L-NMMA Research Articles

Intermolecular energy migration via homoFRET captures the modulation in the material property of phase-separated biomolecular condensates

Physical properties of biomolecular condensates formed via phase separation of proteins and nucleic acids are associated with cell physiology and disease. Condensate properties can be regulated by several cellular factors including post-translational modifications. Here, we introduce an application of intermolecular energy migration via homo-FRET (Förster resonance energy transfer), a nanometric proximity ruler, to study the modulation in short- and long-range protein-protein interactions leading to the changes in the physical properties of condensates of fluorescently-tagged FUS (Fused in Sarcoma) that is associated with the formation of cytoplasmic and nuclear membraneless organelles. We show that homoFRET captures modulations in condensate properties of FUS by RNA, ATP, and post-translational arginine methylation. We also extend the homoFRET methodology to study the in-situ formation of cytoplasmic stress granules in mammalian cells. Our studies highlight the broad applicability of homoFRET as a potent generic tool for studying intracellular phase transitions involved in function and disease.

Targeting proteins to lysosomes with a chemical inducer of arginine methylation.

Targeting proteins to lysosomes with a chemical inducer of arginine methylation.

Methylarginine targeting chimeras for lysosomal degradation of intracellular proteins.

A paradigm shift in drug development is the discovery of small molecules that harness the ubiquitin-proteasomal pathway to eliminate pathogenic proteins. Here we provide a modality for targeted protein degradation in lysosomes. We exploit an endogenous lysosomal pathway whereby protein arginine methyltransferases (PRMTs) initiate substrate degradation via arginine methylation. We developed a heterobifunctional small molecule, methylarginine targeting chimera (MrTAC), that recruits PRMT1 to a target protein for induced degradation in lysosomes. MrTAC compounds degraded substrates across cell lines, timescales and doses. MrTAC degradation required target protein methylation for subsequent lysosomal delivery via microautophagy. A library of MrTAC molecules exemplified the generality of MrTAC to degrade known targets and neo-substrates-glycogen synthase kinase 3β, MYC, bromodomain-containing protein 4 and histone deacetylase 6. MrTAC selectively degraded target proteins and drove biological loss-of-function phenotypes in survival, transcription and proliferation. Collectively, MrTAC demonstrates the utility of endogenous lysosomal proteolysis in the generation of a new class of small molecule degraders.

Transcript splicing optimizes the thymic self-antigen repertoire to suppress autoimmunity.

Immunological self-tolerance is established in the thymus by the expression of virtually all self-antigens, including tissue-restricted antigens (TRAs) and cell-type-restricted antigens (CRAs). Despite a wealth of knowledge about the transcriptional regulation of TRA genes, posttranscriptional regulation remains poorly understood. Here, we show that protein arginine methylation plays an essential role in central immune tolerance by maximizing the self-antigen repertoire in medullary thymic epithelial cells (mTECs). Protein arginine methyltransferase-5 (Prmt5) was required for pre-mRNA splicing of certain key genes in tolerance induction, including Aire as well as various genes encoding TRAs. Mice lacking Prmt5 specifically in thymic epithelial cells exhibited an altered thymic T cell selection, leading to the breakdown of immune tolerance accompanied by both autoimmune responses and enhanced antitumor immunity. Thus, arginine methylation and transcript splicing are essential for establishing immune tolerance and may serve as a therapeutic target in autoimmune diseases as well as cancer immunotherapy.

PRMT1-mediated arginine methylation promotes YAP activation and hepatocellular carcinoma proliferation.

The activity of Hippo signaling is commonly dysregulated in various human malignancies, including hepatocellular carcinoma (HCC). YAP, the key effector of Hippo pathway, is regulated through several posttranslational modifications. However, the mechanism by which YAP is regulated by arginine methylation remains unknown. In this study, immunoprecipitation and mass spectrometry were used to identify the arginine methylation site of YAP in HCC cells. The transcriptional activity of YAP and TEAD were further characterized by real-time qPCR and immunofluorescence assay, and a subcutaneous and orthotopic tumor mouse model was used to assess the effect of PRMT1-knockdown on HCC tumor growth. The result of mass spectrometry analysis identified that YAP was methylated at arginine 124. Moreover, we found that arginine methyltransferase PRMT1 interacted with YAP to mediate its arginine methylation, thus inhibited YAP phosphorylation and promoted YAP activity in the nucleus. PRMT1 was up-regulated in HCC tissues and positively associated with the expressions of YAP target genes. Silencing PRMT1 in HCC cells inhibited cell proliferation and tumor growth, while PRMT1-overexpression promoted HCC growth through YAP methylation. Our study reveals that PRMT1-mediated arginine methylation at R124 is mutually exclusive with YAP S127 phosphorylation, thereby facilitating YAP activity in the nucleus and promoting tumorigenesis in HCC.

Assessing the Role of AtGRP7 Arginine 141, a Target of Dimethylation by PRMT5, in Flowering Time Control and Stress Response.

PROTEIN ARGININE METHYLTRANSFERASES (PRMTs) catalyze arginine (R) methylation that is critical for transcriptional and post-transcriptional gene regulation. In Arabidopsis, PRMT5 that catalyzes symmetric R dimethylation is best characterized. PRMT5 mutants are late-flowering and show altered responses to environmental stress. Among PRMT5 targets are Arabidopsis thaliana GLYCINE RICH RNA BINDING PROTEIN 7 (AtGRP7) and AtGRP8 that promote the transition to flowering. AtGRP7 R141 has been shown to be modified by PRMT5. Here, we tested whether this symmetric dimethylation of R141 is important for AtGRP7's physiological role in flowering time control. We constructed AtGRP7 mutant variants with non-methylable R141 (R141A, R141K). Genomic clones containing these variants complemented the late-flowering phenotype of the grp7-1 mutant to the same extent as wild-type AtGRP7. Furthermore, overexpression of AtGRP7 R141A or R141K promoted flowering similar to overexpression of the wild-type protein. Thus, flowering time does not depend on R141 and its modification. However, germination experiments showed that R141 contributes to the activity of AtGRP7 in response to abiotic stress reactions mediated by abscisic acid during early development. Immunoprecipitation of AtGRP7-GFP in the prmt5 background revealed that antibodies against dimethylated arginine still recognized AtGRP7, suggesting that additional methyltransferases may be responsible for modification of AtGRP7.

A novel arginine methylation-associated lncRNA signature effectively predicts prognosis in breast cancer patients.

Breast cancer (BC) is a disease highly associated with epigenetic modification, and arginine methylation is particularly important in its genetic regulation. However, the role of arginine methylation related lncRNAs in breast cancer has not been studied. First, we identified the related lncRNAs (from TCGA database) according to the differentially expressed genes related to arginine methylation in breast cancer. Then the lncRNAs related to protein arginine methylation were obtained by regression analysis, and the risk score model was constructed. Finally, the cell experiment and subcutaneous tumor model verified that the arginine methylation related lncRNA z68871.1 in the model had a significant effect on the proliferation and invasion of breast cancer cells. In conclusion, we successfully constructed an arginine methylation related lncRNA model, which has strong predictive ability. At the same time, this study provides an experimental basis for exploring the mechanism of arginine methylation in BC and helps to find new biomarkers of BC.

Arginine methylation of the p30 C/EBPα oncoprotein regulates progenitor proliferation and myeloid differentiation

Arginine methylation of the p30 C/EBPα oncoprotein regulates progenitor proliferation and myeloid differentiation

Epigenetic reader ZMYND11 noncanonical function restricts HNRNPA1-mediated stress granule formation and oncogenic activity

Epigenetic readers frequently affect gene regulation, correlate with disease prognosis, and hold significant potential as therapeutic targets for cancer. Zinc finger MYND-type containing 11 (ZMYND11) is notably recognized for reading the epigenetic marker H3.3K36me3; however, its broader functions and mechanisms of action in cancer remain underexplored. Here, we report that ZMYND11 downregulation is prevalent across various cancers and profoundly correlates with poorer outcomes in prostate cancer patients. Depletion of ZMYND11 promotes tumor cell growth, migration, and invasion in vitro, as well as tumor formation and metastasis in vivo. Mechanistically, we discover that ZMYND11 exhibits tumor suppressive roles by recognizing arginine-194-methylated HNRNPA1 dependent on its MYND domain, thereby retaining HNRNPA1 in the nucleus and preventing the formation of stress granules in the cytoplasm. Furthermore, ZMYND11 counteracts the HNRNPA1-driven increase in the PKM2/PKM1 ratio, thus mitigating the aggressive tumor phenotype promoted by PKM2. Remarkably, ZMYND11 recognition of HNRNPA1 can be disrupted by pharmaceutical inhibition of the arginine methyltransferase PRMT5. Tumors with low ZMYND11 expression show sensitivity to PRMT5 inhibitors. Taken together, our findings uncover a previously unexplored noncanonical role of ZMYND11 as a nonhistone methylation reader and underscore the critical importance of arginine methylation in the ZMYND11-HNRNPA1 interaction for restraining tumor progression, thereby proposing novel therapeutic targets and potential biomarkers for cancer treatment.

Hetero-oligomeric interaction as a new regulatory mechanism for protein arginine methyltransferases.

Protein arginine methylation is a versatile post-translational protein modification that has notable cellular roles such as transcriptional activation or repression, cell signaling, cell cycle regulation, and DNA damage response. However, in spite of their extensive significance in the biological system, there is still a significant gap in understanding of the entire function of the protein arginine methyltransferases (PRMTs). It has been well-established that PRMTs form homo-oligomeric complexes to be catalytically active, but in recent years, several studies have showcased evidence that different members of PRMTs can have cross-talk with one another to form hetero-oligomeric complexes. Additionally, these heteromeric complexes have distinct roles separate from their homomeric counterparts. Here, we review and highlight the discovery of the heterodimerization of PRMTs and discuss the biological implications of these hetero-oligomeric interactions.

Neurodevelopmental functions and activities of the KAT3 class of lysine acetyltransferases.

The human lysine acetyltransferases KAT3A (CREBBP) and KAT3B (EP300) are essential enzymes in gene regulation in the nucleus. Their ubiquitous expression in metazoan cell types controls cell proliferation and differentiation during development. This comprehensive review delves into the biological roles of KAT3A and KAT3B in neurodevelopment, shedding light on how alterations in their regulation or activity can potentially contribute to a spectrum of neurodegenerative diseases (e.g. Huntington's and Alzheimer's). We explore the pathophysiological implications of KAT3 function loss in these disorders, considering their conserved protein domains and biochemical functions in chromatin regulation. The discussion also underscores the crucial role of KAT3 proteins and their substrates in supporting the integration of key cell signaling pathways. Furthermore, the narrative highlights the interdependence of KAT3-mediated lysine acetylation with lysine methylation and arginine methylation. From a cellular perspective, KAT3-dependent signal integration at subnuclear domains is mediated by liquid-liquid phase separation in response to KAT3-mediated lysine acetylation. The disruption of these finely tuned regulatory processes underscores their pathological roles in neurodegeneration. This review also points to the exciting potential for future research in this field, inspiring further investigation and discovery in the area of neurodevelopment and neurodegenerative diseases.

Arginine Methylation of DDX3 by PRMT1 Mediates Mitochondrial Homeostasis to Promote Breast Cancer Metastasis.

Dysregulated mitochondrial dynamics and metabolism play important roles in tumorigenesis. Metastasizing tumor cells predominantly utilize mitochondrial metabolism, and regulators of metabolic reprogramming may provide reliable biomarkers for diagnosing cancer metastasis. Here, we identified a type I arginine methyltransferase-DEAD-box polypeptide 3, X-linked (PRMT1-DDX3) axis that promotes breast cancer metastasis by coordinating mitochondrial biogenesis and mitophagy to ensure mitochondrial quality control. Mechanistically, PRMT1 induces arginine methylation of DDX3, which enhances its protein stability and prevents proteasomal degradation. DDX3 mediates mitochondrial homeostasis by translocating to mitochondria where it facilitates phosphatase and tensin homology-induced kinase 1 translation in response to mitochondrial stress. Inhibition of DDX3 suppresses mitochondrial biogenesis and mitophagy, resulting in diminished cancer stemness and metastatic properties. Overall, this study uncovers a mechanism by which the PRMT1-DDX3 axis regulates mitochondrial homeostasis to support breast cancer metastasis, suggesting strategies for targeting metabolic vulnerabilities to treat metastatic breast cancer. Significance: DDX3 is stabilized by PRMT1-mediated arginine methylation and coordinates mitophagy and mitochondrial biogenesis by upregulating PINK1 to facilitate breast cancer progression.

R-Methylation in Plants: A Key Regulator of Plant Development and Response to the Environment.

Although arginine methylation (R-methylation) is one of the most important post-translational modifications (PTMs) conserved in eukaryotes, it has not been studied to the same extent as phosphorylation and ubiquitylation. Technical constraints, which are in the process of being resolved, may partly explain this lack of success. Our knowledge of R-methylation has recently evolved considerably, particularly in metazoans, where misregulation of the enzymes that deposit this PTM is implicated in several diseases and cancers. Indeed, the roles of R-methylation have been highlighted through the analyses of the main actors of this pathway: the PRMT writer enzymes, the TUDOR reader proteins, and potential "eraser" enzymes. In contrast, R-methylation has been much less studied in plants. Even so, it has been shown that R-methylation in plants, as in animals, regulates housekeeping processes such as transcription, RNA silencing, splicing, ribosome biogenesis, and DNA damage. R-methylation has recently been highlighted in the regulation of membrane-free organelles in animals, but this role has not yet been demonstrated in plants. The identified R-met targets modulate key biological processes such as flowering, shoot and root development, and responses to abiotic and biotic stresses. Finally, arginine demethylases activity has mostly been identified in vitro, so further studies are needed to unravel the mechanism of arginine demethylation.

330 Growth performance, incidence of scours, and plasma metabolites in beef × dairy crossbred calves transported at 2, 4, 8 and 14 d of age

Abstract Transportation can compromise health and production performance in young calves. Although widely accepted that the younger calves are, the greater the probability of negative outcomes, there still is a lack of evidence-based recommendations on the minimum age at transportation. Our objective was to evaluate the impact of transport age on growth performance, incidence of scours, and plasma metabolites over the first 28 d post-transport in Charolais × Holstein crossbred calves. Calves (n = 36) ages 2, 4, 8 and 14-d old were transported in summer from a commercial farm to an on-campus calf rearing facility (410 miles). Calf body weight (BW) was recorded before departure, on arrival and d 28 post-transport. Dry matter intake (DMI) and fecal scores were recorded daily. Blood samples collected prior to transportation, on arrival, and d 7 and 28 post-transport were used for targeted metabolomics analysis (Biocrates MxP Quant 500 kit). Growth performance and fecal score data were analyzed using PROC MIXED (SAS) whereas R was used for predictive modeling of metabolomics data. Over the 28-d period post-transport, DMI was greater (P < 0.01) for 14 d than 2 and 4 d old calves; however, it did not differ between 2, 4, and 8 d old calves. Average daily gain (ADG) and feed efficiency (FE) were also greater (P < 0.01) for 14 d than 2, 4, and 8 d old calves. During wk 1 post-transport, the total number of days calves had scours was least (P < 0.01) for 2 and 14 d, intermediate for 4 d, and greatest for 8 d old calves. During wk 2, the total number of days with scours was less (P < 0.01) for 8 and 14 d (average of 2 d) compared with 2 and 4 d old calves (5 d). Additionally, the total number of days calves had scours during the first 2 wk post-transport tended (P = 0.08) to be greater for 2, 4, and 8 d than 14 d old calves. However, during wk 3 and 4 post-transport, there were no differences (P ≥ 0.58) in the total number of days with scours across transport age. Arginine methylation, d 7 of measurements, carnitine uptake defect, phosphatidic acid 16:0:18:2, methylenetetrahydrofolate reductase, sodium, triglyceride 18:2:34:4, threonine, α-aminobutyric acid, and glycolithocholic acid 3-sulfate were the most important features in terms of predicting the occurrence of scours. Overall, DMI, ADG and FE were less for calves transported at 2, 4, and 8 d than at 14 d of age, which could partially be attributed to differences in the incidence rate of scours. In addition, several plasma metabolites could be good predictors of scours in transported calves.

Structure, Function, and Activity of Small Molecule and Peptide Inhibitors of Protein Arginine Methyltransferase 1.

Protein arginine N-methyltransferases (PRMT) are a family of S-adenosyl-l-methionine (SAM)-dependent enzymes that transfer methyl-groups to the ω-N of arginyl residues in proteins. PRMTs are involved in regulating gene expression, RNA splicing, and other activities. PRMT1 is responsible for most cellular arginine methylation, and its dysregulation is involved in many cancers. Accordingly, many groups have targeted PRMT1 using small molecules and peptide inhibitors. In this Perspective, we discuss the structure and function of selected peptide and small molecule inhibitors of PRMT1. We examine inhibitors that target the substrate arginyl peptide, SAM, or both binding sites, and the type of inhibition that results. Small molecules, and peptides that are bisubstrate, and/or PRMT transition state mimic inhibitors as well as inhibitors that alkylate PRMTs will be discussed. We define a structure-activity relationship for the aromatic/heteroaromatic N-methylethylenediamine inhibitors of PRMT1 and review current progress of PRMT1 inhibitors in clinical trials.

Histone Arginine Methylation as a Regulator of Gene Expression in the Dehydrating African Clawed Frog (Xenopus laevis).

The African clawed frog (Xenopus laevis) endures prolonged periods of dehydration while estivating underground during the dry season. Epigenetic modifications play crucial roles in regulating gene expression in response to environmental changes. The elucidation of epigenetic changes relevant to survival could serve as a basis for further studies on organ preservation under extreme stress. The current study examined the relative protein levels of key enzymes involved in the arginine methylation of histones in the liver and kidney tissues of control versus dehydrated (35 ± 1%) X. laevis through immunoblotting. Protein arginine methyltransferases (PRMT) 4, 5, and 6 showed significant protein level decreases of 35 ± 3%, 71 ± 7%, and 25 ± 5%, respectively, in the liver tissues of the dehydrated frogs relative to controls. In contrast, PRMT7 exhibited an increase of 36 ± 4%. Similarly, the methylated histone markers H3R2m2a, H3R8m2a, and H3R8m2s were downregulated by 34 ± 11%, 15 ± 4%, and 42 ± 12%, respectively, in the livers of dehydrated frogs compared to controls. By contrast, the kidneys of dehydrated frogs showed an upregulation of histone markers. H3R2m2a, H3R8m2a, H3R8m2s, and H4R3m2a were significantly increased by 126 ± 12%, 112 ± 7%, 47 ± 13%, and 13 ± 3%, respectively. These changes can play vital roles in the metabolic reorganization of X. laevis during dehydration, and are likely to increase the chances of survival. In turn, the tissue-specific regulation of the histone arginine methylation mechanism suggests the importance of epigenetic regulation in the adaptation of X. laevis for whole-body dehydration.

Protein arginine methyltransferase 6 mediates antiviral immunity in plants.

Viral suppressor RNA silencing (VSR) is essential for successful infection. Nucleotide-binding and leucine-rich repeat (NLR)-based and autophagy-mediated immune responses have been reported to target VSR as counter-defense strategies. Here, we report a protein arginine methyltransferase 6 (PRMT6)-mediated defense mechanism targeting VSR. The knockout and overexpression of PRMT6 in tomato plants lead to enhanced and reduced disease symptoms, respectively, during tomato bush stunt virus (TBSV) infection. PRMT6 interacts with and inhibits the VSR function of TBSV P19 by methylating its key arginine residues R43 and R115, thereby reducing its dimerization and small RNA-binding activities. Analysis of the natural tomato population reveals that two major alleles associated with high and low levels of PRMT6 expression are significantly associated with high and low levels of viral resistance, respectively. Our study establishes PRMT6-mediated arginine methylation of VSR as a mechanism of plant immunity against viruses.

Inhibition of PRMT5 moderately suppresses prostate cancer growth in vivo but enhances its response to immunotherapy

Protein arginine methylation is a common post-translational modification (PTM) catalyzed by nine protein arginine methyltransferases (PRMTs). As the major symmetric arginine methyltransferase that methylates both histone and non-histone substrates, PRMT5 plays key roles in a number of biological processes critical for development and tumorigenesis. PRMT5 overexpression has been reported in multiple cancer types including prostate cancer (PCa), but the exact biological and mechanistic understanding of PRMT5 in aggressive PCa remains ill-defined. Here, we show that PRMT5 is upregulated in PCa, correlates with worse patient survival, promotes corrupted RNA splicing, and functionally cooperates with an array of pro-tumorigenic pathways to enhance oncogenesis. PRMT5 inhibition via either genetic knockdown or pharmacological inhibition reduces stemness with paralleled differentiation and arrests cell cycle progression without causing appreciable apoptosis. Strikingly, the severity of antitumor effect of PRMT5 inhibition correlates with disease aggressiveness, with AR+ PCa being less affected. Molecular characterization pinpoints MYC, but not (or at least to a lesser degree) AR, as the main partner of PRMT5 to form a positive feedback loop to exacerbate malignancy in both AR+ and AR— PCa cells. Inspired by the surprising finding that PRMT5 negatively correlates with tumor immune infiltration and transcriptionally suppresses an immune-gene program, we further show that although PRMT5 inhibitor (PRMT5i) EPZ015666 or anti-PD-1 immunotherapy alone exhibits limited antitumor effects, combination of PRMT5i with anti-PD-1 displays superior efficacy in inhibiting castration-resistant PCa (CRPC) in vivo. Finally, to expand the potential use of PRMT5i through a synthetic lethality concept, we also perform a global CRISPR/Cas9 knockout screen to unravel that many clinical-grade drugs of known oncogenic pathways can be repurposed to target CRPC when used in combination with PRMT5i at low doses. Collectively, our findings establish a rationale to exploit PRMT5i in combination with immunotherapy or other targeted therapies to treat aggressive PCa.

A Chemoenzymatic Method Enables Global Enrichment and Characterization of Protein Arginine Methylation.

Arginine methylation is one of the most important post-translational modifications involved in the regulation of numerous biological processes. To better understand the biological significance of arginine methylation, enrichment methods need to be developed to analyze the methylated proteome at large-scale. Unfortunately, the prevailing enrichment method based on immunoaffinity purification can only enrich a subset of them due to the lack of pan-specific antibodies. Therefore, it is crucial to develop a stable and efficient antibody-free approach for the global analysis of arginine methylation. In this study, we developed a chemoenzymatic method for the simultaneous identification of mono- and dimethylated arginine. Totally, we identified 1006 arginine methylation events in Jurkat T cells, corresponding to 645 dimethylated sites and 361 monomethylated sites. We further applied the developed approach to global identification of the substrate proteins regulated by type I protein arginine methyltransferases (PRMTs) and identified 49 substrate proteins of type I PRMTs, which will facilitate a better understanding of PRMTs-regulated biological processes. Given the robust performance of this method, it would have broad application in methylproteomics analysis.

Protein Arginine Methylation of the Translation Initiation Factor eIF1A Increases Usage of a Near-cognate Start Codon.

Protein arginine methylation has emerged as a key post-translational modification responsible for many facets of eukaryotic gene expression. To better understand the extent of this modification in cellular pathways, we carried out bioorthogonal methylation profiling in Saccharomyces cerevisiae to comprehensively identify the in vivo substrates of the major yeast protein arginine methyltransferase Hmt1. Gene ontology analysis of candidate substrates revealed an enrichment of proteins involved in the process of translation. We verified one such factor, eIF1A, by in vitro methylation. Three sites on eIF1A were found to be responsible for its methylation: R13, R14, and R62, with varied capacity by which each site contributed to the overall methylation capacity in vitro. To determine the role of methylation in eIF1A function, we used a battery of arginine-to-alanine substitution mutants to evaluate translation fidelity in these mutants. Our data show that substitution mutants at R13 and R14 in the N-terminal tail improved the fidelity of start codon recognition in an initiation fidelity assay. Overall, our data suggest that Hmt1-mediated methylation of eIF1A fine-tunes the fidelity of start codon recognition for proper translation initiation.