Protein Arginine Methylation Research Articles

The Influence of Arginine Methylation in Immunity and Inflammation.

Exploration in the field of epigenetics has revealed that protein arginine methyltransferases (PRMTs) contribute to disease, and this has given way to the development of specific small molecule compounds that inhibit arginine methylation. Protein arginine methylation is known to regulate fundamental cellular processes, such as transcription; pre-mRNA splicing and other RNA processing mechanisms; signal transduction, including the anti-viral response; and cellular metabolism. PRMTs are also implicated in the regulation of physiological processes, including embryonic development, myogenesis, and the immune system. Finally, the dysregulation of PRMTs is apparent in cancer, neurodegeneration, muscular disorders, and during inflammation. Herein, we review the functions of PRMTs in immunity and inflammation. We also discuss recent progress with PRMTs regarding the modulation of gene expression related to T and B lymphocyte differentiation, germinal center dynamics, and anti-viral signaling responses, as well as the clinical relevance of using PRMT inhibitors alone or in combination with other drugs to treat cancer, immune, and inflammatory-related diseases.

Protein Arginine Methylation: An Emerging Modification in Cancer Immunity and Immunotherapy.

In recent years, protein arginine methyltransferases (PRMTs) have emerged as new members of a gene expression regulator family in eukaryotes, and are associated with cancer pathogenesis and progression. Cancer immunotherapy has significantly improved cancer treatment in terms of overall survival and quality of life. Protein arginine methylation is an epigenetic modification function not only in transcription, RNA processing, and signal transduction cascades, but also in many cancer-immunity cycle processes. Arginine methylation is involved in the activation of anti-cancer immunity and the regulation of immunotherapy efficacy. In this review, we summarize the most up-to-date information on regulatory molecular mechanisms and different underlying arginine methylation signaling pathways in innate and adaptive immune responses during cancer. We also outline the potential of PRMT-inhibitors as effective combinatorial treatments with immunotherapy.

PRMT5 Interacting Partners and Substrates in Oligodendrocyte Lineage Cells.

The protein arginine methyl transferase PRMT5 is an enzyme expressed in oligodendrocyte lineage cells and responsible for the symmetric methylation of arginine residues on histone tails. Previous work from our laboratory identified PRMT5 as critical for myelination, due to its transcriptional regulation of genes involved in survival and early stages of differentiation. However, besides its nuclear localization, PRMT5 is found at high levels in the cytoplasm of several cell types, including oligodendrocyte progenitor cells (OPCs) and yet, its interacting partners in this lineage, remain elusive. By using mass spectrometry on protein eluates from extracts generated from primary oligodendrocyte lineage cells and immunoprecipitated with PRMT5 antibodies, we identified 1196 proteins as PRMT5 interacting partners. These proteins were related to molecular functions such as RNA binding, ribosomal structure, cadherin and actin binding, nucleotide and protein binding, and GTP and GTPase activity. We then investigated PRMT5 substrates using iTRAQ-based proteomics on cytosolic and nuclear protein extracts from CRISPR-PRMT5 knockdown immortalized oligodendrocyte progenitors compared to CRISPR-EGFP controls. This analysis identified a similar number of peptides in the two subcellular fractions and a total number of 57 proteins with statistically decreased symmetric methylation of arginine residues in the CRISPR-PRMT5 knockdown compared to control. Several PRMT5 substrates were in common with cancer cell lines and related to RNA processing, splicing and transcription. In addition, we detected ten oligodendrocyte lineage specific substrates, corresponding to proteins with high expression levels in neural tissue. They included: PRC2C, a proline-rich protein involved in methyl-RNA binding, HNRPD an RNA binding protein involved in regulation of RNA stability, nuclear proteins involved in transcription and other proteins related to migration and actin cytoskeleton. Together, these results highlight a cell-specific role of PRMT5 in OPC in regulating several other cellular processes, besides RNA splicing and metabolism.

Genome-wide identification, characterization and function analysis of PRMT family in relation to fruit ripening in banana

Protein arginine methylation is an important post-translational modification involved in different cellular processes, such as transcriptional regulation, RNA processing, DNA repair and signaling. Protein arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs). However, little information on the involvement of PRMT-mediated protein arginine methylation in fruit ripening is available. Here, we identified and cloned nine PRMTs from the banana genome and characterized their phylogenetic relationship, conserved domains, gene structure and promoter cis-element. In addition, subcellular localization of MaPRMT proteins, expression profiles of MaPRMTs and protein arginine methylation levels during banana fruit ripening also were investigated. All the nine MaPRMTs were localized to cytoplasm and nucleus, suggesting that they might be involved in the histone and non-histone arginine methylation modification. The transcriptional levels of MaPRMT4B, MaPRMT6A and MaPRMT6B were significantly upregulated as fruit ripening proceeded. The upregulation was promoted by ethylene treatment, but postponed by 1-MCP treatment. Furthermore, the levels of protein arginine methylation, including asymmetric (aDMA) and symmetric (sDMA) dimethylarginine, were upregulated in banana during fruit ripening. These results suggested that PRMTs-mediated protein arginine methylation were implicated in banana fruit ripening.

Coactivator-associated arginine methyltransferase 1 controls oligodendrocyte differentiation in the corpus callosum during early brain development.

Protein arginine methylation has been recognized as one of key posttranslational modifications for refined protein functions, mediated by protein arginine methyltransferases (Prmts). Coactivator-associated arginine methyltransferase (Carm1, also known as Prmt4) participates in various cellular events, such as cell survival, proliferation, and differentiation through its protein arginine methylation activities. Carm1 regulates cell proliferation of a neuronal cell line and is reportedly expressed in the mammalian brain. However, its detailed function in the central nervous system, particularly in glial cells, remains largely unexplored. In this study, Carm1 exhibited relatively high expression in oligodendrocyte (OL) lineage cells present in the corpus callosum of the developing brain, followed by a remarkable downregulation after active myelination. The suppression of Carm1 activity by inhibitors in isolated oligodendrocyte precursor cells (OPCs) reduced the number of Ki67-expressing and BrdU-incorporated proliferating cells. Furthermore, Carm1 inactivation attenuated OL differentiation, as determined by the expression of Plp, a reliable myelin-related marker. It also impaired the extension of OL processes, accompanied by a significant reduction in gene expression related to OL differentiation and myelination, such as Sox10, Cnp, Myrf, and Mbp. In addition, OLs co-cultured with embryonic dorsal root ganglia neurons demonstrated that Carm1 activity is required for the appropriate formation of myelin processes and myelin sheaths around neuronal axons, and the induction of the clustering of Caspr, a node of Ranvier structural molecule. Thus, we propose that Carm1 is an essential molecule for the development of OPCs and OLs during brain development.

Protein Arginine Methyltransferase 1 and its Dynamic Regulation Associated with Cellular Processes and Diseases

Post-translational modifications (PTMs) of proteins influence protein degradation, protein-protein interactions, expression of genes, and intracellular signal transduction, and thereby regulate major life processes. Among the PTMs occurring within the cytoplasm and nucleus, the most commonly studied one is the arginine methylation of proteins catalyzed by PRMTs. PRMT1 is the most excellent and extensively studied member of the PRMT family. PRMT1 occurs in various isoforms, and the unique sequence splicing of each of these isoforms encodes differential proteins that exhibit different cellular localization, substrate specificity, and enzyme activity. In addition to methylating histones, PRMT1 also methylates a large number of non-histone substrates that regulate a broad range of cellular processes. In recent years, research has been revealing an increasing number of pathological diseases caused by the misregulation and aberrant expression of PRMT1, demonstrating the potential of PRMT1 as an effective biomarker for drug targets. In this context, the present study discusses the structural characteristics and the biological functions of PRMT1.

Deactivation by S-Glutathionylation overrules activation by PRMT1-dependent asymmetrical di-methylation in PFKFB3

To understand PFKFB3 control by covalent modifications, the structure/function effect of protein arginine methyl transferase 1-dependent asymmetric di-methylations at Arg131 and Arg134 (N-CH3) and its relationship to S-glutathionylation at Cys206 (S-Gsh) was investigated. Distinctly from the report that N-CH3 is for protection of PFKFB3 from the APC/C-Cdh-mediated polyubiquitination and proteolysis, an increase in the activity for Fru-2,6-P2 production was shown from a molecular simulation and in-vitro tests. The simulation suggested that N-CH3 would uncouple the Fru-6-P entry turn (-130TRERRH-) from its coupling to the p-helix (-204DKCDRD-) by disabling the interaction between Arg131/134 and Asp207. The uncoupling consequently is likely to facilitate the Fru-6-P binding by enhancing the conformational flexibility. Confirming the simulation, N-CH3 was shown to cause a 5-fold increase in the specific activity (kcat/Km) mostly through a 4-fold decrease in Kms for Fru-6-P. A similar extent of activation was induced by Asp207àA mutagenesis, which disables the coupling, while the activation by N-CH3 was almost abolished by Arg131àA mutagenesis. More interestingly, PFKFB3 with N-CH3 could be additionally S-glutathionylated at Cys206, when oxidative stress is elevated. When modified by both N-CH3 and S-Gsh, the activity was decreased, as if there was no N-CH3 at all, suggesting that the deactivation completely overrules the activation. When HeLa cells were treated for the dual modifications of PFKFB3, the overruling deactivation effect of S-Gsh was prevalent, causing decreases in Fru-2,6-P2 levels and increases in glycolytic flux redirected to the pentose phosphate pathway. As a result, the levels of NADPH and reduced glutathione were markedly elevated, enhancing cell viability under the conditions of elevated oxidative stress. Altogether, it is suggested that the functional effect of S-Gsh, which represents a mechanism for survival under detrimental oxidative stress, dominates over the effect of N-CH3, which has been suggested as a mechanism for growth.

Brain Regions Show Different Metabolic and Protein Arginine Methylation Phenotypes in Frontotemporal Dementias and Alzheimer's Disease

Brain Regions Show Different Metabolic and Protein Arginine Methylation Phenotypes in Frontotemporal Dementias and Alzheimer's Disease

Structure, Activity and Function of the PRMT2 Protein Arginine Methyltransferase.

PRMT2 belongs to the protein arginine methyltransferase (PRMT) family, which catalyzes the arginine methylation of target proteins. As a type I enzyme, PRMT2 produces asymmetric dimethyl arginine and has been shown to have weak methyltransferase activity on histone substrates in vitro, suggesting that its authentic substrates have not yet been found. PRMT2 contains the canonical PRMT methylation core and a unique Src homology 3 domain. Studies have demonstrated its clear implication in many different cellular processes. PRMT2 acts as a coactivator of several nuclear hormone receptors and is known to interact with a multitude of splicing-related proteins. Furthermore, PRMT2 is aberrantly expressed in several cancer types, including breast cancer and glioblastoma. These reports highlight the crucial role played by PRMT2 and the need for a better characterization of its activity and cellular functions.

Cholecalciferol and metformin protect against lipopolysaccharide-induced endothelial dysfunction and senescence by modulating sirtuin-1 and protein arginine methyltransferase-1

Endothelial cell activation through nuclear factor-kappa-B (NFkB) and mitogen-activated protein kinases leads to increased biosynthesis of pro-inflammatory mediators, cellular injury and vascular inflammation under lipopolysaccharide (LPS) exposure. Recent studies report that LPS up-regulated global methyltransferase activity. In this study, we observed that a combination treatment with metformin (MET) and cholecalciferol (VD) blocked the LPS-induced S-adenosylmethionine (SAM)-dependent methyltransferase (SDM) activity in Eahy926 cells. We found that LPS challenge (i) increased arginine methylation through up-regulated protein arginine methyltransferase-1 (PRMT1) mRNA, intracellular concentrations of asymmetric dimethylarginine (ADMA) and homocysteine (HCY); (ii) up-regulated cell senescence through mitigated sirtuin-1 (SIRT1) mRNA, nicotinamide adenine dinucleotide (NAD+) concentration, telomerase activity and total antioxidant capacity; and (iii) lead to endothelial dysfunction through compromised nitric oxide (NOx) production. However, these LPS-mediated cellular events in Eahy926 cells were restored by the synergistic effect of MET and VD. Taken together, this study identified that the dual compound effect inhibits LPS-induced protein arginine methylation, endothelial senescence and dysfunction through the components of epigenetic machinery, SIRT1 and PRMT1, which is a previously unidentified function of the test compounds. In silico results identified the presence of vitamin D response element (VDRE) sequence on PRMT1 suggesting that VDR could regulate PRMT1 gene expression. Further characterization of the cellular events associated with the dual compound challenge, using gene silencing approach or adenoviral constructs for SIRT1 and/or PRMT1 under inflammatory stress, could identify therapeutic strategies to address the endothelial consequences in vascular inflammation-mediated atherosclerosis.

Naturally occurring cancer-associated mutations disrupt oligomerization and activity of protein arginine methyltransferase 1 (PRMT1)

Protein arginine methylation is a posttranslational modification catalyzed by the protein arginine methyltransferase (PRMT) enzyme family. Dysregulated protein arginine methylation is linked to cancer and a variety of other human diseases. PRMT1 is the predominant PRMT isoform in mammalian cells and acts in pathways regulating transcription, DNA repair, apoptosis, and cell proliferation. PRMT1 dimer formation, which is required for methyltransferase activity, is mediated by interactions between a structure called the dimerization arm on one monomer and a surface of the Rossman Fold of the other monomer. Given the link between PRMT1 dysregulation and disease and the link between PRMT1 dimerization and activity, we searched the Catalogue of Somatic Mutations in Cancer (COSMIC) database to identify potential inactivating mutations occurring in the PRMT1 dimerization arm. We identified three mutations that correspond to W215L, Y220N, and M224V substitutions in human PRMT1V2 (isoform 1) (W197L, Y202N, M206V in rat PRMT1V1). Using a combination of site-directed mutagenesis, analytical ultracentrifugation, native PAGE, and activity assays, we found that these conservative substitutions surprisingly disrupt oligomer formation and substantially impair both S-adenosyl-L-methionine (AdoMet) binding and methyltransferase activity. Molecular dynamics simulations suggest that these substitutions introduce novel interactions within the dimerization arm that lock it in a conformation not conducive to dimer formation. These findings provide a clear, if putative, rationale for the contribution of these mutations to impaired arginine methylation in cells and corresponding health consequences.

PRMT7: A Pivotal Arginine Methyltransferase in Stem Cells and Development.

Protein arginine methylation is a posttranslational modification catalyzed by protein arginine methyltransferases (PRMTs), which play critical roles in many biological processes. To date, nine PRMT family members, namely, PRMT1, 2, 3, 4, 5, 6, 7, 8, and 9, have been identified in mammals. Among them, PRMT7 is a type III PRMT that can only catalyze the formation of monomethylarginine and plays pivotal roles in several kinds of stem cells. It has been reported that PRMT7 is closely associated with embryonic stem cells, induced pluripotent stem cells, muscle stem cells, and human cancer stem cells. PRMT7 deficiency or mutation led to severe developmental delay in mice and humans, which is possibly due to its crucial functions in stem cells. Here, we surveyed and summarized the studies on PRMT7 in stem cells and development in mice and humans and herein provide a discussion of the underlying molecular mechanisms. Furthermore, we also discuss the roles of PRMT7 in cancer, adipogenesis, male reproduction, cellular stress, and cellular senescence, as well as the future perspectives of PRMT7-related studies. Overall, PRMT7 mediates the proliferation and differentiation of stem cells. Deficiency or mutation of PRMT7 causes developmental delay, including defects in skeletal muscle, bone, adipose tissues, neuron, and male reproduction. A better understanding of the roles of PRMT7 in stem cells and development as well as the underlying mechanisms will provide information for the development of strategies for in-depth research of PRMT7 and stem cells as well as their applications in life sciences and medicine.

PRMT1 Confers Resistance to Olaparib via Modulating MYC Signaling in Triple-Negative Breast Cancer.

Treatment of triple-negative breast cancer (TNBC) remains an unmet clinical need owing to its lack of an efficient therapeutic target. The targeting of DNA repair by poly(ADP-ribose) polymerase (PARP) inhibitors has shown benefit for patients with the BRCA variation. However, sensitivities to the PARP inhibitors were reported regardless of BRCA status. Thus, exploring the underlying mechanisms is imperative. Herein, we identified that breast cancer cells with an elevated expression of protein arginine methyl transferase 1 (PRMT1) was associated with therapeutic sensitivity to the PARP inhibitor olaparib. The results of cell viability and colony formation assays indicated that the suppression of PRMT1 by small hairpin RNA or by the chemical inhibitor increased sensitivity to olaparib in human TNBC MDA-MB-231 and BT549 cells. Bioinformatic analysis revealed that PRMT1 expression was significantly associated with the MYC signature, and TNBC cells with higher PRMT1 and the MYC signature were associated with therapeutic sensitivity to olaparib. Mechanistic studies further demonstrated that knockdown of PRMT1 reduced the c-Myc protein level and downregulated the expression of MYC downstream targets, whereas overexpression of PRMT1 enhanced c-Myc protein expression. Moreover, the overexpression of PRMT1 promoted c-Myc protein stability, and the inhibition of PRMT1 downregulated c-Myc protein stability. Accordingly, the knockdown of PRMT1 inhibited homologous recombination gene expression. These data indicate that PRMT1 is instrumental in regulating DNA repair, at least in part, by modulating c-Myc signaling. Our data highlighted the PRMT1/c-Myc network as a potential therapeutic target in patients with TNBC.

Arginine methylation by PRMT2 promotes IFN-β production through TLR4/IRF3 signaling pathway

A balance between the positive and negative regulation of toll-like receptor (TLR) signaling pathways is required to avoid detrimental and inappropriate inflammatory responses. Although some protein post-translational modifications (PTMs) such as phosphorylation and ubiquitination have been demonstrated to potently modulate innate immune responses, the role of methylation, an important PTM, control of TLR4 signaling pathway remains unclear. In this study, we found that protein arginine methyltransferase 1, 2 and 3 (PRMT1, 2 and 3) were recruited to methylate TLR4-CD (cytoplasmic domain) after lipopolysaccharide (LPS) stimulation respectively, but the effect of PRMT2 on arginine methylation of TLR4-CD is the most significant among above three PRMTs, which prompted us to focus on PRMT2. Reduction of PRMT2 expression down-regulated arginine (R) methylation level of TLR4 with or without LPS treatment. Methionine 115 (M115) mediated PRMT2 catalyzed-arginine methylation of TLR4 on R731 and R812. Furthermore, PRMT1, 2 and 3 was recruited to methylate interferon regulatory factor 3 (IRF3) after LPS stimulation respectively, but the effect of PRMT2 on arginine methylation of IRF3 is the most significant among the above three PRMTs. Arginine methylation of TLR4 on R812 or arginine methylation of IRF3 on R285 mediated the interaction between TLR4 and IRF3 respectively. Arginine methylation of IRF3 on R285 induced by LPS led to its dimerization and promoted its translocation from the cytoplasm to the nucleus. In addition, the enhancement of arginine methylation of TLR4 induced by PRMT1 or 2 increased IRF3 transcription activity with or without LPS treatment, while PRMT2 with histidine 112 glutamine (H112Q) or methionine 115 isoleucine (M115I) mutation and TLR4 with arginine 812 lysine (R812K) mutation decreased it. Arginine methylation of TLR4 on R812 or PRMT2 enhanced interferon-β (IFN-β) production. Our study reveals a critical role for PRMT2 and protein arginine methylation in the enhancement of IFN-β production via TLR4/IRF3 signaling pathway and may provide a therapeutic strategy to control endotoxemia.

The protein arginine methyltransferase PRMT1 promotes TBK1 activation through asymmetric arginine methylation.

TBK1 is an essential kinase for the innate immune response against viral infection. However, the key molecular mechanisms regulating the TBK1 activation remain elusive. Here, we identify PRMT1, a type I protein arginine methyltransferase, as an essential regulator of TBK1 activation. PRMT1 directly interacts with TBK1 and catalyzes asymmetric methylation of R54, R134, and R228 on TBK1. This modification enhances TBK1 oligomerization after viral infection, which subsequently promotes TBK1 phosphorylation and downstream type I interferon production. More important, myeloid-specific Prmt1 knockout mice are more susceptible to infection with DNA and RNA viruses than Prmt1fl/fl mice. Our findings reveal insights into the molecular regulation of TBK1 activation and demonstrate the essential function of protein arginine methylation in innate antiviral immunity.

Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells.

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine members that can monomethylate or symmetrically/asymmetrically dimethylate arginine residues. Several antibodies recognizing different types of arginine methylation of various proteins are available; thus, providing tools for the development of PRMT activity biomarker assays. PRMT antibody-based assays are challenging due to overlapping substrates and motif-based antibody specificities. These issues and the experimental setup to investigate the arginine methylation contributed by individual PRMTs are discussed. Through the careful selection of the representative substrates that are biomarkers for eight out of nine PRMTs, a panel of PRMT activity assays were designed. Here, the protocols for cellular assays quantitatively measuring the enzymatic activity of individual members of the PRMT family in cells are reported. The advantage of the described methods is their straightforward performance in any lab with cell culture and fluorescent western blot capabilities. The substrate specificity and chosen antibody reliability were fully validated with knockdown and overexpression approaches. In addition to detailed guidelines of the assay biomarkers and antibodies, information on the use of an inhibitor tool compound collection for PRMTs is also provided.

Profiling and Validation of Live‐Cell Protein Methylation with Engineered Enzymes and Methionine Analogues

Protein methyltransferases (PMTs) regulate many aspects of normal and disease processes through substrate methylation, with S-adenosyl-L-methionine (SAM) as a cofactor. It has been challenging to elucidate cellular protein lysine and arginine methylation because these modifications barely alter physical properties of target proteins and often are context dependent, transient, and substoichiometric. To reveal bona fide methylation events associated with specific PMT activities in native contexts, we developed the live-cell Bioorthogonal Profiling of Protein Methylation (lcBPPM) technology, in which the substrates of specific PMTs are labeled by engineered PMTs inside living cells, with in situ-synthesized SAM analogues as cofactors. The biorthogonality of this technology is achieved because these SAM analogue cofactors can only be processed by the engineered PMTs-and not native PMTs-to modify the substrates with distinct chemical groups. Here, we describe the latest lcBPPM protocol and its application to reveal proteome-wide methylation and validate specific methylation events. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Live-cell labeling of substrates of protein methyltransferases GLP1 and PRMT1 with lcBPPM-feasible enzymes and SAM analogue precursors Support Protocol: Gram-scale synthesis of Hey-Met Basic Protocol 2: Click labeling of lcBPPM cell lysates with a biotin-azide probe Alternate Protocol: Click labeling of small-scale lcBPPM cell lysates with a TAMRA-azide dye for in-gel fluorescence visualization Basic Protocol 3: Enrichment of biotinylated lcBPPM proteome with streptavidin beads Basic Protocol 4: Proteome-wide identification of lcBPPM targets with mass spectrometry Basic Protocol 5: Validation of individual lcBPPM targets by western blot.

HDAC6 Substrate Discovery Using Proteomics-Based Substrate Trapping: HDAC6 Deacetylates PRMT5 to Influence Methyltransferase Activity

Histone deacetylase 6 (HDAC6) is upregulated in a variety of tumor cell lines and has been linked to many cellular processes, such as cell signaling, protein degradation, cell survival, and cell motility. HDAC6 is an enzyme that deacetylates the acetyllysine residues of protein substrates, and the discovery of HDAC6 substrates, including tubulin, has revealed many roles of HDAC6 in cell biology. Unfortunately, among the wide variety of acetylated proteins in the cell, only a few are verified as HDAC6 substrates, which limits the full characterization of HDAC6 cellular functions. Substrate trapping mutants were recently established as a tool to discover unanticipated substrates of histone deacetylase 1 (HDAC1). In this study, we applied the trapping approach to identify potential HDAC6 substrates. Among the high confidence protein hits after trapping, protein arginine methyl transferase 5 (PRMT5) was successfully validated as a novel HDAC6 substrate. PRMT5 acetylation enhanced its methyltransferase activity and symmetrical dimethylation of downstream substrates, revealing possible crosstalk between acetylation and methylation. Substrate trapping represents a powerful, systematic, and unbiased approach to discover substrates of HDAC6.

Abstract 1431: Role of emt biomarkers in mediating osimertinib resistance in non-small cell lung cancer

Abstract Osimeritinib (OR), a Tyrosine Kinase Inhibitor is a first-line therapy in EGFR-mutant NSCLC patients. Resistance to OR treatment may occur due to acquired C797S mutations, MET amplifications and ALK rearrangements and other mechanisms not clearly defined. EMT may be one of the reasons for TKI resistance. EMT is a process by which epithelial cells acquire mesenchymal characteristics that increase invasiveness. We hypothesize that loss of E-cadherin during EMT, results in abnormal localization of p120 catenin, an E-cadherin regulating protein, and Kaiso factor, a repressor of Wnt signaling, promoting oncogenesis. Protein arginine methyl transferase 1 (PRMT1) an EMT regulator catalyzes methylation of Twist1, which represses E-cadherin. However, their role in OR resistance is still unknown. We have made parental (P) NSCLC cells, H2170, H358, H1975 and H3255 resistant to OR. MTT assays were performed on the parental cells and OR resistant cells which showed that the H2170OR, H3255OR, H358OR, H1975OR cells had IC50 for OR, 2.8-14 fold higher in comparison to parental cells, respectively. We further performed qPCR to study the modulation of EMT biomarkers in OR cells as compared to parental cells and found upregulation of PRMT1, p120 catenin and Kaiso factor by 1.5-3.67 fold respectively in H1975 cells and 1.5-5.73 fold in H3255 cells. Immunofluorescence studies in H1975OR cells for expression of PRMT1 showed that there was 3.18 fold increase as compared to 2.21 fold increase in Erlotinib resistant H1975 (H1975ER) and 4.77 fold increase in H3255OR as compared to 2.27 fold increase in H3255ER. After transfection with p120 siRNA we found that OR efficacy was increased by 33% in comparison to mock siRNA and OR treated cells. The percentage wound closure after 24 hours was found to be 26.2% in the p120-catenin siRNA transfected cells as compared to 7.4%.in mock siRNA transfected cells (p<0.05) indicating its role in EMT and cell migration. Furthermore, our results showed increased expression of p120 catenin in smokers with 50% of smokers showing high expression compared to 0% and 5.8% of quit smokers and nonsmokers indicating that high p-120 expression was associated with smoking (P=0.002). In conclusion, biomarkers like PRMT1 may mediate the process of EMT by methylation of Twist1 and upregulation of p120 that prevents transcriptional repression of Kaiso factor target genes, promoting EMT in OR and ER resistant cells. p-120 expression was found to be higher in smokers compared to quit/nonsmokers indicating that it may have a potential role in lung cancer. Citation Format: Kavya Sri Racherla, Katrina Dovalovsky, Neelu Puri. Role of emt biomarkers in mediating osimertinib resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1431.

Love is in the hair: arginine methylation of human hair proteins as novel cardiovascular biomarkers

Cardiovascular disease is the major cause of death worldwide. Extensive cardiovascular biomarkers are available using blood tests but very few, if any, investigations have described non-invasive tests for cardiovascular biomarkers based on readily available hair samples. Here we show, first, that human hair proteins are post-translationally modified by arginine methylation (ArgMe). Using western blot, proteomic data mining and mass spectrometry, we identify several ArgMe events in hair proteins and we show that keratin-83 is extensively modified by ArgMe in the human hair. Second, using a preliminary cohort (n = 18) of heterogenous healthy donors, we show that the levels of protein ArgMe in hair correlate with serum concentrations of a well-established cardiovascular biomarker, asymmetric dimethylarginine (ADMA). Compared to blood collection, hair sampling is cheaper, simpler, requires minimal training and carries less health and safety and ethical risks. For these reasons, developing the potential of hair protein ArgMe as clinically useful cardiovascular biomarkers through further research could be useful in future prevention and diagnosis of cardiovascular disease.