolar Epithelial ◽  
Leukotriene A4

Leukotriene A4 (LTA4) hydrolase catalyzes the final step in leukotriene B4 (LTB4) synthesis. In addition to its role in LTB4 synthesis, the enzyme possesses aminopeptidase activity. In this study, we sought to define the subcellular distribution of LTA4 hydrolase in alveolar epithelial cells, which lack 5-lipoxygenase and do not synthesize LTA4. Immunohistochemical staining localized LTA4 hydrolase in the nucleus of type II but not type I alveolar epithelial cells of normal mouse, human, and rat lungs. Nuclear localization of LTA4 hydrolase was also demonstrated in proliferating type II-like A549 cells. The apparent redistribution of LTA4 hydrolase from the nucleus to the cytoplasm during type II-to-type I cell differentiation in vivo was recapitulated in vitro. Surprisingly, this change in localization of LTA4 hydrolase did not affect the capacity of isolated cells to convert LTA4 to LTB4. However, proliferation of A549 cells was inhibited by the aminopeptidase inhibitor bestatin. Nuclear accumulation of LTA4 hydrolase was also conspicuous in epithelial cells during alveolar repair following bleomycin-induced acute lung injury in mice, as well as in hyperplastic type II cells associated with fibrotic lung tissues from patients with idiopathic pulmonary fibrosis. These results show for the first time that LTA4 hydrolase can be accumulated in the nucleus of type II alveolar epithelial cells and that redistribution of the enzyme to the cytoplasm occurs with differentiation to the type I phenotype. Furthermore, the aminopeptidase activity of LTA4 hydrolase within the nucleus may play a role in promoting epithelial cell growth.

scholarly journals Influence of Culture Substrates on Morphology and Function of Pulmonary Alveolar Cells In Vitro

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 675
Author(s):  
Chiara Emma Campiglio ◽  
Marina Figliuzzi ◽  
Sara Silvani ◽  
Matteo Tironi ◽  
Sara Conti ◽  
...  
Keyword(s):  
Cell Morphology ◽  
A549 Cells ◽  
Alveolar Type ◽  
Binding Motif ◽  
Type I ◽  
Type Ii ◽  
Alveolar Cells ◽  
And Function ◽  
Cells Cultured ◽  
In Cells

Cell’s microenvironment has been shown to exert influence on cell behavior. In particular, matrix-cell interactions strongly impact cell morphology and function. The purpose of this study was to analyze the influence of different culture substrate materials on phenotype and functional properties of lung epithelial adenocarcinoma (A549) cells. A549 cells were seeded onto two different biocompatible, commercially available substrates: a polyester coverslip (Thermanox™ Coverslips), that was used as cell culture plate control, and a polydimethylsiloxane membrane (PDMS, Elastosil® Film) investigated in this study as alternative material for A549 cells culture. The two substrates influenced cell morphology and the actin cytoskeleton organization. Further, the Yes-associated protein (YAP) and its transcriptional coactivator PDZ-binding motif (TAZ) were translocated to the nucleus in A549 cells cultured on polyester substrate, yet it remained mostly cytosolic in cells on PDMS substrate. By SEM analysis, we observed that cells grown on Elastosil® Film maintained an alveolar Type II cell morphology. Immunofluorescence staining for surfactant-C revealing a high expression of surfactant-C in cells cultured on Elastosil® Film, but not in cells cultured on Thermanox™ Coverslips. A549 cells grown onto Elastosil® Film exhibited morphology and functionality that suggest retainment of alveolar epithelial Type II phenotype, while A549 cells grown onto conventional plastic substrates acquired an alveolar Type I phenotype.

scholarly journals Effects of a single cleavage in insulin-like growth factors I and II on binding to receptors, carrier proteins and antibodies

1990 ◽  
Vol 266 (2) ◽  
pp. 513-520 ◽  
Author(s):  
J Jansen ◽  
S C van Buul-Offers ◽  
C M Hoogerbrugge ◽  
J L Van Den Brande
Keyword(s):  
Cell Membranes ◽  
Binding Proteins ◽  
Type I ◽  
Type Ii ◽  
Placental Cell ◽  
Basic Peptide ◽  
Receptor Sites ◽  
Common Receptor ◽  
Igf I ◽  
A Minor

Two somatomedin-like peptides were extracted from Cohn fraction IV of human plasma and brought to homogeneity: one focused at pH 7.8 and the other at pH less than 5.6. Each consisted of two peptide chains interlinked by disulphide bonds. The basic peptide was identical to insulin-like growth factor I (IGF-I) and had a single cleavage in the C-domain before Arg37 [IGF-I(Arg36cl)]. The acid peptide showed identity with IGF-II, with a cleavage in the B-domain before Arg30 [IGF-II(Ser29cl)]. The effects of these cleavages on the characteristics of binding to type I and type II receptor sites, to binding proteins and to antibodies was studied. Binding of IGF-I(Arg36cl) to antibodies directed against the B-domain or against the AD-domain of IGF-I was the same as IGF-I binding. Thus the cleavage does not influence these antigenic sites. In contrast, binding of IGF-I(Arg36cl) to the type I receptor on human and bovine placental cell membranes was markedly decreased compared with IGF-I binding. Binding to the insulin receptor on human placental cell membranes was slightly diminished, whereas the interaction with specific type II receptors on bovine placental cell membranes was unaffected. There was only a minor influence of the cleavage on the region involved in binding to binding proteins. The cleavage in IGF-II(Ser29cl) diminished binding to antibodies directed against the C-domain of IGF-II, compared with binding of IGF-II itself. Binding to receptors (type I and type II) was changed less profoundly. With 125I-labelled IGF-II(Ser29cl), less insulin was needed in order to obtain 50% displacement of the tracer compared with displacement of 125I-labelled IGF-II. The cleaved form of IGF-II probably has a greater affinity towards the common receptor population than does native IGF-II. Binding to binding proteins was not affected by the cleavage in IGF-II.

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