RESUMO
Glycine- and arginine-rich (GAR) motifs, commonly found in RNA-binding and -processing proteins, can be symmetrically (SDMA) or asymmetrically (ADMA) dimethylated at the arginine residue by protein arginine methyltransferases. Arginine-methylated protein motifs are usually read by Tudor domain-containing proteins. Here, using a GFP-Trap, we identify a non-Tudor domain protein, squamous cell carcinoma antigen recognized by T cells 3 (SART3), as a reader for SDMA-marked GAR motifs. Structural analysis and mutagenesis of SART3 show that aromatic residues lining a groove between two adjacent aromatic-rich half-a-tetratricopeptide (HAT) repeat domains are essential for SART3 to recognize and bind to SDMA-marked GAR motif peptides, as well as for the interaction between SART3 and the GAR-motif-containing proteins fibrillarin and coilin. Further, we show that the loss of this reader ability affects RNA splicing. Overall, our findings broaden the range of potential SDMA readers to include HAT domains.
Assuntos
Motivos de Aminoácidos , Arginina , Glicina , Arginina/metabolismo , Arginina/química , Humanos , Glicina/metabolismo , Glicina/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Ligação Proteica , Splicing de RNA , Células HEK293 , Metilação , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/química , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/químicaRESUMO
Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.
Assuntos
Fosfotirosina , Proteínas Tirosina Quinases , Especificidade por Substrato , Tirosina , Animais , Humanos , Motivos de Aminoácidos , Evolução Molecular , Espectrometria de Massas , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteoma/química , Proteoma/metabolismo , Proteômica , Transdução de Sinais , Domínios de Homologia de src , Tirosina/metabolismo , Tirosina/químicaRESUMO
Multiple cancers exhibit aberrant protein arginine methylation by both type I arginine methyltransferases, predominately protein arginine methyltransferase 1 (PRMT1) and to a lesser extent PRMT4, and by type II PRMTs, predominately PRMT5. Here, we perform targeted proteomics following inhibition of PRMT1, PRMT4, and PRMT5 across 12 cancer cell lines. We find that inhibition of type I and II PRMTs suppresses phosphorylated and total ATR in cancer cells. Loss of ATR from PRMT inhibition results in defective DNA replication stress response activation, including from PARP inhibitors. Inhibition of type I and II PRMTs is synergistic with PARP inhibition regardless of homologous recombination function, but type I PRMT inhibition is more toxic to non-malignant cells. Finally, we demonstrate that the combination of PARP and PRMT5 inhibition improves survival in both BRCA-mutant and wild-type patient-derived xenografts without toxicity. Taken together, these results demonstrate that PRMT5 inhibition may be a well-tolerated approach to sensitize tumors to PARP inhibition.
Assuntos
Neoplasias , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Neoplasias/tratamento farmacológico , Linhagem Celular , Replicação do DNA , Arginina/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/uso terapêutico , Proteínas Repressoras/metabolismoRESUMO
Staphylococcal nuclease Tudor domain containing 1 (SND1) protein is an oncogene that 'reads' methylarginine marks through its Tudor domain. Specifically, it recognizes methylation marks deposited by protein arginine methyltransferase 5 (PRMT5), which is also known to promote tumorigenesis. Although SND1 can drive hepatocellular carcinoma (HCC), it is unclear whether the SND1 Tudor domain is needed to promote HCC. We sought to identify the biological role of the SND1 Tudor domain in normal and tumorigenic settings by developing two genetically engineered SND1 mouse models, an Snd1 knockout (Snd1 KO) and an Snd1 Tudor domain-mutated (Snd1 KI) mouse, whose mutant SND1 can no longer recognize PRMT5-catalyzed methylarginine marks. Quantitative PCR analysis of normal, KO, and KI liver samples revealed a role for the SND1 Tudor domain in regulating the expression of genes encoding major acute phase proteins, which could provide mechanistic insight into SND1 function in a tumor setting. Prior studies indicated that ectopic overexpression of SND1 in the mouse liver dramatically accelerates the development of diethylnitrosamine (DEN)-induced HCC. Thus, we tested the combined effects of DEN and SND1 loss or mutation on the development of HCC. We found that both Snd1 KO and Snd1 KI mice were partially protected against malignant tumor development following exposure to DEN. These results support the development of small molecule inhibitors that target the SND1 Tudor domain or the use of upstream PRMT5 inhibitors, as novel treatments for HCC.
Assuntos
Carcinoma Hepatocelular , Endonucleases , Neoplasias Hepáticas , Animais , Camundongos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Endonucleases/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição , Predisposição Genética para DoençaRESUMO
Coactivator-associated arginine methyltransferase 1 (CARM1) is an arginine methyltransferase that posttranslationally modifies proteins that regulate multiple levels of RNA production and processing. Its substrates include histones, transcription factors, coregulators of transcription, and splicing factors. CARM1 is overexpressed in many different cancer types, and often promotes transcription factor programs that are co-opted as drivers of the transformed cell state, a process known as transcription factor addiction. Targeting these oncogenic transcription factor pathways is difficult but could be addressed by removing the activity of the key coactivators on which they rely. CARM1 is ubiquitously expressed, and its KO is less detrimental in embryonic development than deletion of the arginine methyltransferases protein arginine methyltransferase 1 and protein arginine methyltransferase 5, suggesting that therapeutic targeting of CARM1 may be well tolerated. Here, we will summarize the normal in vivo functions of CARM1 that have been gleaned from mouse studies, expand on the transcriptional pathways that are regulated by CARM1, and finally highlight recent studies that have identified oncogenic properties of CARM1 in different biological settings. This review is meant to kindle an interest in the development of human drug therapies targeting CARM1, as there are currently no CARM1 inhibitors available for use in clinical trials.
Assuntos
Neoplasias , Proteína-Arginina N-Metiltransferases , Animais , Humanos , Camundongos , Sistemas de Liberação de Medicamentos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fatores de Transcrição/metabolismo , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
The mammalian target of rapamycin complex1 (mTORC1) is a central regulator of metabolism and cell growth by sensing diverse environmental signals, including amino acids. The GATOR2 complex is a key component linking amino acid signals to mTORC1. Here, we identify protein arginine methyltransferase 1 (PRMT1) as a critical regulator of GATOR2. In response to amino acids, cyclin-dependent kinase 5 (CDK5) phosphorylates PRMT1 at S307 to promote PRMT1 translocation from nucleus to cytoplasm and lysosome, which in turn methylates WDR24, an essential component of GATOR2, to activate the mTORC1 pathway. Disruption of the CDK5-PRMT1-WDR24 axis suppresses hepatocellular carcinoma (HCC) cell proliferation and xenograft tumor growth. High PRMT1 protein expression is associated with elevated mTORC1 signaling in patients with HCC. Thus, our study dissects a phosphorylation- and arginine methylation-dependent regulatory mechanism of mTORC1 activation and tumor growth and provides a molecular basis to target this pathway for cancer therapy.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Aminoácidos/metabolismo , Quinase 5 Dependente de Ciclina , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
The N-terminal half of PHF2 harbors both a plant homeodomain (PHD) and a Jumonji domain. The PHD recognizes both histone H3 trimethylated at lysine 4 and methylated nonhistone proteins including vaccinia-related kinase 1 (VRK1). The Jumonji domain erases the repressive dimethylation mark from histone H3 lysine 9 (H3K9me2) at select promoters. The N-terminal amino acid sequences of H3 (AR2TK4) and VRK1 (PR2VK4) bear an arginine at position 2 and lysine at position 4. Here, we show that the PHF2 N-terminal half binds to H3 and VRK1 peptides containing K4me3, with dissociation constants (KD values) of 160 nM and 42 nM, respectively, which are 4 × and 21 × lower (and higher affinities) than for the isolated PHD domain of PHF2. X-ray crystallography revealed that the K4me3-containing peptide is positioned within the PHD and Jumonji interface, with the positively charged R2 residue engaging acidic residues of the PHD and Jumonji domains and with the K4me3 moiety encircled by aromatic residues from both domains. We suggest that the micromolar binding affinities commonly observed for isolated methyl-lysine reader domains could be improved via additional functional interactions within the same polypeptide or its binding partners.
Assuntos
Histonas , Proteínas de Homeodomínio , Lisina , Histonas/química , Lisina/química , Metilação , Peptídeos/química , Ligação Proteica , Domínios Proteicos , Proteínas de Homeodomínio/químicaRESUMO
The coactivator associated arginine methyltransferase (CARM1) promotes transcription, as its name implies. It does so by modifying histones and chromatin bound proteins. We identified nuclear factor I B (NFIB) as a CARM1 substrate and show that this transcription factor utilizes CARM1 as a coactivator. Biochemical studies reveal that tripartite motif 29 (TRIM29) is an effector molecule for methylated NFIB. Importantly, NFIB harbors both oncogenic and metastatic activities, and is often overexpressed in small cell lung cancer (SCLC). Here, we explore the possibility that CARM1 methylation of NFIB is important for its transforming activity. Using a SCLC mouse model, we show that both CARM1 and the CARM1 methylation site on NFIB are critical for the rapid onset of SCLC. Furthermore, CARM1 and methylated NFIB are responsible for maintaining similar open chromatin states in tumors. Together, these findings suggest that CARM1 might be a therapeutic target for SCLC.
Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Animais , Camundongos , Fatores de Transcrição NFI , Proteína-Arginina N-Metiltransferases/metabolismo , CromatinaRESUMO
Gliomas are one of the most common and lethal brain tumors among adults. One process that contributes to glioma progression and recurrence is the epithelial to mesenchymal transition (EMT). EMT is regulated by a set of defined transcription factors which tightly regulate this process, among them is the basic helix-loop-helix family member, TWIST1. Here we show that TWIST1 is methylated on lysine-33 at chromatin by SETD6, a methyltransferase with expression levels correlating with poor survival in glioma patients. RNA-seq analysis in U251 glioma cells suggested that both SETD6 and TWIST1 regulate cell adhesion and migration processes. We further show that TWIST1 methylation attenuates the expression of the long-non-coding RNA, LINC-PINT, thereby promoting EMT in glioma. Mechanistically, TWIST1 methylation represses the transcription of LINC-PINT by increasing the occupancy of EZH2 and the catalysis of the repressive H3K27me3 mark at the LINC-PINT locus. Under un-methylated conditions, TWIST1 dissociates from the LINC-PINT locus, allowing the expression of LINC-PINT which leads to increased cell adhesion and decreased cell migration. Together, our findings unravel a new mechanistic dimension for selective expression of LINC-PINT mediated by TWIST1 methylation.
Assuntos
Glioma , Proteínas Metiltransferases , RNA Longo não Codificante , Proteína 1 Relacionada a Twist , Humanos , Transição Epitelial-Mesenquimal , Proteínas Nucleares/genética , Proteínas Metiltransferases/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Glioma/metabolismo , Glioma/patologia , RNA Longo não Codificante/metabolismo , Linhagem Celular TumoralRESUMO
Protein arginine methyltransferases (PRMT) are a widely expressed class of enzymes responsible for catalyzing arginine methylation on numerous protein substrates. Among them, type I PRMTs are responsible for generating asymmetric dimethylarginine. By controlling multiple basic cellular processes, such as DNA damage responses, transcriptional regulation, and mRNA splicing, type I PRMTs contribute to cancer initiation and progression. A type I PRMT inhibitor, GSK3368715, has been developed and has entered clinical trials for solid and hematologic malignancies. Although type I PRMTs have been reported to play roles in modulating immune cell function, the immunologic role of tumor-intrinsic pathways controlled by type I PRMTs remains uncharacterized. Here, our The Cancer Genome Atlas dataset analysis revealed that expression of type I PRMTs associated with poor clinical response and decreased immune infiltration in patients with melanoma. In cancer cell lines, inhibition of type I PRMTs induced an IFN gene signature, amplified responses to IFN and innate immune signaling, and decreased expression of the immunosuppressive cytokine VEGF. In immunocompetent mouse tumor models, including a model of T-cell exclusion that represents a common mechanism of anti-programmed cell death protein 1 (PD-1) resistance in humans, type I PRMT inhibition increased T-cell infiltration, produced durable responses dependent on CD8+ T cells, and enhanced efficacy of anti-PD-1 therapy. These data indicate that type I PRMT inhibition exhibits immunomodulatory properties and synergizes with immune checkpoint blockade (ICB) to induce durable antitumor responses in a T cell-dependent manner, suggesting that type I PRMT inhibition can potentiate an antitumor immunity in refractory settings.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Proteína-Arginina N-Metiltransferases , Animais , Arginina , Humanos , Imunidade , Camundongos , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
Canonical targeting of Polycomb repressive complex 1 (PRC1) to repress developmental genes is mediated by cell-type-specific, paralogous chromobox (CBX) proteins (CBX2, 4, 6, 7, and 8). Based on their central role in silencing and their dysregulation associated with human disease including cancer, CBX proteins are attractive targets for small-molecule chemical probe development. Here, we have used a quantitative and target-specific cellular assay to discover a potent positive allosteric modulator (PAM) of CBX8. The PAM activity of UNC7040 antagonizes H3K27me3 binding by CBX8 while increasing interactions with nucleic acids. We show that treatment with UNC7040 leads to efficient and selective eviction of CBX8-containing PRC1 from chromatin, loss of silencing, and reduced proliferation across different cancer cell lines. Our discovery and characterization of UNC7040 not only reveals the most cellularly potent CBX8-specific chemical probe to date, but also corroborates a mechanism of Polycomb regulation by non-specific CBX nucleotide binding activity.
Assuntos
Neoplasias , Complexo Repressor Polycomb 1 , Proteínas de Ciclo Celular/metabolismo , Cromatina , Histonas/metabolismo , Humanos , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Ligação ProteicaRESUMO
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.
Assuntos
Polietilenoglicóis , Proteína-Arginina N-Metiltransferases , Animais , Anticorpos/genética , Arginina/metabolismo , Mamíferos/metabolismo , Metilação , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
Repair of genetic damage is coordinated in the context of chromatin, so cells dynamically modulate accessibility at DNA breaks for the recruitment of DNA damage response (DDR) factors. The identification of chromatin factors with roles in DDR has mostly relied on loss-of-function screens while lacking robust high-throughput systems to study DNA repair. In this study, we have developed two high-throughput systems that allow the study of DNA repair kinetics and the recruitment of factors to double-strand breaks in a 384-well plate format. Using a customized gain-of-function open-reading frame library ("ChromORFeome" library), we identify chromatin factors with putative roles in the DDR. Among these, we find the PHF20 factor is excluded from DNA breaks, affecting DNA repair by competing with 53BP1 recruitment. Adaptable for genetic perturbations, small-molecule screens, and large-scale analysis of DNA repair, these resources can aid our understanding and manipulation of DNA repair.
Assuntos
Cromatina/genética , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Histonas/metabolismo , Fases de Leitura Aberta , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Cromatina/metabolismo , Enzimas Reparadoras do DNA/genética , Ensaios de Triagem em Larga Escala , Histonas/genética , Humanos , Cinética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genéticaRESUMO
SPINDOC is tightly associated with the histone H3K4me3 effector protein SPIN1. To gain a better understanding of the biological roles of SPINDOC, we identified its interacting proteins. Unexpectedly, SPINDOC forms two mutually exclusive protein complexes, one with SPIN1 and the other with PARP1. Consistent with its ability to directly interact with PARP1, SPINDOC expression is induced by DNA damage, likely by KLF4, and recruited to DNA lesions with dynamics that follows PARP1. In SPINDOC knockout cells, the levels of PARylation are reduced, in both the absence and presence of DNA damage. The SPINDOC/PARP1 interaction promotes the clearance of PARP1 from damaged DNA, and also impacts the expression of known transcriptional targets of PARP1. To address the in vivo roles of SPINDOC in PARP1 regulation, we generate SPINDOC knockout mice, which are viable, but slightly smaller than their wildtype counterparts. The KO mice display reduced levels of PARylation and, like PARP1 KO mice, are hypersensitive to IR-induced DNA damage. The findings identify a SPIN1-independent role for SPINDOC in the regulation of PARP1-mediated PARylation and the DNA damage response.
Assuntos
Proteínas Correpressoras/metabolismo , Neoplasias/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Animais , Linhagem Celular , Dano ao DNA , Reparo do DNA , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Neoplasias/genética , Neoplasias/patologia , Domínios e Motivos de Interação entre ProteínasRESUMO
Arginine methylation is an essential post-translational modification (PTM) deposited by protein arginine methyltransferases (PRMTs) and recognized by Tudor domain-containing proteins. Of the nine mammalian PRMTs, PRMT5 is the primary enzyme responsible for the deposition of symmetric arginine methylation marks in cells. The staphylococcal nuclease and Tudor domain-containing 1 (SND1) effector protein is a key reader of the marks deposited by PRMT5. Both PRMT5 and SND1 are broadly expressed and their deregulation is reported to be associated with a range of disease phenotypes, including cancer. Hepatocellular carcinoma (HCC) is an example of a cancer type that often displays elevated PRMT5 and SND1 levels, and there is evidence that hyperactivation of this axis is oncogenic. Importantly, this pathway can be tempered with small-molecule inhibitors that target PRMT5, offering a therapeutic node for cancer, such as HCC, that display high PRMT5-SND1 axis activity. Here we summarize the known activities of this writer-reader pair, with a focus on their biological roles in HCC. This will help establish a foundation for treating HCC with PRMT5 inhibitors and also identify potential biomarkers that could predict sensitivity to this type of therapy.
RESUMO
DNA replication forks use multiple mechanisms to deal with replication stress, but how the choice of mechanisms is made is still poorly understood. Here, we show that CARM1 associates with replication forks and reduces fork speed independently of its methyltransferase activity. The speeding of replication forks in CARM1-deficient cells requires RECQ1, which resolves reversed forks, and RAD18, which promotes translesion synthesis. Loss of CARM1 reduces fork reversal and increases single-stranded DNA (ssDNA) gaps but allows cells to tolerate higher replication stress. Mechanistically, CARM1 interacts with PARP1 and promotes PARylation at replication forks. In vitro, CARM1 stimulates PARP1 activity by enhancing its DNA binding and acts jointly with HPF1 to activate PARP1. Thus, by stimulating PARP1, CARM1 slows replication forks and promotes the use of fork reversal in the stress response, revealing that CARM1 and PARP1 function as a regulatory module at forks to control fork speed and the choice of stress response mechanisms.
Assuntos
Quebras de DNA de Cadeia Simples , Replicação do DNA , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Proteína-Arginina N-Metiltransferases/genética , RecQ Helicases/genética , RecQ Helicases/metabolismoRESUMO
The chromatin-binding E3 ubiquitin ligase ubiquitin-like with PHD and RING finger domains 1 (UHRF1) contributes to the maintenance of aberrant DNA methylation patterning in cancer cells through multivalent histone and DNA recognition. The tandem Tudor domain (TTD) of UHRF1 is well-characterized as a reader of lysine 9 di- and tri-methylation on histone H3 (H3K9me2/me3) and, more recently, lysine 126 di- and tri-methylation on DNA ligase 1 (LIG1K126me2/me3). However, the functional significance and selectivity of these interactions remain unclear. In this study, we used protein domain microarrays to search for additional readers of LIG1K126me2, the preferred methyl state bound by the UHRF1 TTD. We show that the UHRF1 TTD binds LIG1K126me2 with high affinity and selectivity compared to other known methyllysine readers. Notably, and unlike H3K9me2/me3, the UHRF1 plant homeodomain (PHD) and its N-terminal linker (L2) do not contribute to multivalent LIG1K126me2 recognition along with the TTD. To test the functional significance of this interaction, we designed a LIG1K126me2 cell-penetrating peptide (CPP). Consistent with LIG1 knockdown, uptake of the CPP had no significant effect on the propagation of DNA methylation patterning across the genomes of bulk populations from high-resolution analysis of several cancer cell lines. Further, we did not detect significant changes in DNA methylation patterning from bulk cell populations after chemical or genetic disruption of lysine methyltransferase activity associated with LIG1K126me2 and H3K9me2. Collectively, these studies identify UHRF1 as a selective reader of LIG1K126me2 in vitro and further implicate the histone and non-histone methyllysine reader activity of the UHRF1 TTD as a dispensable domain function for cancer cell DNA methylation maintenance.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Código das Histonas , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/química , Epigênese Genética , Células HCT116 , Células HeLa , Histonas/química , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Processamento de Proteína Pós-Traducional , Domínio Tudor , Ubiquitina-Proteína Ligases/químicaRESUMO
The aberrant expression of protein arginine methyltransferase 5 (PRMT5) has been associated with multiple cancers. Using the proteolysis targeting chimera technology, we discovered a first-in-class PRMT5 degrader 15 (MS4322). Here, we report the design, synthesis, and characterization of compound 15 and two structurally similar controls 17 (MS4370) and 21 (MS4369), with impaired binding to the von Hippel-Lindau E3 ligase and PRMT5, respectively. Compound 15, but not 17 and 21, effectively reduced the PRMT5 protein level in MCF-7 cells. Our mechanism studies indicate that compound 15 degraded PRMT5 in an E3 ligase- and proteasome-dependent manner. Compound 15 also effectively reduced the PRMT5 protein level and inhibited growth in multiple cancer cell lines. Moreover, compound 15 was highly selective for PRMT5 in a global proteomic study and exhibited good plasma exposure in mice. Collectively, compound 15 and its two controls 17 and 21 are valuable chemical tools for exploring the PRMT5 functions in health and disease.
Assuntos
Dipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Dipeptídeos/síntese química , Dipeptídeos/farmacocinética , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Humanos , Ligantes , Masculino , Camundongos , Estrutura Molecular , Proteína-Arginina N-Metiltransferases/metabolismo , Proteólise/efeitos dos fármacos , Relação Estrutura-Atividade , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
PRMT5 participates in various cellular processes, including transcription regulation, signal transduction, mRNA splicing, and DNA repair; however, its mechanism of regulation is poorly understood. Here, we demonstrate that PRMT5 is phosphorylated at residue Y324 by Src kinase, a negative regulator of its activity. Either phosphorylation or substitution of the Y324 residue suppresses PRMT5 activity by preventing its binding with the methyl donor S-adenosyl-L-methionine. Additionally, we show that PRMT5 activity is associated with non-homologous end joining (NHEJ) repair by methylating and stabilizing p53-binding protein 1 (53BP1), which promotes cellular survival after DNA damage. Src-mediated phosphorylation of PRMT5 and the subsequent inhibition of its activity during the DNA damage process blocks NHEJ repair, leading to apoptotic cell death. Altogether, our findings suggest that PRMT5 regulates DNA repair through Src-mediated Y324 phosphorylation in response to DNA damage.
Assuntos
Neoplasias/genética , Proteína-Arginina N-Metiltransferases/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Quinases da Família src/genética , Células A549 , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , Metilação de DNA/genética , Células HeLa , Histonas/genética , Humanos , Células MCF-7 , Neoplasias/patologia , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional/genéticaRESUMO
Somatic mutations affecting CREBBP and EP300 are a hallmark of diffuse large B-cell lymphoma (DLBCL). These mutations are frequently monoallelic, within the histone acetyltransferase (HAT) domain and usually mutually exclusive, suggesting that they might affect a common pathway, and their residual WT expression is required for cell survival. Using in vitro and in vivo models, we found that inhibition of CARM1 activity (CARM1i) slows DLBCL growth, and that the levels of sensitivity are positively correlated with the CREBBP/EP300 mutation load. Conversely, treatment of DLBCLs that do not have CREBBP/EP300 mutations with CARM1i and a CBP/p300 inhibitor revealed a strong synergistic effect. Our mechanistic data show that CARM1i further reduces the HAT activity of CBP genome wide and downregulates CBP-target genes in DLBCL cells, resulting in a synthetic lethality that leverages the mutational status of CREBBP/EP300 as a biomarker for the use of small-molecule inhibitors of CARM1 in DLBCL and other cancers.