Prevalent Post-translational Modification Research Articles

E3 ubiquitin ligase HECW2: a promising target for tumour therapy.

Ubiquitination is a prevalent post-translational modification that plays a crucial role in a wide range of pathophysiological processes, including cell proliferation, apoptosis, autophagy, immune response, and DNA damage repair. Among the enzymes involved in ubiquitination, E3 ubiquitin ligases are particularly significant, serving as key regulators of numerous diseases, including tumours. This review focuses on HECW2 (HECT, C2, and WW domain-containing E3 ubiquitin protein ligase 2, also known as NEDL2), providing a comprehensive overview of its interactors and its pathological roles in tumorous cancer and other diseases. The insights gained from this review may contribute to the development of novel treatment strategies for various diseases, particularly tumours.

Direct mapping of tyrosine sulfation states in native peptides by nanopore.

Sulfation is considered the most prevalent post-translational modification (PTM) on tyrosine; however, its importance is frequently undervalued due to difficulties in direct and unambiguous determination from phosphorylation. Here we present a sequence-independent strategy to directly map and quantify the tyrosine sulfation states in universal native peptides using an engineered protein nanopore. Molecular dynamics simulations and nanopore mutations reveal specific interactions between tyrosine sulfation and the engineered nanopore, dominating identification across diverse peptide sequences. We show a nanopore framework to discover tyrosine sulfation in unknown peptide fragments digested from a native protein and determine the sequence of the sulfated fragment based on current blockade enhancement induced by sulfation. Moreover, our method allows direct observation of peptide sulfation in ultra-low abundance, down to 1%, and distinguishes it from isobaric phosphorylation. This sequence-independent strategy suggests the potential of nanopore to explore specific PTMs in real-life samples and at the omics level.

K128 ubiquitination constrains RAS activity by expanding its binding interface with GAP proteins

The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis.

Nε-lysine acetylation of the histone-like protein HBsu influences antibiotic survival and persistence in Bacillus subtilis.

Nε-lysine acetylation is recognized as a prevalent post-translational modification (PTM) that regulates proteins across all three domains of life. In Bacillus subtilis, the histone-like protein HBsu is acetylated at seven sites, which regulates DNA compaction and the process of sporulation. In Mycobacteria, DNA compaction is a survival strategy in response antibiotic exposure. Acetylation of the HBsu ortholog HupB decondenses the chromosome to escape this drug-induced, non-growing state, and in addition, regulates the formation of drug-tolerant subpopulations by altering gene expression. We hypothesized that the acetylation of HBsu plays similar regulatory roles. First, we measured nucleoid area by fluorescence microscopy and in agreement, we found that wild-type cells compacted their nucleoids upon kanamycin exposure, but not exposure to tetracycline. We analyzed a collection of HBsu mutants that contain lysine substitutions that mimic the acetylated (glutamine) or unacetylated (arginine) forms of the protein. Our findings indicate that some level of acetylation is required at K3 for a proper response and K75 must be deacetylated. Next, we performed time-kill assays of wild-type and mutant strains in the presence of different antibiotics and found that interfering with HBsu acetylation led to faster killing rates. Finally, we examined the persistent subpopulation and found that altering the acetylation status of HBsu led to an increase in persister cell formation. In addition, we found that most of the deacetylation-mimic mutants, which have compacted nucleoids, were delayed in resuming growth following removal of the antibiotic, suggesting that acetylation is required to escape the persistent state. Together, this data adds an additional regulatory role for HBsu acetylation and further supports the existence of a histone-like code in bacteria.

N-Glycosylation as a Modulator of Protein Conformation and Assembly in Disease.

Glycosylation, a prevalent post-translational modification, plays a pivotal role in regulating intricate cellular processes by covalently attaching glycans to macromolecules. Dysregulated glycosylation is linked to a spectrum of diseases, encompassing cancer, neurodegenerative disorders, congenital disorders, infections, and inflammation. This review delves into the intricate interplay between glycosylation and protein conformation, with a specific focus on the profound impact of N-glycans on the selection of distinct protein conformations characterized by distinct interactomes-namely, protein assemblies-under normal and pathological conditions across various diseases. We begin by examining the spike protein of the SARS virus, illustrating how N-glycans regulate the infectivity of pathogenic agents. Subsequently, we utilize the prion protein and the chaperone glucose-regulated protein 94 as examples, exploring instances where N-glycosylation transforms physiological protein structures into disease-associated forms. Unraveling these connections provides valuable insights into potential therapeutic avenues and a deeper comprehension of the molecular intricacies that underlie disease conditions. This exploration of glycosylation's influence on protein conformation effectively bridges the gap between the glycome and disease, offering a comprehensive perspective on the therapeutic implications of targeting conformational mutants and their pathologic assemblies in various diseases. The goal is to unravel the nuances of these post-translational modifications, shedding light on how they contribute to the intricate interplay between protein conformation, assembly, and disease.

A resource database for protein kinase substrate sequence-preference motifs based on large-scale mass spectrometry data

BackgroundProtein phosphorylation is one of the most prevalent posttranslational modifications involved in molecular control of cellular processes, and is mediated by over 520 protein kinases in humans and other mammals. Identification of the protein kinases responsible for phosphorylation events is key to understanding signaling pathways. Unbiased phosphoproteomics experiments have generated a wealth of data that can be used to identify protein kinase targets and their preferred substrate sequences.MethodsThis study utilized prior data from mass spectrometry-based studies identifying sites of protein phosphorylation after in vitro incubation of protein mixtures with recombinant protein kinases. PTM-Logo software was used with these data to generate position-dependent Shannon information matrices and sequence motif ‘logos’. Webpages were constructed for facile access to logos for each kinase and a new stand-alone application was written in Python that uses the position-dependent Shannon information matrices to identify kinases most likely to phosphorylate a particular phosphorylation site.ResultsA database of kinase substrate target preference logos allows browsing, searching, or downloading target motif data for each protein kinase (https://esbl.nhlbi.nih.gov/Databases/Kinase_Logos/). These logos were combined with phylogenetic analysis of protein kinase catalytic sequences to reveal substrate preference patterns specific to particular groups of kinases (https://esbl.nhlbi.nih.gov/Databases/Kinase_Logos/KinaseTree.html). A stand-alone program, KinasePredictor, is provided (https://esbl.nhlbi.nih.gov/Databases/Kinase_Logos/KinasePredictor.html). It takes as input, amino-acid sequences surrounding a given phosphorylation site and generates a ranked list of protein kinases most likely to phosphorylate that site.ConclusionsThis study provides three new resources for protein kinase characterization. It provides a tool for prediction of kinase-substrate interactions, which in combination with other types of data (co-localization, etc.), can predict which kinases are likely responsible for a given phosphorylation event in a given tissue.-q3N7pjxaL8wVBVRsJ9wBTVideo

Role of O-GlcNAcylation in Central Nervous System Development and Injuries: A Systematic Review.

The development of central nervous system (CNS) can form perceptual, memory, and cognitive functions, while injuries to CNS often lead to severe neurological dysfunction and even death. As one of the prevalent post-translational modifications (PTMs), O-GlcNAcylation has recently attracted great attentions due to its functions in regulating the activity, subcellular localization, and stability of target proteins. It has been indicated that O-GlcNAcylation could interact with phosphorylation, ubiquitination, and methylation to jointly regulate the function and activity of proteins. Furthermore, a growing number of studies have suggested that O-GlcNAcylation played an important role in the CNS. During development, O-GlcNAcylation participated in the neurogenesis, neuronal development, and neuronal function. In addition, O-GlcNAcylation was involved in the progress of CNS injuries including ischemic stroke, subarachnoid hemorrhage (SAH), and intracerebral hemorrhage (ICH) and played a crucial role in the improvement of brain damage such as attenuating cognitive impairment, inhibiting neuroinflammation, suppressing endoplasmic reticulum (ER) stress, and maintaining blood-brain barrier (BBB) integrity. Therefore, O-GlcNAcylation showed great promise as a potential target in CNS development and injuries. In this article, we presented a review highlighting the role of O-GlcNAcylation in CNS development and injuries. Hence, on the basis of these properties and effects, intervention with O-GlcNAcylation may be developed as therapeutic agents for CNS diseases.

Enrichment of phosphopeptides by arginine-functionalized magnetic chitosan nanoparticles.

One of the most crucial and prevalent post-translational modifications is the phosphorylation of proteins. The study and examination of protein phosphorylation hold immense importance in comprehending disease mechanisms and discovering novel biomarkers. However, the inherent low abundance, low ionization efficiency, and coexistence with non phosphopeptides seriously affect the direct analysis of phosphopeptides by mass spectrometry. In order to tackle these problems, it is necessary to carry out selective enrichment of phosphopeptides prior to conducting mass spectrometry analysis. Herein, magnetic chitosan nanoparticles were developed by incorporating arginine, and were then utilized for phosphopeptide enrichment. A tryptic digest of β-casein was chosen as the standard substance. After enrichment, combined with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), the detection limit of the method was 0.4 fmol. The synthesized magnetic material demonstrated great potential in the detection of phosphopeptides in complex samples, as proven by its successful application in detecting phosphopeptides in skim milk and human saliva samples.

M6A methyltransferase METTL3 contributes to sympathetic hyperactivity post-MI via promoting TRAF6-dependent mitochondrial ROS production

ObjectivesN6-methyladenosine (m6A) is the most prevalent post-translational modification in eukaryotic mRNA. Recently, m6A editing modified by methyltransferase-like enzyme 3 (METTL3), the core m6A methyltransferase, has been demonstrated to be involved in cardiac sympathetic hyperactivity. This study aimed to clarify the effects and underlying mechanisms of METTL3 in the paraventricular nucleus (PVN) in mediating sympathetic activity following myocardial infarction (MI). MethodsWe established rat MI models by left anterior descending coronary artery ligation. m6A quantification was performed.The expression of METTL3 and its downstream gene, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), were determined. The functional role of METTL3 in sympathetic hyperactivity and electrical conduction stability were verified by assessing renal sympathetic nerve activity (RSNA), norepinephrine (NE) levels, and programmed electrical stimulation. Rescue experiments were also conducted. The mechanism by which m6A is involved in mitochondrial reactive oxygen species (mROS) production, mediated by TRAF6/ECSIT pathway, was explored in lipopolysaccharide (LPS) treated primary microglial cells. ResultsMETTL3 was predominantly localized in the microglia and significantly increased within the PVN at 3 days post-MI. Inhibition of METTL3 decreased m6A levels, TRAF6 expression, and mROS production; downregulated sympathoexcitation, indicated by attenuated NE concentration and RSNA; decreased the incidence of ventricular tachycardia or fibrillation; and improved cardiac function. Mechanistically, downregulation of METTL3 prevented TRAF6 translocation to the mitochondria in the microglia and subsequent TRAF6/ECSIT pathway activation, resulting in decreased mROS production. ConclusionsThis study demonstrates that METTL3-mediated m6A modification promotes sympathetic hyperactivity through TRAF6/ECSIT pathway and mitochondrial oxidative stress in the PVN, thereby leading to ventricular arrhythmias post-MI.

Systematic review: Glycomics as diagnostic markers for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer with one of the highest cancer-related mortality rates worldwide. Early diagnosis is crucial for improving the therapeutic options and reducing the disease-related mortality. To investigate serum N-glycomics as diagnostic markers for HCC. We performed a comprehensive search in PubMed, EMBASE, Web of Science and Scopus through August 17, 2023. Eligible studies assessed the potential use of serum N-glycomics as diagnostic biomarkers for HCC. Study selection, data extraction and quality assessment were performed by two independent reviewers. Of the 48 articles included, 11 evaluated the utility of N-glycomics for the diagnosis of HCC in whole serum while the remaining articles focused on specific protein glycoforms or protein levels. Of these specific proteins, haptoglobin, alpha-fetoprotein (AFP), kininogen (Kin), α-1-antitrypsin and Golgi protein 73 (GP73) were the most frequently studied. Increased levels of fucosylation and branching presented as the most prevalent post-translational modifications of glycoproteins in patients with HCC compared to controls. Notably, glycomics-based biomarkers may provide a clinical benefit for the diagnosis of early HCC, as several algorithms achieved AUCs between 0.92-0.97. However, these were based on single studies with limited sample sizes and should therefore be validated. Alterations in serum N-glycomics, characterised by increased levels of fucosylation and branching, have potential as diagnostic biomarkers for HCC. Optimisation of study design, patient selection and analysing techniques are needed before clinical implementation will be possible.

Integration of high-pH fractionation and cotton-HILIC enrichment approaches for analyzing N-glycopeptides

Glycosylation, a prevalent post-translational modification, plays a significant role in diverse biological processes and exhibits associations with various diseases, including cancer. Among the different types of glycosylation, N-glycosylation is particularly prominent. Glycosylation analysis also plays a crucial role in understanding the efficacy of protein biopharmaceuticals, as the majority of these drugs are glycoproteins. The precise detection and characterization of N-glycosylation require a series of enrichment steps. On the other hand, peptide fragmentation methods constitute a crucial step in reducing sample complexity. Moreover, enrichment strategies coupled with mass spectrometry are essential for the analysis and identification of N-glycopeptides. This research aims to enhance the detection methods for N-glycopeptides and N-glycoproteins by employing strategies that involve peptide fractionation and glycopeptide enrichment. This study targeted to assess and compare the efficacy of an integration method and a direct enrichment approach in terms of identifying glycopeptides from the glycoproteome of human plasma. The results demonstrate that the integration method detects 212 N-glycopeptides and 88 N-glycoproteins, while Cotton-HILIC direct enrichment detects 88 N-glycopeptides and 41 N-glycoproteins. In conclusion, the employment of Cotton-HILIC enrichment with high pH fractionation exhibits greater qualitative abilities in characterizing the structures and functions of glycopeptides.

Protein arginine methylation in transcription and epigenetic regulation

Arginine methylation is a prevalent post-translational modification found in all eukaryotic systems. It involves the addition of a methyl group to the guanidino nitrogen atoms of arginine residues within proteins, and this process is catalyzed by a family of enzymes called protein arginine methyltransferases (PRMTs). In mammals, there exist nine PRMTs (PRMT1–9) that catalyze three distinct types of arginine methylation: monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine. These modifications play critical roles in numerous fundamental cellular processes, including transcription, RNA metabolism, genome maintenance, and signaling transduction. Aberrations in protein arginine methylation have been implicated in various human diseases, such as neurodevelopmental disorders and cancer. This review offers a general overview of arginine methylation, covering its deposition, its impact on protein function, and the diverse regulatory mechanisms involved. We specifically focus on an in-depth view of the role of arginine methylation in transcription and the epigenetic regulation of gene expression. Readers are directed towards additional reviews that encompass other aspects of arginine methylation biology.

Proteome-wide Profiling of Asymmetric Dimethylated Arginine in Human Breast Tumors.

Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) is a prevalent post-translational modification (PTM) that regulates diverse cellular processes. Aberrant expression of type I PRMTs that catalyze asymmetric arginine dimethylation (ADMA) is often found in cancer, though little is known about the ADMA status of substrate proteins in tumors. Using LC-MS/MS along with pan-specific ADMA antibodies, we performed global mapping of ADMA in five patient-derived xenograft (PDX) tumors representing different subtypes of human breast cancer. In total, 403 methylated sites from 213 proteins were identified, including 322 novel sites when compared to the PhosphositesPlus database. Moreover, using peptide arrays in vitro, approximately 70% of the putative substrates were validated to be methylated by PRMT1, PRMT4, and PRMT6. Notably, when compared with our previously identified ADMA sites from breast cancer cell lines, only 75 ADMA sites overlapped between cell lines and PDX tumors. Collectively, this study provides a useful resource for both PRMT and breast cancer communities for further exploitation of the functions of PRMT dysregulation during breast cancer progression.

Ring Finger Protein 34 Facilitates Nervous Necrosis Virus Evasion of Antiviral Innate Immunity by Targeting TBK1 and IRF3 for Ubiquitination and Degradation in Teleost Fish.

Ubiquitination, as one of the most prevalent posttranslational modifications of proteins, enables a tight control of host immune responses. Many viruses hijack the host ubiquitin system to regulate host antiviral responses for their survival. Here, we found that the fish pathogen nervous necrosis virus (NNV) recruited Lateolabrax japonicus E3 ubiquitin ligase ring finger protein 34 (LjRNF34) to inhibit the RIG-I-like receptor (RLR)-mediated interferon (IFN) response via ubiquitinating Lateolabrax japonicus TANK-binding kinase 1 (LjTBK1) and interferon regulatory factor 3 (LjIRF3). Ectopic expression of LjRNF34 greatly enhanced NNV replication and prevented IFN production, while deficiency of LjRNF34 led to the opposite effect. Furthermore, LjRNF34 targeted LjTBK1 and LjIRF3 via its RING domain. Of note, the interactions between LjRNF34 and LjTBK1 or LjIRF3 were conserved in different cellular models derived from fish. Mechanically, LjRNF34 promoted K27- and K48-linked ubiquitination and degradation of LjTBK1 and LjIRF3, which in turn diminished LjTBK1-induced translocation of LjIRF3 from the cytoplasm to the nucleus. Ultimately, NNV capsid protein (CP) was found to bind with LjRNF34, CP induced LjTBK1 and LjIRF3 degradation, and IFN suppression depended on LjRNF34. Our finding demonstrates a novel mechanism by which NNV CP evaded host innate immunity via LjRNF34 and provides a potential drug target for the control of NNV infection. IMPORTANCE Ubiquitination plays an essential role in the regulation of innate immune responses to pathogens. NNV, a type of RNA virus, is the causal agent of a highly destructive disease in a variety of marine and freshwater fish. A previous study reported NNV could hijack the ubiquitin system to manipulate the host's immune responses; however, how NNV utilizes ubiquitination to facilitate its own replication is not well understood. Here, we identified a novel distinct role of E3 ubiquitin ligase LjRNF34 as an IFN antagonist to promote NNV infection. NNV capsid protein utilized LjRNF34 to target LjTBK1 and LjIRF3 for K27- and K48-linked ubiquitination and degradation. Importantly, the interactions between LjRNF34 and CP, LjTBK1, or LjIRF3 are conserved in different cellular models derived from fish, suggesting it is a general immune evasion strategy exploited by NNV to target the IFN response via RNF34.

Paradoxes of Cellular SUMOylation Regulation: A Role of Biomolecular Condensates?

Protein SUMOylation is a major post-translational modification essential for maintaining cellular homeostasis. SUMOylation has long been associated with stress responses as a diverse array of cellular stress signals are known to trigger rapid alternations in global protein SUMOylation. In addition, while there are large families of ubiquitination enzymes, all small ubiquitin-like modifiers (SUMOs) are conjugated by a set of enzymatic machinery comprising one heterodimeric SUMO-activating enzyme, a single SUMO-conjugating enzyme, and a small number of SUMO protein ligases and SUMO-specific proteases. How a few SUMOylation enzymes specifically modify thousands of functional targets in response to diverse cellular stresses remains an enigma. Here we review recent progress toward understanding the mechanisms of SUMO regulation, particularly the potential roles of liquid-liquid phase separation/biomolecular condensates in regulating cellular SUMOylation during cellular stresses. In addition, we discuss the role of protein SUMOylation in pathogenesis and the development of novel therapeutics targeting SUMOylation. SIGNIFICANCE STATEMENT: Protein SUMOylation is one of the most prevalent post-translational modifications and plays a vital role in maintaining cellular homeostasis in response to stresses. Protein SUMOylation has been implicated in human pathogenesis, such as cancer, cardiovascular diseases, neurodegeneration, and infection. After more than a quarter century of extensive research, intriguing enigmas remain regarding the mechanism of cellular SUMOylation regulation and the therapeutic potential of targeting SUMOylation.

Protein SUMOylation and phase separation: partners in stress?

Protein SUMOylation is one of the most prevalent post-translational modifications (PTMs) and important for maintaining cellular homeostasis in response to various cellular stresses. Emerging evidence reveals the role of liquid–liquid phase separation (LLPS)/biomolecular condensates in cellular SUMOylation, potentially solving a puzzle regarding the cellular mechanism of SUMOylation regulation.

Novel Online Three-Dimensional Separation Expands the Detectable Functional Landscape of Cellular Phosphoproteome.

Protein phosphorylation is a prevalent post-translational modification that regulates essentially every aspect of cellular processes. Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an extensive offline sample fractionation and a phosphopeptide enrichment method is a best practice for deep phosphoproteome profiling, but balancing throughput and profiling depth remains a practical challenge. We present an online three-dimensional separation method for ultradeep phosphoproteome profiling that combines an online two-dimensional liquid chromatography separation and an additional gas-phase separation. This method identified over 100,000 phosphopeptides (>60,000 phosphosites) in HeLa cells during 1.5 days of data acquisition, and the largest HeLa cell phosphoproteome significantly expanded the detectable functional landscape of cellular phosphoproteome.

Identification and Analysis of Six Phosphorylation Sites Within the Xenopus laevis Linker Histone H1.0 C-Terminal Domain Indicate Distinct Effects on Nucleosome Structure

As a key structural component of the chromatin of higher eukaryotes, linker histones (H1s) are involved in stabilizing the folding of extended nucleosome arrays into higher-order chromatin structures and function as a gene-specific regulator of transcription in vivo. The H1 C-terminal domain (CTD) is essential for high-affinity binding of linker histones to chromatin and stabilization of higher-order chromatin structure. Importantly, the H1 CTD is an intrinsically disordered domain that undergoes a drastic condensation upon binding to nucleosomes. Moreover, although phosphorylation is a prevalent post-translational modification within the H1 CTD, exactly where this modification is installed and how phosphorylation influences the structure of the H1 CTD remains unclear for many H1s. Using novel mass spectrometry techniques, we identified six phosphorylation sites within the CTD of the archetypal linker histone Xenopus H1.0. We then analyzed nucleosome-dependent CTD condensation and H1-dependent linker DNA organization for H1.0 in which the phosphorylated serine residues were replaced by glutamic acid residues (phosphomimics) in six independent mutants. We find that phosphomimetics at residues S117E, S155E, S181E, S188E, and S192E resulted in a significant reduction in nucleosome-bound H1.0 CTD condensation compared with unphosphorylated H1.0, whereas S130E did not alter CTD structure. Furthermore, we found distinct effects among the phosphomimetics on H1-dependent linker DNA trajectory, indicating unique mechanisms by which this modification can influence H1 CTD condensation. These results bring to light a novel role for linker histone phosphorylation in directly altering the structure of nucleosome-bound H1 and a potential novel mechanism for its effects on chromatin structure and function.

Ubiquitin and Ubiquitin-like Proteins in Cancer, Neurodegenerative Disorders, and Heart Diseases.

Post-translational modification (PTM) is an essential mechanism for enhancing the functional diversity of proteins and adjusting their signaling networks. The reversible conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubls) to cellular proteins is among the most prevalent PTM, which modulates various cellular and physiological processes by altering the activity, stability, localization, trafficking, or interaction networks of its target molecules. The Ub/Ubl modification is tightly regulated as a multi-step enzymatic process by enzymes specific to this family. There is growing evidence that the dysregulation of Ub/Ubl modifications is associated with various diseases, providing new targets for drug development. In this review, we summarize the recent progress in understanding the roles and therapeutic targets of the Ub and Ubl systems in the onset and progression of human diseases, including cancer, neurodegenerative disorders, and heart diseases.

Pan-methylarginine antibody generation using PEG linked GAR motifs as antigens.

Arginine methylation is a prevalent posttranslational modification which is deposited by a family of protein arginine methyltransferases (PRMTs), and is found in three different forms in mammalian cells: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Pan-methylarginine antibodies are critical for identifying proteins that are methylated on arginine residues, and are also used for evaluating signaling pathways that modulate this methyltransferase activity. Although good pan-MMA, -ADMA and -SDMA antibodies have been developed over the years, there is still room for improvement. Here we use a novel antigen approach, which involves the separation of short methylated motifs with inert polyethylene glycol (PEG) linkers, to generate a set of pan antibodies to the full range of methylarginine marks. Using these antibodies, we observed substrate scavenging by PRMT1, when PRMT5 activity is blocked. Specifically, we find that the splicing factor SmD1 displays increased ADMA levels upon PRMT5 inhibitor treatment. Furthermore, when the catalysis of both SDMA and ADMA is blocked with small molecule inhibitors, we demonstrate that SmD1 and SMN no longer interact. This could partially explain the synergistic effect of PRMT5 and type I PRMT inhibition on RNA splicing and cancer cell growth.