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
Proper protein arginine methylation by protein arginine methyltransferase 1 (PRMT1) is critical for maintaining cellular health, while dysregulation is often associated with disease. How the activity of PRMT1 is regulated is therefore paramount, but is not clearly understood. Several studies have observed higher order oligomeric species of PRMT1, but it is unclear if these exist at physiological concentrations and there is confusion in the literature about how oligomerization affects activity. We therefore sought to determine which oligomeric species of PRMT1 are physiologically relevant, and quantitatively correlate activity with specific oligomer forms. Through quantitative western blotting, we determined that concentrations of PRMT1 available in a variety of human cell lines are in the sub-micromolar to low micromolar range. Isothermal spectral shift binding data were modeled to a monomer/dimer/tetramer equilibrium with an EC50 for tetramer dissociation of ~20 nM. A combination of sedimentation velocity and Native polyacrylamide gel electrophoresis experiments directly confirmed that the major oligomeric species of PRMT1 at physiological concentrations would be dimers and tetramers. Surprisingly, the methyltransferase activity of a dimeric PRMT1 variant is similar to wild type, tetrameric PRMT1 with some purified substrates, but dimer and tetramer forms of PRMT1 show differences in catalytic efficiencies and substrate specificity for other substrates. Our results define an oligomerization paradigm for PRMT1, show that the biophysical characteristics of PRMT1 are poised to support a monomer/dimer/tetramer equilibrium in vivo, and suggest that the oligomeric state of PRMT1 could be used to regulate substrate specificity.
Assuntos
Multimerização Proteica , Proteína-Arginina N-Metiltransferases , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Humanos , Especificidade por Substrato , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , MetilaçãoRESUMO
Invasion and migration are the key hallmarks of cancer, and aggressive growth is a major factor contributing to treatment failure and poor prognosis in glioblastoma. Protein arginine methyltransferase 6 (PRMT6), as an epigenetic regulator, has been confirmed to promote the malignant proliferation of glioblastoma cells in previous studies. However, the effects of PRMT6 on glioblastoma cell invasion and migration and its underlying mechanisms remain elusive. Here, we report that PRMT6 functions as a driver element for tumor cell invasion and migration in glioblastoma. Bioinformatics analysis and glioma sample detection results demonstrated that PRMT6 is highly expressed in mesenchymal subtype or invasive gliomas, and is significantly negatively correlated with their prognosis. Inhibition of PRMT6 (using PRMT6 shRNA or inhibitor EPZ020411) reduces glioblastoma cell invasion and migration in vitro, whereas overexpression of PRMT6 produces opposite effects. Then, we identified that PRMT6 maintains the protein stability of EZH2 by inhibiting the degradation of EZH2 protein, thereby mediating the invasion and migration of glioblastoma cells. Further mechanistic investigations found that PRMT6 inhibits the transcription of TRAF6 by activating the histone methylation mark (H3R2me2a), and reducing the interaction between TRAF6 and EZH2 to enhance the protein stability of EZH2 in glioblastoma cells. Xenograft tumor assay and HE staining results showed that the expression of PRMT6 could promote the invasion of glioblastoma cells in vivo, the immunohistochemical staining results of mouse brain tissue tumor sections also confirmed the regulatory relationship between PRMT6, TRAF6, and EZH2. Our findings illustrate that PRMT6 suppresses TRAF6 transcription via H3R2me2a to enhance the protein stability of EZH2 to facilitate glioblastoma cell invasion and migration. Blocking the PRMT6-TRAF6-EZH2 axis is a promising strategy for inhibiting glioblastoma cell invasion and migration.
Assuntos
Movimento Celular , Proteína Potenciadora do Homólogo 2 de Zeste , Glioblastoma , Invasividade Neoplásica , Estabilidade Proteica , Proteína-Arginina N-Metiltransferases , Ubiquitinação , Humanos , Glioblastoma/patologia , Glioblastoma/metabolismo , Glioblastoma/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Animais , Linhagem Celular Tumoral , Camundongos , Fator 6 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Camundongos Nus , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/genética , Regulação Neoplásica da Expressão Gênica , Masculino , Proteólise , Feminino , Camundongos Endogâmicos BALB C , Proteínas NuclearesRESUMO
PRMT5 is a widely expressed arginine methyltransferase that regulates processes involved in tumor cell proliferation and survival. In the study described here, we investigated whether PRMT5 provides a target for tumor radiosensitization. Knockdown of PRMT5 using siRNA enhanced the radiosensitivity of a panel of cell lines corresponding to tumor types typically treated with radiotherapy. To extend these studies to an experimental therapeutic setting, the PRMT5 inhibitor LLY-283 was used. Exposure of the tumor cell lines to LLY-283 decreased PRMT5 activity and enhanced their radiosensitivity. This increase in radiosensitivity was accompanied by an inhibition of DNA double-strand break repair as determined by γH2AX foci and neutral comet analyses. For a normal fibroblast cell line, although LLY-283 reduced PRMT5 activity, it had no effect on their radiosensitivity. Transcriptome analysis of U251 cells showed that LLY-283 treatment reduced the expression of genes and altered the mRNA splicing pattern of genes involved in the DNA damage response. Subcutaneous xenografts were then used to evaluate the in vivo response to LLY-283 and radiation. Treatment of mice with LLY-283 decreased tumor PRMT5 activity and significantly enhanced the radiation-induced growth delay. These results suggest that PRMT5 is a tumor selective target for radiosensitization.
Assuntos
Proteína-Arginina N-Metiltransferases , Tolerância a Radiação , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Animais , Humanos , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/genética , Linhagem Celular Tumoral , Camundongos , Reparo do DNA , Proliferação de Células/efeitos da radiação , Ensaios Antitumorais Modelo de Xenoenxerto , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Camundongos NusRESUMO
Non-alcoholic fatty liver disease (NAFLD) has emerged as the leading cause of chronic liver disease worldwide. Caspase 8 and FADD-like apoptosis regulator (CFLAR) has been identified as a potent factor in mitigating non-alcoholic steatohepatitis (NASH) by inhibiting the N-terminal dimerization of apoptosis signal-regulating kinase 1 (ASK1). While arginine methyltransferase 1 (PRMT1) was previously reported to be associated with increased hepatic glucose production, its involvement in hepatic lipid metabolism remains largely unexplored. The interaction between PRMT1 and CFLAR and the methylation of CFLAR were verified by Co-IP and immunoblotting assays. Recombinant adenoviruses were generated for overexpression or knockdown of PRMT1 in hepatocytes. The role of PRMT1 in NAFLD was investigated in normal and high-fat diet-induced obese mice. In this study, we found a significant upregulation of PRMT1 and downregulation of CFLAR after 48h of fasting, while the latter significantly rebounded after 12h of refeeding. The expression of PRMT1 increased in the livers of mice fed a methionine choline-deficient (MCD) diet and in hepatocytes challenged with oleic acid (OA)/palmitic acid (PA). Overexpression of PRMT1 not only inhibited the expression of genes involved in fatty acid oxidation (FAO) and promoted the expression of genes involved in fatty acid synthesis (FAS), resulting in increased triglyceride accumulation in primary hepatocytes, but also enhanced the gluconeogenesis of primary hepatocytes. Conversely, knockdown of hepatic PRMT1 significantly alleviated MCD diet-induced hepatic lipid metabolism abnormalities and liver injury in vivo, possibly through the upregulation of CFLAR protein levels. Knockdown of PRMT1 suppressed the expression of genes related to FAS and enhanced the expression of genes involved in FAO, causing decreased triglyceride accumulation in OA/PA-treated primary hepatocytes in vitro. Although short-term overexpression of PRMT1 had no significant effect on hepatic triglyceride levels under physiological conditions, it resulted in increased serum triglyceride and fasting blood glucose levels in normal C57BL/6J mice. More importantly, PRMT1 was observed to interact with and methylate CFLAR, ultimately leading to its ubiquitination-mediated protein degradation. This process subsequently triggered the activation of c-Jun N-terminal kinase 1 (JNK1) and lipid deposition in primary hepatocytes. Together, these results suggested that PRMT1-mediated methylation of CFLAR plays a critical role in hepatic lipid metabolism. Targeting PRMT1 for drug design may represent a promising strategy for the treatment of NAFLD.
Assuntos
Hepatócitos , Metabolismo dos Lipídeos , Fígado , Hepatopatia Gordurosa não Alcoólica , Proteína-Arginina N-Metiltransferases , Animais , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Fígado/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Hepatócitos/metabolismo , Metilação , Masculino , Dieta Hiperlipídica/efeitos adversos , Humanos , Camundongos Endogâmicos C57BL , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genéticaRESUMO
Purpose: Due to intrinsic defensive response, ferroptosis-activating targeted therapy fails to achieve satisfactory clinical benefits. Though p62-Keap1-Nrf2 axis is activated to form a negative feedback loop during ferroptosis induction, how p62 is activated remains largely unknown. Methods: MTS assay was applied to measure cell growth. Lipid ROS was detected with C11-BODIPY reagent by flow cytometer. Quantitative real-time PCR (qPCR) and western blotting were performed to determine mRNA and protein level. Immunofluorescence (IF) was performed to examine the distribution of proteins. Fluorescence recovery after photobleaching (FRAP) was adopted to evaluate p62 phase separation. Immunoprecipitation (IP), co-IP and Proximal ligation assay (PLA) were performed to detected protein posttranslational modifications and protein-protein interactions. Tumor xenograft model was employed to inspect in vivo growth of pancreatic cancer cells. Results: Upon ferroptosis induction, Nuclear Factor E2 Related Factor 2 (Nrf2) protein and its downstream genes such as HMOX1 and NQO1 were upregulated. Knockdown of p62 significantly reversed Nrf2 upregulation and Keap1 decrease after ferroptosis induction. Knockdown of either p62 or Nrf2 remarkably sensitized ferroptosis induction. Due to augmented p62 phase separation, formation of p62 bodies were increased to recruit Keap1 after ferroptosis induction. Protein arginine methyltransferase 6 (PRMT6) mediated asymmetric dimethylarginine (ADMA) of p62 to increase its oligomerization, promoting p62 phase separation and p62 body formation. Knockdown of p62 or PRMT6 notably sensitized pancreatic cancer cells to ferroptosis both in vitro and in vivo through suppressing Nrf2 signaling. Conclusion: During ferroptosis induction, PRMT6 mediated p62 ADMA to promote its phase separation, sequestering Keap1 to activate Nrf2 signaling and inhibit ferroptosis. Therefore, targeting PRMT6-mediated p62 ADMA could be a new option to sensitize ferroptosis for cancer treatment.
Assuntos
Arginina , Ferroptose , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2 , Proteína-Arginina N-Metiltransferases , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Humanos , Animais , Arginina/metabolismo , Arginina/análogos & derivados , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Camundongos , Linhagem Celular Tumoral , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Retroalimentação Fisiológica , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Proteína Sequestossoma-1/metabolismo , Proteína Sequestossoma-1/genética , Camundongos Nus , Transdução de Sinais , Separação de Fases , Proteínas de Ligação a RNARESUMO
OBJECTIVES: Obesity related glomerulopathy (ORG) is induced by obesity, but the pathogenesis remains unclear. This study aims to investigate the expression of early growth response protein 3 (EGR3) in the renal cortex tissues of ORG patients and high-fat diet-induced obese mice, and to further explore the molecular mechanism of EGR3 in inhibiting palmitic acid (PA) induced human podocyte inflammatory damage. METHODS: Renal cortex tissues were collected from ORG patients (n=6) who have been excluded from kidney damage caused by other diseases and confirmed by histopathology, and from obese mice induced by high-fat diet (n=10). Human and mouse podocytes were intervened with 150 µmol/L PA for 48 hours. EGR3 was overexpressed or silenced in human podocytes. Enzyme linked immunosorbent assay (ELISA) was used to detcet the levels of interleukin-6 (IL-6) and interleukin-1ß (IL-1ß). Real-time RT-PCR was used to detect the mRNA expressions of EGR3, podocytes molecular markers nephrosis 1 (NPHS1), nephrosis 2 (NPHS2), podocalyxin (PODXL), and podoplanin (PDPN). RNA-seq was performed to detect differentially expressed genes (DEGs) after human podocytes overexpressing EGR3 and treated with 150 µmol/L PA compared with the control group. Co-immunoprecipitation (Co-IP) combined with liquid chromatography tandem mass spectrometry (LC-MS) was used to detect potential interacting proteins of EGR3 and the intersected with the RNA-seq results. Co-IP confirmed the interaction between EGR3 and protein arginine methyltransferases 1 (PRMT1), after silencing EGR3 and PRMT1 inhibitor intervention, the secretion of IL-6 and IL-1ß in PA-induced podocytes was detected. Western blotting was used to detect the expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) after overexpression or silencing of EGR3. RESULTS: EGR3 was significantly upregulated in renal cortex tissues of ORG patients and high-fat diet-induced obese mice (both P<0.01). In addition, after treating with 150 µmol/L PA for 48 hours, the expression of EGR3 in human and mouse podocytes was significantly upregulated (both P<0.05). Overexpression or silencing of EGR3 in human podocytes inhibited or promoted the secretion of IL-6 and IL-1ß in the cell culture supernatant after PA intervention, respectively, and upregulated or downregulated the expression of NPHS1, PODXL, NPHS2,and PDPN (all P<0.05). RNA-seq showed a total of 988 DEGs, and Co-IP+LC-MS identified a total of 238 proteins that may interact with EGR3. Co-IP confirmed that PRMT1 was an interacting protein with EGR3. Furthermore, PRMT1 inhibitors could partially reduce PA-induced IL-6 and IL-1ß secretion after EGR3 silencing in human podocytes (both P<0.05). Overexpression or silencing of EGR3 negatively regulated the expression of PRMT1 and p-STAT3. CONCLUSIONS: EGR3 may reduce ORG podocyte inflammatory damage by inhibiting the PRMT1/p-STAT3 pathway.
Assuntos
Proteína 3 de Resposta de Crescimento Precoce , Obesidade , Podócitos , Proteína-Arginina N-Metiltransferases , Proteínas Repressoras , Fator de Transcrição STAT3 , Animais , Humanos , Masculino , Camundongos , Dieta Hiperlipídica/efeitos adversos , Proteína 3 de Resposta de Crescimento Precoce/metabolismo , Proteína 3 de Resposta de Crescimento Precoce/genética , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-6/genética , Córtex Renal/metabolismo , Córtex Renal/patologia , Nefropatias/metabolismo , Nefropatias/etiologia , Nefropatias/patologia , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/complicações , Obesidade/metabolismo , Ácido Palmítico/farmacologia , Podócitos/metabolismo , Podócitos/patologia , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais , Fator de Transcrição STAT3/metabolismoRESUMO
Cardiovascular diseases (CVDs) and metabolic disorders stand as formidable challenges that significantly impact the clinical outcomes and living quality for afflicted individuals. An intricate comprehension of the underlying mechanisms is paramount for the development of efficacious therapeutic strategies. Protein arginine methyltransferases (PRMTs), a class of enzymes responsible for the precise regulation of protein methylation, have ascended to pivotal roles and emerged as crucial regulators within the intrinsic pathophysiology of these diseases. Herein, we review recent advancements in research elucidating on the multifaceted involvements of PRMTs in cardiovascular system and metabolic diseases, contributing significantly to deepen our understanding of the pathogenesis and progression of these maladies. In addition, this review provides a comprehensive analysis to unveil the distinctive roles of PRMTs across diverse cell types implicated in cardiovascular and metabolic disorders, which holds great potential to reveal novel therapeutic interventions targeting PRMTs, thus presenting promising perspectives to effectively address the substantial global burden imposed by CVDs and metabolic disorders.
Assuntos
Doenças Cardiovasculares , Doenças Metabólicas , Proteína-Arginina N-Metiltransferases , Humanos , Doenças Cardiovasculares/metabolismo , Doenças Metabólicas/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Animais , MetilaçãoRESUMO
Abnormal epigenetic modifications are involved in the regulation of Warburg effect in tumor cells. Protein arginine methyltransferases (PRMTs) mediate arginine methylation and have critical functions in cellular responses. PRMTs are deregulated in a variety of cancers, but their precise roles in Warburg effect in cancer is largely unknown. Experiments from the current study showed that PRMT1 was highly expressed under conditions of glucose sufficiency. PRMT1 induced an increase in the PKM2/PKM1 ratio through upregulation of PTBP1, in turn, promoting aerobic glycolysis in non-small cell lung cancer (NSCLC). The PRMT1 level in p53-deficient and p53-mutated NSCLC remained relatively unchanged while the expression was reduced in p53 wild-type NSCLC under conditions of glucose insufficiency. Notably, p53 activation under glucose-deficient conditions could suppress USP7 and further accelerate the polyubiquitin-dependent degradation of PRMT1. Melatonin, a hormone that inhibits glucose intake, markedly suppressed cell proliferation of p53 wild-type NSCLC, while a combination of melatonin and the USP7 inhibitor P5091 enhanced the anticancer activity in p53-deficient NSCLC. Our collective findings support a role of PRMT1 in the regulation of Warburg effect in NSCLC. Moreover, combination treatment with melatonin and the USP7 inhibitor showed good efficacy, providing a rationale for the development of PRMT1-based therapy to improve p53-deficient NSCLC outcomes.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Proteínas de Membrana , Proteína-Arginina N-Metiltransferases , Proteínas de Ligação a Hormônio da Tireoide , Hormônios Tireóideos , Proteína Supressora de Tumor p53 , Efeito Warburg em Oncologia , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Humanos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/tratamento farmacológico , Efeito Warburg em Oncologia/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Hormônios Tireóideos/metabolismo , Linhagem Celular Tumoral , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proliferação de Células/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Peptidase 7 Específica de Ubiquitina/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Animais , Glicólise/efeitos dos fármacos , Camundongos Nus , Glucose/metabolismo , Camundongos , Regulação Neoplásica da Expressão Gênica , Células A549 , Proteína de Ligação a Regiões Ricas em PolipirimidinasRESUMO
Gastric cancer (GC) is a common form of cancer and the leading cause of cancer-related deaths worldwide. Chemotherapy is the primary treatment for patients with unresectable or partially resectable GC. However, its adverse effects and chemoresistance greatly restrict its applicability and efficacy. Although HER2-targeted therapy and immunotherapy have been successfully used for GC treatment, their beneficial population is limited. To expand the range of cancer treatments, drug repurposing has emerged as a promising strategy. In this study, we evaluated the potential of Metformin, an oral anti-hyperglycemic agent, to suppress GC progression both in vivo and in vitro. Functional investigations showed that Metformin significantly inhibits GC proliferation and migration. Furthermore, we discovered that Metformin bound and disrupted STAT1 phosphorylation, inhibiting PRMT1 expression and consequently GC progression. In conclusion, our study not only provides further evidence for the anti-GC role of Metformin but also identifies the direct target mediating the tumor-inhibitory effects of Metformin in GC.
Assuntos
Metformina , Camundongos Nus , Proteína-Arginina N-Metiltransferases , Proteínas Repressoras , Fator de Transcrição STAT1 , Neoplasias Gástricas , Animais , Humanos , Masculino , Camundongos , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Metformina/uso terapêutico , Camundongos Endogâmicos BALB C , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/antagonistas & inibidores , Fator de Transcrição STAT1/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma. The oncogene product Tax of HTLV-I is thought to play crucial roles in leukemogenesis by promoting proliferation of the virus-infected cells through activation of growth-promoting genes. These genes code for growth factors and their receptors, cytokines, cell adhesion molecules, growth signal transducers, transcription factors and cell cycle regulators. We show here that Tax activates the gene coding for coactivator-associated arginine methyltransferase 1 (CARM1), which epigenetically enhances gene expression through methylation of histones. Tax activated the Carm1 gene and increased protein expression, not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs). Tax increased R17-methylated histone H3 on the target gene IL-2Rα, concomitant with increased expression of CARM1. Short hairpin RNA (shRNA)-mediated knockdown of CARM1 decreased Tax-mediated induction of IL-2Rα and Cyclin D2 gene expression, reduced E2F activation and inhibited cell cycle progression. Tax acted via response elements in intron 1 of the Carm1 gene, through the NF-κB pathway. These results suggest that Tax-mediated activation of the Carm1 gene contributes to leukemogenic target-gene expression and cell cycle progression, identifying the first epigenetic target gene for Tax-mediated trans-activation in cell growth promotion.
Assuntos
Produtos do Gene tax , Vírus Linfotrópico T Tipo 1 Humano , Proteína-Arginina N-Metiltransferases , Humanos , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Produtos do Gene tax/genética , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/genética , Ciclina D2/genética , Ciclina D2/metabolismo , Ativação Transcricional , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , NF-kappa B/metabolismo , NF-kappa B/genética , Histonas/metabolismo , Histonas/genética , Epigênese Genética , Células JurkatRESUMO
Arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and is involved in various cellular processes, including cancer development. PRMT2 expression is increased in several cancer types although its role in acute myeloid leukemia (AML) remains unknown. Here, we investigate the role of PRMT2 in a cohort of patients with AML, PRMT2 knockout AML cell lines as well as a Prmt2 knockout mouse model. In patients, low PRMT2 expressors are enriched for inflammatory signatures, including the NF-κB pathway, and show inferior survival. In keeping with a role for PRMT2 in control of inflammatory signaling, bone marrow-derived macrophages from Prmt2 KO mice display increased pro-inflammatory cytokine signaling upon LPS treatment. In PRMT2-depleted AML cell lines, aberrant inflammatory signaling has been linked to overproduction of IL6, resulting from a deregulation of the NF-κB signaling pathway, therefore leading to hyperactivation of STAT3. Together, these findings identify PRMT2 as a key regulator of inflammation in AML.
Assuntos
Inflamação , Leucemia Mieloide Aguda , Camundongos Knockout , NF-kappa B , Proteína-Arginina N-Metiltransferases , Transdução de Sinais , Animais , Feminino , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Inflamação/metabolismo , Inflamação/genética , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genéticaRESUMO
The dynamic regulation of mitochondria through fission and fusion is essential for maintaining cellular homeostasis. In this study, we discovered a role of coactivator-associated arginine methyltransferase 1 (CARM1) in mitochondrial dynamics. CARM1 methylates specific residues (R403 and R634) on dynamin-related protein 1 (DRP1). Methylated DRP1 interacts with mitochondrial fission factor (Mff) and forms self-assembly on the outer mitochondrial membrane, thereby triggering fission, reducing oxygen consumption, and increasing reactive oxygen species (ROS) production. This sets in motion a feedback loop that facilitates the translocation of CARM1 from the nucleus to the cytoplasm, enhancing DRP1 methylation and ROS production through mitochondrial fragmentation. Consequently, ROS reinforces the CARM1-DRP1-ROS axis, resulting in cellular senescence. Depletion of CARM1 or DRP1 impedes cellular senescence by reducing ROS accumulation. The uncovering of the above-described mechanism fills a missing piece in the vicious cycle of ROS-induced senescence and contributes to a better understanding of the aging process.
Assuntos
Senescência Celular , Citoplasma , Dinaminas , Dinâmica Mitocondrial , Proteína-Arginina N-Metiltransferases , Espécies Reativas de Oxigênio , Dinaminas/metabolismo , Dinaminas/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Humanos , Espécies Reativas de Oxigênio/metabolismo , Metilação , Citoplasma/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas de MembranaRESUMO
DNA replication stress (RS) is a widespread phenomenon in carcinogenesis, causing genomic instability and extensive chromatin alterations. DNA damage leads to activation of innate immune signaling, but little is known about transcriptional regulators mediating such signaling upon RS. Using a chemical screen, we identified protein arginine methyltransferase 5 (PRMT5) as a key mediator of RS-dependent induction of interferon-stimulated genes (ISGs). This response is also associated with reactivation of endogenous retroviruses (ERVs). Using quantitative mass spectrometry, we identify proteins with PRMT5-dependent symmetric dimethylarginine (SDMA) modification induced upon RS. Among these, we show that PRMT5 targets and modulates the activity of ZNF326, a zinc finger protein essential for ISG response. Our data demonstrate a role for PRMT5-mediated SDMA in the context of RS-induced transcriptional induction, affecting physiological homeostasis and cancer therapy.
Assuntos
Replicação do DNA , Imunidade Inata , Proteína-Arginina N-Metiltransferases , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Humanos , Transdução de Sinais , Arginina/metabolismo , Arginina/análogos & derivados , Estresse Fisiológico , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Dano ao DNA , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Cancer cells are often addicted to serine synthesis to support growth. How serine synthesis is regulated in cancer is not well understood. We recently demonstrated protein arginine methyltransferase 1 (PRMT1) is upregulated in hepatocellular carcinoma (HCC) to methylate and activate phosphoglycerate dehydrogenase (PHGDH), thereby promoting serine synthesis. However, the mechanisms underlying PRMT1 upregulation and regulation of PRMT1-PHGDH axis remain unclear. Here, we show the E3 ubiquitin ligase F-box-only protein 7 (FBXO7) inhibits serine synthesis in HCC by binding PRMT1, inducing lysine 37 ubiquitination, and promoting proteosomal degradation of PRMT1. FBXO7-mediated PRMT1 downregulation cripples PHGDH arginine methylation and activation, resulting in impaired serine synthesis, accumulation of reactive oxygen species (ROS), and inhibition of HCC cell growth. Notably, FBXO7 is significantly downregulated in human HCC tissues, and inversely associated with PRMT1 protein and PHGDH methylation level. Overall, our study provides mechanistic insights into the regulation of cancer serine synthesis by FBXO7-PRMT1-PHGDH axis, and will facilitate the development of serine-targeting strategies for cancer therapy.
Assuntos
Carcinoma Hepatocelular , Proteínas F-Box , Neoplasias Hepáticas , Fosfoglicerato Desidrogenase , Proteína-Arginina N-Metiltransferases , Serina , Ubiquitinação , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Humanos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Serina/metabolismo , Serina/biossíntese , Fosfoglicerato Desidrogenase/metabolismo , Fosfoglicerato Desidrogenase/genética , Linhagem Celular Tumoral , Animais , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Camundongos , Proliferação de Células , Metilação , Regulação Neoplásica da Expressão Gênica , Camundongos Nus , Masculino , Células HEK293 , Feminino , Células Hep G2RESUMO
ß-Thalassemia is an inherited genetic disorder associated with ß-globin chain synthesis, which ultimately becomes anemia. Adenosine-2,3-dialdehyde, by inhibiting arginine methyl transferase 5 (PRMT5), can induce fetal hemoglobin (HbF) levels. Hence, the materialization of PRMT5 inhibitors is considered a promising therapy in the management of ß-thalassemia. This study conducted a virtual screening of certain compounds similar to 5'-deoxy-5'methyladenosine (3XV) using the PubChem database. The top 10 compounds were chosen based on the best docking scores, while their interactions with the PRMT5 active site were analyzed. Further, the top two compounds demonstrating the lowest binding energy were subjected to drug-likeness analysis and pharmacokinetic property predictions, followed by molecular dynamics simulation studies. Based on the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) score and molecular interactions, (3R,4S)-2-(6-aminopurin-9-yl)-5-[(4-ethylcyclohexyl)sulfanylmethyl]oxolane-3,4-diol (TOP1) and 2-(6-Aminopurin-9-yl)-5-[(6-aminopurin-9-yl)methylsulfanylmethyl]oxolane-3,4-diol (TOP2) were identified as potential hit compounds, while TOP1 exhibited higher binding affinity and stabler binding capabilities than TOP2 during molecular dynamics simulation (MDS) analysis. Taken together, the outcomes of our study could aid researchers in identifying promising PRMT5 inhibitors. Moreover, further investigations through in vivo and in vitro experiments would unquestionably confirm that this compound could be employed as a therapeutic drug in the management of ß-thalassemia.
Assuntos
Inibidores Enzimáticos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteína-Arginina N-Metiltransferases , Talassemia beta , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/metabolismo , Talassemia beta/tratamento farmacológico , Humanos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Descoberta de Drogas , Ligação Proteica , Domínio Catalítico , Adenosina/análogos & derivados , Adenosina/química , Adenosina/farmacologiaRESUMO
Gastric cancer is a most malignancy in digestive tract worldwide. This study aimed to investigate the roles of protein arginine methyltransferase 6 (PRMT6) in gastric cancer. Immunohistochemistry was performed to detect PRMT6 expression in gastric tumors. Real-time transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to detected mRNA levels. Protein expression was determined using western blot. Gastric cancer cells were co-cultured with CD8+ T cells. Colony formation assay was performed to detect cell proliferation. Flow cytometry was performed to determine CD8+ T cell function and tumor cell apoptosis. PRMT6 was overexpressed in gastric tumors. High level of PRMT6 predicted poor outcomes of gastric cancer patients and inhibition of CD8+ T cell infiltration. PRMT6 promoted proliferation of CD8+ T cells and enhanced its tumor killing ability. Moreover, PRMT6 upregulated annexin A1 (ANXA1) and promoted ANXA1 protein stability. ANXA1 overexpression suppressed the proliferation of CD8+ T cells and promoted tumor cell survival. PRMT6 functions as an oncogene in gastric cancer. PRMT6-mediated protein stability inhibits the infiltration of CD8+ T cells, resulting in immune evasion of gastric cancer. The PRMT6-ANXA1 may be a promising strategy for gastric cancer.
Assuntos
Anexina A1 , Linfócitos T CD8-Positivos , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Proteína-Arginina N-Metiltransferases , Neoplasias Gástricas , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/metabolismo , Humanos , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Anexina A1/genética , Anexina A1/metabolismo , Linhagem Celular Tumoral , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Regulação para Cima , Apoptose , Evasão Tumoral/genética , Masculino , Evasão da Resposta Imune , Feminino , Proteínas NuclearesRESUMO
Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that deposits asymmetrical dimethylation marks on both histone and nonhistone substrates. The regulatory role of CARM1 in transcription was first identified in estrogen receptor positive (ER+) breast cancer. Since then, the mechanism of CARM1 in activating ER-target genes has been further interrogated. CARM1 is expressed at the highest level in ER negative (ER-) breast cancer and higher expression correlates with poor prognosis, suggesting an oncogenic role of CARM1. Indeed, in ER- breast cancer, CARM1 can promote proliferation and metastasis at least partly through methylation of proteins and activation of oncogenes. In this review, we summarize the mechanisms of transcriptional activation by CARM1 in breast cancer. The methyltransferase activity of CARM1 is important for many of its functions; here, we also highlight the nonenzymatic roles of CARM1. We also cover the biological processes regulated by CARM1 that are often deregulated in cancer and the ways to harness CARM1 in cancer treatment.
Assuntos
Neoplasias da Mama , Proteína-Arginina N-Metiltransferases , Humanos , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Ativação Transcricional , AnimaisRESUMO
Background: Colorectal cancer (CRC) is one of the most frequently diagnosed cancers. In many cases, the poor prognosis of advanced CRC is associated with resistance to treatment with chemotherapeutic drugs such as 5-Fluorouracil (5-FU). The epithelial-to-mesenchymal transition (EMT) and dysregulation in protein methylation are two mechanisms associated with chemoresistance in many cancers. This study looked into the effect of 5-FU dose escalation on EMT and protein methylation in CRC. Materials and Methods: HCT-116, Caco-2, and DLD-1 CRC cell lines were exposed to dose escalation treatment of 5-FU. The motility and invasive potentials of the cells before and after treatment with 5-FU were investigated through wound healing and invasion assays. This was followed by a Western blot which analyzed the protein expressions of the epithelial marker E-cadherin, mesenchymal marker vimentin, and the EMT transcription factor (EMT-TF), the snail family transcriptional repressor 1 (Snail) in the parental and desensitized cells. Western blotting was also conducted to study the protein expressions of the protein methyltransferases (PMTs), Euchromatic histone lysine methyltransferase 2 (EHMT2/G9A), protein arginine methyltransferase (PRMT5), and SET domain containing 7/9 (SETD7/9) along with the global lysine and arginine methylation profiles. Results: The dose escalation method generated 5-FU desensitized CRC cells with distinct morphological features and increased tolerance to high doses of 5-FU. The 5-FU desensitized cells experienced a decrease in migration and invasion when compared to the parental cells. This was reflected in the observed reduction in E-cadherin, vimentin, and Snail in the desensitized cell lines. Additionally, the protein expressions of EHMT2/G9A, PRMT5, and SETD7/9 also decreased in the desensitized cells and global protein lysine and arginine methylation became dysregulated with 5-FU treatment. Conclusion: This study showed that continuous, dose-escalation treatment of 5-FU in CRC cells generated 5-FU desensitized cancer cells that seemed to be less aggressive than parental cells.
Assuntos
Movimento Celular , Neoplasias Colorretais , Transição Epitelial-Mesenquimal , Fluoruracila , Humanos , Fluoruracila/farmacologia , Fluoruracila/administração & dosagem , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Movimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Antimetabólitos Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Relação Dose-Resposta a Droga , Metiltransferases/metabolismo , Metiltransferases/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Metilação , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genéticaRESUMO
Background: The dysregulation of Isocitrate dehydrogenase (IDH) and the subsequent production of 2-Hydroxyglutrate (2HG) may alter the expression of epigenetic proteins in Grade 4 astrocytoma. The interplay mechanism between IDH, O-6-methylguanine-DNA methyltransferase (MGMT)-promoter methylation, and protein methyltransferase proteins-5 (PRMT5) activity, with tumor progression has never been described. Methods: A retrospective cohort of 34 patients with G4 astrocytoma is classified into IDH-mutant and IDH-wildtype tumors. Both groups were tested for MGMT-promoter methylation and PRMT5 through methylation-specific and gene expression PCR analysis. Inter-cohort statistical significance was evaluated. Results: Both IDH-mutant WHO grade 4 astrocytomas (n = 22, 64.7%) and IDH-wildtype glioblastomas (n = 12, 35.3%) had upregulated PRMT5 gene expression except in one case. Out of the 22 IDH-mutant tumors, 10 (45.5%) tumors showed MGMT-promoter methylation and 12 (54.5%) tumors had unmethylated MGMT. All IDH-wildtype tumors had unmethylated MGMT. There was a statistically significant relationship between MGMT-promoter methylation and IDH in G4 astrocytoma (p-value = 0.006). Statistically significant differences in progression-free survival (PFS) were also observed among all G4 astrocytomas that expressed PRMT5 and received either temozolomide (TMZ) or TMZ plus other chemotherapies, regardless of their IDH or MGMT-methylation status (p-value=0.0014). Specifically, IDH-mutant tumors that had upregulated PRMT5 activity and MGMT-promoter methylation, who received only TMZ, have exhibited longer PFS. Conclusions: The relationship between PRMT5, MGMT-promoter, and IDH is not tri-directional. However, accumulation of D2-hydroxyglutarate (2-HG), which partially activates 2-OG-dependent deoxygenase, may not affect their activities. In IDH-wildtype glioblastomas, the 2HG-2OG pathway is typically inactive, leading to PRMT5 upregulation. TMZ alone, compared to TMZ-plus, can increase PFS in upregulated PRMT5 tumors. Thus, using a PRMT5 inhibitor in G4 astrocytomas may help in tumor regression.